Molecular investigation of genetic variation to
improve uniformity of cauliflower production
Ida Ayu Astarini
B.Sc. (Hort), Bogor Agricultural University, West Java, Indonesia
M.Sc. (Hort), The University of Western Australia, Perth, WA
This thesis is presented for the degree of
Doctor of Philosophy
of The University of Western Australia
School of Plant Biology
Faculty of Natural and Agricultural Sciences
2006
Abstract
Cauliflower production in Australia is export oriented. The industry aims for
uniform, high quality product and is based on Fi hybrid cauliflower cultivars. However
non-uniform products still occur. The lack of uniformity may be due to genetic
variation evident in the seed, seedling or production stage.
There are many cauliflower cultivars released each year and there is a need to
correctly identify cultivars for seed companies and growers. W e developed
fingerprinting keys using molecular markers to correctly identify cultivars at any stage,
and therefore without the need for field trials. Australian Fi hybrid cultivars and open
pollinated cultivars of Indonesia were assessed genetically using R A P D markers.
Genetic variation within and between cultivars of both countries were investigated.
Dendograms were constructed using Neighbor-Joining analysis based on Phylogenetic
Analysis Using Parsimony (PAUP). D N A fingerprinting keys were developed and
genetic relationships among cultivars were determined. Comparison between
Indonesian and Australian based cultivars indicated that Indonesian cultivars have
unique genotypes and would be good sources of genes for future crop improvement.
Results proved that R A P D markers can be used for the routine identification of
cauliflower cultivars.
Hybrid cauliflowers have been developed to exploit heterosis and to improve
uniformity of production. However, 'morphological sib' plants, assumed to be self
inbred, often contaminate hybrid seed lots in the SI system and contribute up to 2 0 % of
the total harvest. Sibs produce very small and non-marketable curds. Whilst self
inbreeding is not possible in the C M S system, plants that look like sibs often occur up to
4 0 % of the crop. In this study, microsatellite markers for male and female cauliflower
parent lines of both SI and C M S systems were developed. T w o pairs of markers were
chosen for purity testing of F] hybrid seeds. Microsatellite analysis, glasshouse and
field trials confirmed that morphological sib plants from the SI system were not always
self-inbred. In contrast, most self inbred plants showed normal growth. All
morphological sibs from the C M S system showed hybrid bands. This suggests that
morphological sibs were not always due to selfing but possibly to an interaction
between genetic and environmental factors and this requires further investigation.
Variation in curd maturity which results in spread of harvesting time is another
problem in the cauliflower industry and contributes to up to a 2 0 % loss. This
phenomenon prevents the use of harvest machinery and increases cost of harvesting as
i
several manual harvests are required. Morphological variation from seed to harvest is
due to genetic variation interacting with environmental conditions and here the genetic
factors were investigated using R A P D markers. Multivariate analysis based on
principle coordinates analysis was employed to correlate morphological traits with
molecular markers across cultivars. Markers associated with seed weight, germination
rate, shoot length, root length, fresh weight and dry weight were identified.
In summary, successful application of molecular markers to screen cauliflower
plants in every stage of production, from choosing the right cultivar, screening for
particular traits to reduce seedling variability, and screening for abnormality will
significantly improve uniformity in cauliflower production.
ii
Declaration
I declare that this thesis contains no experimental materials that have been previously
presented for any degree at any other university or institution.
I and my supervisors A/Prof Julie Plummer, Dr Guijun Yan and Ms Rachel Lancaster
discussed and decided the research topics and techniques. The preparation of this
thesis, including published papers and manuscript was done by myself with feedback
from m y supervisors.
March, 2006
Ida Ayu Astarini
iii
Dedication
I dedicated this thesis to the memory of my father, the late Dr Ida Bagus
Astawa, M.P.H. Although you never lived long enough to see me through
this study, your encouragement has always been a guiding light in my life.
IV
Acknowledgments
First and foremost, I express my sincere thanks to my principal supervisor
Associate Professor Julie Plummer for her advice, patience, time, effort and supervision
during m y study. There is so much I learnt from her, not only supervision for m y
projects, but also beyond m y PhD study. Sincere thanks to m y supervisors Dr Guijun
Yan for his encouragement and guidance throughout m y study and M s Rachel Lancaster
for her suggestions and valuable feedback on m y projects, comprehensive information
on cauliflower production and access to Manjimup Horticulture Research Institute.
Thorough and detailed supervision from all of them has made m y study a success.
Financial support was generous and I would like to thank AusAID and
Department of Agriculture Western Australia for supporting this study. M y special
thanks to M s Rhonda Haskell, the AusAID liaison officer for being very supportive and
helpful during m y study.
I thank M s Anouska Cousins for technical guidance, Dr Matthew Nelson for
advice on microsatellite technique and help to make m y visit to Dutch seed companies
possible. Thank you to Associate Professor Wallace Cowling for useful advice and
comments on m y projects. Also, the Plant Breeding and Molecular Genetics Discussion
Group has broadened up m y knowledge about plant breeding. A Field trial in Manjimup
would not have been possible without the kind assistance from M r John Doust, Dr
Kristen Stirling, M r David Tooke and Grazi.
Thank you to Henderson Seeds, Enza Zaden Australia, South Pacific Seeds,
Syngenta Seeds, Lefroy Valley Seeds and Bejo Seeds for supplying seeds. Kind help
from Ahmad Rivani, Sitawati, Agus Suryanto, Dewa Okayadnya and Professor IGP
Wirawan during sample collection in Indonesia was greatly appreciated.
M y colleagues in room 2.105, Leida, Nic, Cam, Chris, Claire, Nader, Leila,
Bambang and Sharmin, it has been a fun experience sharing a study room with all of
you. I improved m y English and learned different cultures. For m y fellow Indonesian
students, Ila, Anne, Suzie, Ndari, Iin, Ita, Pharma and Titik, your companionship has
made this study an enjoyable experience.
To m y m u m , thank you for love and support. Special thanks to m y parents in
law, Peranda Gede and Peranda Istri, for always praying for m y success. M y sons,
Guntur and Ari, you are the best kids in the world!
Finally, to m y husband, Ida Bagus Gunawan, your endless support,
understanding and sacrifice has nothing to compare. Thank you!!
v
Thesis Outline
This thesis consists of 8 chapters. The first chapter contains background
information about problems in cauliflower production around the world, with particular
reference to the Western Australian cauliflower industry. This chapter also introduces
the usefulness of molecular techniques in crop improvement programs. The second
chapter, Literature Review, contains details about cauliflower with emphasis on
breeding systems and justification of current molecular techniques used in this study
and successfully applied in Brassica and other vegetable crops.
The research program consists of five projects completed during m y P h D
candidature. These are presented in Chapters 3-7. Chapter 3 is on the development of
a fingerprinting technique using R A P D marker systems on hybrid cultivars commonly
grown in Australia. This paper has been published in the Australian Journal of
Agricultural Research. In Chapter 4, an extension of the R A P D fingerprinting
technique was applied in open pollinated cultivars from Indonesia. The paper was
presented at an International/Australian Society of Horticultural Science conference
entitled "Harnessing the Potential of Horticulture in the Asian-Pacific region in Coolum,
Queensland, 1 - 3 September 2004, where it was awarded 'Young Scientist Award' for
the best student presentation. The paper has been published in Acta Horticulturae
(2005) 694, 149-152. In Chapter 5, genetic distance between Indonesian and Australian
cultivars were revealed. The paper from this chapter has been accepted in Scientia
Horticulturae.
In Chapter 6, the use of R A P D and microsatellites to identify a marker to
distinguish between male and female parent lines, hybrid and non-hybrid (commonly
known as 'sibs') plants was investigated. The manuscript of this chapter is currently
under review in Theoretical and Applied Genetics. In Chapter 7, the association
between molecular markers and morphological traits of cauliflower seedlings was
investigated. A number of associations were found and these may be useful in
molecular-assisted selection in breeding programs. A manuscript of this chapter is
currently under review with the Australian Journal of Agricultural Research.
Chapter 8 is the General Discussion of the thesis. All chapters/papers are
brought together and justify the molecular techniques employed to assist in improving
uniformity of cauliflower production.
vi
List of Publications, Conferences Attended and Awards
Publications
Astarini IA, Plummer JA, Yan G, Lancaster R A (2006) 'Sib' plants in hybrid
cauliflowers may be hybrid or self-inbred progeny. Proceeding 13 Australasian
Plant Breeding Conference. Christchurch, N e w Zealand (accepted).
Astarini IA, Plummer JA, Yan G, Lancaster R A (2006) Molecular markers correlated
with morphological traits in cauliflower seedlings. Australian Journal of
Agricultural Research (under review).
Astarini IA, Plummer JA, Yan G, Lancaster R A (2006) Identification of 'sib' plants in
hybrid cauliflowers using microsatellite markers. Theoretical and Applied
Genetics (under review).
Astarini IA, Plummer JA, Yan G, Lancaster R A (2006) Genetic diversity of Indonesia
cauliflowers and their relationship with Australian grown hybrid cultivars.
Scientia Horticulturae 108, 143-150.
Astarini IA, Plummer JA, Yan G, Lancaster R A (2005) Genetic diversity of open
pollinated cauliflower cultivars in Indonesia. Acta Horticulturae 694, 149-152.
Astarini IA, Plummer JA, Yan G, Lancaster R A (2004) Fingerprinting of cauliflower
cultivars using R A P D markers. Australian Journal of Agricultural Research 55,
117-124.
Conferences attended and visits
1. The 12th Australasian Plant Breeding Conference, 15-20 September 2002, Perth,
Western Australia
2. International/Australian Society of Horticultural Science (ISHS/AUSHS)
Conference: Harnessing the Potential of Horticulture in the Asian-Pacific
Region, 1-3 September 2004, Coolum, Queensland.
3. ComBio 2004,26-30 September 2004, Perth, Western Australia.
4. International Society of Horticultural Sciences (ISHS) Symposium on Brassicas,
24-28 October 2004, Daejeon, South Korea.
5. Visits to Seminis Vegetable Seeds and Enza Zaden, Enkhuizen, The
Netherlands, 1-3 November 2004.
vii
Awards
1. Young Scientist Award (2004). The best student presentation in ISHS/AUSHS
conference: Harnessing the Potential of Horticulture in the Asian-Pacific
Region, Coolum, Queensland.
2. Mary Janet Lindsay of Yanchep Travel Award (2004). Faculty of Natural and
Agricultural Science, The University of Western Australia.
viii
Table of Contents
Abstract i
Declaration iii
Dedication iv
Acknowledgments v
Thesis Outline vi
List of Publications, Conferences Attended and Awards vii Publications vii Conferences attended and visits vii Awards viii
Table of Contents ix
List of Tables xi
List of Figures xiii
Chapter 1 1
General Introduction 1 Background 2 Hypotheses 5 Objectives 6
Chapter 2 7
Literature Review 7 Cauliflower 8
Origin, distribution and taxonomy 8 Cultivars 9 Seedling production 12
Brassica breeding 14 Breeding objectives 14 Floral biology, pollination and seed production 14 Breeding systems 15 Genetic purity in Fi hybrid cultivars 20
Molecular Markers technology and its applications 22 Types ofDNA markers 23 Other markers, marker combinations 25 Application of molecular markers 25
Chapter 3 Error! Bookmark not defined.
Fingerprinting of Cauliflower Cultivars using R A P D Markers Error! Bookmark not defined. Abstract Error! Bookmark not defined. Introduction Error! Bookmark not defined. Materials and Methods Error! Bookmark not defined.
Results Error! Bookmark not defined. Discussion Error! Bookmark not defined. Acknowledgements Error! Bookmark not defined. References Error! Bookmark not defined.
ix
Chapter 4 Error! Bookmark not defined.
Genetic Diversity of Open Pollinated Cauliflower Cultivars in Indonesia Error! Bookmark not defined.
Abstract Error! Bookmark not defined. Introduction Error! Bookmark not defined. Material And Methods Error! Bookmark not defined. Results And Discussion Error! Bookmark not defined. Conclusions Error! Bookmark not defined. Acknowledgements Error! Bookmark not defined. Literature cited Error! Bookmark not defined.
Chapter 5 Error! Bookmark not defined.
Genetic Diversity of Indonesian Cauliflower Cultivars and Their Relationships with Hybrid Cultivars Grown in Australia Error! Bookmark not defined.
Abstract Error! Bookmark not defined. Introduction Error! Bookmark not defined. Materials and methods Error! Bookmark not defined. Results Error! Bookmark not defined. Discussion Error! Bookmark not defined. Acknowledgements Error! Bookmark not defined. References Error! Bookmark not defined.
Chapter 6 64
Identification of 'Sib' Plants in Hybrid Cauliflowers using Microsatellite Markers 64 Abstract 65 Introduction 66 Materials and methods 67 Results 70 Discussions 72 Acknowledgments 75 References 75
Chapter 7 80
Molecular Markers Correlated with Seedling Traits in Cauliflower Varieties 80
Abstract 81 Introduction 81 Materials and Methods 82
Results 84 Discussion 86 Acknowledgments 89 References 89
Chapter 8 98
General Discussion 98
Chapter 9 103
References 103
List of Tables
Chapter 2
Table 1. Types of genetic markers commonly generated using P C R method (Rafalski
and Tingey, 1993) 26
Chapter 3
Table 1. Decamer primers used in this study. Primers were synthesised by Life Technologies. Primers marked with an asterisk (*) generated polymorphisms
33
Table 2. Useful R A P D markers for the identification of 25 cauliflower cultivars. Polymorphic bands are described as Primer code followed by molecular weight indicated by number of base pairs. Bands were P=Present or A = absent 34
Table 3. Pairwise differences between 25 cultivars of cauliflowers. The data were calculated using Phylogenetic Analysis Using Parsimony based on R A P D bands from all the random primers tested 36
Chapter 5
Table 1. R A P D markers for the identification of 12 cauliflower cultivars. Bands are described as primer code followed by molecular weight. Bands were present (P) or absent (A) 59-61
Table 2. Pairwise distances between cauliflower cultivars. The data were calculated using Phylogenetic Analysis Using Parsimony based on R A P D bands from all primers tested 62-63
Chapter 6
Table 1. Number of plants grown (n), number of morphological sibs (nm) and the proportion, number of genetic sibs (ng) and the proportion, plant height, leaf number and curd weight of each cauliflower lines in Field Trial 2. Genetic sib was confirmed using microsatellites analysis, where only one female band was present. * Proportion of genetic sibs on normal plants was based on 20 plants tested, except for selfed SI, 10 plants tested. **Proportion of genetic sibs of sib plants = ng/nm, also expressed as a percentage. N = narrow, W = w a v y (Fig 2) 78
Chapter 7
Table 1. Cauliflower cultivars and their characteristics. SI=Self incompatibility breeding system, CMS=cytoplasmic male sterility, W = week. Plant characteristics were provided by Seed Companies 92
Table 2. Comparison of morphological characteristics observed at 7 days and 6 weeks. Values are mean ± s.e. It = length, Germ = germination, # = number, wt= weight, F W = fresh weight, D W = dry weight, d= day, w = week 93
Table 3. Correlation coefficient (r) of seedlings traits across cultivars. wt = weight, It = length, Germ = germination, # = number, F W = fresh weight, D W = dry weight, d=day, w = week 94
XI
Table 4. Correlation between seed weight with other seedling traits within cultivars. F W = fresh weight, Germ = germination, d=day, w = week 95
Table 5. Significance of markers associated with higher or lower values for seed weight, germination rate (Germ), shoot length (It), root length, total length and fresh weight (FW) at 7 days (d) for 21 cultivars. Values given are/?-values..96
Table 6. Significance of markers associated with higher or lower values for fresh weight (FW), dry weight ( D W ) , leaf number (#), shoot length (It) and root length at harvest after 6 weeks (w) for 21 cultivars. Values given are upvalues 96
xii
List of Figures
Chapter 2
Figure 1. Genomes of Brassica (U, 1935) 9
Figure 2. Comparison of breeding programs to produce hybrid vegetable and seed commodities. M F = male fertile, M S = male sterile, C M S = Cytoplasmic male sterility, Rf = nuclear restoration gene for C M S trait, - = absence of trait, + = presence of trait (Makaroff, 1995) 19
Chapter 3
Figure 1. R A P D amplification profiles obtained with primer UBC106. Standard bands are indicated by arrows. Molecular weight of standard are indicated in base pairs. M , 100 bp D N A ladder; C, Control lane; 1, Monarch; 2, Donner; 3, M3444; 4, Cauldron; 5, Gibralter; 6, Chaser; 7, CF535; 8, SPS716; 9, Liberty; 10, Omeo; 11, G389; 12, Advantage; 13, CLF33902; 14, CF536; 15, G376; 16, Virgin; 17, J3195; 18, Sirente; 19, Morpheus; 20, Fremont; 21, Plana; 22, Alabama; 23, SPS3074; 24, Celeste; 25, Discovery 33
Figure 2. Fingerprinting key for cauliflower cultivars generated from R A P D markers. Bold indicates that the cultivars has been differentiated 35
Figure 3. Dendogram of 25 cauliflower cultivars, constructed using P A U P based on Neighbor-Joining (NJ) analysis. The numbers at each node represent NJ coefficient of differences 37
Chapter 4
Figure 1. Dendogram of Indonesian cauliflower cultivars, constructed by unweighted pair-group method with arithmetic averages ( U P G M A ) based on total character differences. Numbers above branches represent branch length and numbers below branches indicate bootstrap values 43
Chapter 5
Figure 1. R A P D amplification profiles of 12 cultivars obtained with primer SL-01 and SL-08. Standard bands are indicated by arrows. M , marker ladder, 1, Harli; 2, Blaster; 3, Broad; 4, Manalagi, 5, Gembel; 6, Bandung; 7, Malang; 8, Bedugul; 9, Atlantis; 10, Omeo; 11, Monarch; 12, Plana; M , Marker ladder..55
Figure 2. Fingerprinting key for cauliflower cultivars. Bold indicates that the cultivar has been differentiated. Markers=primer-number of base pairs, P= Present, A=Absent 56
Figure 3. Dendogram of Indonesian cultivars ('Harli', 'Broad', 'Bandung', 'Gembel', 'Malang', 'Blaster', 'Manalagi', 'Bedugul'), Australian-bred cultivars ('Atlantis', 'Omeo') and European-bred cultivars ('Monarch', 'Plana'), constructed by unweighted pair-group method with arithmetic averages ( U P G M A ) based on total character differences. Numbers adjacent to cultivars indicate collection number. Numbers above branches represent branch length and numbers below branches indicated bootstrap values 57-58
xiii
Chapter 6
Figure 1. Reproductive organs of two types of CMS flowers compared to normal flowers (petals removed), a = shrunken anthers, b = petaloid anthers, c = normal anthers 79
Figure 2. Abnormal plant types (8-weeks old) observed in the field, a = wavy leaf (W), b = narrow leaf (N), c = blind apex, d = normal 79
Figure 3. Banding patterns of male parent ($), female parent (?), Fi hybrids (H), manual crosses (C) and self pollinated plants (S) using primer Nal2-E06b on SI system plants. M L = Marker Ladder. *= abnormal plant such that H*= abnormal hybrid, C*= abnormal crosses, S*= abnormal selfed 79
Chapter 7
Figure 1. a. Seedling size variation at day 7, from lightest to heaviest (left to right), b. Largest and smallest seedlings at 6 weeks, c. Biggest and smallest root mass at 6 weeks 97
Figure 2. R A P D profiles of 21 cauliflower cultivars using primer U B C 106. A, Markers for root length, total length at day 7, shoot length, fresh weight and dry weight at week 6. M , Marker Ladder; 1, Plana; 2, Donner; 3, Discovery; 4, Fremont; 5, Monarch; 6, CF0284; 7, Virgin; 8, Morpheus; 9, Cauldron; 10, Arctic; 11, Atlantis; 12, L3368; 13, Belot; 14, Lateman; 15, Phanter; 16, Jerez; 17, Fandango; 18, Megan; 19, Delfur; 20, SPS 716; 21, Omeo 97
xiv
Chapter 1
General Introduction
Background
Cauliflower (Brassica oleraceae var. botrytis, Family Brassicaceae) is a major
vegetable crop in Australia and around the world. World production of cauliflower was
16.4 million tonnes in 2004. The major cauliflower-producing countries are China, India,
Spain, Italy and U S A , and Australia produces about 128 thousand tonnes (FAOSTAT,
2005).
In Western Australia, cauliflower is the fourth most valuable vegetable crop grown
with the majority being exported to Asian countries. More than 8 0 % of Australia's export
cauliflower is grown and packed in the Manjimup district of Western Australia. The
industry is valued at about A U D $ 25 million per year (Lancaster and Burt, 2001; A B S ,
2005; A U S V E G , 2005). Western Australia has the advantages of a suitable climate and
soils, available water, advanced agronomic practices and excellent postharvest handling
facilities which make it an ideal place to supply a high quality produce to domestic and
international markets.
Western Australia has a strong cauliflower industry. Industry members aim for a
high level of customer satisfaction by offering desired varieties, quality and the ability to
consistently supply markets (Lancaster and Pasqual, 1999). However, high labour and
shipping costs have lead to increased economic pressure and competition from other
suppliers such as China and U S A (Mattingley, 2002). To compete in international markets,
all costs must be kept to a minimum and waste product must be minimized, yet around 1 0 %
of hybrid cauliflower crops grown are lost due to non-uniformity. Variation makes the
industry very labour intensive and reduces export income.
Improved uniformity is the first priority of breeding programs in Brassica around
the world (Monteiro and Lunn, 1999). Improving uniformity is a priority research area for
the cauliflower industry (Warren Cauliflower Group, 2003). A high degree of uniformity
in cauliflower quality is required for the domestic and export markets. Greater uniformity
would reduce cultivation and harvesting costs and improve crop quality. Distinctness,
uniformity and stability are criteria that new Fi hybrid varieties have to accomplish before
patent registration (Ruffio-Chable et al, 2000; Raparelli and Menesatti, 2000).
Variation between cauliflowers occurs at all stages in production and reduces
profitability at harvest. Cauliflower shows considerable variation in germination and
harvest date (Hadley and Pearson, 1998). Variation may be caused by genetic or
2
environmental differences or the interaction of these factors. Moreover the curd, which is
an immature inflorescence, is highly perishable and harvest must occur at a specific stage
(Wurr, 1990). Several harvests are commonly required due to plant to plant variation in
maturity and non-uniform curd development (Rubatzky and Yamaguchi, 1996).
The uneven and unpredictable curd development periods of cauliflower are a
problem all over the world (Fujime and Okuda, 1996; Raparelli and Menesatti, 2000;
Ruffio-Chable et al., 2000). In Western Australia, many selective harvests are required at
two to three day intervals to obtain each curd at the optimum quality. Consequently all
picking is done by hand and may extend for up to 30 days. This is very labour intensive as
leaves are folded over cauliflower heads to prevent sun damage and these must be removed
and replaced at each inspection to determine head maturity. M a n y curds are left in the field
as they mature outside the profitable picking time range (Shellabear, 1994). This variation
also prevents the use of mechanical harvesting systems. A grower's profit is reduced first
by the unpicked curds and secondly by the high labour costs required for several sweeps of
checking, covering and harvesting the curds. Less variable plants would improve harvest
uniformity and reduce losses from early and late maturity dates where harvestable numbers
are small and uneconomic. A uniform cauliflower field would also make grading much
easier and cheaper.
Several approaches have been taken to address the existing variation, such as
agronomic management (improved approaches to nutrition, irrigation) and physiological
approaches (vernalisation, temperature and hormonal control) with limited success (Stirling
and Lancaster, 2005; Charsley, 1998). The alternative approach, examining genetic aspects
requires further attention. This approach will benefit and complement existing
management options.
Variation begins at the seed and seedling stage and these differences in growth are
exacerbated during field production. Elimination of variation at the seed or seedling stage
would greatly enhance uniformity of the crop. Identification of specific molecular markers
associated with seedling traits would assist in the selection of superior, uniform seedlings.
Associations between seedling characters and genetic data exist in radish (Raphanus
sativus) (Pradhan et al., 2004b). Molecular markers linked with particular traits are
available for Arabidopsis and the related Brassica oilseeds (Allonso-Blanco et ah, 1999;
Barret et al., 1998; Somers et al., 2001). Associations and molecular markers linked with
3
cauliflower seedling traits would be of considerable value in cauliflower and will be
investigated.
Cauliflower identification is important for accurate classification of cultivars.
Intensive breeding in cauliflower has resulted in difficulties and irregularities in their
classification and cultivar identification. Problems with plant classification based on
morphological characters occur in cauliflower and broccoli (Malatesta and Davey, 1996).
Identification based on morphological characters is also time consuming and requires
expensive field trials and evaluation. Molecular ( D N A ) markers have an advantage over
other tests for cultivar identification, in that D N A is not affected by environmental factors
or the developmental stage of the plant. Molecular markers can be used to study
cauliflower diversity and to develop fingerprinting keys for selected cultivars.
The use of Fi hybrids, which can produce a genetically uniform population is still
developing. T w o breeding systems for hybrid production are currently employed, self
incompatibility and cytoplasmic male sterility. However, hybrid varieties usually contain
up to 2 0 % 'sib' plants, thought to be from self-inbred seed (Holland and McNeilly, 1985;
Crockett et al, 2000; Crockett et al., 2002). Sibs can be smaller and have darker green leaf
colour than the hybrid, be taller and have paler green leaf colour, or have much weaker
growth habit than the hybrid. Sib plants usually flower very early and produce a very small
curd which is not suitable for export (Lancaster and Burt, 2001). Registration of new
varieties allows a m a x i m u m of 5 % sibs, and seed company breeders need to meet this
criteria (Ruffio-Chable et al, 2000; Harvey and Smith, 1987).
There are several possible causes of sibs which are assumed to be non-hybrid plants
(Holland and M c Neilly, 1985). This includes pollinating vector behaviour, the presence of
S-allele modifying genes, inappropriately matched parents and the effect of temperature
and humidity within the crop. Brassica oleraceae possesses a single locus, multi-allelic,
sporophytic incompatibility system. Breeder's lines are usually maintained by bud
pollination. All incompatibility alleles are not equally effective and varying amounts of
self-fertilization m a y occur in inbred lines homozygous for S-alleles. In addition, the
incompatibility reaction m a y be weakened by environmental factors such as high
temperature. The ratio of selfing to crossing m a y be affected by the behavior of pollinating
insects and the availability of foreign pollen (Wills et al, 1980). These factors mean that
methods to screen for sibs or potential sibs would be desirable.
4
Aberrant plants, which are also unsuitable for harvest, are often observed in
cauliflower production utilizing the Cytoplasmic Male Sterility ( C M S ) breeding system.
Phenotypes of aberrant plants predominantly involve the modification of curd size, leaf
shape, size and thickness (Ruffio-Chable et al, 2000). Occurrence is more frequent at the
end of the vegetative cycle. Aberrations occur in up to 4 0 % of plants in cauliflower
varieties. Again a method to screen for potential aberrant plants would be desirable.
To address the existing problems, knowledge of molecular genetics in cauliflower is
required. M a n y molecular techniques can be used to define identity, purity and stability of
a given plant. The range of techniques available for immediate application varies, with
major species offering a wide range of established molecular markers and minor or less
well studied species requiring evaluation of potential marker systems. The choice of
method for any particular application will depend upon the difficulty of the distinction to be
made and factors such as time, facilities and funds available. The nature of the sample may
determine the techniques to be used (Henry, 1997). The techniques currently available
include R A P D (Random Amplified Polymorphic D N A ) , R F L P (Restriction Fragment
Length Polymorphism), A F L P (Amplified Fragment Length Polymorphism), S S R (Simple
Sequence Repeats), R N A (Ribonucleic Acid) and protein analysis. Scientists have used
R A P D markers to investigate seed purity of hybrid cabbage and broccoli (Crockett et al,
2000; 2002), which are the same species as cauliflower. It would be very useful to apply
molecular techniques to seed purity studies in cauliflower.
Hypotheses
The overall hypothesis of this thesis is that variation in cauliflower production is
due to genetic and environmental factors. Here the genetic factors were investigated. This
can be divided into four specific hypotheses:
1. Hybrid cultivars have a narrow or limited genetic base resulting in a very similar
morphological appearance among cultivars. Cultivar identification should be based
on genetic make up instead of morphology. Variation can be detected on a genetic
level, which is independent of the environment.
2. Different breeding systems result in different degrees of variation in their progeny.
Variation within open pollinated cultivars is greater than control pollinated
cultivars. Greater variation occurs between distantly related cultivars.
5
3. The occurrence of sibs in hybrid seed is mainly due to genetic factors, i.e. self
pollination of the female parent. Development of molecular markers for
identification of sib plants will allow selection for more uniform crops.
4. Morphological variation at the seedling stage is in part caused by genetic variation
and genes controlling morphological traits can be identified using molecular
markers.
Objectives
These hypotheses reflect four specific aims of this study:
1. T o fingerprint the c o m m o n cauliflower cultivars using molecular markers (Chapter
3,4).
2. To study diversity/variation in open pollinated cultivars and genetic distance
between Australian grown and Indonesian grown cultivars (Chapter 4, 5).
3. To develop markers for the identification of sib and normal plants (Chapter 6).
4. To search for molecular markers associated with genes controlling morphological
traits related to plant growth and development (Chapter 7).
6
Chapter 2
Literature Review
Cauliflower
Origin, distribution and taxonomy
Brassica contains around 40 species. This genus has great commercial value and
plays a major role in feeding the world population. Brassicas include nutritious vegetables,
mustards and oil seeds, animal feed, cover crops and weeds (Rubatzky and Yamaguchi,
1996).
Six of the most important Brassica species are closely interrelated. B. rapalB.
campestris (turnip, Chinese cabbage), Brassica nigra (black mustard) and B. oleracea
(cabbage, cauliflower, broccoli, kale, kohlrabi, Brussel sprouts) are monogenomic. Their
genomic composition has been labelled as A, B and C. The other three are amphidiploid
species, B. juncea (leaf mustard), B. napus (rutabaga, oil rape) and B. carinata (Abyssinian
mustard) identified as A B , A C and B C respectively (Rubatzky and Yamaguchi, 1996).
According to the widely accepted scheme known as the U-triangle (Figure 1), the three
Brassica amphidiploids have been derived from interspecific hybridisation between the
three diploid species (Song et al, 1996).
Large genetic diversity exists among and within the three cultivated amphidiploid
species. Based on genetic diversity, B. napus seems to be the most ancient amphidiploid,
followed by B. juncea and B. carinata. T w o major factors are responsible for the genetic
diversity within amphidiploids, one is multiple hybridizations between different diploid
parents and the other is genome modification after polyploidization.
Cauliflower appears to have been domesticated in the Mediterranean region. The
first written description of cauliflower appeared in 1544 and Italy is widely regarded as the
centre of diversity of cultivated B. oleracea (Rubatzky and Yamaguchi, 1996; Massie et al,
1996; Wien and Wurr, 1997). The simple cultivated variety of the 16th Century possessed a
small, quick-bolting inflorescence. Today, specially adapted varieties and spatial and
temporal distribution of production allow year-round supply (Sauer, 1993).
8
B. oleracea, n = 9
Cabbage, Cauliflower, Broccoli
CC
n = 9 n = 9
BBCC
B. carinata, 2n = 34
Abyssinian
mustard
AACC
B. napus, 2n = 38
.Canola
BB
B. nigra,
2n=16
Black mustard
AABB
B. juncea,
2n = 36
Leaf mustard
Figure 1. Genomes of Brassica (U, 1935).
AA
B. campestris
2n = 20
Chinese cabbage
Cauliflower {Brassica oleracea var botrytis) is one of the most popular Brassica
vegetables. It is cultivated worldwide in different climatic conditions, ranging from
temperate to tropical regions and is available year round in the market. Major producers of
cauliflower are India, China, France, Italy, United Kingdom, USA, Spain, Poland,
Germany, Pakistan (Sharma et al, 2004) and Australia (Lancaster and Burt, 2001). They
are also cultivated in tropical zones of Africa, Central and South America, and Oceania
(Sauer, 1993).
Cultivars
A range of cauliflower cultivars has been developed through breeding and selection,
to produce satisfactory yields in specific environments ranging from tropics to temperate
areas (Wien and Wurr, 1997). Proper variety selection is crucial for cauliflower
production. Cultivars have biological clocks which trigger the curd to develop at a specific
9
time based on plant age and past and present ambient temperature. Depending on the
cultivar, the period of vegetative growth may be only a couple of weeks or more than a
month. Cultivars grown out of their appropriate season will not develop satisfactorily.
C o m m o n defects include bolting (early flowering), riceyness, yellowing, light weight curds
and breaking-apart of the florets. These occur due to inappropriate planting period for a
given variety, curds exposed to sunlight or when the crop grows during adverse weather
(Mayberry, 2000).
Important cultivar variables are curd weight, size, shape, compactness, surface
texture and colour. Traditionally pure white curds are preferred, although cultivars
producing cream, purple, green and orange curds are also grown (Rubatzky and
Yamaguchi, 1996). In Australia, curd size for export must be heavier than 0.5 kg, with the
best size being approximately 1 kg - 1.2 kg. In addition, curds with the following defects
are discarded: leaves in the curds, surface dirt, bruising, severe shape distortion, excessive
furriness or 'riceyness', unevenness, yellow, brown or pink discolouration and insect and
disease damage (Lancaster and Burt, 2001).
Cauliflower cultivars can be grouped into three major maturity types: early (for
summer and early autumn harvest), intermediate (late autumn and early winter) and late
(winter and spring). Late-maturing types require vernalization for curd initiation. Some of
the major cultivar include: 1. Italian cultivars of varied curd form and colour, grown as
annuals and biennials, for example Jezi, Romanesco, Flora and Blanca, 2. Northern
European cultivars, grown as annuals during summer and autumn, for example Alpha and
Snowball, 3. Northwestern European cultivars grown as biennials for late winter and spring
harvesting, for example Roscoff and St Malo, 4. Australian cultivars grown as annuals
using cultivars mostly developed from European sources, 5. Asian cultivars grown as
annuals and adapted for high temperature regions, often lacking in uniformity, curd
compactness and colour, for example Patna (Rubatzky and Yamaguchi, 1996).
Most cauliflowers available in the Australian market are Fi hybrid cultivars. These
varieties result from intensive breeding programs conducted by seed companies throughout
the world. Seed companies in developed countries such as those in Europe and Northern
America often exchange their breeding materials. Consequently, most hybrid varieties are
very closely related and the same variety may be released under different names.
10
Since hybrid varieties are very closely related and difficult to distinguish
morphologically, there is a need for more accurate identification of the varieties released to
the market. Identification based on morphological characters is time consuming and
requires expensive field trials and evaluation. Methods using molecular markers may
provide an accurate fingerprinting method as they are based on genetic information and are
not affected by environmental factors (Henry, 1997).
Most cauliflower cultivars available in Indonesia are open pollinated. There is little
information about cultivars or landraces in Indonesia. Cultivars may have been introduced
from India during the Dutch period in Indonesia more than a hundred years ago. These
cultivars became locally adapted. They are grown in highlands throughout the country,
mainly for domestic consumption.
Before introducing any vegetable species to the tropics, it is necessary to identify
the optimal growth temperatures and photoperiodic requirements to ensure they are
compatible with local climatic conditions. It is also important to utilise existing local
varieties of the species to be introduced, even though the quality of the produce m a y not be
high. This is because such traditional, local varieties m a y have an important role to play in
future plant improvement schemes since they represent an irreplaceable source of genetic
variability (Messiaen, 1992). More importantly, local varieties often have a good level of
resistance to pests and diseases (William et al. 1991).
The lack of adequate taxonomy has seriously affected the systematic collection and
assessment of cauliflower genetic resources (cultivars). This has had two damaging effects.
Firstly, many genotypes may have become extinct because the range of variation in
cauliflower was unknown to genetic conservationists. Secondly, the genetic variation of
cauliflower and its closest relatives has not been exploited by breeders, instead a
disproportionate effort m a y have been spent making wide crosses within B. oleracea or
even with other species, in order to introduce desirable traits into cauliflower. For example,
attempts to backcross resistance to the clubroot disease into cauliflower from cabbage were
unsuccessful because of the difficulty in regaining an acceptable cauliflower phenotype
(Sharma et al, 2004). There is a need for the adoption of molecular techniques for cultivar
identification and their application in selection of superior characters and individuals for
future breeding programs.
11
Seedling production
Cauliflower is commercially propagated from seeds usually by a specialist nursery.
Seeds are commonly sown in individual cell trays. Growing conditions are closely
controlled to ensure morphologically uniform seedlings are produced. Seedlings are ready
for transplanting into the field when they have three to four true leaves and can be
transplanted until they are 7 or 8 weeks old. Planting older seedlings increases the
likelihood of premature curd production (Madhavi and Gosh, 1998). Weekly to fortnightly
plantings are usually made to ensure continuity of supply throughout the season. Specialist
nurseries provide good quality plants, reduce risk of soil diseases, reduce transplanting
shock and variation in seedling size leading to greater uniformity at maturity (Lancaster and
Burt, 2001).
Seed and seedling variation
It is important to begin with high quality seed that will produce a uniform stand of
vigorous seedlings. Seedling uniformity is critical since variation in vigour results in
shading of small plants and slower plant growth (Webster, 1964). Variation begins at the
seed and seedling stage and differences in growth are exagerated during field production.
Minimising or elimination of variation at the seed or seedling stage would greatly enhance
uniformity of the crop.
Seed size variation influences early seedling performance and subsequent adult
growth (Bretagnolle et al, 1995). Seed size m a y vary within species, among populations,
within populations, individuals and within fruits in an individual plant. In Alliariapetiolata
(Brassicaceae), individual seed weight varies 2.5-fold to 7.5-fold within populations and
nearly eightfold among populations (Susko and Lovett-Doust, 2000).
Variation in seed weight is caused by environmental, maternal or genetic factors.
Environmental effects include differences in temperature, light, water and nutrient levels
(Gutterman, 2000). Position of seeds in the mother plants affects seed weight. Within an
infructescence of fruit, individual seed mass decreases from basal fruits to distal fruits.
Furthermore, seed mass decreases within fruits from basal to distal seed positions (Susko
and Lovett-Doust, 2000). Basal fruits within an infructescence and basal seeds within fruits
may behave as strong sinks for limited parental resources, such as nutrients and
photosynthate. Thus, competition for limited resources m a y influence the maturity of a
seed, as well as the mass of the seed. Early-initiated, basal fruits produce larger seeds than
12
fruits in the middle or the tip of an infructescence. Cauliflower plants produce many
inflorescences in a plant with many siliques (pods) within an inflorescence. Approximately
16 seeds are produced in a silique. Variation in seed weight may occur within a silique and
within an inflorescence. The detected differences may be due to genetic and/or
environmental factors (Susko and Lovett-Doust, 2000).
Genetic factors are also involved in seed size though to date these are poorly
understood. At least 11 seed size and seed weight quantitative trait loci contribute to seed
size variation by affecting both cell number and cell size in Arabidopsis (Alonso-Blanco et
al, 1999). The gene AP2 (APETALA2) plays an important role in the control of seed mass
and seed yield, by affecting seed size, embryo size, seed weight and the accumulation of
seed reserves. AP2 acts in the maternal sporophyte and endosperm perhaps by influencing
source-sink relations, and it is required for normal seed coat development (Jofuku et al,
2005; Ohto et al, 2005). In tomato, Sw4.1 was described as a major Q T L for seed weight
variation (Orsi and Tanksley, 2005). Genes or QTLs for large seed size are unknown in B.
oleracea. The advantages associated with larger seeds and the potential for increasing yield
through seed size indicate the importance of identifying the genes involved in the
determination of seed size and seed mass. Three ISSR markers are found linked with low
seed weight in wheat (Ammiraju et al, 2001), 13 SSR markers are associated with seed
size in soybean (Hoeck et al, 2003), 11 R L F P loci for mungbean (Humpry et al, 2005) and
4 Q T L for seed weight have been identified based on R A P D , ISSR and phenotypic markers
in chickpea, (Cho et al, 2002). Association of seedling traits and molecular markers in
cauliflower were investigated.
Effect of seed weight variation on seedling traits
Seed weight can greatly affect seedling traits including germination (Van Molken et
al, 2005) and seedling size (Schaal, 1980). Germination responses depend on the species
(Baskin and Baskin, 1998) and rate and percentage germination can increase, decrease or
remain unaffected by differences in seed size. In Alliaria petiolata, smaller seeds
germinate earlier but larger seedlings produce higher total plant biomass (Susko and
Lovett-Doust, 2000). In Cakile edentula (Zhang, 1993) and Erodium brachycarpum
(Stamp, 1990), small seeds germinate earlier than large seeds. This may be because small
seeds have greater access to water as a result of their higher surface to volume ratios.
Hence, small seeds imbibe water faster and germinate sooner. In Arabidopsis, the RGL2
13
gene is responsible for seed germination and it probably functions as an integrator of
environment and endogenous cues to control seed germination (Lee et al, 2002).
Seed size and seed weight are important determinants in seedling dry matter and
seedling leaf area in Cercis canadensis (Couvillon, 2002). Larger seeds produce larger
seedlings, with greater fresh and dry weight and leaf area than small seeds. Small seed size
may influence other aspects early seedling growth and establishment. In Pastinaca sative
seedlings, the maximum ratio of root length to total leaf area is negatively related to seed
weight at 10 and 20 days after emergence (Hendrix et al, 1991).
In a number of Brassica oleracea species, differences in seed vigour contribute to
differences in seed germination and seedling variability. Seeds with high vigour usually
germinate fast and produce more uniform seedlings. Seeds with low vigour, as a result of
ageing, germinate more slowly and produce smaller and more variable seedlings (Powell et
al, 1991). Genes or Q T L s for seed and seedlings traits are unknown in Brassica oleracea.
However, in other Brassica, R A P D markers have been linked to seed coat colour in
Brassica napus and B. rapa-alboglabra (Somers et al, 2001; Heneen and Jorgensen, 2001).
In conclusion, genetic and environmental factors contribute to seed and seedling
variation in plants. Identification of specific molecular markers associated with seedling
traits such as seed weight, germination rate, fresh weight, dry weight, shoot length and root
length, would greatly assist in the selection of superior, uniform seedlings.
Brassica breeding
Breeding objectives
Breeding objectives can be addressed to satisfy both growers and consumers. These
need be considered in terms of crop production and product improvement. The main
criteria for crop production are yield, resistance to disease or environmental stress,
uniformity and continuity of cropping. Breeding for appearance, commercial quality, shelf
life, taste and nutritional value is part of product improvement. The most important
objective is crop uniformity which makes grading much easier and reduces harvest time
(Monteiro and Lunn, 1999).
Floral biology, pollination and seed production
Cauliflower flowers are typical of the Brassicaceae family, with four sepals, four
symmetrical yellow petals and six stamens (2 short and 4 long). Anthers rarely dehisce
14
before flower opening, even though the stigma may be level with the anthers within the
flower bud (Crisp and Tapsell, 1993; Sharma et al, 2004).
Cauliflower is a cross-pollinated crop, mainly pollinated by insects. Honeybees are
the usual pollinating agents, although bumble bees and flies may also be responsible for
pollination. Wind can also be the pollinating agent. The stigma of Brassica is receptive 5
days before and 4 days after flower opening. The period from pollination to fertilization
generally takes 24 - 48 hours, depending on temperature, with the ideal temperature being
12-18°C. Higher day temperatures cause pollen sterility, resulting in poor seed
development. Pod maturity for harvest of pods may require 50-90 days from the date of
flowering, depending on climatic conditions. The fruit is a siliqua but often called a pod.
The seeds are small, globular, smooth and dark brown. There are normally 1 2 - 2 0 seeds
per pod and nearly 350 seeds weigh one gram (Sharma et al, 2004).
Breeding systems
Controlled pollination is essential for production of hybrid seed. Plants grown from
hybrid seeds benefit from the heterotic effect of crossing two genetically distinct breeding
lines. Heterosis or hybrid vigour is the increased vigour of plants when compared with
parents. The agronomic performance of the hybrid progeny is superior to both parents in
terms of yield, vigour, adaptability and uniformity. In order to produce hybrid seed
uncontaminated with self-pollinated seed, control methods need to be developed to stop self
pollination (Bhalla and Singh, 1999). The two major methods applied for the production of
Fi hybrids in Brassica are self-incompatibility (SI) and cytoplasmic male sterility (CMS).
Open pollination is still employed, particularly in developing countries (Williams et al,
1991).
Self-incompatibility system in Brassica
Self-incompatibility (SI) is a natural mechanism that prevents self-fertilization and
promotes outcrossing and m a x i m u m recombination in Angiosperms (Watanabe and Hinata,
1999). Brassica oleracea posseses a single locus, multi-allelic, sporophytic incompatibility
system. Phenotype of the pollen is determined by the diploid genotype of the pollen
producer, the sporophyte. Pollen rejection occurs when both the pollen and the pistil
exhibit the same S-phenotype, although they m a y have different genotypes (Bateman,
1955). All incompatibility alleles are not equally effective and varying amounts of self-
15
fertilization may occur in inbred lines homozygous for S-alleles. Not all species naturally
possess SI (McCubbin and Dickinson, 1997). In addition, the incompatibility reaction may
be weakened by environmental factors such as high temperature and high humidity (Zur et
al, 2003). A weakening in self-incompatibility is likely to increase selfing and selfed seed,
especially during the non-coincident phases of flowering (Gowers, 2000).
Developing self-incompatible breeding lines for hybrid seed production is costly
since the stabilisation of inbred parental lines requires several generations of selfing, and
the maintenance of breeding lines is labour intensive. In order to self SI plants, the
mechanism needs to be overcome or avoided. The incompatibility system becomes
operative two or three days before anthesis, so self-incompatible plants can be self-
pollinated by opening immature buds and placing pollen on the exposed stigma (Crisp and
Tapsell, 1993). Alternatively the stigma can be removed and pollen placed on the cut
surface of the style in bud stage. This avoids the SI mechanism which is located in the
stigma. Bud pollination is commonly used to overcome self-incompatibility in cauliflower
(Hallidri and Pertena, 2002). Sodium chloride solution and carbon dioxide can break down
the incompatibility mechanism in Brassica rapa (Mohring et al, 1999).
SI is widely used in the production of Fi hybrids in vegetable Brassica oleracea
(Sharma et al, 2004). All of the new hybrid Brussels sprouts, cabbages and kales owe their
origins to breeding programs employing SI. Unfortunately, there is potential for
breakdown of self-incompatibility due to adverse environmental factors in the hybrid seed
production field resulting in contamination of hybrid seed with selfed seed, commonly
known as sibs (Bhalla and Singh, 1999).
Cytoplasmic Male Sterility (CMS)
Cytoplasmic male sterility ( C M S ) is a convenient method for the production of
hybrid seeds. It is a more advanced and reliable system than SI. C M S occurs in a wide
variety of higher plants and is characterized by a very low level or complete absence of
pollen production. C M S is caused by different factors linked to mitochondria and it is
assumed to be a consequence of mitochondrial dysfunction (Araya et al, 1998). In C M S ,
sterility is carried out by the cytoplasm and therefore through the maternal line.
C M S systems have been characterized by the restorer genes required to overcome
them and to provide male-fertile progeny in the male-sterile cytoplasm. Female fertility is
generally not affected by C M S , so that male-sterile plants can set seed if viable pollen is
16
provided (McVetty, 1997). The affected organs and tissues in C M S plants are the stamens
(anther and filament) and pollen grain (microspores). Abnormal behaviour of the tapetum
in the anthers is frequently identified with C M S (McVetty, 1997).
There are several ways to create C M S . C M S can arise spontaneously in breeding
lines, following mutagenesis, as a result of wide crosses or through interspecific exchange
of nuclear and cytoplasmic genomes (Schnable and Wise, 1998).
Types of CMS
At present, there are several C M S systems (Schnable and Wise, 1998; Makaroff,
1995). These include:
1. "Pol" (Polima). This system was identified in China, in Polish Brassica napus cv Polima.
This C M S system is relatively but not completely temperature stable. The availability of
maintainer and restorer lines of the pol cytoplasm, in addition to the relative temperature
stability of the male-sterile phenotype, has made the pol cytoplasm one of the most
advantageous C M S systems for the production of hybrid rapeseed.
2. Ctr (Bronowsky). This is the most recent system found in B. napus. It arose in the F2
generation of triazine resistant lines Tower x Bronowski. This system is temperature
unstable.
3. Nap (SHIGA). First discovered in B. napus cytoplasm following crossing of two Japanese
varieties, Hokuriku x Isuzu. This system is unstable at high temperatures (26 - 30°C),
when it reverts to fertility and therefore it is unsuitable for most field locations.
4. Nigra. This system has been transferred to broccoli and cauliflower and other B. oleracea
vegetables and rapeseed. It has very stable sterility in B. oleracea but not in B. napus. It
also has problems with seed set. The anthers develop as petals (petaloid sterility).
5. Tour (tournefotii). This C M S system resulted from spontaneous interspecific hybridisation
between B. tournefortii and B. juncea.
6. Mur. This system was found after transferring B. napus nuclei into Diplotaxis muralis
cytoplasm. It has been transferred to turnip (B. rapa) and rapeseed. The sterility is
complete and stable.
7. Ogu. This C M S was found by Ogura in a Japanese radish variety. Fi hybrid seed
production in B. oleracea has been achieved using this system.
17
C M S has not been found in cauliflower but it has been introduced from several
sources. Commercial Fi hybrid production has been achieved in B. oleracea using the
improved "Ogura" cytoplasm obtained by Pelletier et al. (1989). F, hybrids of various B.
oleracea types (cauliflower, sauerkraut cabbage, garden cabbage, savoy cabbage) have
been registered (Leviel, 1998).
Morphological changes associated with male sterility
Achievement of an effective C M S system has often been impeded by difficulties
such as the instability of the male sterility, the absence of maintainer or restorer lines,
chlorophyll deficiencies and deformed flowers which lack nectarines (Delourme and Budar,
1999). In some forms of C M S , the absence of functional nectarines prevents commercial
production of Fi hybrid seed using normal insect pollinators.
In the Ogura system, low temperature induces chlorosis in the seedling stage, which
is expressed in the field grown Fi commercial crop as a loss of vigour. The Ogura source
has also been associated with low seed set and poor curd quality in Fi cauliflower. In B.
napus and B. oleracea, which use the Ogura system, chlorophyll deficiency is corrected by
obtaining hybrids via protoplast fusion (Delourme and Budar, 1999). In the nap, pol and
tour C M S , flowers are characterized by narrow petals but this has not been found or
reported in Brassica oleracea, C M S system.
The stability of male sterility is largely dependent on the environment or maintainer
lines. Fertile pollen grains may be produced at temperatures higher than 25 - 30°C in nap
and polima systems or at low temperatures depending on the maintainer lines in the polima
system (Delourme and Budar, 1999). In the field this could lead to selfing and production
of self-inbred plants, which will contaminate hybrid seed production. Temperature effect
on particular C M S system requires further investigation.
CMS for production of commercial hybrids seed in Brassica
In developing a C M S system for breeding purposes, it is important to consider the
crop. Breeding programs for the generation of Fi hybrids of a seed commodity, such as
canola, are different from those that are used to develop vegetable hybrids like broccoli,
cabbage or cauliflower. Production of Fi hybrid seed for the seed commodity requires the
development of both maintainer lines, which maintain the C M S trait and male fertility
restorer lines, which contain nuclear genes that suppress the C M S trait and result in male-
18
fertile plants. Production of Fi hybrid vegetable seed only requires the development of
maintainer lines because vegetable crops are grown for the plant and not the seed
(Makaroff, 1995). A simplified breeding program for the two different crop types is
outlined in Figure 2.
Vegetable commodity Seed commodity
1. C M S line x maintainer line
(MS.CMS+, Rf-) (MF:CMS-, Rf-)
1 hybrid seed sold to farmers
(MS:CMS+, Rf-)
1. C M S line x maintainer line
(MS:CMS+, Rf-) (MF:CMS-, Rf-)
1 2. C M S line x restorer line
(MS:CMS+, Rf-) (MF:CMS+/-, Rf+)
i hybrid seed sold to farmers
(MF:CMS+, Rf+)
Figure 2. Comparison of breeding programs to produce hybrid vegetable and seed commodities. M F = male fertile, M S = male sterile, C M S = Cytoplasmic male sterility, Rf = nuclear restoration gene for C M S trait, - = absence of trait, + = presence of trait
(Makaroff, 1995).
Use of CMS lines is gaining more commercial status, since there should be no risk
of selfed seed. The male sterile lines in commercial Fi production are planted with the
pollen parent in the ratio of 2:1 or 3:1 or 4:1 depending upon varietal characters. In
selecting the pollen parent, factors that need to be considered besides good combining
ability are similarity in morphological characters including plant height and synchrony of
flowering with the male sterile plant. Immediately after pod setting, the pollen parent is
removed to avoid mixture and provide sufficient space for the female plant to produce
hybrid seed (Sharma et al, 2004; Frankel and Galun, 1977).
Seed production in open-pollinated cultivars
Several techniques are used to produce basic seeds from selected plants of open-
pollinated cultivars. In all cases the mother plants are grown in their normal season and
19
final selections are made after confirmation of plant characters and curd quality. W h e n
environmental conditions in the field are expected to remain favourable for further flower
development, anthesis and seed maturity, the selected mother plants can be left in situ. This
is the normal practice in northern Europe and North America for early summer
cauliflowers. It is also a c o m m o n method in Asian countries (Sharma et al, 2004).
Genetic purity in Fj hybrid cultivars
High uniformity has been almost impossible to achieve with open-pollinated
varieties owing to the cross-pollinating nature of brassicas. The introduction of Fi hybrids
is a major advance. Fi hybrid cultivars are the result of crossing two inbred lines which
have been maintained under the control of plant breeders and are known to produce a
desirable hybrid. The advantages of Fi hybrid cultivars include uniformity, increased
vigour, earliness, high yield and resistance to specific pests and pathogens (Crisp and
Tapsell, 1993).
In theory, all plants in an Fi hybrid cultivar resemble each other exactly. Fi hybrid
seed production necessitates the use of a hybridization control system. In Brassica
oleracea such systems exploit either self-incompatibility (SI) or cytoplasmic male sterility
( C M S ) (Ruffio-Chable et al, 1993). With the SI system, due to some self-pollination of
the female parent used in the cross, some plants which are not Fi hybrids may occur and
they are usually morphologically different. These off-types in an Fi hybrid are assumed to
be the result of accidental self-pollination of the female parent and are generally known as
'sibs' (Hodgkin, 1981).
Sibs are a worldwide problem in vegetable Brassica. The key characteristics
associated with sibs are not identifiable in the seedlings stage and therefore they are planted
in the field. Sibs have weak plant habit and produce small, unmarketable curds (Holland
and McNeilly 1985; Lancaster and Burt, 2001). The limit for off type plants in registration
of new varieties of cauliflower is 5 % (Ruffio-Chable et al, 2000). This has been difficult
to meet with some desirable selections. Seed companies cannot afford to market hybrid
seed with appreciable amounts of sib seeds (Hodgkin, 1981).
In addition to the problems associated with sibs in an Fi hybrid seed lot, there are
increased production costs compared to open-pollinated cultivars. The development of the
initial breeding program, subsequent maintenance of the inbred parents, extra land required
for male parents, care with sowing, isolation and harvesting, high labour input for manual
20
emasculation of female flowers, lower seed yield per unit of land and high cost of seeds for
farmers all add to the cost of Fi hybrid seed production (George, 1999).
It is assumed that it is possible to identify sibs by their distinctive plant phenotype
and these will be here referred to as 'morphological sibs'. Also it is assumed that sibs are
self inbred, therefore are genetically determined and could be identified by genetic markers.
Here plants from a self inbred parent will be referred to as 'genetic sibs'.
CMS-based seed parent lines do not produce pollen and thus do not risk self-
pollination (Bhalla and Singh, 1999). Fi cauliflower hybrid derived from C M S system
often produce developmentally aberrant plants, which are unsuitable for harvest.
Phenotypes of aberrant plants mainly involve the modification of three characters: leaf
shape, size and thickness (Ruffio-Chable et al, 2000; Fujime and Okuda, 1996). The
proportion of aberrant plants ranges from 5 % to 4 0 % in cauliflower production.
The high cost associated with hybrid seed production and losses during harvest has
lead many studies to determine sib or other types of aberrant in the Fi harvest. M u c h
research has gone into methods to identify sibs but a definitive method is still required for
cauliflower and other brassica vegetables. For example, image analysis (Fitzgerald et al,
1997), high-pressured liquid chromatography (HPLC) (Mennella et al, 1996), isozyme
analysis (Harvey and Smith, 1987; Zheng and Liu, 1994) and analysis of ploidy levels by
flow cytometry (Ruffio-Chable et al, 2000). The basic features of the image analyses
system include image capture and digitalization, image processing into pixel intensity
numbers and image analyses using mathematical tools to sort and compare images.
Accuracy of this technique is limited by genetic relatedness of cultivars and heterogeneity
of characters within cultivars (Cooke, 1999).
Seed or cotyledon extracts from a seed lot are analysed for isozymes of acid
phosphatase by P A G E of Fi hybrid Brussels sprout varieties (Harvey and Smith, 1987).
Seeds that show homozygous alleles are regarded as sibs. The level of sib content of bulk
seed lots can be above 10%. There are some limitations to isozyme analysis, such as
insufficient polymorphisms among closely-related genotypes and variations affected by
environmental factors, seed vigour and growing stage (Meng et al, 1998).
Molecular marker techniques such as R A P D and R F L P have also been employed in
other brassica vegetables. There are up to 1 4 % of morphological sibs in cabbage (Crockett
et al. 2000) and up to 4 5 % morphological sibs in broccoli observed in the field (Crockett et
al. 2002). A similar proportion of genetic sibs were identified using R A P D technique on
21
seed lots suggested the genetic sibs would have been morphological sibs and therefore
causal, but this has yet to be proved. Thus, there is a need to develop a purity test for
cauliflower and other brassicas to prevent unacceptable levels of sib and aberrant seed
being released in the market.
Improving crop purity standards (environmental factors)
Members of the Brassica oleracea group, i.e. cabbage, cauliflower, Brussels
sprouts, broccoli, kale and other wild allies freely cross with each other, generating
potential causes of variation via genetic pollution/impurity. To maintain seed purity, seed
producers need to consider a range of factors (Stewart, 2002).
The first and key factor is isolation distance. Proper isolation among crops and
fields of different cultivars within each crop are required. A n isolation border of 3000 m
for breeder seed and 1500 m for certified seed is recommended. This is to minimize pollen
flow caused by honeybees, bumble bees and wind (Sharma et al, 2004). Field history of
Brassica seed production and Brassica weeds need to be recorded and taken into account.
Brassica seeds m a y survive 10-15 years in the soil so a cropping history free of Brassica
will be required for a clean crop. The overlap of flowering time with nearby crops, off-
types and weeds also needs to be considered. A s flowering time of a Brassica crops is
quite extended, there is a large window of opportunity for an overlap of flowering. The
propensity of the crop to accept outside pollen is another factor. This increases from self-
compatible to self-incompatible to hybrid plants. Furthermore, C M S hybrid types would
have an even greater propensity to accept outside pollen. Seed need to be screened for size
to remove any potential hybrids. Dressing to remove small seed can significantly reduce
the contamination from other species but is unlikely to affect the contamination caused by
pollen of the same species. Harvest machinery cleanliness also needs to be maintained. All
machinery and seed boxes must be completely free of seed.
To summarise, impurity in Fi hybrid cauliflower may be caused by genetic and
environmental factors and agronomic and postharvest management. Genetic factors were
investigated in this thesis.
Molecular Markers technology and its applications
Theoretically, all molecular techniques can be used to define identity, purity and
stability of a given plant but some techniques are better suited for particular species or
22
cultivars. The range of techniques available for immediate application varies, with major
species offering a wide range of established molecular markers and minor or less well-
studied species requiring evaluation of potential marker systems (Table 1). The choice of a
method for any particular application will depend upon the difficulty of the distinction to be
made and factors such as time, facilities and funds available. The nature of the sample will
vary and the appropriate techniques to be used will require investigation (Henry, 1997).
Types of DNA markers
D N A markers can be classified into hybridization and P C R based techniques.
RFLP is the major hybridization-based marker system, whereas PCR-based markers
include R A P D , AFLP, CAPS, EST, S C A R and SSR. Besides these, high-throughput
techniques of micro-arrays have been developed, which employ both P C R and
hybridization and these can be used to simultaneously analyse a large number of loci
(Lakshmikumaran et al, 2003).
RFLP (Restriction fragment length polymorphism)
RFLP markers remain extremely useful in research applications. These markers
provide co-dominant bands where heterozygote can be distinguished from homozygote
bands that are easily interpreted and amenable to population genetic analysis (Nybom,
2001; Rafalski and Tingey, 1993).
The utility of RLFP is hampered by the large amount of D N A required (5-10 ug) for
restriction digestion and southern blotting. Further, the requirement of a radioactive isotope
makes the analysis relatively expensive and hazardous. The assay is time-consuming and
labour-intensive, often producing low numbers of polymorphic bands (Nybom, 2001;
Rafalski and Tingey, 1993).
RAPD (Random amplified polymorphic DNA)
R A P D technique has been adopted most widely. R A P D markers are preferred for
their ease of use. The key innovation of this method is the use of D N A amplification to
generate genetic markers that require no prior knowledge of the target D N A sequence. The
technique uses P C R with short oligonucleotide primers of arbitrary (random) sequence to
generate genetic markers (William et al, 1990). R A P D is attractive for breeding
applications because it requires only a small amount of D N A (15-25 ng), a non radioactive
23
assay that can be performed in several hours and a simple experimental set up (Rafalski et
al, 1994).
The main issue associated with the use of this technique is to ensure reproducibility
of amplification profiles. Both the quantity and quality of the template D N A preparation
substantially influences results. Besides template D N A , magnesium concentration, cycling
temperatures and times, and base composition of the primer affect the success of R A P D
analyses (Henry, 1997). These factors need to be carefully controlled to ensure reproducible
results. R A P D is also dominant markers, meaning that in a segregating population the
homozygote of the parental type cannot be distinguished from the heterozygote (Nybom,
2001).
Simple Sequence Repeats (SSRs or Microsatellites)
Tandem arrays of short nucleotide repeats from 1 to 5 bases per unit are usually
called microsatellites or SSRs (simple sequence repeats). In particular, the dinucleotide
repeats (AC)n, (AG)n and (AT)n are abundant and highly polymorphic in eukaryotic
genomes (Rafalsky and Tingey, 1993).
In microsatellite analysis, a P C R is conducted using primers that are complementary
to the D N A sequences of regions flanking a highly variable microsatellite. The resulting
polymorphic bands are caused by differences in the number of D N A repeat units and
provide locus-specific, co-dominantly inherited bands with a high level of polymorphism.
SSRs have gained increasing importance in plant genetics and breeding. High abundance
and extensive polymorphism make them an ideal marker system for genetic mapping and
the characterization of germplasm, particularly in very closely related and inbreeding
species (Plieske and Struss, 2001; Saal et al, 2001). A major drawback is the time-
consuming and expensive development of suitable primers which are usually transferable
only among related species (Nybom, 2001).
Microsatellites of Brassica species are well documented. A large number of
microsatellites from rape seed (B. napus) have been identified and characterized. Many
rape seed microsatellite flanking primer pairs are functional in the A and C genome species
within the genus Brassica, but are not useful as markers for a wide range of species in the
family Brassicacea (Saal et al, 2001). Cauliflowers are among diploid C genome species
and therefore may utilize microsatellite information established in B. napus.
24
AFLP (Amplifiedfragment length polymorphism)
A F L P method combines both RFLP and R A P D . There are two rounds of
amplification, the first with primers that will amplify numerous fragments and then a
second with more specific primers. Numerous bands are produced for each primer pair.
These bands are dominantly inherited just like R A P D . The main difference between A F L P
and R A P D is that A F L P yields more bands but is technically more demanding. Thus A F L P
is cost-effective in situations where many analyses need to be performed (Henry, 1997;
Nybom, 2001). The main disadvantage of A F L P is the difficulty in identifying
homologous markers (alleles), rendering this method less useful for studies that require
precise assignment of allelic states, such as heterozygosity analyses (Mueller and
Wolfenbarger, 1999).
ISSR (Inter simple sequence repeat)
ISSR involves the use of microsatellite sequences as primers in a polymerase chain
reaction to generate multilocus markers. It is a simple and quick method that combines
most of the advantages of SSRs and A F L P to the universality of random amplified
polymorphic D N A (RAPD). ISSR markers are highly polymorphic and are useful in
studies on genetic diversity, phylogeny, gene tagging, genome mapping and evolutionary
biology (Reddy et al, 2002).
ISSR have high reproducibility possibly due to the use of longer primers (16-25
mers) as compared to R A P D primers (10-mers), which permit the use of high annealing
temperature (45-60°C) leading to higher stringency (Reddy et al, 2002). ISSR segregate
mostly as dominant markers following simple Mendelian inheritance (Gupta et al, 1994).
Other markers, marker combinations
Other markers such as C A P S and various modifications and combinations of the
above described methods (Table 1) have been developed and proven useful (Gupta et al,
1999; Wolfe and Liston, 1998).
Application of molecular markers
Cultivar identification
The analysis of genetic variation or diversity in plants has been traditionally
assessed by analysis of morphological or biochemical traits. The assessment of phenotype
25
may not be a reliable measure of genetic difference because of the influence of environment
on gene expression. The analysis of plant D N A allows the direct assessment of variation in
genotype (Henry, 1997).
The type of molecular method used to measure genetic distances in plants will vary
depending upon the magnitude of the genetic differences being assessed. Techniques such
as R A P D analysis may be useful for distinguishing different genotypes within a plant
cultivar while sequence analysis of the ribosomal genes may allow species or higher level
analysis (Henry, 1997).
Table 1. Types of genetic markers commonly generated using P C R method (Rafalski and
Tingey, 1993).
Charac
teristics
Principle
Type of
polymorphis
m
Inheritance
Need prior
sequence
knowledge?
Need radio-
labelling?
R A P D
Arbitrary
primers used
for D N A
amplification
Nucleotide
substitutions
indels
Dominant
No
No
SSR
Site-specific
amplification
of SSRs
Changes in
number of units
in repeated
motif
Codominant
Yes
Sometimes
Genetic Markers
ISSR
Non-specific
amplification
of SSRs
Nucleotide
substitutions
indels
Dominant
No
No
AFLP
Amplification
of fragment
length
polymorphisms
Nucleotide
substitutions
indels
Dominant
No
Sometimes
CAPS, MRSP
Restriction
digest of PCR
amplification
Nucleotide
substitutions
indels
Codominant
Yes
No
R A P D has been widely used to study genetic diversity in Brassica. Geraci et al.
(2001) utilized R A P D markers to assess genetic complexity of Sicilian populations of
Brassica using bulked seeds. They reveal that R A P D technique is useful to confirm
morphological differences among populations and to differentiate extremely similar
populations. Similarly, Cansian and Echeverrigaray (2000) successfully discriminated 16
26
commercial cabbage cultivars using R A P D analysis. Yuan et al. (2004) employed R A P D
markers to study genetic diversity among populations and breeding lines in Brassica napus.
R A P D analyses of individuals of open pollinated cultivars and landraces of collard in the
United States indicated that intra-population genetic variance accounts for as much
variation as that observed between populations (Farnham, 1996). The R A P D technique has
been utilized to discriminate between 14 broccoli and 12 cauliflower cultivars from the U S
and European seed companies. Amplification products from only four random primers
were sufficient to differentiate between cultivars (Hu and Quiros, 1991).
In other vegetable crops, R A P D technology is a rapid, precise and sensitive
technique for identification of pea genotypes (Pisum sativum) (Samec and Nasinec, 1996),
melon (Cucumis melo) (Staub et al, 2004), chicory (Chicorium intybum) (Bellamy et al,
1996) and radish (Raphanus sativus) (Pradhan et al, 2004a).
Besides R A P D , other markers such as AFLP, ISSR and SSR have been employed
for cultivar fingerprinting. A F L P was successfully used to estimate levels of genetic
diversity among Brassica crop species, particularly B. carinata, B. juncea, B. nigra
(Warwick and Soleimani, 2001) and B. rapa (Zhao et al, 2005). ISSR is used for genetic
diversity studies in several Brassica species and Arabidopsis (Bornet and Branchard, 2004).
SSR markers are also used to study the genetic relationships of Brassica vegetables
(Tongue and Griffiths, 2004) and B. napus (Lowe et al, 2004).
D N A fingerprinting techniques exhibit a great potential as a tool for a wide range of
areas in plants, including genotype identification, population genetics, taxonomy and plant
breeding. It appears that R A P D is a powerful technique for cultivar distinction and
therefore was used in this study to distinguish Fi hybrid cultivars commonly grown in
Australia and open pollinated cultivars (landraces) from Indonesia.
Genetic purity analyses
Determination of genetic purity of Fi hybrid seeds is a quality control requirement
in the production of hybrid Brassica vegetable seeds. R A P D technique is employed to
determine levels of self-inbred plants (genetic 'sibs') in a number of hybrid Brassica crops
such as cabbage (Crockett et al, 2000), broccoli (Crockett et al, 2002), Chinese cabbage
(Meng et al, 1998) and cauliflower (Boury et al, 1992) and other vegetable crop such as
chicory (Bellamy et al, 1996).
27
To test for the level of genetic sibs, D N A was isolated from seed or germinated
seeds and then examined using R A P D analyses. Sib contamination percentage obtained by
R A P D analyses was similar to that from field trials. This shows R A P D analyses can be
used for seed purity testing of commercial hybrid seeds (Crockett et al, 2000; 2002, Meng
etal, 1998).
Application of other molecular techniques to detect self-inbred seed in Fi hybrids
has not been reported. In this study, R A P D and SSR techniques were employed to
distinguish parent lines and use the markers generated to differentiate sib and hybrid plants
at the genetic level and compare these with morphological sibs (phenotypically identified
sib plants).
Markers association with morphological traits
Molecular marker associations with agronomically important traits have been
reported in a number of plants such as wheat, soybean, mungbean and Brassica. R A P D
technique is mainly used to find associations, with some reports using other techniques
such as RFLP, ISSR and SSR.
Molecular markers associated with seed and seedling traits using R A P D markers
were reported in radish (Pradhan et al, 2004b). A number of studies have identified R A P D
markers linked with seed coat colour in Brassica napus (Somers et al, 2001), linoleic acid
in Brassica napus (Jourdren et al, 1996), rust resistance in B. juncea (Prabhu et al, 1998),
leaf shape, period to bolting and self incompatibility in B. campestris (Nozaki et al, 1997)
and siliqua shatter resistance in B. rapa (Mongkolporn et al, 2003).
In crop plants, 3 ISSR markers are associated with low seed size in wheat
(Ammiraju et al, 2001), and 13 SSR markers were identified as being associated with seed
size in soybean. R L F P analyses on mungbean (Vigna radiata) revealed 11 loci for seed
weight. T w o of them co-localised with hard seededness (Humpry et al, 2005). RLFP
markers associated with seed weight also exist for soybean (Glycine max). U p to nine
independent loci were associated with seed weight in two different populations (Mian et al,
1996).
Bulk segregant analysis is commonly used to link molecular markers with
phenotypic traits to generate linkage maps. A well-defined linkage map can describe the
linkage relationships of genetically characterized markers with traits of interest (Bert and
Lydiate, 2003; Quijada et al, 2004). Recently a different approach based on statistical
28
analysis was introduced to find associations between molecular markers and morphological
traits (Pradhan et al, 2004b). The new approach employing multivariate analysis and
generating correlations based on principal coordinate analyis is expected to be simpler and
quicker as there is no need for segregating population and a huge number of primers to be
screened. The use of multivariate analysis was explored in this thesis to find links between
seedling traits and molecular markers.
In summary, there are a range of molecular marker techniques currently available
for research in plant genetics and breeding. The marker techniques will be applied in this
study for cultivar identification, purity testing and finding associations between molecular
markers and morphological traits.
29
Chapter 3
Fingerprinting of Cauliflower Cultivars using RAPD Markers
This chapter has been published in Australian Journal of Agricultural Research and is
presented in its P D F format. Citation: Astarini IA, Plummer JA, Lancaster R A , Yan G
(2004) Fingerprinting of cauliflower cultivars using R A P D markers. Australian Journal of
Agricultural Research 55:117-124.
CS1R0 PUBLISHING
www.publish.csiro.au/joumals/ajar Australian Journal of Agricultural Research, 2004, 55, 117-124
Fingerprinting of cauliflower cultivars using R A P D markers
Ida A. AstariniA'C, Julie A. Plummet, Rachel A. Lancaster^, and Guijun YanA
APlant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, W A 6009, Australia.
BDepartment of Agriculture Western Australia, Bunbury District Office, P O Box 1231, Bunbury, W A 6231, Australia.
Corresponding author; email: [email protected]
Abstract. Randomly amplified polymorphic D N A (RAPD) was used to investigate genetic relationships among 25 cultivars of cauliflower (Brassica oleracea var. botrytis L.). Forty decamer primers were examined, among which 15 primers produced polymorphism. Twenty-five polymorphic bands were observed, ranging in size from 428 to 1646 bp. A fingerprinting key was generated using these polymorphic bands. A dendogram was constructed using neighbour-joining analysis based on phylogenetic analysis using parsimony (PAUP). Results indicate that R A P D markers can be used for the routine identification of cauliflower cultivars within B. oleracea var. botrytis L.
Additional keywords: Brassica oleracea var. botrytis L., DNA markers, DNA polymorphism, genetic relationships.
Introduction
Cauliflower is one of the most important commercial crops of Brassica oleracea. Many open-pollinated cultivars and F, hybrids are commercialised around the world and new cultivars are continuously being released. Traditional cultivar identification in cauliflower, as in other crops, is based on a laborious evaluation of phenological and morphological characteristics (Cansian and Echeverrigaray 2000). Other techniques such as ion-exchange high performance liquid chromatography (IE-HPLC) (Mennella et al. 1996) and isozyme analysis (Zheng and Liu 1994) have also been attempted for varietal identification in Brassica.
Cauliflower identification is important for accurate classification of the cultivar, for example, when there is controversy over ownership with regard to Plant Breeder's Rights. Problems with plant classification based on morphological characters have been reported in cauliflower and broccoli (Malatesta and Davey 1996). Intensive breeding within these crops has resulted in irregularities in their classification and cultivar identification. Identification based on morphological characters is also time consuming and requires expensive field trials and evaluation. Morphological differences may be due to environmental influences on plants. Environmental factors such as space, amount of irradiance, temperature, water, and mineral nutrition affect physiological processes and morphology of plants (Kumar et al. 1998).
DNA-based markers have an advantage over other tests for cultivar identification, in that D N A is not affected by environmental factors or the developmental stage of the
© CSIRO 2004
plant. In recent years, identification of Brassica cultivars has been attempted using D N A markers such as restriction fragment length polymorphisms (RFLPs) (Dos Santos et al. 1994), randomly amplified polymorphic D N A sequences (RAPDs) (Williams et al. 1990), microsatellites (Charters etal. 1996), and amplified fragment length polymorphisms (AFLP) (Das et al. 1999).
Despite the development of the newer techniques, R A P D methodologies have retained their advantage in that they are fast, require no radioactive handling facilities, and the costs are relatively low. In Brassica, R A P D markers are considered to be as efficient as R F L P markers for estimating intraspecific genetic relationships among genotypes (Dos Santos et al. 1994). R A P D has been used successfully for genetic fingerprinting in B. oleracea and B. rapa (Kresovich et al. 1992), B. oleracea var. capitata (Phippen et al. 1994), B. rapa ssp. pekinensis (Lamboy et al. 1994), and B. napus (Dulson et al. 1998). R A P D markers have been used to analyse genetic variability among cultivar collections of cauliflower, cabbage, and kale populations in France (Margale et al. 1995). R A P D also appears to be a useful tool to confirm the U triangle relationship between diploid and amphidiploid Brassica taxa (Demeke et al. 1992).
Limited work has been reported on the identification and evaluation of genetic relationships between cultivars or germplasm entries of cauliflower. R A P D markers have been used to quickly estimate genetic distances between cauliflower cultivars in France (Boury et al. 1992) and between broccoli and cauliflower cultivars in the U S A (Hu and Quiros 1991). A pairwise distance matrix was developed
10.1071/AR03012 0004-9409/04/020117
31
18 Australian Journal of Agricultural Research I. A. Astarini et al.
sing the computer program phylogenetic analysis using
arsimony ( P A U P ) to determine the relationships a m o n g
ultivars.
A fingerprinting key using R A P D markers has not been
sported for cauliflower cultivars. A key could be based on
ie same principals as conventional classification keys.
however, conventional keys use morphological
laracteristics, whereas D N A markers could be used in a
ngerprinting key. Markers could be developed from
mding patterns of polymerase chain reaction ( P C R )
roducts from each cultivar.
The aim of this study was to provide a protocol for routine
lentification of cauliflower cultivars within B. oleracea var.
otrytis L. using R A P D markers based on a simple
ngerprinting key and to determine the genetic relationships
nong these cultivars.
Iaterials and methods
'ant material
ie plant material used in this study included 18 cauliflower cultivars ithin B. oleracea var. botrytis L., grown in a variety trial in the field at e Department of Agriculture Western Australia Horticultural ;search Institute at Manjimup, and seedlings of 7 other cultivars pplied by G & S Seedling Nursery, The Seedling Factory, and South icific Seeds. The cultivars were Advantage, Alabama, Cauldron,
:leste, CF535, CF536, Chaser, CLF33902, Discovery, Donner, emont, Gibralter, G376, G389, J3195, Liberty, Monarch, Morpheus, 3444, Omeo, Plana, Sirente, SPS716, SPS3074, and Virgin. These
ltivars were supplied by Henderson Seeds, Lefroy Valley Seeds,
'ngenta Seeds, South Pacific Seeds, and Yates Vegetable Seeds. iltivars were selected by seed companies as the most likely to be
ccessfiil in the Manjimup district, which is the major region in
estern Australia for the production of export cauliflowers. Individual
if samples were collected from the field, transported to the laboratory i ice, and then stored at -80°C.
VA extraction
^A was extracted from leaf samples following the C T A B method
scribed by (Yan et al. 2002). Leaf tissue (1 g) from 4 young leaves
is ground in liquid nitrogen using a mortar and pestle. The extract was
msferred to 50-mL centrifuge tubes containing 10 m L C T A B
traction buffer [ 2 % CTAB, 100 m M TRIS (pH 8), 20 m M EDTA,
1M NaCl], and |J-mercaptoethanoI (20 uL) was then added. The
xture was swirled gently and incubated in a waterbath at 60°C for 15
n. Chloroformrisoamyl alcohol (24:1, 10 m L ) was added, mixed
:11, and centrifuged at 2236G for 20 min at 20°C. The supernatant was
llected in a fresh centrifuge tube and an equal amount of isopropanol
is added, mixed well and then refrigerated at -20°C for 2 h. The lution was then centrifuged at 2236G for 10 min at 20°C to separate
: D N A pellet. Isopropanol was poured off and the pellet was washed th 7 0 % and then 100% ethanol. The pellet was then dryed in a
siccator at 37°C for 30 min. The pellet was resuspended in 500 uL '• buffer [10 m M TRIS-HC1 (pH 8) and 1 m M E D T A (pH 8)] and then > uL RNAse (10 ng/mL) was added to remove R N A contamination,
i the tube was tapped gently to mix it thoroughly. The mixture was
itedat37°Cforatleast3h.
IA quantification
e D N A extract was diluted with sterile deionised water to 1/250 and
'A concentration was measured using a U V absorbance D U R 640
spectrophotometer (Beckman, U S A ) at 260 ran. Readings were taken 3 times for each sample and the average calculated. Absorbance at
260 n m was used to calculate the D N A concentration in the sample
(Eqn 1) and the ratio between absorbance at 260 nm and 280 nm was used to estimate D N A purity. The required D N A concentration
(60 ng/uL) was prepared for each genotype using injection water.
Absorbance at 260 nm x 250 * 50 = fig/mL DNA cone. (1)
RAPD analysis
Forty arbitrary decamer primers were examined for P C R amplification. All primers were synthesised by Life Technologies customer primer
program and published sequences are indicated in Table 1. The P C R reaction was performed in a final volume of 25 uL containing injection water, 1 x Taq polymerase buffer (Promega), 1.5 units of Taq polymerase (Promega), 0.05 m M of each dNTP (dATP, dCTP, dGTP,
dTTP; Promega), 1 p.M of primer, 1.5 m M MgCl2, and 120 ng template
D N A . A negative P C R tube containing all components except genomic D N A was used with each primer to check for contamination. P C R was performed in an iCycler (Bio-Rad, U S A ) using the following cycling
program: 10 times 5-s cycles of denaturation at 94°C, annealing at 35°C for 30 s, elongation at 72°C for 1 min, 25 times 5-s cycles of denaturation at 94°C, annealing at 45°C for 30 s, elongation at 72°C for
1 min, and finally 1 cycle including an elongation step at 72°C for 2 min. The iCycler was programmed to retain the samples at 4°C until they were collected and stored at-20°C.
Gel electrophoresis
Each sample of R A P D products (10 uL) was mixed with 6 x gel loading
buffer (2 uL) a nd loaded onto an agarose (1.5% w/v) gel for electrophoresis (Bio-Rad, N S W ) in 1 x T A E buffer (50 x T A E buffer
contains 242 g TRIS base, 57.1 g glacial acetic acid, 100 m L 0 . 5 M EDTA,
and distilled water to 1 L) at 60 V for 2 h. A 100-bp D N A ladder (5 pX of D N A ladder and 1 uL of gel loading buffer, Promega) was included
on both sides as a molecular standard. Amplification products separated
by gels were stained in ethidium bromide solution (2 uL Etbr/100 m L 1 x T A E buffer) for 30 min and then photographs were taken under U V
light using a digital camera (Kodak D C 120) and the images were recorded with a Macintosh Kodak ID 2.0 computer program.
Analysis of data
A data matrix was created from photographs of gels by scoring 1 for present bands or 0 for absent bands. The size of amplification products
in base pairs was estimated using the D N A marker with bands of known
molecular weight. The regression of distance run against the molecular
weight of each band of the 100-bp D N A ladder was used to calculate the equivalent molecular weight in base pairs for each band. Only
clearly scorable bands with the size between 300 and 1700 bp were
included in the analysis. A pairwise distance matrix was generated based on total and mean
R A P D band differences in PAUP, using a Power Macintosh 7600/120
(Swofford 1993). The data were subsequently used to construct a
dendogram using neighbour-joining analysis. To test the validity of phylogenetic relationships revealed by neighbour-joining, the data were
also used to generate fingerprinting keys.
Results
All 40 primers produced multiple PCR fragments in each
cultivar. Ninety bands were scored, of which 25 were
polymorphic. Fifteen out of the 4 0 primers tested had
polymorphic bands (Table 1). N o bands were observed in the
control lane (Fig. 1).
32
Fingerprinting cauliflower using RAPD markers Australian Journal of Agricultural Research 119
Table 1. Decamer primers used in this study Primers were synthesised by Life Technologies. Primers marked with
an asterisk (*) generated polymorphisms
Primer name
AOI A
A02 A
A03 A
*A04A
D12B
D20B
Hong-H
LISA-1
LISA-2
•OPA-07C
•OPB-04D
OPB-08C
*OPB-12D
•OPH-01E
OPH-03E
OPH-06E
OPH-09E
*UBC-106F
UBC-I27F
UBC-147F
Nucleotide sequence (5' -> y)
AAGACGACGG
AATCCGCTGG
AGTCGGCCCA
AACAGGGCAG
CACCGTATCC
ACCCGGTCAC
GTCACTGCTC
GGCCTTGAGT
GGTCCTCAGG
GAAACGGGTG
GGACTGGAGT
GTCCACACGG
CCTTGACGCA
GGTCGGAGAA
AGACGTCCAC
ACGCATCGTG
TGTAGCTGGG
CGTCTGCCCG
ATCTGGCAGC
GTGCGTCCTC
Primer name
*UBC-250F
SF-04
•SF-06
SF-08
SF-09
SF-13
SF-17
•SL-01
SL-03
SL-07
•SL-08
SL-12
•SK-01
SK-02
•SK.-03
•SK-09
*SK-14
SK-17
SK-18
•SK-19
Nucleotide sequence
(S'-+3')
CGACAGTCCC
GGTGATCAGG
GGGAATTCGG
GGGATATCGG
CCAAGCTTCC
GGCTGCAGAA
AACCCGGGAA
GGCATGACCT
CCAGCAGCTT
AGGCGGGACC
AGCAGGTGGA
GGGCGGTACT
CATTCGAGCC
GTCTCCGCAA
CCAGCTTAGG
CCCTACCGAC
CCCGCTACAC
CCCAGCTGTG
CCTAGTCGAG
CACAGGCGGA
Obtained from: A Hu and Quiros (1991); BBoury el al. (1992); cCrockett et al. (2002); "Crockett et al. (2002); EMeng el al. (1998); FMailer and May (1999).
The molecular weight of bands amplified from 15 primers (Table 1) ranged from 428 to 1646 bp (Table 2). Two to 6 bands were scored per primer. A minimum of 12 markers (coded as primer-number of base pairs: A04-1427, OPA07-498, OPB04-797, OPB12-1159, OPH01-1646, OPH01-1460, SL01-1062, SL08-698, SK14-1294, SK14-575, UBC106-525, and UBC250-839) obtained from 10 primers was required to distinguish between cultivars. A fingerprinting key was developed for 18 cultivars (Fig. 2), but 7 cultivars could not be separated. Cultivar CLF33902 showed identical R A P D profiles to G376, as did cultivar Plana with Alabama, cultivar Donner with SPS3074 and with Sirente. A dendogram showing the relationship among cultivars was generated (Fig. 3). Three major clusters were obtained. Monarch had a very distant relationship to other cultivars. Cultivars CLF33902 and G376 were very closely related, as revealed by their neighbour-joining (NJ) coefficient being zero. Zero NJ coefficient was also obtained between cultivars Plana and Alabama, and between cultivars Donner, SPS3074, and Sirente.
The pairwise distance matrix generated by the P A U P program was used to quantify the differences among all
M l 2 3 4 5 1 1 1 1
6 7 8 9 10 1 2 3 4 1 1 1 1 1 2 2 2 2 2 5 6 7 8 9 20 1 2 3 4 5 C M
Fig. 1. R A P D amplification profiles obtained with primer UBC106. Standard bands are indicated by arrows. Molecular weights of the standard are indicated in base pairs. M, 100 bp D N A ladder; C, control lane; 1, Monarch; 2, Donner; 3, M3444; 4, Cauldron; 5, Gibralter; 6, Chaser; 7, CF535; 8, SPS716; 9, Liberty; 10, Omeo; 11, G389; 12, Advantage; 13, CLF33902; 14.CF536; 15.G376; 16, Virgin; 17.J3195; 18, Sirente; 19, Morpheus; 20, Fremont; 21, Plana; 22, Alabama; 23, SPS3074; 24, Celeste; 25, Discovery.
33
Australian Journal of Agricultural Research
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Fingerprinting cauliflower using R A P D markers A ustralian Journal of Agricultural Research 121
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A
Cauldron, Chaser, CF535, Liberty, G389, Advantage, CLF33902, G376, J3195, Plana. Alabama, Celeste, Morpheus, Discovery Liberty, G389, Advantage, CLF33902, G376 CLF33902, G376 Liberty, G389, Advantage P Advantage A Liberty, G389 P Liberty A G389 Cauldron, Chaser, CF535, J3195, Plana Alabama, Celeste. Morpheus, Discovery J3195 Cauldron, Chaser, CF535, Plana, Alabama, Celeste, Morpheus, Discovery
2. U B C 106-525
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Chaser, Plana, Alabama, Morpheus Chaser Plana, Alabama, Morpheus P Morpheus A Plana, Alabama Cauldron, CF535, Celeste, Discovery Discovery Cauldron, CF535, Celeste P SPS3075 A Cauldron, CF535 P Cauldron A CFS3S
Monarch, Donner, M3444, Gibralter, SPS716. Omeo, CF536, Virgin, Fremont, SPS3074, Sirente Monarch, M3444, SPS716, Fremont M3444.SPS716 SPS716 M3444 Monarch, Fremont P Fremont A Monarch Donner, Gibralter, Omeo, CF536, Virgin, SPS3074, Sirente Omeo Donner, Gibralter, CF536, Virgin, SPS3074, Sirente P Donner, Virgin, SPS3074, Sirente P Virgin A Donner, SPS3074, Sirente A Gibralter, CF536 P Gibralter A CF536
Fig. 2. Fingerprinting key for cauliflower cultivars generated from R A P D markers.
Bold indicates that the cultivar has been differentiated.
cultivars (Table 3). The pairwise difference between cultivars
ranged from 0 to 16 markers.
Discussion
Eighteen cultivars were differentiated in the fingerprinting key, showing that each of these cultivars had a different genetic background. Seven other cultivars were clustered together into 3 different groups, indicating that they are closely related. Cultivars from the same company were often clustered together, possibly indicating similar parentage and a high level of genetic similarity. Cultivar Donner was the same as SPS3074, which was the breeding line number
(South Pacific Seeds, pers. comm.). Most crops are given a commercial name upon release, eliminating the requirement for identification numbers (Noli et al. 1999).
Cultivars CLF33902 and G376 were obtained from different seed companies; however, they may have similar parental lines. The same germplasm may be used in different breeding programs, resulting in similar progeny and released varieties (Cansian and Echeverrigaray 2000). It should be noted that a small number of genes could make a substantial difference to the performance of a variety. This is highlighted, for example, in herbicide, pest, or disease resistance. R A P D , by definition, uses
35
Australian Journal of Agricultural Research I. A. Astarini et al.
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Fingerprinting cauliflower using R A P D markers Australian Journal of Agricultural Research 123
0.5
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— Monarch
Fig. 3. Dendogram of 25 cauliflower cultivars, constructed using
PAUP based on neighbour-joining (NJ) analysis. The numbers at each node represent NJ coefficient of differences.
randomly marked sections of the genome and any associated genes are not necessarily critical in distinguishing cultivar performance (Heneen and Jorgensen 2001). Also note that the R A P D technique tends to provide only dominant markers. Individuals containing 2 copies of an allele are not distinguished quantitatively from those containing only 1 copy (Williams et al. 1990).
The dendogram also indicated possible genetic relationships among cultivars (Fig. 3) and similarities of these relationships with economic traits and source of seeds were also noticed. For example, in trials in Manjimup, W A , in 2002, Monarch was clearly distinguished from other cultivars. High similarity between Donner and Sirente corresponded to the morphology of these cultivars. Both Donner and Sirente are recommended for spring and autumn harvest and both have pure white curds (McArthur 2001). In contrast, Plana, Discovery, and Fremont, which were in different groups of the dendogram, are all summer cultivars (McArthur 1999).
The neighbour-joining coefficient varied between 0.1 and 6.4, which means that the genetic diversity ranged from little
to reasonably wide. A few cultivars appeared to have the same or similar parent lines. Cauliflower has been bred for a long time, and the present day cultivars have a very narrow genetic base. This is typical of many present-day crops. Following R A P D analysis on cabbage and pea, Jaccard's coefficient ranged from 0.72 to 0.87 and from 0.49 to 0.98, respectively, indicating little genetic diversity. Many cabbage or pea cultivars have the same ancestors (Samec and Nasinec 1996; Cansian and Echeverrigaray 2000).
Identification of cauliflower cultivars with R A P D markers has been reported by H u and Quiros (1991). They used 12 cultivars from 4 American seed companies. Successful cultivar identification by diagnostic markers was developed using 4 primers (AOl, A 0 2 , A 0 3 , and A04). With our cultivars, which came from European, Australian, and N e w Zealand based seed companies, only A 0 4 produced polymorphic bands. This may indicate that different seed companies have used parent lines with different genetic background. However, these studies indicated that a RAPD-based key encompassing all worldwide cultivars is a feasible goal for future research.
Bulked D N A samples were used in this experiment in order to detect the c o m m o n genetic base of each cultivar. Bulked D N A is commonly used for R A P D analysis, e.g. in cauliflower, cabbage, and kale local cultivars from France (Margale et al. 1995), B. napus cultivars (Dulson et al. 1998; Mailer and May 1999), and Sicilian wild populations of Brassica (Geraci et al. 2001). Use of bulked samples may allow identification of a distinctive profile of R A P D markers. Moreover, the large number of populations in Brassica collections makes genetic diversity studies based on plant-to-plant analysis impracticable.
Hybrid cultivars used in this experiment were produced using C M S (cytoplasmic male sterility) and SI (self-incompatibility) systems. In the C M S systems, male sterile plants are used as the female parent and a specific maintainer line is used as the male parent. C M S allows the production of 1 0 0 % hybrid seed (Ruffio-Chable et al. 2000). Self-incompatibility avoids the production of self-inbred plants and is commonly used for hybrid seed production of cauliflower, cabbage, and broccoli (Crockett et al. 2002).
In Brassica, R A P D markers are considered to be as efficient as R F L P markers for estimating genetic relation-ships among genotypes. A study on 45 B. oleracea genotypes indicated that R A P D provides a level of resolution equivalent to RFLPs for determination of the genetic relationships among genotypes (Dos Santos et al. 1994).
The present study demonstrated that R A P D analysis provides a simple and reliable method for cultivar identification. Using R A P D markers to identify genetic diversity within B. oleracea var. botrytis L. is important, providing breeders with genetic information for the improvement of crops. Identification of genetic diversity/similarity may help in selection of appropriate
37
;4 Australian Journal of Agricultural Research I. A. Astarini et al.
eeding lines. Future work to systematically identify R A P D
arkers associated with economic traits, origin, and general
;netic diversity would be beneficial.
cknovvledgments
re thank AusAID for providing a scholarship to Ida Ayu
starini. Thanks also to Henderson Seeds, Lefroy Valley
;eds, Syngenta Seeds, South Pacific Seeds, and Yates
jgetable Seeds for providing seeds, and to G & S Seedlings
id The Seedlings Factory for providing s o m e seedlings for
is project. This project was supported by grants from the
estern Australia Department of Agriculture and Plant
lology, The University of Western Australia.
eferences
iury S, Lutz I, Gavalda M-C, Guidet F, Schlesser A (1992)
Empreintes genetiques du chou-fleur par R A P D et verification de la
purete hybride FI d'un lot de semences. Agronomie 12, 669-681.
nsian RL, Echeverrigaray S (2000) Discrimination among cultivars
of cabbage using randomly amplified polymorphic D N A markers.
HortScience 35, 1155-1158. larters Y M , Robertson A, Wilkinson MJ, Ramsay G (1996) P C R
analysis of oilseed rape cultivars (Brassica napus L. ssp. oleifera) using 5'-anchored simple sequence repeat (SSR) primers. Theoretical and Applied Genetics 92, 442—447. doi: 10.1007/S001220050147 ockett PA, Singh M B , Lee C K , Bhalla PL (2002) Genetic purity analysis of hybrid broccoli (Brassica oleracea var. italica) seeds using R A P D PCR. Australian Journal of Agricultural Research 53,
51-54. doi:10.1071/AR01022 s S, Rajagopal J, Bhatia S, Srivastava PS, Lakshmikumaran M
(1999) Assessment of genetic variation within Brassica campestris
cultivars using amplified fragment length polymorphism and
random amplification of polymorphic D N A markers. Journal of
Biosciences 24,433-440. meke T, Adams RP, Chibbar R (1992) Potential taxonomic use of
random amplified polymorphic D N A (RAPD): a case study in Brassica. Theoretical and Applied Genetics 84, 990-994.
s Santos JBD, Nienhuis J, Skroch P, Tivang J, Slocum M K (1994) Comparison of R A P D and RFLP genetic markers in determining
genetic similarity among Brassica oleracea L. genotypes.
Theoretical and Applied Genetics 87, 909-915. lson J, Kott LS, Ripley V L (1998) Efficacy of bulked D N A samples
for R A P D D N A fingerprinting of genetically complex Brassica
napus cultivars. Euphytica 102, 65-70.
doi:10.1023/A:1018378304701 raci A, Divaret I, Raimondo FM, Chevre A M (2001) Genetic
relationships between Sicilian wild populations of Brassica
analysed with R A P D markers. Plant Breeding 120, 193-196.
doi: 10.1046/J. 1439-0523.2001.00589.X
leen W K , Jorgensen R B (2001) Cytology, R A P D , and seed colour of progeny plants from Brassica rapa-alboglabra aneuploids and
development of monosomic addition lines. Genome 44,1007-1021. J, Quires CF (1991) Identification of broccoli and cauliflower
cultivars with R A P D markers. Plant Cell Reports 10, 505-511. sovich S, Williams JGK, McFerson JR, Routman EJ, Schaal B A
(1992) Characterization of genetic identities and relationships of
Brassica oleracea L. via a random amplified polymorphic D N A
assay. Theoretical and Applied Genetics 85, 190-196.
http://www.publish.
Kumar PP, Yau JCK, Goh CJ (1998) Genetic analyses of Heliconia
species and cultivars with randomly amplified polymorhic D N A (RAPD) markers. Journal of the American Society for Horticultural
Science 123, 91-97. Lamboy WF, McFerson JR, Li R, Kresovich S (1994) Relationships
among Chinese vegetable brassicas using R A P D markers.
Cruciferae Newsletter 16,44—44. Mailer RJ, May C E (1999) Heterogeneity of random amplified
polymorphic D N A sequences in individual seedlings and bulked
samples of four cultivars of Brassica napus. Plant Breeding 118,
465^170. doi: 10.1046/J. 1439-0523.1999.00428.X Malatesta M , Davey JC (1996) Cultivar identification within broccoli,
Brassica oleracea L. var. italica Plenk and cauliflower, Brassica
oleacea var. botrytis L. Acta Horticulturae 407, 109-113. Margate E, Herve Y, H u J, Quiros C F (1995) Determination of genetic
variability by R A P D markers in cauliflower, cabbage and kale local cultivars from France. Genetic Resources and Crop Evolution 42,
281-289. McArthur S (1999) Winter newsletter. South Pacific Seeds,
Christchurch, N Z . McArthur S (2001) Winter newsletter. South Pacific Seeds,
Christchurch, N Z . Meng X, Hong M , Zhang W, Wang D (1998) A fast procedure for
genetic purity determination of head Chinese cabbage hybrid seed based on R A P D markers. Seed Science and Technology 26,
829-833. Mennella G.Iori A, Sanaja VO, Magnifico V (1996) Broccoli and
cauliflower cultivars identification through IE-HPLC seed protein
analysis. Acta Horticulturae 407, 115-121, Noli E, Conti S, Maccaferri M , Sanguineti M C (1999) Molecular
characterization of tomato cultivars. Seed Science and Technology 27, 1-10.
Phippen W B , Kresovich S, McFerson JR (1994) Assessing genetic
identity and relatedness in cabbage with RAPDs. Cruciferae Newsletter 16, 46-46.
Ruffio-Chable V, Chatelet P, Thomas G (2000) Developmentally
'aberrant' plants in F, hybrids of Brassica oleracea. Acta Horticulturae 539, 89-94.
Samec P, Nasinec V (1996) The use of R A P D technique for the
identification and classification of Pisum sativum L. genotypes.
Euphytica 89, 229-234. Swofford D L (1993) 'PAUP: Phylogenetic analysis using parsimony,
version 3.1.' (Illinois Natural History Survey: Champaign, IL) Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey S V (1990)
D N A polymorphisms amplified by arbitrary primers are useful as
genetic markers. Nucleic Acids Research 18, 6531-6535. Yan G, Shan F, Plummer JA (2002) Genetic relationship within Boron ia
(Rutaceae) as revealed by karyotype analysis and R A P D molecular
markers. Plant Systematics and Evolution 233, 147-161. Zheng XY, Liu Y (1994) Inbred testing of Chinese cabbage F( varieties
by peroxidase and esterase isozyme analysis. Acta Horticulture
Sinica 21, 65-70.
Manuscript received 9 January 2003, accepted 1 September 2003
csiro.au/journals/aj ar
38
Chapter 4
Genetic Diversity of Open Pollinated Cauliflower
Cultivars in Indonesia
This chapter has been presented in an International/Australian Society of Horticultural
Sciences (ISHS/AUSHS) conference entitled: Harnessing the potential of horticulture in the
Asian-Pacific region. The paper has been published in Acta Horticulturae and is presented
here in the published format. Citation: Astarini IA, Plummer JA, Yan G, Lancaster R A
(2005) Genetic diversity of open pollinated cauliflower cultivars in Indonesia. Acta
Horticulturae 694, 149-152.
Genetic diversity of open pollinated cauliflower cultivars in Indonesia
LA. Astarini, J.A. Plummer and G.Yan R.A. Lancaster Plant Biology, F N A S Department of Agriculture Western Australia The University of Western Australia Bunbury District Office 35 Stirling Highway, Crawley W A 6009 P O Box 1231 Bunbury, W A 6231, Australia Australia
Keywords: Brassica oleracea, DNA fingerprinting, DNA markers, genetic relationships.
Abstract Eight cauliflower cultivars collected from three production regions in
Indonesia were evaluated using R A P D markers. The objectives of this study were to investigate genetic variation and relationships between cultivars and to evaluate variation within cultivars as all of them are open-pollinated. D N A was extracted using Nucleon Phytopure Plant D N A extraction kit, followed by treatment with RNAse. D N A polymorphism generated from 10 polymorphic primers was used to construct a dendogram using the unweighted pair-group method with arithmetic averages ( U P G M A ) . The R A P D technique indicated that variation occurred both within and between cultivars.
INTRODUCTION Cauliflower is gaining popularity as a vegetable crop in Indonesia. Cauliflowers
attract middle and upper income Indonesian, because the price is higher than other vegetables available in the market. Cauliflower is produced in cooler highland regions across The Indonesian archipelago. Lembang, Malang and Bedugul are central production areas for vegetables in West Java, East Java and Bali respectively. In Bali, cauliflowers are grown to supply the tourist industry.
Most cauliflower cultivars available in Indonesia are open-pollinated (OP) lines. Therefore, variation in product frequently occurs. Variation is found in curd size, maturity time and resistance to diseases such as club root. Little is known about the genetic make up of these cultivars and there has never been a systematic evaluation of cauliflower cultivars in Indonesia. The first step in crop improvement in developing countries should be a full assessment of the local materials (Williams et al, 1991).
The objectives of this study were to determine the diversity present among Indonesian cultivars and their relationships, to evaluate within cultivar variation and to examine the potential of Indonesian cauliflowers as new sources in Brassica oleracea gene
pools.
MATERIAL AND METHODS
Plant materials and DNA extraction Eight cauliflower cultivars were collected, they are 'Harli', 'Broad' and 'Blaster'
(from West Java), 'Manalagi', 'Bandung' and 'Gembel' (from East Java), 'Malang' and 'Bedugul' (from Bali). Since liquid nitrogen was not available at Biotechnology Laboratory, Udayana University, Bali, leaf samples, together with mortars and pestles were frozen in the -80°C freezer for 30 minute before grinding. Once the leaves were frozen, they were ground immediately into powder. D N A was extracted using Nucleon Phytopure
Proc. IS on Hort. in Asian-Pacific Region 40 (149)
Ed. R. Drew Acta Hort. 694, ISHS 2005
Plant D N A extraction kit (Amersham Biosciences, UK). Purified D N A was kept in 1.5 ml tubes in a cool container and brought to Perth, Western Australia for analysis.
R A P D analysis
Three cultivars ('Blaster', 'Manalagi' and 'Bedugul) were chosen for inter-variety differences with eight individuals for each cultivar. To test between cultivars variation, D N A from 4 individuals of were bulked randomly. Ten arbitrary decamer primers were screened for PCR amplification. Primer name and nucleotide sequence (5' —• 3') were: SK-01 (CATTCGAGCC), SK-14 (CCCGCTACAC), SK-19 ( C A C A G G C G G A ) , SL-01 (GGCATGACCT), SL-08 ( A G C A G G T G G A ) , SF-06 (GGGAATTCGG), UBC-106 (CGTCTGCCCG), UBC-250 (CGACAGTCCC), OPA-07 ( G A A A C G G G T G ) , OPB-04 (GGACTGGAGT). All primers were synthesized by Life Technologies customer primer
program. The P C R reaction was performed in a final volume of 15 uL containing injection water, lx Taq polymerase buffer (Promega), 1 unit of Taq polymerase (Promega), 0.05 m M
of each dNTP (dATP, dCTP, dGTP, dTTP; Promega), 1 u M of primer, 1.5 m M MgCl2 and 40 ng template D N A . P C R was performed in an iCycler ™ (Bio-Rad, USA) using the following cycling program: 10 times 5 s cycles of denaturation at 94°C, annealing at 35°C for 30 s, elongation at 72°C for 1 min, 25 times 5 s cycles of denaturation at 94°C, annealing at 45°C for 30 s, elongation at 72°C for 1 min and finally 1 cycle included an elongation step at 72°C for 2 min. The iCycler was programmed to retain the samples at 4°C until they were collected and stored at -20°C. The P C R products were examined using 1.8 % agarose gel electrophoresis in T A E buffer and stained with ethidium bromide. A 100 bp ladder (Promega) was used as a size marker.
Data analysis A data matrix was created based on photographs of gels by scoring 1 for present
bands or 0 for absent bands. The molecular weight in base pairs for each band was estimated using regression of distance run against the molecular weight of the 100 bp D N A ladder. A pairwise distance matrix was generated based on total R A P D band differences in PAUP (Phylogenetic Analysis Using Parsimony), using a Power Macintosh 7600/120 (Swofford, 1993). The data was subsequently used to construct a dendogram using U P G M A analysis.
RESULTS AND DISCUSSION All ten R A P D primers produced polymorphic bands. These primers have proved
useful in distinguishing Australian cultivars (Astarini et al, 2004). This experiment confirmed that R A P D technique is reproducible and is a reliable, rapid method for D N A
fingerprinting. A total of 65 bands were scored and 34 of these were polymorphic. The molecular
weight of amplified bands ranged from 380 to 1800 base pairs. Four to eight bands were scored per primer. A dendogram showing the relationship between and within cultivars was generated (Figure 1). Two major clusters were obtained. 'Bedugul' has a distant relationship to other cultivars. 'Bedugul' was cultivated and bred locally in Bali. This indicating 'Bedugul' may have different origins from the rest of the cultivars.
'Harli' and 'Broad' were cultivated in Lembang, West Java. These cultivars may have been introduced from India in the 19th century and have been reproduced locally since then. 'Blaster' and 'Manalagi' are intermixed in the dendogram, suggesting that these cultivars are similar, although they are cultivated in different regions, West Java and East
Proc. IS on Hort. in Asian-Pacific Region 41 (150)
Ed. R. Drew Acta Hort. 694, ISHS 2005
Java respectively. Gene flow is more likely to occur within the island than between islands. 'Malang', another Bali line, had a close relationship with 'Gembel' from East Java. A number of cauliflower growers in Bali have bought cauliflower seeds from East Java, and this explains the similarity between these two cultivars.
There was substantial within variety variation and almost all individuals were separated on the dendogram. Seed production of each variety is done by local farmers and variation within variety maybe due to cross-pollination, as isolation of plants during seed production is poor. Populations have been selected and traditionally multiplied by growers and they will therefore possess genetic adaptation to local conditions. This variability may contribute to diversity for breeding purposes.
The sale of seeds among local farmers, including lots of commercial seed from seed companies, m ay also contribute to genetic variability. In this region, cauliflowers are planted around vegetable gardens and seeds are often collected from the best individuals without specific varietal isolation. Frequent intercrossing among different local varieties in the same area may increase the genetic variability of populations from the same region. In West Java, cauliflowers are planted as intercropping plants, usually with chilli or spring onion.
CONCLUSIONS In conclusion, the variability among cauliflower cultivars could be related primarily
to their geographical origin. Identification of genetic diversity with molecular markers may help in selection of appropriate breeding lines and the time for new variety development can be reduced.
ACKNOWLEDGEMENTS W e would like to thank AusAID for providing a scholarship to Ida Astarini. Thanks
also to Pak Ah m a d Rivani, Dr. Ir. Agus Suryanto M S , Ir. Sitawati, M S and Pak D e w a Okayadnya for providing information about cauliflower production in Indonesia and provision of field grown cauliflowers. Sincere thanks to Prof. I G.P. Wirawan for permitting Ida Astarini to extract D N A in his Biotechnology Lab in Bali. This project was supported by grants from The Department of Agriculture Western Australia and Plant Biology, The University of Western Australia.
Literature cited Astarini, I.A., Plummer, J.A., Lancaster, R.A. and Yan, G. 2004. Fingerprinting of
cauliflower cultivars using R A P D markers. Australian Journal of Agricultural Research
55:117-124. Swofford, D. L. 1993. 'PAUP:Phylogenetic analysis using Parsimony, version 3.1' (Illinois
Natural History Survey: Champaign, Illinois) Williams, C.N., Uzo, J.O. and Peregrine, W.T.H. 1991. Vegetable Production in the
Tropics. Intermediate Tropical Agriculture Series. Longman Scientific and
Technical, U K .
Proc. IS on Hort. in Asian-Pacific Region
Ed. R Drew Acta Hort. 694, ISHS 2005
42(151)
3.0
0
0 5
0.5
2.1
2.3 51
2.6 77
1.9
0 3.0
3.0
1.6 53
0.2
0.3 55
0.5 72
2.5
2.5
0
0
0.5
1.7
1.0 59
1.5 60
0.2
1.0
1.0
U.5
0.5
0.2
1.3
1.2
u
0
0.5
0.5
1.5
1.5
0.8
6.2
0.8
0.6
0.5
i..\j
2.0
3.0
1.0 ZR
3.5
<£.U
2.0
1.1
3.4 9 3
3.0
30
1.5
1.5
Harli
Broad
Bandung
Gembel
Malang
Blaster-1
Blaster-2
Manalagi-2
Blaster-3
Manalagi-4
Manalagi-7
Manalagi-1
Manalagi-3
Manalagi-8
Manalagi-5
Manalagi-6
Blaster-4
Blaster-6
Blaster-7
Blaster-8
Blaster-5
Bedugul-1
Bedugul-2
Bedugul-7
Bedugul-8
Bedugul-3
Bedugul-4
Bedugul-5
Bedugul-6
Fig. 1. Dendogram of Indonesian cauliflower cultivars, constructed by unweighted pair-
group method with arithmetic averages ( U P G M A ) based on total character differences.
Numbers above branches represent branch length and numbers below branches indicate
bootstrap values.
Proc. IS on Hort. in Asian-Pacific Region Ed. R. Drew Acta Hort. 694, ISHS 2005
43 (152)
Chapter 5
Genetic Diversity of Indonesian Cauliflower
Cultivars and Their Relationships with Hybrid
Cultivars Grown in Australia
This chapter has been accepted and currently in press in Scientia Horticulturae and is
presented here in its submitted format. Citation: Astarini IA, Plummer JA, Yan G,
Lancaster R A (2006) Genetic Diversity of Indonesian Cauliflowers and Their
Relationships with Hybrid Cultivars Grown in Australia. Scientia Horticulturae (in
press)
Genetic diversity of Indonesian cauliflower cultivars and their
relationships with hybrid cultivars grown in Australia
Ida A. AstariniAB, Julie A. PlummerA, Rachel A. Lancaster0, and Guijun Y a n A
School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of
Western Australia, 35 Stirling Highway, Crawley W A 6009, Australia.
Author for correspondence: Ida Ayu Astarini, email: arin(a).student.uwa. edu.au
Fax: +61 8 6488 1108, Phone: +61 8 6488 1992
department of Agriculture, Western Australia, Bunbury District Office, P O Box 1231
Bunbury, W A 6231, Australia.
Abstract
The objectives of this study were to investigate genetic variation and relationships
between Indonesia, Australian and European based cultivars and to evaluate variation
within Indonesia cultivars as all cultivars are open-pollinated. Eight cauliflower cultivars
collected from three production regions in Indonesia and four Fi hybrids cultivars grown in
Australia were evaluated using R A P D and ISSR markers. D N A polymorphisms generated
from 10 polymorphic R A P D primers were used to construct a dendogram using the
unweighted pair-group method with arithmetic averages ( U P G M A ) and to generate a
fingerprinting key. ISSR marker analysis using 14 primers were attempted but D N A
polymorphisms could not be clearly identified. The R A P D technique indicated that
variation occurred both within and between Indonesian cultivars. Comparison between
Indonesian, Australian and European based cultivars showed that Indonesian cultivars have
unique genotypes and would be good sources of genes for future crop improvement.
Key words: Brassica oleracea; D N A fingerprinting; D N A markers; ISSR, R A P D .
1. Introduction
Cauliflower is gaining popularity as a vegetable crop in Indonesia. Cauliflowers are
mainly consumed by middle and upper income Indonesians, because they are still less
common and only seasonally available, and therefore attract a higher price compared to
traditional vegetables available in the market. Cauliflower is produced in cool highland
regions across the Indonesian archipelago. Lembang, Malang and Bedugul are central
45
production areas for vegetables in West Java, East Java and Bali respectively. In Bali,
cauliflowers are grown to supply the tourist industry.
Most cauliflower cultivars available in Indonesia are open-pollinated lines.
Therefore variation in product frequently occurs. Variation is found in curd size, maturity
time and resistance to diseases such as club root. Little is known about the genetic make up
of these cultivars and there has never been a systematic evaluation of cauliflower cultivars
in Indonesia.
Major constraints to increasing cauliflower production in Indonesia include lack of
varieties adapted to tropical conditions, inadequate supplies of seeds of improved varieties
and a high incidence of pests and diseases (Asandhi and Sastrosiswojo, 1988; Darmawan
and Pasandaran, 2000). Indonesian research stations in collaboration with overseas seed
companies and growers from major cauliflowers growing areas have tested introduced
cultivars in Indonesia. To date these cultivars have not been successful and breeding
cauliflower for tropical conditions is required.
The first step in crop improvement in developing countries should be a full
assessment of the local materials, including collection, evaluation and molecular
characterization of germplasm lines. Often, local varieties are of excellent quality and
flavour, have a good level of resistance to pests and diseases and may be superior to exotic
materials (Williams et al, 1991)
Cauliflower cultivars grown in Australia are mainly Fi hybrid cultivars, which have
a limited genetic source. In the long term, there is a need for new or novel genes for
cultivar improvement. It is essential that w e avoid loosing genetic variation and many
Western cultivars were highly bred (Sharma et al., 2004). Investigating genetic distance
between Indonesian and Australian grown cultivars will provide useful information for
future breeding programs in both countries.
Inter Simple Sequence Repeat (ISSR) and Random Amplified Polymorphic D N A
( R A P D ) were employed to investigate the genetic diversity. ISSR markers are highly
polymorphic and are useful for genetic diversity, phylogeny, genome mapping and
evolutionary studies (Reddy et al., 2002). It is a simple and quick method, although
optimisation of the P C R reaction of each primer needs to be tested before applying the
technique (Pharmawati et al., 2004). R A P D has been widely used to study genetic
relationships between cultivars in Brassica (Astarini et al, 2004, H u and Quiros, 1991;
Divaret et al., 1999; Mailer and May, 1999), radish (Pradhan et al., 2004), Cucumis spp.
46
(Zhuang et al., 2004), and banana (Onguso et al., 2004). This is because the technique is
simple, requires small amounts of D N A , does not require information on D N A sequence
and it is economical (William et al., 1990). The numbers of R A P D primers utilized to
determine relationships between cultivars varies. H u and Quiros (1991) could distinguish
14 broccoli and 12 cauliflower cultivars using 4 primers that produce 40 bands. Mailer and
May (1999) also employed 4 primers producing 61 bands to differentiate four Brassica
napus cultivars. Genetic diversity of some core collections of Brassica oleracea were
determined using 8 primers that produced R A P D bands ranging from 39 to 57 depended on
sources of D N A (Divaret et al., 1999). In radish, 5 primers producing 52 bands were
sufficient to distinguish cultivars (Pradhan et al., 2004) and 19 R A P D primers were
employed to characterize banana cultivars in Kenya (Onguso et al., 2004).
The objectives of this study were to investigate genetic variation and relationships
between Indonesian and Australian and European-based cultivars and to fingerprint
Indonesia cultivars based on D N A markers.
2. Materials and methods
2.1. Plant materials and DNA extraction
Eight cauliflower cultivars were collected from Indonesia, they are 'Harli', 'Broad'
and 'Blaster' from West Java, 'Manalagi', 'Bandung' and 'Gembel' from East Java, and
'Malang' and 'Bedugul' from Bali. Since liquid nitrogen was not available, leaf samples,
together with mortars and pestles were frozen in -80°C freezer for 30 min to maintain low
temperature during grinding of leaf samples into fine powder. It is important to keep the
materials, mortars and pestles cold to prevent D N A degradation. D N A was extracted using
a Nucleon Phytopure Plant D N A extraction kit (Amersham Biosciences,
Buckinghamshire). Purified D N A was kept in 1.5 ml tubes in a cool container and brought
to Perth, Western Australia for analysis. D N A of 4 hybrid cultivars commonly grown in
Australia, 'Atlantis' and 'Omeo' (from an Australian-based breeding program), 'Monarch'
and 'Plana' (from a European-based breeding program) were extracted at The University of
Western Australia.
2.2. ISSR analysis
Fourteen ISSR primers (UBC, Vancouver) were screened for polymerase chain
reaction (PCR) amplification. The optimum annealing temperature for P C R was
47
determined for each primer. The PCR reaction was performed in a final volume of 15 uL
containing injection water, lx Taq polymerase buffer (Promega, California), 1 unit of Taq
polymerase (Promega, California), 0.05 m M of each dNTP (dATP, dCTP, dGTP, dTTP;
Promega, California), 0.3 u M of primer, 1.5 m M MgCl2 and 40 ng template D N A . D N A
amplifications were performed in an iCycler™ (Bio-Rad, California) using the following
cycling program: 15 min at 95°C for initial activation step, followed by 45 cycles of 30 s at
94°C, 45 s at annealing temperature (50°C, 54°C depending on the primers used) and a 2
min extension at 72°C. The iCycler was programmed to retain the samples at 4°C until they
were collected and stored at -20°C. P C R amplification was repeated 2 times using separate
D N A samples.
2.3. RAPD analysis
Three cultivars ('Blaster', 'Manalagi' and 'Bedugul') were chosen to study inter-
variety variations with 8 individuals from each cultivar. To test between cultivar variation,
D N A was bulked from 4 individuals for the 5 other Indonesia cultivars and 4 Australian
grown hybrid cultivars. Ten R A P D primers suitable for use in cauliflowers (Astarini et al,
2004) were employed for PCR amplification. Primer names and nucleotide sequences
were: SK-01 (CATTCGAGCC), SK-14 (CCCGCTACAC), SK-19 ( C A C A G G C G G A ) , SL-
01 (GGCATGACCT), SL-08 ( A G C A G G T G G A ) , SF-06 ( G G G A A T T C G G ) , UBC-106
(CGTCTGCCCG), UBC-250 (CGACAGTCCC), OPA-07 ( G A A A C G G G T G ) , OPB-04
(GGACTGGAGT). All primers were synthesized by Life Technologies Inc. (Madison)
customer primer program. The P C R reaction was performed in a final volume of 15 uL
containing injection water, lx Taq polymerase buffer (Promega, California), 1 unit of Taq
polymerase (Promega, California), 0.05 m M of each dNTP (dATP, dCTP, dGTP, dTTP;
Promega, California), 1 u M of primer, 1.5 m M MgCl2 and 40 ng template D N A . P C R was
performed in an iCycler ™ (Bio-Rad, California) using the following cycling program: 10
times 5 s cycles of denaturation at 94°C, annealing at 35°C for 30 s, elongation at 72°C for
1 min, 25 times 5 s cycles of denaturation at 94°C, annealing at 45°C for 30 s, elongation at
72°C for 1 min and finally 1 cycle including an elongation step at 72°C for 2 min. The
iCycler was programmed to retain the samples at 4°C until they were collected and stored at
-20°C. R A P D analysis on each primer was repeated 2 times using separate D N A samples
to confirm results.
48
2.4. Gel Electrophoresis
Each sample of R A P D or ISSR products (10 uL) was mixed with 6x gel loading
buffer (2 uL) and loaded onto an agarose gel (1.8 % w/v) for electrophoresis (Bio-Rad,
California) in lx T A E buffer (50x T A E buffer contains 242 g Tris base, 57.1 g glacial
acetic acid, 100 m L 0.5 M E D T A and distilled water to 1 L) at 80 volt for 1.5 h. Ethidium
bromide solution (2 uL Etbr/100 m L lx T A E buffer) was added to the gel. A 100 bp D N A
ladder (Promega, California), 5 uL of D N A ladder and 1 uL of gel loading buffer was
included on one sides of the gel as a molecular standard. Amplification products separated
by gels were photographed under U V light using a Kodak D C 120 digital camera and the
images were recorded with a Macintosh Kodak ID 2.0 computer program.
2.5. Data analysis
A data matrix was created based on photographs of gels by scoring 1 for present
bands or 0 for absent bands. The molecular weight in base pairs for each band was
estimated using regression of distance run against the molecular weight of the 100 bp D N A
ladder. Only clearly scorable bands were included in the analysis. A pairwise distance
matrix was generated based on total R A P D band differences in P A U P (Phylogenetic
Analysis Using Parsimony), using a Power Macintosh 7600/120 (Swofford, 1993). The
data were subsequently used to construct a dendogram using unweighted pair-group
method with arithmetic averages ( U P G M A ) . Bootstrap analysis was done based on 1000
reiterates to show the degree of confidence of each branch/node on the dendogram. The
higher bootstrap value (presented in percent), the higher degree of confidence (Swofford,
1993). To help the molecular identification of the cultivar tested, the data were also used to
generate fingerprinting keys.
3. Results
ISSR analysis was difficult to conduct. Only one primer ( G A C A ) 4 produced clear
bands but no polymorphisms were observed, four primers produced faint bands and nine
others did not produce bands.
All ten R A P D primers produced polymorphic bands. R A P D technique was
reproducible and was a reliable, rapid method for D N A fingerprinting. A total of 65 bands
were scored and 46 of these were polymorphic (Table 1). Four to eight bands were scored
per primer and molecular weight ranged from 250 to 1915 base pairs (Figure 1).
49
A fingerprinting key was developed for 10 cultivars (Figure 2). A minimum of 8
markers (coded as primer-number of base pairs): OPB04-1172, OPB04-760, OPB04-330,
OPA07-733, OPA07-509, UBC250-935.4, SL01-1915 and SL01-1076 obtained from 4
primers were required to distinguish between cultivars (Table 1). T w o cultivars, 'Blaster'
and 'Manalagi' could not be separated, as they had identical R A P D profiles.
A dendogram showing the relationship between and within cultivars was generated
(Figure 3). T w o major clusters were obtained, Australian grown cultivars were clustered
and separated from Indonesian cultivars and this was supported by high Bootstrap values of
88%. Australian-bred and European-bred cultivars were much more closely related and
were clustered together. 'Bedugul' had a distant relationship with other Indonesian
cultivars, while 'Manalagi' and 'Blaster' were intermixed. 'Harli', 'Broad', 'Bandung',
'Gembel' and 'Malang' were closely related.
Pairwise distance matrix produced from the P A U P program was used to quantify
differences among cultivars (Table 2). The pairwise difference between cultivars ranged
from 0 to 34. 'Atlantis' (Australian grown hybrid cultivar) tended to have the greatest
difference from Indonesian cultivars.
4. Discussion
Australian grown varieties are all hybrid cultivars from breeding programs based in
either European or/and Australian seed companies. These hybrids have been highly bred,
resulting in narrow genetic diversity. Replacement of open-pollinated cultivars with Fi
hybrids of a narrow genetic base has resulted in the genetic erosion of cauliflower cultivars.
In the long term, genetic variability in the form of landraces and primitive types will
disappear unless efforts are made to define and preserve them (Sharma et al., 2004).
Three ISSR primers which proved useful to generate polymorphisms in Brassica
oleracea (Leroy et al., 2000) and 11 primers useful for Leucadendron (Pharmawati et al.,
2004) were employed in this study. However, only one primer from B. oleracea produced
clear ISSR patterns. Primers from Leucadendron resulted in smear bands or did not
produce bands at all. The smeared ISSR bands obtained indicated that P C R conditions
need to be further refined. Smeared ISSR patterns are c o m m o n but not useful (Gupta et al.,
2000, Pharmawati et al., 2004). Annealing temperatures are usually the main factors
affecting pattern quality and reproducibility of ISSR fingerprints and are primer-specific
50
(Bomet and Branchard, 2001). Different annealing temperatures have been tested for each
primer in this experiment but no clear bands were obtained.
A fingerprinting key is useful for correct identification of cultivars. Correct
identification of a cultivar is an important step in a breeding program, to ensure the right
line for breeding purposes is chosen and not the same line under different names.
Fingerprinting keys can also be used for protection of new cultivars. Similarly, a
dendogram is a practical way to show relationships among cultivars tested. W h e n using
new or distinct materials for a breeding program, the dendogram will show the distance
between new or distinct materials and existing cultivars. This will assist plant breeders in
choosing which cultivars will be used in their breeding program.
In this study, R A P D showed clear bands and a high level of polymorphism. In
R A P D technique, it is important to maintain consistent reaction conditions that have been
optimised for reproducible D N A amplification. Several factors including template D N A
concentration, magnesium concentration, primer annealing temperature, primer length and
primer base composition all affect the reaction (William et al., 1990) and were carefully
controlled.
The distant relationships between Indonesian and Australian grown cultivars
suggest that the two gene pools have been separated and there has been no or limited gene
flow between the pools. For future breeding programs, the available cultivars could be
used as genetic sources for broadening the development of new cultivars, including hybrid
cultivars suitable for warmer climates.
'Harli' and 'Broad' were cultivated in Lembang, West Java. These cultivars may
have been introduced from India in the 19th century and have been reproduced locally since
then (Rukmana, 1994). 'Blaster' and 'Manalagi' were intermixed in the dendogram and
could not be separated by the fingerprinting key, suggesting that these cultivars are similar.
Although they are cultivated in different regions, West Java and East Java respectively,
gene flow is more likely to occur within an island than between islands. 'Malang', another
Balinese line, had a close relationship with 'Gembel' from East Java. A number of
cauliflower growers in Bali have purchased cauliflower seeds from East Java (Okayadnya,
pers. c o m m ) and this would explain the close similarity and perhaps past cross pollination
between these two cultivars.
There was substantial within-variety variation in the Indonesian cultivars and almost
all individuals were separated on the dendogram. Seed production of each variety is carried
51
out by local farmers and variation within variety maybe due to cross-pollination, as there is
little effort to isolate plants during seed production. Populations have been selected and
traditionally multiplied by growers and they will therefore possess genetic adaptation to
local conditions. This variation m a y contribute to diversity for breeding purposes.
The sale of seeds among local farmers, including lots of commercial seed from seed
companies, m a y also contribute to genetic variability. In West Java, cauliflowers are
intercropped, usually with chilli or spring onion. Cauliflower seeds are usually collected
from the best individuals without specific varietal isolation. Frequent intercrossing among
different local varieties in the same area may increase the genetic variability of populations
from the same region.
In Indonesia, most cauliflower seeds available for growers are open pollinated. The
main hindrance to the adoption of Fj hybrids by growers is their lack of availability and
cost. Attempts to introduce hybrid cultivars have not been successful. The current hybrid
cultivars have poor performance in the field, produce very small non-harvestable curds and
sometimes pink coloured curds due to high temperature (Grubben, 1977; Okayadnya,
pers.comm). This suggests that the introduced cultivars are not adapted to warm tropical
climates, even though they were cultivated in the cooler mountainous regions. The
temperature in Indonesia varies depending on altitude and distance from the sea. Average
temperatures are 28°C near the coasts and around 22°C in the mountains. Indonesia has an
average relative humidity between 7 0 % and 9 0 % . Therefore, there is tremendous potential
for the development of hybrid cauliflower cultivars for tropical countries such as Indonesia.
Identification of genetic diversity with molecular markers m a y help in selection of
appropriate breeding lines and the time for new variety development can be reduced. In the
future, research in the tropics should be aimed primarily to develop hybrid cultivars and
seed production technologies that provide better adapted varieties that will increase the
availability and quality of vegetables (Darmawan and Pasandaran, 2000).
In conclusion, the variability among cauliflower cultivars could be related primarily
to their geographical origin. This study demonstrated that Indonesian cauliflower cultivars
have unique genotypes that could be used for future breeding program.
52
Acknowledgements
W e thank AusAID for providing a scholarship to Ida Ayu Astarini. Thanks also to
M r Ahmad Rivani, Dr. Ir. Agus Suryanto M S , Ir. Sitawati, M S and M r Dewa Okayadnya
for provision of field grown cauliflowers in Indonesia. Sincere thanks to Prof. I. G. P.
Wirawan for permitting Ida Astarini to extract D N A in his Biotechnology Laboratory in
Bali. This project was supported by grants from The Department of Agriculture Western
Australia and Plant Biology, The University of Western Australia.
References
Asandhi, A. A., Sastrosiswojo, S., 1988. Research on vegetable in Indonesia. In: McLean,
B. T. (Ed.). Vegetable Research in Southeast Asia. Asian Vegetable Research and
Development Center, Taipei.
Astarini, I. A., Plummer, J. A., Lancaster, R. A., Yan, G., 2004. Fingerprinting of
cauliflower cultivars using R A P D markers. Aust. J. Agric. Res. 55, 117-124.
Bornet, B., Branchard, M., 2001. Nonanchored inter simple sequence repeat (ISSR)
markers: reproducible and specific tools for genome fingerprinting. Plant Moi. Biol.
Reporter 19,209-215.
Darmawan, D. A., Pasandaran, E., 2000. Indonesia, dynamics of vegetable production,
distribution and consumption in Asia. In: AH, M . (Ed.), Asian Vegetable Research and
Development Center, Taiwan.
Divaret, I., Margale, E., Thomas, G., 1999. R A P D markers on seed bulks efficiently assess
the genetic diversity of a Brassica oleracea L. collection. Theor. Appl. Genet. 98,
1029-1035.
Grubben, G. J. H., 1977. Tropical vegetable and their genetic resources. International
Board for Plant Genetic Resources, Rome.
Gupta, P. K., Varshney, R. K., 2000. The development and use of microsattelite markers
for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica 113,
163-185.
Hu, J., Quiros, C. F. 1991. Identification of broccoli and cauliflower cultivars with R A P D
markers. Plant Cell Reports 10, 505-511.
Leroy, X. J., Leon, K., Branchard, M., 2000. Characterisation oi Brassica oleracea L. by
microsatellite primers. Plant Syst. Evol. 225, 235-240.
53
Mailer, R. J., May, C. E., 1999. Heterogeneity of random amplified polymorphic D N A
sequences in individual seedlings and bulked samples of four cultivars of Brassica
napus. Plant Breeding 118, 465-470.
Onguso, J. M., Kahangi, E. M., Ndiritu, D. W., Mizutami, F., 2004. Genetic
characterization of cultivated bananas and plantains in Kenya by R A P D markers. Sci.
Hort. 99, 9-20.
Pharmawati, M., Yan, G., McFarlane, I. J., 2004. Application of R A P D and ISSR markers
to analyse molecular relationship in Grevillea (Proteaceae). Aust. Syst. Bot. 17,49-61.
Pradhan, A., Yan, G., Plummer, J. A., 2004. Development of D N A fingerprinting keys for
the identification of radish cultivars. Aust. J. Exp. Agric. 44, 95-102.
Reddy, M. P., Sarla, N., Siddiq, E. A., 2002. Inter simple sequence repeat (ISSR)
polymorphism and its application in plant breeding. Euphytica 128, 9-17.
Rukmana, R., 1994. Budidaya Kubis Bunga dan Brokoli. Penerbit Kanisius, Yogyakarta.
Sharma, S. R., Singh, P.K., Chable, V., Tripathi, S. K., 2004. A review of hybrid
cauliflower development. Journal of N e w Seeds 6, 151-193.
Swofford, D. L., 1993. PAUP: Phylogenetic analysis using Parsimony, version 3.1 Illinois
Natural History Survey, Champaign, Illinois.
William, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A., Tingey, S. V., 1990. D N A
polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic
Acids Res. 18,6531-6535.
Williams, C. N., Uzo, J. O., Peregrine, W . T. H., 1991. Vegetable production in the tropics.
Intermediate Tropical Agriculture Series. Longman Scientific and Technical, Essex.
Zhuang, F. Y., Chen, J. F., Staub, J. E., Qian, C. T., 2004. Assessment of genetic
relationships among Cucumis spp. by SSR and R A P D marker analysis. Plant Breeding
123, 167-172.
54
Figure 1
RAPD amplification profiles of 12 cultivars obtained with primer SL-01 and SL-08.
Standard bands are indicated by arrows. M, marker ladder, 1, Harli; 2, Blaster; 3, Broad; 4,
Manalagi, 5, Gembel; 6, Bandung; 7, Malang; 8, Bedugul; 9, Atlantis; 10, Omeo; 11,
Monarch; 12, Plana; M, Marker ladder.
1 23 4 56 7 8910 1112 123 4567 8 9 10 11 12 M
^^m9- -tUUft
Hi r f
A 4lilf4|W«i
1500 bp
1000 bp
500 bp
55
Figure 3
Dendogram of Indonesian cultivars ('Harli', 'Broad', 'Bandung', 'Gembel', 'Malang',
'Blaster', 'Manalagi', 'Bedugul'), Australian-bred cultivars ('Atlantis', 'Omeo') and
European-bred cultivars ('Monarch', 'Plana'), constructed by unweighted pair-group
method with arithmetic averages ( U P G M A ) based on total character differences. Numbers
adjacent to cultivars indicate collection number. Numbers above branches represent branch
length and numbers below branches indicated bootstrap values.
57
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Chapter 6
Identification of 'Sib' Plants in Hybrid
Cauliflowers using Microsatellite Markers
This chapter was presented in the 4th International Society of Horticultural Sciences
(ISHS) Symposium on Brassica in Dejeon, South Korea, October 2004. Manuscript of
this chapter was submitted to Theoretical and Applied Genetics and is currently under
review. Paper is presented here in its submitted format.
IDENTIFICATION OF 'SIB' PLANTS IN HYBRID CAULDTLOWERS USING
MICROSATELLITE AND RAPD MARKERS
Ida A. Astarini^, Julie A. PlummerA, Rachel A. Lancaster0 and Guijun Y a n A
APlant Biology, Faculty of Natural and Agricultural Sciences, The University of
Western Australia, 35 Stirling Highway, Crawley W A 6009, Australia.
BAuthor for correspondence: Ida A y u Astarini
email: [email protected] Fax: +61 8 6488 1108, Phone: +61 8 6488 1992
department of Agriculture, Western Australia, Bunbury District Office, P O Box 1231
Bunbury, W A 6231, Australia.
Abstract Hybrid cauliflowers have been developed to obtain heterosis and to improve
uniformity of production. T w o breeding systems are commonly employed, self-
incompatibility (SI) and cytoplasmic male sterility (CMS). Sibs, assumed to be self
inbred, often contaminate hybrid seed lots in the SI system and whilst self inbreeding is
not possible in the C M S system, plants that look like sibs occur. The objective of this
study was to develop microsatellite markers for male and female cauliflower parent
lines of both SI and C M S systems and to use them to identify sibs and aberrant plants in
Fi hybrids. Fifty six microsatellite primers were screened and 8 primers produced co-
dominant markers in parent plants and two markers were chosen for purity testing of Fj
hybrid seeds. Controlled pollinations were conducted in the glasshouse to produce
hybrid and selfed-seeds. These seeds were grown in a field trial to identify
morphologically normal and sib plants and to assess the reliability of microsatellite
markers in detecting sibs and aberrant plants. Microsatellite analysis of morphological
sib plants from the SI system revealed that sibs were not always self-inbred, in contrast,
most self inbred plants showed normal growth. Similarly, all morphological sibs from
the C M S system had hybrid bands. This suggests that morphological sibs were not due
to selfing but possibly to an interaction between genetic and environmental factors and
this requires further investigation.
Keywords Brassica oleracea var. botrytis, self-inbred, self-incompatibility,
cytoplasmic male sterility, S S R (simple sequence repeats).
65
Introduction
Identifying breeding lines and determining Fi hybrid purity are important quality
controls in vegetable breeding and seed production. Fi hybrids have been developed to
improve the uniformity of Brassica crops. In cauliflower, Fi hybrid selection aims for
earliness, high yield, better curd quality with regard to compactness and colour, uniform
maturity, and resistance to insects, diseases and unfavourable weather conditions (Crisp
andTapsell, 1993).
T w o systems have been employed for hybrid seed production, cytoplasmic male
sterility ( C M S ) and self-incompatibility (SI). Both systems were developed to prevent
self-pollination. M a n y commercial hybrids of cauliflower utilising SI system have a
substantial proportion of abnormal plants usually known as 'sibs', non-hybrid plants, or
selfing of parent plants. It is assumed that it is possible to identify sibs by their
distinctive plant phenotype and these will be here referred to as morphological sibs.
Compared to the hybrid, morphological sibs can be smaller and have darker green
leaves, have much weaker growth habit or be taller, with paler green leaves. Leaves are
unusually narrow or wavy. Curd size is usually small and not marketable (Holland and
McNeilly 1985). It is therefore necessary to carry out quality control on all hybrid seed
harvests to prevent unacceptable levels of sib seed being released in the market
(Crockett, 2002).
Fi cauliflower hybrid derived from C M S system often produce aberrant plants,
which are unsuitable for harvest. Phenotypes of aberrant plants mainly involve the
modification of three characters: leaf shape, size and thickness (Ruffio-Chable et al.
2000; Fujime and Okuda 1996). In cauliflower varieties, the proportion of aberration
ranges from 5 % to 4 0 % . The main concern is the economic consequence for growers.
M a n y attempts have been made to identify and screen out morphological sib or
aberrant plants in hybrid populations. Automated phenotypic examination such as
image analysis on Brussels sprout and cabbage seedlings, have been developed
(Fitzgerald 1997). This technique is not a universal approach as morphological
differences m a y be difficult to detect and are affected by environmental factors.
It is assumed that sibs are self inbred and therefore are genetically determined
and could be identified by genetic markers. Here plants from a self inbred parent will
be referred to as genetic sibs. Isoenzymes of acid phosphatase using P A G E can detect
genetic sib content in Fi hybrid Brussels sprout varieties (Harvey and Smith 1987).
Isozymes are also used in seed purity testing of Chinese cabbage (Brassica campestris)
66
(Zheng and Liu 1994). There are some limitations to isozyme analysis, such as
insufficient polymorphisms among closely related genotypes, and variation affected by
environmental factors, seed vigour and growing stage (Meng et al. 1998). Ploidy levels
have been examined by flow cytometry, but they do not reveal any correlation between
ploidy level (haploid, diploid or tetraploid) and abnormalities observed in plants
(Ruffio-Chable et al. 2000).
Molecular techniques such as R A P D have been employed to determine genetic
purity in Brassica oleracea crops such as cauliflower (Boury et al. 1992), broccoli
(Crockett et al. 2002) and cabbage (Crockett et al. 2000) and Brassica campestris such
as Chinese cabbage (Meng et al. 1998). R A P D P C R was successfully employed to
identify female and male parent lines and detect genetic sibs in cabbage and broccoli
(Crockett et al. 2000; 2002). The proportion of genetic sib contamination was similar to
that of morphological sib observed in the field trial.
In recent years microsatellites, also referred to as simple sequence repeats
(SSR), have gained increasing importance in plant genetics and breeding. High
abundance and extensive polymorphism make them an ideal marker system for genetic
mapping and characterization of germplasm, in particular in very closely related and
inbreeding species (Saal et al. 2001). Microsatellites are highly variable with regard to
repeat number and show co-dominant inheritance. Therefore they are considered
suitable for use in genetic purity testing in hybrid populations of crops such as
cauliflower.
Microsatellites of Brassica species are well documented. A large number of
microsatellites from rapeseed canola (Brassica napus) have been identified and
characterized. M a n y B. napus microsatellite flanking primer pairs are functional in the
A and C genome species within the genus Brassica, but are not useful as markers for a
wide range of species in the family Brassicacea (Saal et al. 2001). However, some
primers m a y be useful as markers for cauliflower, which has C genomes.
The hypothesis in this study was that morphological sibs are equal to genetic
sibs, a view held by both the farming and scientific community. The aim of this study
was to generate R A P D and microsatellite markers for discrimination of cauliflower
parent lines and the subsequent identification of genetic sibs in Fi hybrid cauliflower.
Materials and methods
Plant materials
67
A commercial cultivar 'Discovery' was provided by South Pacific Seeds Pty Ltd. This
hybrid cultivar is produced using the SI system. T w o pairs of parent lines and their
hybrid progenies were provided by Henderson Seeds Pty Ltd. One pair utilised the SI
system (5038 SI) and another pair utilised the C M S system (5038 C M S ) . Seeds were
stored at 4°C until required.
Field Trial 1
Plants of 'Discovery' were grown under standard commercial field conditions at the
Horticultural Research Institute, Western Australian Department of Agriculture at
Manjimup, from September to December 2001. Leaf morphology and curd weight were
observed. Leaf samples were collected from 10 normal plants and all sib plants at
harvest and were stored at -20°C until required for R A P D analysis.
Controlled pollination in glasshouse grown plants
Cauliflower seeds supplied by Henderson Seed were germinated using the top of paper
method (ISTA 2003). Twenty seeds of each parent line were germinated on top of filter
paper (2 x Whatman N o 1) in 15 cm petri dishes filled with 15 ml distilled water. Seed
were germinated in a 20°C growth chamber with a 12h/12h light/dark regime for 7 days.
Germinated seeds were transferred to a glasshouse at day 7. Ten vigorous,
uniform seedlings were chosen. The seedlings were maintained in 100 m m diameter
pots for 6 weeks and then transferred to 255 m m diameter pots until mature. Plants
were regularly watered and fertilized. Glasshouse temperature ranged from 20°C to
27°C. N o mature plants were morphological sibs.
Crossing was done on both SI and C M S pairs. One hundred flowers from 4
plants were cross-pollinated for each pair. Flowers were bagged after pollination and
seed set was recorded. Selfing is difficult in SI female parent lines since plants are self
incompatible. T w o methods for overcoming self incompatibility in SI systems were
investigated. In the first method, selfing was performed on the 5038 SI line, 10 min
after spraying both stigma and stamens of open flowers with 5 % salt (NaCl) solution
(Fu et al. 1992). The second method used bud pollination, where immature, non-
receptive stigmas (1-2 days prior to anthesis) were pollinated with mature pollen of the
same plant (Hallidri and Pertena 2002).
To induce pollen fertility in C M S female parent lines, four plants at flower bud
stage were kept in a 30°C growth chamber for two weeks. T w o plants were maintained
in the glasshouse as controls. Plants were self pollinated under both conditions. Flower
68
morphology and pollen production was observed. Pollen viability was examined using
the fluorescein diacetate staining method (Heslop-Harrison and Heslop-Harrison 1970).
Counts of pollen were made under the microscope. All seed produced from crossing
and selfing were collected and stored at room temperature until required for Field Trial
2. Leaf samples of female and male parents of SI and C M S lines were collected for
molecular analysis.
Field Trial 2
Parent lines, hybrid seeds and plants from controlled pollination (crossed and selfed
seeds obtained from the glasshouse) were grown at a seedling nursery in Manjimup.
Seed was sown manually, one seed per cell and each line was allocated to a separate
tray. After 6 weeks, seedlings were transplanted to the field at the Manjimup
Horticultural Research Institute, Western Australian Department of Agriculture, at a
spacing of 40 c m between plants within a row and 80 c m between rows. They were
grown under standard commercial field conditions.
Plants were observed at 2, 9 and at harvest at 13 weeks after transplanting.
Observations included normal and unusual characteristics of leaves, plant height, leaf
number and curd weight of normal and morphological sib plants. The difference
between normal and sibs were analysed using paired two sample t-XesX on the mean
values of plants from different lines using Genstat 7.0. Leaf samples of normal and
morphological sib plants were collected and stored at -20°C for molecular analysis.
Molecular analysis
T w o types of molecular markers, R A P D and microsatellite were examined for their
suitability in testing for parent lines, self and hybrid plants.
RAPD analysis
Twelve normal hybrids and 12 sibs of 'Discovery' were chosen for D N A extraction.
D N A was extracted following the C T A B method (Astarini et al. 2004). R A P D P C R
was performed following the method described in Astarini et al. (2004).
Microsatellite analysis
First step in microsatellite analysis was to find markers for parent lines. Eight
individual plants of each parent line were chosen for this purpose. Fifty six
microsatellite primers suitable for Brassica C genome were screened for P C R
69
amplification. Canola Breeders Western Australia (Perth) provided 50 primers and 6
other primers were synthesized by Life Technologies Inc. (Madison) customer primer
program. The next step, specific markers that able to distinguish parent lines was then
tested on hybrids, self inbred and morphological sib plants to differentiate true hybrids
and genetic sibs. Twenty normal plants of each line, self-inbred and all morphological
sibs were examined. The P C R reaction was performed in a final volume of 20 uL
containing injection water, 10 uL AmpliTaq Gold P C R Master Mix (Applied
Biosystems, Foster City, C A ) , 0.5 u M of each forward and reverse primer and 40 ng
template D N A .
P C R was performed in an iCycler ™ (Bio-Rad, Hercules, C A ) using the
following cycling program : 1 cycle of 5 min at 94°C, 40 cycles of denaturation at 94°C
for 1 min, annealing at 53°C for 2 min, elongation at 72°C for 2 min, and finally 1 cycle
of elongation step at 72°C for 10 min. The iCycler was programmed to retain the
samples at 4°C until collected and stored at -20°C.
Each sample of P C R product (10 uL) was mixed with 6x gel loading buffer (1
uL) and loaded onto an agarose ( 4 % w/v) gel for electrophoresis (Bio-Rad, Hercules,
C A ) in lx T A E buffer at 100 volts for 2 hours. Fifty times T A E buffer contained 242 g
Tris base, 57.1 g glacial acetic acid, 100 m L 0.5 M E D T A and distilled water to 1 L. A
100 bp D N A ladder (5 uL) and 1 uL of gel loading buffer (Promega, Madison), was
included on both sides of the gel as a molecular standard. Ethidium bromide solution (2
uL/100 m L ) was incorporated into the gel. Amplification products separated by gels
were then photographed under U V light using a digital camera (Kodak D C 120) and the
images were recorded with a Macintosh Kodak ID 2.0 computer program.
Results
Controlled pollination
Crosses were 1 0 0 % successful in both pairs of the C M S and SI parent lines. Some
fertilisation (16%) occurred following selfing of the SI female line using bud
pollination, while salt spray treatment only produced empty pods with no seed.
T w o types of female C M S parent line plants were observed. The first type, had
flowers with reduced and shrunken stamens (Fig. la). A n average of 5.3 ± 0.5
pollen/microscope field was counted, and 1 2 % pollen was viable. The second type, had
flowers with petaloid stamens (Fig. lb). N o pollen was observed in anthers of this
flower type and so self pollination was not possible. Non-CMS plants had flowers with
70
abundant fertile pollen, 46 ± 2.8 pollen/microscope field, and 8 8 % of pollen was viable
(Fig. lc).
Maintaining the female C M S line in the 30°C growth chamber did not induce
pollen fertility. Pollen viability decreased from 1 2 % to 5 % with high temperature
treatment. N o seed formation was observed on any C M S plants.
Field Trial 1
O n 'Discovery', 1.2% sibs were observed. Average curd weight of normal plants was
1148 ± 13 g, compared to 101 ± 22 g on sib plants.
Field Trial 2
Two weeks after transplanting (8 week-old seedlings), abnormal seedlings were
distinguishable from normal seedlings by their unusual morphology. Abnormal
seedlings observed in the field had wavy leaves (Fig. 2a), narrow leaves (Fig. 2b), or
blind apexes (Fig. 2c). Abnormal plants with wavy leaves or narrow leaves were noted
as morphological sibs.
At 9 weeks after transplanting, average plant height and leaf number were vary
between lines (Table 1). Significance differences were found between normal plants
and morphological sibs on plant height (p value < 0.001) and leaf number (p value =
0.014).
At harvest, all morphological sibs observed at 8-week old seedlings (2 weeks
after transplanting) continued to have unusual vegetative growth and produced small
curds. Sib plants produced very small curds, below 200 g, compared to more than 700 g
of normal hybrid plants (Table 1). Most self-inbred plants (92%) exhibited normal
growth in the field and produced curd around 417 g. Morphological sibs found in all
lines tested except male SI parent, with the proportion of 1.3% to 8%.
RAPD analysis
N o polymorphic bands were obtained from 36 primers tested during R A P D analysis of
'Discovery'. The same banding patterns were observed on normal and sib plants grown
in Field Trial 1.
Microsatellite analysis
Out of 56 microsatellite primers screened, 8 primers had polymorphic bands.
Furthermore, only two primers were suitable as specific markers for both 5038 C M S
71
and 5038 SI. These primers, Nal2-E06b (Forward: C A T A T A G G G G A A T C A T C A T
C G G C , Reverse: A G A C C A A T T A G C A T C T C G C C ) and O110-G08 (Forward: T G C T
T A A T T G A T T A G G G C A G , Reverse: T T A C C T C A T C A G G T G G A G G C ) showed
distinct co-dominant bands between female and male parents.
Microsatellite analysis of normal Fi hybrids and manual crosses from the SI and
C M S systems had both female and male bands present, confirming they were all true
hybrids. All self-inbred plants only had the female band indicating they were genuinely
self-inbred (Fig. 3). Microsatellite analysis on morphological sibs on parent plants
revealed no genetic sibs on the parent plants.
Microsatellite analysis of morphological sib plants from the Fi hybrids and
manual crosses of the SI system indicated that 3 3 % and 3 8 % plants respectively (Table
1), only had the female band, and were therefore genetically self inbred, whilst the
remainder had both female and male bands. All morphological sibs in Fi hybrids and
crosses from the C M S system had male and female bands (Fig. 3).
Discussions
It has been assumed that abnormal plants in Fi hybrid utilising SI systems were due by
self-inbreeding and were known as sibs. There are up to 1 4 % sibs in cabbage (Crocket
et al 2000) and 45%> sibs in broccoli (Crocket et al 2002). Our studies show that plants
with sib characteristics occurred not only in Fi hybrid lines, but also in parents and
selfed lines. Morphological sib plants varied from 1.2% in 'Discovery' to 8 % in
manually crossed 5038 SI and selfed 5038 SI. This proved that sibs are not always self
inbred.
Barriers to selfing were not overcome in the C M S line and so no selfed seeds
were produced. Exposure to 30°C did not induce fertility of pollen on C M S cauliflower
lines, which means the C M S line was stable at high temperatures. C M S lines in
Brassica are derived from a number of systems such as 'Pol', 'Ctr', 'Nap', 'Ogura',
Anand and 'Nigra' (Makaroff, 1995). Stability of pollen sterility in C M S lines depends
on their systems. 'Ctr' and 'Nap' are temperature unstable, 'Pol' is relatively stable but
can be broken down at 24°-30°C while 'Ogura' and 'Nigra' have very stable sterility.
The cauliflowers C M S lines used here may be derived from a very stable system or may
need a lower temperature range to break pollen sterility (Ruffio-Chable et al. 1993).
Unusual flower morphology is often observed in C M S plants (Cardi and Earle
1997). In the cauliflower C M S plants grown here, two distinct types were observed
within one line, flowers with petaloid stamens and flowers with shrunken stamens (Fig
72
1). Petaloid stamens are also found in B. oleracea (Cardi and Earle 1997) and B. juncea
(Malik et al. 1999). The petaloid stamens did not produce any pollen, so could not
produce any self-inbred plants. The shrunken stamens had dehiscent anthers and
produced pollen with 1 2 % viability. Only a small amount of pollen was produced, an
average of 5 pollen/microscope field, compared to 45 pollen/microscopic field in non-
C M S plants. This indicated that although the chances were small, it may be possible for
C M S parent plants to produce self-inbred plants as not all flowers were sterile.
The C M S breeding system was developed to eliminate the possibility of sibs or
inbred seed being produced, as found in less efficient systems, such as SI. However
plants with unusual growth were observed in Fi hybrid cauliflowers derived from the
C M S system. Our glasshouse, field trial and microsatellite examination confirmed that
these abnormalities were not due to self-inbreeding. Abnormal growth may require an
environmental trigger. Ruffio-Chable et al. (2000) suggest the abnormality in C M S
hybrids is due to the occurrence of epigenetic phenomena during plant development
causing modifications in gene expression. The causes of these abnormalities should be
further investigated.
Self-incompatibility mechanisms were overcome. Self incompatibility
mechanisms are often undeveloped at the bud stage in B. oleracea (Crisp and Tapsell
1993). Here placing mature pollen on immature stigmas overcame the incompatibility
mechanisms in cauliflower and allowed successful pollination, fertilization and
production of viable seeds. This enabled analysis of self inbred plants from female SI
lines of cauliflowers.
Overcoming self incompatibility with 5 % salt spray 10 minutes before selfing
was not successful in cauliflower (B. oleracea). The same or a similar technique was
suitable for overcoming incompatibility in B. napus (Fu et al. 1992) and B. campestris
(Monteiro et al. 1988). Salt spray inactivates the incompatibility substance on the
stigmatic surface and allows pollen to germinate and penetrate the stigma and style
resulting in fertilization and seed formation in other Brassica species. Further
investigation on the appropriate stage at which flowers should be sprayed and
optimising salt concentration may improve pollination success in cauliflower.
Unusual growth habits in cauliflower were easily distinguished from normal
growth in 8-weeks old plants in the field (2 weeks after transplanting). Particular
abnormal growth types, such as plants with a blind apex or wavy leaves, were obvious
during transplanting at 6 weeks. Early detection and removal of abnormal plants would
be useful to avoid further profit loss due to production costs and labour costs at harvest.
73
Growers m a y be able to detect and discard abnormal seedlings during transplanting at 6
weeks.
Self-pollinated SI plants produced the same height and leaf shape as Fi hybrid
plants and produced marketable size curds. Morphologically, they could not be
distinguished from normal hybrid plants. Thus abnormalities in cauliflower hybrid are
not caused by self-inbreeding, which has been assumed to be the cause of
morphological sibs.
All R A P D primers trialed produced clear and scorable bands, indicating the
technique was easy to conduct. R A P D primers, including several of those examined
produce polymorphism in other Brassica crops including Chinese cabbage {Brassica
rapa) (Meng et al. 1998). This technique could also identify and distinguish sibs in
cabbage {B. oleracea var. capitata) (Crockett et al. 2000) and broccoli (B. oleracea var.
italica) (Crockett et al. 2002). This technique however was not able to generate markers
for genetic sibs in the Fi hybrid cauliflower 'Discovery'.
Parent lines were not available for R A P D analysis of 'Discovery', therefore it
was difficult to distinguish true hybrids from self inbred plants as there was no genetic
information on the parents. It is useful to develop genetic markers for the parent lines
prior to identification of self-inbred plants from their progeny.
R A P D is a dominant marker and employs random primers (Henry, 1997). This
marker m a y not be suitable for distinguishing specific male and female bands, which are
usually co-dominant (Saal et al. 2001), especially when parent lines are closely related.
It was therefore necessary to investigate co-dominant markers, such as microsatellites.
Microsatellite analysis was a powerful technique which could be used to
distinguish specific male and female bands and to detect true hybrids and self-inbred
plants. Difficulties m a y be encountered in screening for appropriate primers but the
selection process was reduced by choosing primers from a closely related species with
similar genome. There are hundreds of Brassica microsatellites publicly available
(Bond et al. 2004) which can be used in Brassica vegetables.
In cabbage and broccoli, Crockett et al. (2000; 2002) found a similar proportion
of morphological sibs (abnormal plants observed in the field) and genetic sibs (detected
using R A P D markers). They therefore assumed that self inbreeding was the genetic
cause of morphologically identifiable sibs. In this experiment, morphological sibs
identified in the field were not the same plants as genetic sibs as revealed by
microsatellite analysis (Fig. 3). Thus self-inbreeding was not the only cause of
morphological sibs in cauliflower production.
74
In conclusion, microsatellite analysis was a reliable technique for identification
of parent line, hybrid and self-inbred plants. The lack of uniformity in hybrid
cauliflowers observed in the field was not solely due to contamination by self inbred
plants (genetic sibs). Self inbred parents did produce seed which grew into normal
plants. Morphological sibs were not all self-inbred. Thus the small size plants usually
referred to as sibs were not only from self-inbred parent lines. Environmental triggers
or other genes may be involved in this abnormal plant formation. The molecular
evidence that sib is not the only cause of contamination in hybrid cauliflower is new and
is further supported by the evidence that controlled self-pollinated plants exhibited
normal growth in the field.
Acknowledgments W e thank AusAID for providing a scholarship to Ida Astarini.
Thanks to South Pacific Seeds and Henderson Seeds for providing seeds. Sincere
thanks to M s Anouska Cousins, Dr Matthew Nelson and Associate Professor Wallace
Cowling for technical advice and provision of primers, M r John Doust, Dr Kristen
Stirling, M r David Tooke and M r Grazi Giadresco for invaluable assistance during the
field trial at Manjimup. Financial assistance from Horticulture Australia Limtd and
Department of Agriculture Western Australia are gratefully acknowledged.
References
Astarini IA, Plummer JA, Lancaster R A , Yan G (2004) Fingerprinting of cauliflower
cultivars using R A P D markers. Aust J Agric Res 55:117-124.
Bond JM, M o g g RJ, Squire G R, Johnstone C (2004) Microsatellite amplification in
Brassica napus cultivars: Cultivar variability and relationship to a long-term feral
population. Euphytica 139:173-178.
Boury S, Lutz I, Gavalda M-C, Guidet F, Schlesser A (1992) Genetic fingerprinting in
cauliflower by the R A P D method and determination of the level of inbreeding in a
set of Fi hybrid seeds. Agronomie 12:669-681.
Cardi T and Earle E D (1997) Production of new C M S Brassica oleracea by transfer of
'Anand' cytoplasm from B. rapa through protoplast fusion. Theor Appl Genet
94:204-212
Crisp P and Tapsell C R (1993) Cauliflower, Brassica oleracea L. In: Kalloo G, Bergh
B O (Eds). Genetic Improvement of Vegetables Crops. Pergamon Press, Oxford.
Crockett PA, Bhalla PL, Lee CK, Singh M B (2000) R A P D analysis of seed purity in a
commercial hybrid cabbage {Brassica oleraceae var. capitata) cultivar. Genome
43:317-321.
75
Crockett PA, Singh M B , Lee CK, Bhalla PL (2002) Genetic purity analysis of hybrid
broccoli {Brassica oleracea var. italica) seeds using R A P D PCR. Aust J Agric Res
53:51-54.
Fitzgerald D M , Barry D, Dawson PR, Cassells A C (1997) The application of image
analysis in determining sib proportion and aberrant characterization in Fj hybrid
Brassica population. Seed Sci Technol 25:503-509.
Fu T, Ping S, Xiaoniu Y, Guangsheng Y (1992) Overcoming self-incompatibility of
Brassica napus by salt (NaCl) spray. Plant Breeding 109:255-258.
Fujime Y, Okuda N (1996) The physiology of flowering in Brassicas, especially about
cauliflower and broccoli. Acta Hort 407:247-254.
Hallidri M , Pertena D (2002) Self-incompatibility test in cabbage {B. oleracea var
capitata). Acta Hort 579:117-122.
Harvey E, Smith B M (1987) A recent survey of sib content in Fi hybrid Brussel sprout
varieties. Cruciferae Newsl 12:122-123.
Henry RJ (1997) Practical Applications of Plant Molecular Biology. Chapman and
Hall, London.
Heslop-Harrison J, Heslop-Harrison Y (1970) Evaluation of pollen viability by
enzymatically-induced fluorescence; intracellular hydrolysis of fluorescein
diacetate. Stain Tech 45:115-120.
Holland RL, McNeilly T (1985) Genotype environment interaction and sib content in
Fi hybrid Brussels sprouts. Euphytica 34:371-376.
International Seed Testing Association (ISTA) (2003) International Rules for Seed
Testing. Bassersdorf, CH-Switzerland.
Makaroff C A (1995) Cytoplasmic male sterility in Brassica species. In Levings III CS,
Vasil IK (Eds). The Molecular Biology of Plant Mitochondria. Kluwer Academic
Publ. London.
Malik M , Vyas P, Rangaswamy N S , Shivanna K R (1999) Development of two new
cytoplasmic male-sterile lines in B. juncea through wide hybridization. Plant
Breeding 118:75-78.
Meng X, M a H, Zhang W , Wang D (1998) A fast procedure for genetic purity
determination of head Chinese cabbage purity seed based on R A P D markers. Seed
Sci Technol 26:829-833.
Monteiro A A , Gabelman W H , William P H (1988) Use of sodium chloride solution to
overcome self-incompatibility in Brassica campestris. HortSci 23:876-877.
76
Ruffio-Chable V, Chatelet P, Thomas G (2000) Developmental^ "Aberrant" plants in
Fi hybrids of Brassica oleracea. Acta Hort 539:89-94.
Ruffio-Chable V, Bellis H and Herve Y (1993) A dominant gene for male sterility in
cauliflower {Brassica oleracea var botrytis): phenotype expression, inheritance, and
use in FI hybrid production. Euphytica 67:9-17.
Saal B, Plieske J, Quiros C, Struss D (2001) Microsatellite markers for genome
analysis in Brassica. II. Assignment of rapeseed microsatellites to the A and C
genomes and genetic mapping in Brassica oleracea L. Theor Appl Genet 102:695-
699.
Zheng X Y , Liu Y (1994) Inbred testing of Chinese cabbage Fi varieties by peroxidase
and esterase isozyme analysis. Acta Hort Sin 21:65-70.
77
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* * • • -
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79
Chapter 7
Molecular Markers Correlated with Seedling
Traits in Cauliflower Varieties
The manuscript of this chapter was submitted to the Australian Journal of Agricultural
Research on 14 November 2005 and is presented here as the submitted format.
Molecular markers correlated with seedling traits in cauliflower
varieties
Ida A. AstariniAB, Julie A. P l u m m e t , Rachel A. Lancaster* and Guijun Y a n A
APlant Biology, Faculty of Natural and Agricultural Sciences, The University of
Western Australia, 35 Stirling Highway, Crawley W A 6009, Australia.
Correspondence: Ida Astarini [email protected] Fax: +61 8 6488 1108
cDepartment of Agriculture, Western Australia, Bunbury District Office, P O Box 1231
Bunbury, W A 6231, Australia.
Abstract. Cauliflower production is hindered by variation in curd quality and maturity.
Morphological variation from seed to harvest is due to genetic variation interacting with
environmental conditions and here the genetic factors were investigated. The aim of
this study was to search for D N A markers linked to seedling traits, facilitating earlier
selection for cauliflower production. Cauliflower seed lines were germinated in Petri
dishes (20°C, 7 days) and seedlings were transferred to pots and grown under
glasshouse conditions. Seed weight and various seedling characters were measured
until harvest at 6 weeks. D N A was extracted using C T A B method and R A P D markers
were identified using 17 primers. Multivariate analysis based on principle coordinates
analysis was employed to correlate morphological traits with molecular markers across
cultivars. Markers associated with seed weight, germination rate, shoot length, root
length, fresh weight and dry weight were identified.
Additional key words: Brassica oleracea var. botrytis, R A P D markers.
Introduction
Western Australia produces 8 5 % of Australia's export cauliflowers and to remain
internationally competitive, growers need to improve the uniformity of curd maturity
and size (Stirling and Lancaster 2005). Curds must be picked within one day of
maturity and variation in harvest time increases the number of picks, which is labour
intensive and hinders the use of harvest machinery. These variations are determined by
genetic and environmental factors. Several methods can be employed to reduce
variation including screening for uniform seed weight and size and management
practices. Agronomic studies, such as increasing plant density and irrigation application
(Stirling and Lancaster 2005), have partially reduced variation but genetic approaches
are also required.
81
Variation begins at the seed and seedling stage and these differences in growth
are exacerbated during field production. Elimination of variation at the seed or seedling
stage would greatly enhance uniformity of the crop. Identification of specific molecular
markers associated with morphological traits would greatly assist in the selection of
superior, uniform seedlings.
Nowadays, molecular marker techniques have become practical for cultivar
identification and selection for breeding purposes (Hu and Quiros 1991). The use of
molecular markers for morphological traits will assist in removing undesirable traits
such as small or malformed seedlings and assist in the selection of more vigorous and
uniform seedlings. A correlation between morphological traits and molecular markers
has been achieved for a few species. Leaf shape, period to bolting and leaf hairiness in
Brassica campestris have been linked with R A P D markers by Q T L and linkage
analyses (Nozaki et al. 1997). Somers et al. (2001) identified a major gene for yellow
seed coat colour in canola linked to R A P D markers. Hirai et al. (2004) found two
R A P D markers linked to clubroot resistance in Brassica rapa. In radish, morphological
traits correlate with R A P D markers both within and across cultivars (Pradhan et al.
2004).
Molecular markers associated with seedlings traits are potentially useful in
seedling selection for interesting traits. Selection of seedlings very early in their
development for the desired traits m a y provide early selection of desirable seedlings
traits for nursery production and field establishment.
The aim of this study was to search for D N A markers correlated to seedling
traits, facilitating early selection in cauliflower production. Molecular markers
associated with morphological traits will potentially contribute to a wide range of
research. This can provide complementary information in the development of improved
hybrids.
Materials and Methods
Plant materials
Twenty one cauliflower {Brassica oleracea) cultivars were utilized (Table 1). Cultivars
varied in their characteristics and origin; some were produced from C M S (cytoplasmic
male sterility), SI (self incompatibility) or O P (open pollinated) breeding systems, they
included summer, autumn, winter and spring cultivars, they had mini, green or white
curds, some were well-covered curds, they had a short or long period to maturity and
they were sourced from different seed companies. Seeds were provided by Bejo,
82
Syngenta, Enza Zaden (formerly Yates Seeds), South Pacific Seeds and Henderson
Seeds.
Seed germination
Individual seed weight was recorded before sowing. Seeds (25) were sown in four
replicates of 9 cm Petri dishes, on 2 layers of Whatman N o 1 filter paper soaked with 10
ml distilled water (ISTA 2003). Petri dishes were sealed with Parafilm to minimise
evaporation. Seeds were kept at 20°C in a growth chamber under a 12h light/12h dark
regime for 7 days. Seed germination was monitored on a daily basis. Radicle tip
emergence indicated the time of germination for each individual seed. Germination rate
was measured as 1/t, where t was the germination time. Individual seedling vigour was
measured by root length, shoot length, total length and fresh weight on 7 day-old
seedlings.
Seedling growth
Seven-day old seedlings, 5 of each cultivar were transferred from petri dishes to 100
m m pots containing a mix of composted bark, coco peat and river sand (2:1:1).
Seedlings were maintained in the glasshouse for 6 weeks (20-27°C, 7 5 % RH).
Seedlings were watered daily and fertilized with a complete fertilizer (Thrive®) once a
week. Length of the longest leaf (measure from where the leaf joints the stem to leaf
tip) and number of leaves were measured every 2 weeks. Shoot and root length, fresh
weight and dry weight were measured when plants were harvested at 6 weeks.
RAPD analysis
For each cultivar, leaves from 4 plants (approximately 1 mg, F W ) were pooled for D N A
extraction. Genomic D N A was isolated using C T A B method as described by Astarini et
al. (2004).
Twenty arbitrary decamer primers were examined for P C R amplification. All
primers were synthesized by Life Technologies Inc. (Madison) customer primer
program. The P C R reaction was performed in a final volume of 25 uL containing
injection water, lx Taq polymerase buffer, 1.5 units of Taq polymerase, 0.05 m M of
each dNTP (dATP, dCTP, dGTP, dTTP) (Promega, Madison), 1 u M of primer, 1.5 m M
MgCl 2 and 40 ng template D N A . A negative P C R tube containing all components
except genomic D N A was used with each primer to check for contamination. P C R was
performed in an iCycler ™ (Bio-Rad, Hercules, C A ) using the following cycling
program: 10 x 5 s cycles of denaturation at 94°C, annealing at 35°C for 30 s, elongation
at 72°C for 1 min, 25 x 5 s cycles of denaturation at 94°C, annealing at 45°C for 30 s,
83
elongation at 72°C for 1 min and finally 1 cycle included an elongation step at 72°C for
2 min. The iCycler was programmed to retain the samples at 4°C until they were
collected and stored at -20°C.
Each sample of R A P D products (10 uL) was mixed with 6x gel loading buffer
(2 uL) and loaded onto an agarose (1.5 % w/v) gel for electrophoresis (Bio-Rad,
Hercules, C A ) in lx T A E buffer (50x T A E buffer contained 242 g Tris base, 57.1 g
glacial acetic acid, 100 m L 0.5 M E D T A and distilled water to 1 L) at 80 volt for 1 hour
30 min. A 100 bp D N A ladder (Promega, Madison, 5 uL of D N A ladder and 1 uL of
gel loading buffer) was included on both sides as a molecular standard. Ethidium
bromide solution (2 uL Etbr/100 m L lx T A E buffer) was incorporated in the agarose
solution. Gel photographs were taken under U V light using a digital camera (Kodak
D C 120) and the images were recorded with a Macintosh Kodak ID 2.0 computer
program.
Statistical analysis
Bands produced on gels were scored as 0 (absent) and 1 (present). Data set were
analysed using Genstat 7.0 software (Digby et al. 1989). Multivariate analysis based on
principal coordinate analysis was employed to generate -values. The program was run
with both phenotypic and genotypic data sets. The program produced associations
between morphological traits and molecular markers. Strong associations of markers
with traits were identified with high t-values. P-values were calculated based on t-
values and degree of freedom (d.f.) to indicate strength of association. Morphological
traits included in the analysis were seed weight, germination rate, shoot length (1, 2, 4, 6
weeks), root length (1, 6 weeks), total length (1 week), fresh weight (1, 6 weeks),
number of leaves (2, 4, 6 weeks) and dry weight (6 weeks). Correlations between
morphological traits were determined, presented as correlation coefficient (r).
Results
Morphological variation across cultivars
Morphological traits varied across cultivars. Average seed weight ranged from 2.5 m g
in 'Atlantis' to 5.8 m g in 'Delfur' (Table 2). Average germination rate ranged from
0.33 d"1 in 'Arctic' to 0.88 d"1 in 'Belot' and 'Panther'. Overall average of germination
rate was 0.55 d"1 with 1 0 0 % germination of all cultivars within 4 days.
Seedling vigour at 7 days varied across cultivars (Fig. la). Average shoot length
ranged from 1.8 c m to 3.7 cm, 'Morpheus' had the shortest shoots and 'Belot' the
longest. Average root length ranged from 2.6 c m in 'Arctic' and 6.4 cm in 'Megan'.
84
'L3389' had the highest fresh weight of 74 mg, while 'Atlantis' was less than a third of
this (23 m g ) (Table 2).
Seedling vigour measured at 6 weeks indicated the tallest plants were 'Omeo', at
21 cm, while 'Megan' plants only half the length (14.5 cm, Fig lb). 'Plana' had the
highest fresh weight (31.2 g) and dry weight (3.7 g), while 'Belot' had the lowest fresh
weight (11.2 g) and 'Discovery' and 'Panther' had the lowest dry weight (0.8 g).
Average leaf number ranged from 5.8 in 'Donner' to 9 in 'Plana' (Table 2).
Correlations between seed weight and other traits across cultivars
Seed weight was positively correlated with most seedling vigour parameters at day 7 but
not by week 6. The strongest correlation was with fresh weight on day 1 {r = 0.6)
followed by number of leaves at week 4 {r = 0.4). Other traits had weak correlations
with seed weight. These ranged from r = 0.06 for shoot length at week 2 to r = 0.36 for
total length at day 7. There was no correlations found between seed weight and
germination rate, shoot length, leaf number, fresh weight and dry weight at 6 weeks,
where r close to 0 (Table 3).
Correlations among seedling traits across cultivars
Germination rate was positively correlated with shoot length, root length, total length
and fresh weight at day 7 and root length at week 6 (Table 3). Shoot length at day 7 had
a moderate correlation with total length (r = 0.57) and fresh weight (r = 0.44) at day 7.
Overall shoottotal length ratio at day 7 was 1:3, while shootroot ratio was 1:2. Root
length at day 7 had a very strong correlation with total length at day 1 {r = 0.93) (Fig
lc). Overall roottotal length ratio at day 7 was 2:3.
Very strong correlation was also observed between fresh weight and dry weight
at week 6 (r = 0.98), and number of leaves at week 2 with number of leaves at week 4 {r
= 0.77). Shoot length at week 4 strongly correlated with shoot length (r = 0.55), fresh
weight {r = 0.74) and dry weight at week 6 (r = 0.72).
Correlations between individual seed weight and other traits within cultivars
Eight cultivars tested had a positive correlation between seed weight and germination
rate within cultivars {r = 0.13 - 0.99) (Table 4). Thirteen other cultivars had no
correlation between seed weight and germination rate, where r ranging from -0.01 in
'Donner' to -0.99 in 'Plana'.
Correlation between individual seed weight and fresh weight at day 7 was
generally positive and strong, ranging from r = 0.4 in 'Lateman' to r = 0.99 in
'Atlantis'. Only 'Discovery' and 'Fremont' had a negative correlation between seed
weight and fresh weight at day l{r = -0.1 and -0.9) (Table 4).
85
Correlation between individual seed weight and fresh weight and dry weight at
week 6 varied among cultivars. Very strong correlations were found in 'Plana' and
'Megan' (r = 0.8), 'L3668', 'Panther', 'Jerez', 'Cauldron', 'Arctic' {r = 0.9). Some
cultivars had a negative correlation such as 'Belot', 'Discovery' and 'Fremont' (r = -0.5
to -0.9) (Table 4).
Molecular markers associated with morphological traits across cultivars
Out of 20 random primers tested, 17 showed clear polymorphisms, and a total of 88
R A P D bands were used as genetic markers. Only clear and reproducible bands were
used as genetic markers (Fig 2). A number of associations were found between
molecular and morphological traits among 21 cultivars tested. Based on the t-values, a
total of 24 molecular markers were identified as having a strong association with high or
low values for seedling traits.
Seed weight was associated with only one D N A marker SL08-626, which was
linked to lighter seed weight (Table 5). Germination rate associated with 7 markers, 4
of them linked with faster germination (A04-833, A04-185, D20-277, SK01-606).
Markers AO2-1038 and SL03-761 had a moderate association with heavier fresh weight
at day 7. Five markers had a strong association with heavier fresh weight at week 6 and
3 markers associated strongly with lighter fresh weight at week 6. Similarly, 4 markers
had a strong association with heavier dry weight at week 6 and 3 markers strongly
associated with lighter dry weight at 6 weeks (Table 6).
A number of molecular markers were associated across traits. SK01-606
associated with faster germination, longer shoot and total length at day 7. Conversely,
SK01-824 associated with slower germination and lower values of shoot length and
total length at day 7. SKI4-723 and U B C 106-404 associated with shorter root and total
length at day 7. OPH15-1794, SL03-562, UBC106-404 and UBC106-200 were strongly
associated with high values of fresh weight and dry weight at 6 weeks, while SL03-
1047, SL03-791 and SL03-595 were linked with fewer leaves, lower fresh weight, and
lower dry weight.
Discussion
Within and between cultivar variations were observed on all traits measured, even
though almost all cultivars tested were Fi hybrids and they were expected to be very
uniform. Variation occurred in morphological analysis and in molecular genetic
analysis and these was correlated.
86
Seed weight varied within and across cultivars. Seed weight is determined by
several factors such as environment, position of seeds in the mother plants (Gutterman
2000) and genetic factors (Alonso-Blanco et al. 1999). Environmental effects mediated
via the maternal plants contribute to seed weight variation. Nutrients, light and water
regime which the mother plants were subjected to during the growing season affects
seed weight (Fenner 1993).
Cauliflower seed are borne in a silique within a complex inflorescence.
Variation in seed weight both within the fruit and across the inflorescence occurs in
Brasicaceae (Susko and Lovett-Doust 2000). Early-initiated, basal fruits produce larger
seeds than fruits in the middle or the tip of an infructescence and these factors will
interact with genetic effects.
Genetic factors also impact on seed weight variation. In Arabidopsis at least 11
seed size quantitative trait loci contribute to seed size variation. Five loci control seed
size via maternal components affecting ovule number or reproductive resource
allocation in the mother plant. Six other loci are involved specifically in seed
development process (Alonso-Blanco et al. 1999). The APETALA2 gene contributes to
the determination of seed weight and size in Arabidopsis. This gene is required for
normal seed coat development (Jofuku et al. 2005) and also controls embryo cell
number and size (Ohto et al. 2005). In crop plants, 3 ISSR markers are associated with
low seed weight in wheat (Ammiraju et al. 2001) and 13 SSR markers are associated
with seed size in soybean (Hoeck et al. 2003). However, in general w e still know little
about the genetic regulation of seed size.
Seed weight influenced seedling vigour. Across cultivars, seed weight
correlated positively with seedling vigour up to 4 weeks and correlated strongest with
fresh weight at day 7. Positive correlations between seed size and fresh weight in 1-
week-old seedlings are c o m m o n in vegetables (Pradhan 2004; Soffer and Smith 1974).
Within cultivars, strong correlation was found between seed weight and seedling traits,
indicating that variations in seed weight were carried through seedling stage and this
would greatly benefit the successful establishment of individual plants in the nursery
and in the field (Kidson and Westoby 2000).
Smaller seeds germinated earlier in many of the cauliflower cultivars tested,
similar to Alliaria petiolata {Brassicaceae) (Susko and Lovett-Doust 2000), Cakile
edentula (Zhang 1993) and Erodium brachycarpum (Stamp 1990). Small seeds have
greater access to water as a result of their higher surface to volume ratios and so small
seeds m a y imbibe water faster and germinate sooner (Stamp 1990).
87
Whilst maternal and environmental factors affect germinability (Gutterman,
2000), certain genes are also involved in the control of seed germination. In
Arabidopsis, RGL2 regulates seed germination probably by functioning as an integrator
of environment and endogenous cues to control seed germination (Lee et al. 2002).
Germination rate was positively correlated with almost all seedling parameters,
therefore seeds that germinated faster tended to have more vigorous seedlings. In
general, cauliflower seeds examined here germinated within 5 days, much faster than
the ISTA 10 day standard for germination of most Brassica oleracea species (1STA,
2003).
Seedling vigour measurements of 7-day-old seedlings indicated root length was
the major component contributing two thirds to the total length. So within the first
week from germination, growth was biased in favour of root establishment rather than
shoot growth. Root establishment is critical for field establishment.
Seedling vigour at very early age (seedlings before transplanted) carried through
to later growth as shown by strong correlations between number of leaves in week 2 and
week 4, shoot length in week 2, with shoot length in weeks 4 and 6 and with fresh and
dry weight at week 6. There is usually a strong relationship between seed vigour,
seedling vigour and field performance (Kidson and Westoby 2000). Seed vigour
influences crop development and yield in cauliflower (Finch-Savage and McKee, 1990),
beetroot and carrot (Karuna and Aswathaiah, 1989). So, further study of seed and
seedling traits may eventually improve field performance.
A lot of marker associations were identified at 6 weeks, suggesting that seedling
screening/analysis should be performed at 6 weeks. This is a critical time in seedling
development as it marks the developmental stage when seedlings are transferred to the
field.
R A P D markers were employed to investigate the correlations between
morphological traits and genetic markers and consistent bands were found on all
primers tested. Out of 88 markers produced, 24 markers showed association with a
number of seedlings traits across cultivars. The variation observed in certain
morphological traits may be under strong genetic control, as indicated by a strong
association between traits and molecular markers. These potential markers could be
linked to genes controlling these traits and this requires further investigation for their
use in marker-assisted breeding for particular traits.
Several studies have associated R A P D markers with particular traits in Brassica.
Bulk segregrant analysis links R A P D markers with particular traits such as silique
88
shatter resistance in B. rapa (Mongkolporn et al. 2003) and club root resistance in B.
rapa (Hirai et al. 2004). With bulk segregrant analysis, a large number of primers and
populations need to be examined to find associations and validate results. Our method
employed a relatively small number of primers and plants and association was
determined using multivariate analyses based on genetic information generated from
R A P D analyses. This is quite a new approach and has been applied recently on radish
(Pradhan et al. 2004). Although a preliminary phase, this method indicates some
tightness of the association as shown by the range of /^-values. Here w e developed our
range as follow: 0.00 - 0.01 tightly linked, > 0.01 - 0.05 moderate linked, > 0.05 not
linked.
This investigation was a preliminary study in the process of identification of
particular traits using molecular markers. Association of molecular markers with
seedlings traits established here may contribute to marker-assisted selection. Bulk
segregant analysis will be required to examine populations and confirm the reliability of
this simpler alternative technique. Other markers such as SSR and S C A R may also be
employed and compared for their reliability. In the future, marker-assisted selection of
seedling traits could be conducted more efficiently, and this would assist in the
screening of plants at an early stage. This technique can be applied to any traits of
interest.
Acknowledgments W e thank AusAID for providing a scholarship to Ida Astarini.
Sincere thanks to Bejo, Syngenta, Enza Zaden (formerly Yates Seeds), South Pacific
Seeds and Henderson Seeds for providing seeds for this project. Financial assistance
from Department of Agriculture Western Australia is gratefully acknowledged.
References
Alonso-Blanco C, Blankestijn-De Vries H, Hanhart CJ, Koorneef, M (1999) Natural
allelic variation at seed size loci in relation to other life history traits of
Arabidopsis thaliana. Proceedings of the National Academic of Sciences USA
96,4710-4717.
Ammiraju JSS, Dholakia B B , Santra D K , Singh H, Lagu M D , Tamhankar SA, Dhaliwal
H S , Rao V S , Gupta V S , Ranjekar P K (2001) Identification of inter simple
sequence repeat (ISSR) markers associated with seed size in wheat. Theoretical
and Applied Genetics 102, 726-732.
89
Astarini IA, Plummer JA, Lancaster RA, Yan G (2004) Fingerprinting of cauliflower
cultivars using R A P D markers. Australian Journal of Agricultural Research
55,117-124.
Digby P, Galwey N, Lane P (1989) Genstat 7.0, Clarendon Press, Oxford.
Fenner M (1993) Environmental influences on seed size and composition.
Horticultural Reviews 13, 183-213.
Finch-Savage W E , McKee J M T (1990) The influence of seed quality and
pregermination treatment on cauliflower and cabbage transplant production and
field growth. Annals of Applied Biology 116, 365-369.
Gutterman Y (2000) Maternal effects on seed during development. The Ecology of
Regeneration in Plant Communities. Fenner M (ed). C A B International,
Wallingford, UK.
Hirai M , Harada T, Kubo N, Tsukada M , Suwabe K, Matsumoto S (2004) A novel
locus for clubroot resistance in Brassica rapa and its linkage markers.
Theoretical and Applied Genetics 108, 639-643.
Hoeck JA, Fehr W R , Shoemaker RC, Welke GA, Johnson SL, Cianzio SR (2003)
Molecular markers analysis of seed size in soybean. Crop Science 43, 68-74.
Hu J, Quiros CF (1991) Identification of broccoli and cauliflower cultivars with R A P D
markers. Plant Cell Reports 10, 505-511.
International Seed Testing Association (ISTA) (2003) International Rules for Seed
Testing. Bassersdorf, Switzerland.
Jofuku K D , Omidyar PK, Gee Z, Okamura JK (2005) Control of seed mass and seed
yield by the floral homeotic gene APETALA2. Proceedings of the National
Academic of Sciences USA 102, 3117-3122.
Karuna M N , Aswathaiah B (1989) Effect of seed vigour on field performance in
beetroot and carrot. Seeds and Farms Sept-Oct, 40-46.
Kidson R, Westoby M (2000) Seed mass and seedling dimensions in relation to seedling
establishment. Oecologia 125, 11-17.
Mongkolporn O, Kadkol GP, Pang ECK, Taylor PWJ (2003) Identification of R A P D
markers linked to recessive genes conferring siliqua shatter resistance in Brassica
rapa. Plant Breeding 122, 479-484.
Nozaki T, Kumazaki A, Koba T, Ishikawa K, Ikehashi H (1997) Linkage analysis
among loci for RAPDs, isozymes and some agronomic traits in Brassica
campestris L. Euphytica 95, 115-123.
90
Ohto M , Fischer RL, Goldberg R B , Nakamura K, Harada JJ (2005) Control of seed
mass by APETALA2. Proceedings of the National Academic of Sciences USA
102,3123-3128.
Powell A A , Thornton JM, Mitchell JA (1991) Vigour differences in Brassica seed and
their significance to emergence and seedling variability. Journal of Agricultural
Science 116, 369-373.
Pradhan A, Yan G, Plummer JA (2004) Correlation of morphological traits with
molecular markers in radish {Raphanus sativus). Australian Journal of
Experimental Agriculture 44, 813-819.
Soffer H, Smith O E (1974) Studies on lettuce seed quality: IV. Individually measured
embryo and seed charactereistics in relation to continuous plant growth (vigor)
under controlled conditions. Journal of the American Society for Horticultural
Science 99, 270-275.
Somers DJ, Rakow G, Prabhu V K , Friesen K R D (2001) Identification of a major gene
and R A P D markers for yellow seed coat colour in Brassica napus. Genome 44,
1077-1082.
Stamp N E (1990) Production and effect of seed size in a grassland annual {Erodium
brachycarpum, Geraniaceae). American Journal of Botany 11, 874-882.
Stirling K, Lancaster R (2005) Effect of alternative planting configurations on
cauliflower development. Acta Horticulturae (in press).
Susko DJ, Lovett-Doust L (2000) Patterns of seed mass variation and their effects on
seedling traits in Alliaria petiolata (Brassicaceae). American Journal of Botany
87, 56-66.
Zhang J (1993) Seed dimorphism in relation to germination and growth of Cakile
edentula. Canadian Journal of Botany 71, 1231 -123 5.
91
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Table 4. Correlation between seed weight with other seedling traits within
cultivars. F W = fresh weight, G e r m = germination, d=day, w = week, *=
significantly different (p< 0.05), **=higbly significant (p< 0.01), ***= very highly
significant (p< 0.001).
Correlation coefficient (r) between seed weight with traits
Cultivars
Plana
Donner
Discovery
Fremont
Monarch
CF284
Virgin
Morpheus
Cauldron
Arctic
Atlantis
L3368
Belot
Lateman
Panther
Jerez
Fandango
Megan
Delfur
SPS 716
Omeo
FW7d
0.78**
0 87***
-0.60*
-0 98***
0.53
0.73*
0.62*
0.79*
0.69*
0.98***
0 99***
0.59
0.65*
0.40
0.11
0.72*
0.54
0.53
0.62*
0.62*
0.53
Germ rate
-0.01
-0.98***
-0.09
-0 98***
-0.17
-0.51
0.14
0.33
n 90***
0.86***
-0.13
0.84**
-0.58
-0.11
-0.78**
0.83**
-0.22
-0.54
0.34
0.34
-0.54
FW6w
0.78**
-0.02
-0.80**
-0.96***
-0.15
0.58
0.65*
0.06
n 99***
0 99***
0.13
0 99***
-0.56
0.62*
0 97***
0 99***
0.73**
0.81**
0.38
0.38
0.86***
DW6w
0.83**
0.18
-0.87***
-0.99***
-0.88**
0.70*
0.10
0.20
n 99***
0 99***
-0.00
0 99***
-0.49
0.62*
0 97***
0 9c***
0 91***
0.80**
0.37
0.37
0.96***
Table 5. Significance of markers associated with higher or lower values for seed
weight, germination rate (Germ), shoot length (It), root length, total length and
fresh weight (FW) at 7 days (d) for 21 cultivars. Values given are /7-values.
Markers
A02-1038 A04-833 A04-395 A04-185 D20-277 SKO1-606 SL03-761
A04-1059 A04-833 OPA07-707 SKO 1-824 SK14-723 SK19-1443 SL08-626 UBC106-404
Seed wt
n.s. n.s n.s. n.s n.s n.s. n.s
n.s. n.s. n.s. n.s. n.s. n.s. 0.043 n.s.
G e r m rate Markers for
n.s. 0.029 n.s. 0.02 0.02 0.00 n.s
Shoot It Root It • higher values n.s. n.s n.s. n.s n.s 0.001 n.s
n.s. n.s 0.043 n.s n.s n.s. n.s
Markers for lower values 0.02 0.022 0.02 0.001 n.s. n.s. n.s. n.s.
n.s. n.s. n.s. 0.002 n.s. 0.003 n.s. n.s.
n.s. n.s. n.s. n.s. 0.019 n.s. n.s. 0.03
Total It
n.s. n.s n.s. n.s n.s 0.032 n.s
n.s. n.s. n.s. 0.038 0.038 n.s. n.s. 0.023
FW7d
0.016 n.s n.s. n.s n.s n.s. 0.04
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
Table 6. Significance of markers associated with higher or lower values for fresh
weight (FW), dry weight (DW), leaf number (#), shoot length (It) and root length at
harvest after 6 weeks (w) for 21 cultivars. Values given are/>-values.
Markers
D12-1864 D12-1194 D20-1074 OPH15-1794 SL03-562 SK14-723 UBC 106-404 UBC 106-200
A04-833 D12-199 SKO 1-824 SKI 9-953 SL03-1047
SL03-791 SL03-761 SL03-595
FW6w
0.042 n.s. n.s. 0.021 0.000 n.s. 0.000 0.022
n.s. n.s. n.s. n.s. 0.001 0.001 n.s. 0.001
DW6w Markers for
n.s. n.s. n.s. 0.032 0.000 n.s. 0.002 0.026 Markers for
n.s. n.s. n.s. n.s. 0.000 0.000 n.s. 0.000
Leaf# higher values
n.s. 0.03 n.s. n.s. n.s. n.s. n.s. n.s.
• lower values 0.006 0.04 n.s.
n.s. 0.029 0.029 n.s. 0.029
Shoot It
n.s. n.s. 0.025 n.s. n.s. 0.017 0.000 n.s.
n.s.
n.s. n.s.
n.s. n.s. n.s. n.s.
n.s.
Root It
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
n.s.
n.s. 0.009
0.003 n.s. n.s. 0.048
n.s.
96
Fig 1. a. Seedling size variation at day 7, from lightest to heaviest (left to right),
b. Largest and smallest seedlings at 6 weeks, c. Biggest and smallest root mass at 6
weeks.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15161718 19 20 21 M t
ML 1000bp
i."-=.il"lallils'r" ^•|3_5oobP
Fig 2. RAPD profiles of 21 cauliflower cultivars using primer UBC 106. A,
Markers for root length, total length at day 7, shoot length, fresh weight and dry
weight at week 6. M, Marker Ladder; 1, Plana; 2, Donner; 3, Discovery; 4,
Fremont; 5, Monarch; 6, CF0284; 7, Virgin; 8, Morpheus; 9, Cauldron; 10, Arctic;
11, Atlantis; 12, L3368; 13, Belot; 14, Lateman; 15, Phanter; 16, Jerez; 17,
Fandango; 18, Megan; 19, Delfur; 20, SPS 716; 21, Omeo.
97
Chapter 8
General Discussion
Lack of uniformity is a critical issue limiting profitability in the cauliflower
industry. Improving uniformity, machine harvesting and developing new export
markets are regarded as high priority research issues for the cauliflower industry
(Warren Cauliflower Group, 2003). Non-uniform production is caused by variation,
occurring within cultivar seed and in the development of seedlings. This variation is
exacerbated in the field environment to produce plants with uneven curd maturity and
the production of sibs. Procedures were developed to identify sources of genetic
variation with the aim of improving uniformity using molecular genetic techniques.
This thesis demonstrated successful application of molecular marker techniques
to identify cultivars, to develop fingerprinting keys, to reveal genetic relatedness among
cultivars (Chapter 3, 4, 5), to differentiate between male and female parent lines, and
between hybrid and non hybrid plants (Chapter 6), and to link molecular markers with
seedling traits (Chapter 7). Research and findings of this thesis will substantially
increase our ability to visualise and detect sources of genetic variation.
R A P D technique was successful in differentiating 25 Fi hybrid cauliflower
cultivars commonly grown in Australia (Chapter 3). The 25 polymorphic markers
clearly distinguished 18 cultivars and identified genetically identical cultivars. A
minimum of 12 markers obtained from 10 primers was required to separate cultivars
and generate a fingerprinting key.
The ability of R A P D technique to differentiate closely related varieties will be
very useful for the cauliflower industry in Western Australia and around the world. As
the number of cultivars available in the market increases, the ability to distinguish them
on the basis of morphological traits becomes more difficult (Lombard et al, 2000) and
molecular markers are an accurate option, as no environmental factors such as weather,
seasonal and agronomy issues are involved.
The R A P D markers also successfully identified within and between cultivar
variability in open pollinated cultivars from Indonesia (Chapter 4). This technique was
able to differentiate geographical/growing areas of the cultivars. This is the first report
of Indonesian cauliflower cultivar identification using molecular markers and will
certainly encourage further research into cauliflowers, other Brassica vegetables and
other vegetable crops particularly in developing countries.
The study confirmed the ability of R A P D technique to work on genetically
distinct groups of cauliflowers, i.e. control pollinated, Fi hybrids from temperate
regions and open pollinated lines from tropical regions. This indicates the technique
m a y be able to discriminate a wide range of cauliflower cultivars and could be used as a
99
fast method to verify cultivars worldwide. R A P D fingerprinting will be extremely
useful for future cauliflower identification, avoiding possible mislabeling and fraud.
At present, distinctness, uniformity and stability are the criteria of Plant
Breeders's Rights for the purpose of registration of new plant varieties ( U P O V - B M T ,
2002). A s per the guidelines of the International Union for the Protection of N e w
Varieties of Plants, distinctness, uniformity and stability testing is currently primarily
based on essential morphological characters. The distinctness, uniformity and stability
testing based on phenotype and isozyme expression suffers from the limited number of
target traits and genotype by environment interactions when the candidate variety is
evaluated across environments. Cultivar registration based on D N A markers will
provide accuracy as they are not affected by environmental factors. Consequently, it
would significantly reduce costs as no field trials are required to confirm identity. D N A
markers are also independent from the developmental stage of the plant, while many
protein-based markers are, therefore providing more accuracy compared to for example,
isozyme markers that are being used in some crops such as maize, wheat and barley.
The use of D N A markers will be implemented in the future to establish the
distinctness, uniformity and stability of plant variety trials (Nandakumar et al, 2004;
U P O V - B M T , 2002). A D N A marker-based registration test will substantially enhance
the process of discrimination of candidate varieties and hybrids. Information that is
likely to be broadly applicable for cauliflower identification is n o w available for both
narrow genetic based Fi hybrid cultivars and high variability open pollinated cultivars.
Chapters 3 and 4 of this thesis provide valuable information to be considered in the
process of establishing DNA-marker based cultivar registration.
Genetic distance between Indonesian and Australian cultivars was confirmed in
this thesis using R A P D markers (Chapter 5). Comparison between open pollinated and
Fi hybrid cultivars shows that more variation occurs within cultivars in open pollinated
plants. All Indonesian cultivars tested were separated from Australian grown cultivars
confirming they have a long independent breeding history. Distant relationships
between Europe and East Asian Brassica accessions have also been reported in Brassica
rapa (Zhao et al, 2005).
The study opens the opportunity for breeding better cauliflowers in both
countries. Indonesian cultivars will be good resources to broaden genetic diversity and
bring the potential characteristics such as resistance to abiotic and biotic stresses for
cultivar improvement programs. Hybridization of cultivated species with distantly
related species, with unimproved 'wild' relatives or aiming for better productivity and
100
disease resistance has been attempted for a number of crops such as canola (Voss et al,
2000; Brown et al, 1996) and chickpea (Singh et al, 2005; Crosser et al, 2003). For
future work, collection and characterisation of vegetable Brassica and their wild
relatives using R A P D markers would provide important germplasm information for
potential breeding programs both within Australia and world wide. Comprehensive
research should be focused on finding markers for particular traits such as disease
resistance and high yield.
Fi hybrid cultivars were originally established with the aim to improve yield and
uniformity. However, a considerable proportion of non-uniform curds, assumed to be
caused by self-inbred plants are found in all production areas. These are commonly
known as 'sibs'. A n improvement from the 2-20% of losses currently considered to be
'sibs' could benefit growers by at least $330 per hectare, with a potential benefit of
about $3300 per hectare if sib losses were high. In Chapter 6, microsatellite markers
were proved powerful enough to distinguish female and male parent lines, hybrid and
non-hybrid plants. Controlled pollination experiments in the glasshouse, a field
production trial and microsatellite marker examination proved that self-inbreeding was
not the only cause of sibs. It has been observed in the field that poor management
increases the percentage of 'sib-like' aberration (Lancaster, Pers. C o m m ) . It has also
been suggested that aneuploidy, a missing or extra chromosome (Gerard Korevaar;
Duane Falk, Pers. C o m m ) may be responsible. A recent investigation on aberrant
cauliflowers using A F L P and M S A P (Methylation Sensitive Amplification
Polymorphism) indicated a low level of polymorphism between 'normal' and 'aberrant'
plants at the end of the vegetative development, suggesting 'sib-like' aberrations in
cauliflower m a y be under epigenetic control (Salmon et al, 2004).
Future research using cytology examination of chromosome karyotyping would
be useful to explore chromosome abnormalities in sib or aberrant plants. Further
investigation into the interaction between genetic factors and the environment should
also be pursued to find the true cause of the abnormality.
Variation in production begins at the seed and seedling stage. High quality
seeds should have high purity and germination, with good vigour. In Chapter 7,
variation within and between cultivar seedlings traits was revealed. R A P D markers
associated with seedlings characteristics were identified, indicating there are genetic
differences. The interaction of genetic differences and environmental conditions is
expressed in morphological variation. This chapter shows that variations at the early
growth stage are identifiable, screenable and removable. Screening at the seedling stage
101
will assist in breeding and selection of superior seedlings. It will also substantially
reduce seedling variability which will be an important factor in reducing production and
labour costs during growing, reducing losses from early and late harvest dates where
harvestable numbers are small and are considered uneconomic to recover. It is
projected that 1 % increase in exportable yield would represent at least $170 per hectare
return to growers.
Finding associations between molecular markers and seedling morphological
traits is still in its preliminary stage. The idea is to offer a simpler method to identify
genes controlling morphological traits. To confirm the benefit and accuracy of identified
markers, this technique needs to be further tested using currently available methods
such as bulk segregant analyses on recombinant inbred line populations. In the future,
seedling nurseries and breeders would be able to use this information to screen out
unfavorable plants.
In conclusion, procedures for cultivar fingerprinting using R A P D markers,
distinguishing male and female parent lines, hybrids and non-hybrids using
microsatellite markers and finding associations between molecular markers with
morphological traits have been developed in this thesis. Being able to screen cauliflower
plants in every stage of production, from choosing the right cultivar, screening for
particular traits to reduce seedling variability, and screening for abnormality will
significantly improve uniformity in cauliflower production. Molecular techniques
established in this thesis provide a promising and reliable approach to cultivar
identification, purity testing and screening for particular traits.
102
Chapter 9
References
A B S (2005) Australian Bureau of Statistics. Australia.
Alonso-Blanco C, Blankestijn-De Vries H, Hanhart CJ, Koorneef M (1999) Natural allelic variation at seed size loci in relation to other life history traits of Arabidopsis thaliana. Proceedings of the National Academic of Sciences USA 96, 4710-4717.
Ammiraju JSS, Dholakia BB, Santra D K , Singh H, Lagu M D , Tamhankar SA, Dhaliwal HS, Rao VS, Gupta VS, Ranjekar P K (2001) Identification of inter simple sequence repeat (ISSR) markers associated with seed size in wheat. Theoretical and Applied Genetics 102, 726-732.
Araya A, Zabaleta E, Blanc V, Begu D, Hernould M , Mouras A, Litvak S (1998) R N A editing in plant mitochondria, cytoplasmic male sterility and plant breeding. Electronic Journal of Biotechnology 1, 31-39.
Asandhi A A , Sastrosiswojo S (1988) Research on vegetable in Indonesia. In McLean B T (Ed.). Vegetable Research in Southeast Asia. Asian Vegetable Research and
Development Center, Taipei.
Astarini IA, Plummer JA, Lancaster RA, Yan G (2004) Fingerprinting of cauliflower cultivars using R A P D markers. Australian Journal of Agricultural Research 55,
117-124.
A U S V E G (2005) Commodity spotlight, cauliflower. A U S V E G , Victoria, Australia.
Barret P, Delourme R, Foisset N, Renard M (1998) Development of a S C A R (sequence characterized amplified region) marker for molecular tagging of the dwarf BREIZH (Bzh) gene in Brassica napus L. Theoretical and Applied Genetics 97,
828-833.
Bateman AJ (1955) Self-incompatibility systems in Angiosperms. III-Cruciferae.
Heredity 9, 53-68.
Baskin CC, Baskin J M (1998) Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germination. San Diego, Academic Press.
Bellamy A, Vedel F, Bannerot H (1996) Varietal identification in Cichorium intybus L. and determination of genetic purity of Fi hybrid seed samples, based on R A P D
markers. Plant Breeding 115,125-132.
Bert K E , Lydiate DJ (2003) Genetic analysis and genome mapping in Raphanus.
Genome 46, 423-430.
Bhalla PL, Singh M B (1999) Molecular control of male fertility in Brassica. Proceedings of the 10th International Rapeseed Congress, Canberra, Australia.
Bond JM, Mogg RJ, Squire GR, Johnstone C (2004) Microsatellite amplification in Brassica napus cultivars: Cultivar variability and relationship to a long-term feral
population. Euphytica 139, 173-178.
Bornet B, Branchard M (2001) Nonanchored inter simple sequence repeat (ISSR) markers: reproducible and specific tools for genome fingerprinting. Plant
Molecular Biology Reporter 19, 209-215.
Boury S Lutz I, Gavalda M-C, Guidet F, Schlesser A (1992) Empreintes genetiques du chou-fleur par R A P D et verification de la purete hybride Fi d'un lot de semences.
Agronomie 12, 669-681.
104
Bretagnolle F, Thompson JD, Lumaret R (1995) The influence of seed size variation on seed germination and seedling vigour in diploid and tetraploid Dactylis glomerata L. Annals of Botany 76, 607-615.
Brown J, Thill D C , Brown AP, Brammer TA, Nair H (1996) Gene transfer between canola {Brassica napus) and related weed species. In Proceedings of the 8th
Symposium on Environmental Releases of Biotechnology Products: Risk Assessment Methods and Research Progress, Ottawa, Canada.
Cansian RL, Echeverrigaray S (2000) Discrimination among cultivars of cabbage using randomly amplified polymorphic D N A markers. HortScience 35, 1155-1158.
Cardi T and Earle E D (1997) Production of new C M S Brassica oleracea by transfer of 'Anand' cytoplasm from B. rapa through protoplast fusion. Theoretical and Applied Genetics 94, 204-212
Charsley T N (1998) Reducing the Harvest Period of Cauliflower {Brassica oleracea var. botrytis L.) with Pre-treatments of Cold and Gibberellic Acid. B.Sc. thesis. Plant Sciences, Faculty of Agriculture, The University of Western Australia.
Charters Y M , Robertson A, Wilkinson MJ, Ramsay G (1996) P C R analysis of oilseed rape cultivars {Brassica napus L. ssp. oleifera) using 5'-anchored simple sequence repeat (SSR) primers. Theoretical and Applied Genetics 92, 442-447.
Cho S, Kumar J, Shultz JL, Anupama K, Tefera F, Muehlbauer FJ (2002) Mapping genes for double podding and other morphological traits in chickpea. Euphytica 128, 285-292.
Cooke RJ (1999) Modern methods for cultivar verification and the transgenic plant challenge. Seed Science and Technology 27, 669-680.
Couvillon G A (2002) Cercis canadensis L. seed size influences germination rate, seedling dry matter, and seedling leaf area. HortScience 37, 206-207.
Crisp P and Tapsell C R (1993) Cauliflower, Brassica oleracea L. In Kalloo G, Bergh B O (Eds). Genetic Improvement of Vegetables Crops. Pergamon Press, Oxford.
Crockett PA, Bhalla PL, Lee CK, Singh M B (2000) R A P D analysis of seed purity in a commercial hybrid cabbage {Brassica oleraceae var. capitata) cultivar. Genome
43,317-321. "
Crockett PA, Singh M B , Lee CK, Bhalla PL (2002) Genetic purity analysis of hybrid broccoli {Brassica oleracea var italica) seed using R A P D PCR. Australian
Journal of Agricultural Research 53, 51-54.
Croser JS, Ahmad F, Clarke HJ, Sidiqque K H M (2003) Utilisation of wild Cicer in chickpea improvement-progress, constraints, and prospects. Australian Journal of
Agricultural Research 54, 429-444.
Darmawan D A , Pasandaran E (2000) Indonesia, dynamics of vegetable production, distribution and consumption in Asia. In Ali M . (Ed.), Asian Vegetable Research
and Development Center, Taiwan.
Das S, Rajagopal J, Bhatia S, Srivastava PS, Lakshmikumaran M (1999) Assessment of genetic variation within Brassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic D N A markers.
Journal ofBioscience 24, 433-440.
Delourme R, Budar F (1999) Male Sterility. In Gomez-Campo C (Ed.). Biology of
Brassica Coenospecies. Elsevier Science B.V. Amsterdam.
105
Demeke T, Adams RP, Chibbar R (1992) Potential taxonomic use of random amplified polymorphic D N A (RAPD): a case study in Brassica. Theoretical and Applied Genetics 84, 990-994.
Digby P, Galwey N, Lane P (1989) Genstat 7.0, Clarendon Press, Oxford.
Divaret I, Margale E, Thomas G (1999) R A P D markers on seed bulks efficiently assess the genetic diversity of a Brassica oleracea L. collection. Theoretical and Applied Genetics 98,1029-1035.
Dos Santos JBD, Nienhuis J, Skroch P, Tivang J, Slocum M K (1994) Comparison of R A P D and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes. Theoretical and Applied Genetics 87, 909-915.
Dulson J, Kott LS, Ripley V L (1998) Efficacy of bulked D N A samples for R A P D D N A fingerprinting of genetically complex Brassica napus cultivars. Euphytica 102, 65-
70.
F A O S T A T Database. FAO. http://apps.fao.org/page/collection?subset=agricuture. Last
update 14 July 2005.
Farnham M W (1996) Genetic variation among and within United States collard cultivars and landraces as determined by randomly amplified polymorphic D N A markers. Journal of American Society for Horticultural Sciences 121, 374-379.
Fenner M (1993) Environmental influences on seed size and composition. Horticultural
Reviews 13, 183-213.
Finch-Savage W E , McKee J M T (1990) The influence of seed quality and pregermination treatment on cauliflower and cabbage transplant production and
field growth. Annals of Applied Biology 116, 365-369.
Fitzgerald D M , Barry D, Dawson PR, Cassells A C (1997) The application of image analysis in determining sib proportion and aberrant characterization in Fi hybrid
Brassica populations. Seed Science and Technology 25, 503-509.
Frankel R, Galun E (1977) Pollination Mechanism, Reproduction and Plant Breeding.
Springer-Verlag, Berlin, Germany.
Fu T, Ping S, Xiaoniu Y, Guangsheng Y (1992) Overcoming self-incompatibility of
Brassica napus by salt (NaCl) spray. Plant Breeding 109, 255-258.
Fujime Y, Okuda N (1996) The physiology of flowering in Brassicas, especially about
cauliflower and broccoli. Acta Horticulturae 407, 247-254.
George R A T (1999) Vegetable Seed Production. 2nd Ed. CABI Publishing Wallingford,
UK. Geraci A, Divaret I, Raimondo F M , Chevre A M (2001) Genetic relationships between
Sicilian wild populations of Brassica analysed with R A P D markers. Plant
Breeding 120,193-196.
Gowers S (2000) A comparison of methods for hybrid seed production using self-incompatibility in Swedes {Brassica napus ssp. napobrassica). Euphytica 113,
207-210.
Grubben G J H (1977) Tropical Vegetable and Their Genetic Resources. International
Board for Plant Genetic Resources, Rome.
Gupta PK, Varshney R K (2000) The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica
113, 163-185.
106
Gupta PK, Varshney R K , Sharma PC, Ramesh B (1999) Molecular markers and their applications in wheat breeding. Plant Breeding 118, 369-390.
Gupta M , Chyi Y-S, Romero-Severson J, Owen JL (1994) Amplification of D N A markers from evolutionarily diverse genomes using single primers of simple-sequence repeats. Theoretical and Applied Genetics 89, 998-1006.
Gutterman Y (2000) Maternal effects on seed during development. The Ecology of Regeneration in Plant Communities. Fenner M (Ed). CABI Publishing, Wallingford, U K .
Hadley P, Pearson S (1998) Effects of environmental factors on progress to crop maturity in selected Brassica crops. Acta Horticulturae 459, 61-70.
Hallidri M , Pertena D (2002) Self-incompatibility test in cabbage {B. oleracea var capitata). Acta Horticulturae 519, 117-122.
Harvey E, Smith B M (1987) A recent survey of sib content in Fi hybrid Brussels sprout varieties. Cruciferae Newsletter 12, 122-123.
Hendrix SD, Nielsen E, Nielsen T, Schutt M (1991) Are seedlings from small seeds always inferior to seedling from large seeds? Effects of seed biomass on seedling growth in Pastinaca sativa L. New Phytologist 119, 299-305.
Heneen W K , Jorgensen R B (2001) Cytology, R A P D , and seed colour of progeny plants from Brassica rapa-alboglabra aneuploids and development of monosomic
addition lines. Genome 44, 1007-1021.
Henry RJ (1997) Practical Applications of Plant Molecular Biology. Chapman and
Hall, London.
Heslop-Harrison J, Heslop-Harrison Y (1970) Evaluation of pollen viability by enzymatically-induced fluorescence; intracellular hydrolysis of fluorescein
diacetate. Stain Technology 45, 115-120.
Hirai M , Harada T, Kubo N, Tsukada M , Suwabe K, Matsumoto S (2004) A novel locus for clubroot resistance in Brassica rapa and its linkage markers. Theoretical
and Applied Genetics 108, 639-643.
Hodgkin T (1981) Some aspects of sib production in Fi cultivars of Brassica oleracea.
Acta Horticulturae 111, 17-24.
Hoeck JA, Fehr W R , Shoemaker RC, Welke G A , Johnson SL, Cianzio SR (2003) Molecular markers analysis of seed size in soybean. Crop Science 43, 68-74.
Holland RL, McNeilly T (1985) Genotype environment interactions and sib content in
Fi hybrid-brussels sprouts. Euphytica 34, 371-376.
Hu J, Quiros CF (1991) Identification of broccoli and cauliflower cultivars with R A P D
markers. Plant Cell Reports 10, 505-511.
Humpry M E , Lambrides CJ, Chapman SC, Aitken EAB, Imrie BC, Lawn RJ, Mclntyre CL, Liu CJ (2005) Relationships between hard-seedness and seed weight in mungbean {Vigna radiate) assessed by Q T L analysis. Plant Breeding 124, 292-
298.
International Seed Testing Association (ISTA) (2003) International Rules for Seed
Testing. Bassersdorf, CH-Switzerland.
Jofuku K D , Omidyar PK, Gee Z, Okamura JK (2005) Control of seed mass and seed yield by the floral homeotic gene APETALA2. Proceedings of the National
Academic of Sciences USA 102, 3117-3122.
107
Jourdren C, Barret P, Horvais R, Delourme R, Renard M (1996) Identification of R A P D markers linked to linolenic acid genes in rapeseed. Euphytica 90, 351-357.
Karuna M N , Aswathaiah B (1989) Effect of seed vigour on field performance in beetroot and carrot. Seeds and Farms Sept-Oct, 40-46.
Kidson R, Westoby M (2000) Seed mass and seedling dimensions in relation to seedling establishment. Oecologia 125, 11-17.
Kresovich S, Williams JGK, McFerson JR, Routman EJ, Schaal B A (1992) Characterization of genetic identities and relationships oi Brassica oleracea L. via a random amplified polymorphic D N A assay. Theoretical and Applied Genetics 85,190-196.
Kumar PP, Yau JCK, Goh CJ (1998) Genetic analyses of Heliconia species and cultivars with randomly amplified polymorhic D N A (RAPD) markers. Journal of the American Society for Horticultural Science 123, 91-97.
Lamboy W F , McFerson JR, Li R, Kresovich S (1994) Relationships among Chinese vegetable Brassicas using R A P D markers. Cruciferae Newsletter 16, 44.
Lancaster R, Pasqual G (1999) Cauliflowers from Western Australia at a glance. Bulletin 4398. Department of Agriculture, Western Australia.
Lancaster R, Burt J (2001) Cauliflower Production in Western Australia. Bulletin 4521. Department of Agriculture, Western Australia.
Lakshmikumaran M , Mohapatra T, Gupta VS, Ranjekar P K (2003) Molecular markers in improvement of wheat and Brassica. In: Plant Breeding-Mendelian to Molecular Approaches. Jain H K , Kharkwal M C (Eds.). Narosa Publishing House,
N e w Delhi, India.
Lee S, Cheng H, King KE, Wang W , He Y, Hussain A, Lo J, Harberd NP, Peng J (2002) Gibberellin regulates Arabidopsis seed germination via RGL2, a GAI/RGA-like gene whose expression is up regulated following imbibition. Gene
and Development 16, 646-658.
Leroy XJ, Leon K, Branchard M (2000) Characterisation of Brassica oleracea L. by microsatellite primers. Plant Systematics and Evolution 225, 235-240.
Leviel R (1998) La sterilite male chez le chou-fleur. PHM, Revue Horticole 388, 31-33.
Lombard V, Baril CP, Dubreuil P, Blouet F, Zhang D (2000) Genetic relationships and fingerprinting of rapeseed cultivars by AFLP: consequences for varietal
registration. Crop Science 40, 1417-1425.
Lowe AJ, Moule C, Trick M , Edwards KJ (2004) Efficient large-scale development of microsatellites for marker and mapping applications in Brassica crop species. Theoretical and Applied Genetics 108, 1103-1112.
Madhavi DL, Ghosh SP (1998) Cauliflower. In Handbook of Vegetable Science and Technology. Production, Composition, Storage and Processing. Salunkhe DK,
Kadam SS (Eds). Marcel Dekker, Inc. N e w York.
Mailer RJ, M a y C E (1999) Heterogeneity of random amplified polymorphic D N A sequences in individual seedlings and bulked samples of four cultivars of Brassica
napus. Plant Breeding 118, 465-470.
Makaroff C A (1995) Cytoplasmic male sterility in Brassica species. In The Molecular Biology of Plant Mitochondria. Levings III CS, Vasil IK (Eds). Kluwer Academic
Publishers. London.
108
Malatesta M , Davey JC (1996) Cultivar identification within broccoli, Brassica oleracea L. var. italica Plenk and cauliflower, Brassica oleacea var. botrytis L. Acta Horticulturae 407, 109-113.
Malik M, Vyas P, Rangaswamy NS, Shivanna KR (1999) Development of two new cytoplasmic male-sterile lines in B. juncea through wide hybridization. Plant Breeding 118, 75-78.
Margale E, Herve Y, Hu J, Quiros CF (1995) Determination of genetic variability by R A P D markers in cauliflower, cabbage and kale local cultivars from France. Genetic Resources and Crop Evolution 42, 281-289.
Massie IH, Astley D, King GJ (1996) Patterns of genetic diversity and relationships between regional groups and populations of Italian landrace cauliflower and broccoli {Brassica oleracea L. var. botrytis L. and var italica Plenck). Acta Horticulturae 407, 45-53.
Mattingley P (2002) Cauliflowers in Western Australia, an Industry Plan. Department of Agriculture, Western Australia.
Mayberry K S (2000) Sample cost to establish and produce cauliflower. U.C. Cooperative extension. Imperial County, California.
McArthur S (1999) Winter Newsletter. South Pacific Seeds, Christchurch, N e w
Zealand.
McArthur S (2001) Winter Newsletter. South Pacific Seeds, Christchurch, N e w
Zealand.
McCubbin A, Dickinson H (1997) Self-incompatibility. In Pollen Biotechnology for Crop Production and Improvement. Shivanna KR, Sawhney V K (Eds).
Cambridge University Press, N e w York.
McVetty P B E (1997) Cytoplasmic male sterility. In Pollen Biotechnology for Crop Production and Improvement. Shivanna KR, Sawhney V K (Eds). Cambridge
University Press, N e w York.
Meng X, M a H, Zhang W , Wang D (1998) A fast procedure for genetic purity determination of head Chinese cabbage hybrid seed based on R A P D markers.
Research Note. Seed Science and Technology 26, 828-833.
Mennella G, Iori A, Sanaja V O , Magnifico V (1996) Broccoli and cauliflower cultivars identification through IE-HPLC seed protein analysis. Acta Horticulturae 407,
115-121.
Messiaen C M (1992) The Tropical Vegetable Garden. Principles for improvement and increased production with application to the main vegetable types. The Macmillan
Press, London.
Mian M A R , Bailey M A , Tamulonis JP, Shipe ER, Carter Jr TE, Parrott W A , Ashley D A , Hussey RS, Boerma H R (1996) Molecular markers associated with seed weight in two soybean populations. Theoretical and Applied Genetics 93, 1011-
1016
Mohring S Esch E, Wricke G (1999) Breeding hybrid varieties in winter rapeseed using recessive self-incompatibility. Proceedings of the 10th International Rapeseed
Congress, Canberra.
Mongkolporn O, Kadkol GP, Pang ECK, Taylor P W J (2003) Identification of R A P D markers linked to recessive genes conferring siliqua shatter resistance in Brassica
rapa. Plant Breeding 122, 479-484.
109
Monteiro A A , Lunn T (1999) Trends and Perspectives of vegetable Brassica breeding
world-wide, http://www.tropical-seeds.com/tech_forum/pubs_res/brassica.html
Monteiro A A , Gabelman W H , William P H (1988) Use of sodium chloride solution to overcome self-incompatibility in Brassica campestris. HortScience 23, 876-877.
Mueller U G , Wolfenbarger L L (1999) AFLP genotyping and fingerprinting. Tree 14, 389-394
Nandakumar N, Singh A K , Sharma RK, Mohapatra T, Prabhu K V , Zaman F U (2004). Molecular fingerprinting of hybrids and assessment of genetic purity of hybrid seeds in rice using microsatellite markers. Euphytica 136, 257-264.
Noli E, Conti S, Maccaferri M, Sanguineti MC (1999) Molecular characterization of tomato cultivars. Seed Science and Technology 27, 1-10.
Nozaki T, Kumazaki A, Koba T, Ishikawa K, Ikehashi H (1997) Linkage analysis among loci for RAPDs, isozymes and some agronomic traits in Brassica campestris L. Euphytica 95, 115-123.
Nybom H (2001) D N A markers for different aspects of plant breeding research and its applications. Acta Horticulturae 560, 63-66.
Ohto M , Fischer RL, Goldberg RB, Nakamura K, Harada JJ (2005) Control of seed mass by APETALA2. Proceedings of the National Academic of Sciences USA
102,3123-3128.
Onguso JM, Kahangi E M , Ndiritu D W , Mizutami F (2004) Genetic characterization of cultivated bananas and plantains in Kenya by R A P D markers. Scientia
Horticulturae 99, 9-20.
Orsi CH, Tanksley S D (2005) Sw4.1, The major Q T L for seed weight variation in tomato: mapping and characterization during seed development. Plant and Animal
Genomes XIII conference, San Diego, California.
Pelletier G, Ferrault M , Lancelin D, Boulidard L (1989) C M S Brassica oleracea cybrids and their potential for hybrid seed production. 12th Eucarpia Congress, Gottingen.
Pharmawati M , Yan G, McFarlane IJ (2004) Application of R A P D and ISSR markers to analyse molecular relationship in Grevillea (Proteaceae). Australian Systematic
and Botany 11, 49-61.
Phippen W B , Kresovich S, McFerson JR (1994) Assessing genetic identity and relatedness in cabbage with RAPDs. Cruciferae Newsletter 16, 46.
Plieske J, Struss D (2001) Microsatellite markers for genome analysis in Brassica. I. Development in Brassica napus and abundance in Brassicaceae species.
Theoretical and Applied Genetics 102, 689-694.
Powell A A , Thornton JM, Mitchell JA (1991) Vigour differences in Brassica seed and their significance to emergence and seedling variability. Journal of Agricultural
Science 116, 369-373.
Prabhu K V , Somers DJ, Rakow G, Gugel R K (1998) Molecular markers linked to white rust resistance in mustard Brassica juncea. Theoretical and Applied Genetics 97,
865-870.
PradhanA Yan G, Plummer JA (2004a) Development of D N A fingerprinting keys for
the identification of radish cultivars. Australian Journal of Experimental
Agriculture 44, 95-102.
110
Pradhan A, Yan G, Plummer JA (2004b) Correlation of morphological traits with molecular markers in radish {Raphanus sativus). Australian Journal of Experimental Agriculture 44, 813-819.
Quijada PA, Udall JA, Polewicz H, Vogelzang R D , Osborn T C (2004) Phenotypic effects of introgressing French winter germplasm into hybrid spring canola. Crop Science 44, 1982-1989.
Raparelli E, Menesatti P (2000) Quality and Technological Characterization of two Cauliflower hybrids {Brassica oleraceae L. convar. botrytis L.). Acta Horticulturae 539, 109-113.
Rafalski A, Tingey S, Williams J G K (1994) Random amplified D N A (RAPD) markers. Gelvin, S. B. and R. A. Schilperoort (Eds). Plant Molecular Biology Manual. 2nd
Ed. Kluwer Academic Publ. Dordrecht. Section H/4.
Rafalski JA, Tingey S V (1993) Genetic diagnostics in plant breeding: RAPDs, microsatellites and machines. Trends in Genetics 9, 275-280.
Reddy M P , Sarla N, Siddiq E A (2002) Inter simple sequence repeat (ISSR) polymorphism and its application in plant breeding. Euphytica 128, 9-17.
Rubatzky V E , Yamaguchi M (1996) World Vegetables, Principles, production and nutritive values. 2nd Ed. International Thomson Publ. Singapore.
Ruffio-Chable V, Chatelet P, Thomas G (2000) Developmentally "Aberrant" Plants in Fi hybrids oi Brassica oleracea. Acta Horticulturae 539, 89-94.
Ruffio-Chable V, Bellis H and Herve Y (1993) A dominant gene for male sterility in cauliflower {Brassica oleracea var botrytis): phenotype expression, inheritance,
and use in Fi hybrid production. Euphytica 67,9-17.
Rukmana R (1994) Budidaya Kubis Bunga dan Brokoli. Penerbit Kanisius,
Yogyakarta.
Saal B, Plieske J, Quiros C, Struss D (2001) Microsatellite markers for genome analysis in Brassica. II. Assignment of rapeseed microsatellites to the A and C genomes and genetic mapping in Brassica oleracea L. Theoretical and Applied
Genetics 102, 695-699.
Salmon A, Manzanares-Dauleux M , Renard M , Chable V (2004) Epigenetic control of a phenotypic aberration in Brassica oleracea. Joint meeting of the 14 Crucifer Genetics Workshop and the 4th ISHS Symposium on Brassica. South Korea.
Samec P, Nasinec V (1996) The use of R A P D technique for the identification and classification of Pisum sativum L. genotypes. Euphytica 89, 229-234.
Sauer JD (1993) Historical geography of crop plants - a select roster. C R C Press, Boca
Raton, Florida.
Schaal B A (1980) Reproductive capacity and seed size in Lupinus texensis. American
Journal of Botany 67, 703-709.
Schnable PS, Wise R P (1998) The molecular basis of cytoplasmic male sterility and
fertility restoration. Trends in Plant Science 3,175-180.
Sharma SR, Singh PK, Chable V, Tripathi S K (2004) A review of hybrid cauliflower
development. Journal of New Seeds 6, 151-193.
Shellabear M (1994) Export cauliflower improvement project 1993 and 1994. Western Australian Department of Agriculture and Horticultural Research and
Development Corporation, Agriculture Western Australia, Perth.
Ill
Singh S, Gumber R K , Joshi N, Singh K (2005). Introgression from wild Cicer reticulatum to cultivated chickpea for productivity and disease resistance. Plant Breeding 124, 477-480.
Soffer H, Smith O E (1974) Studies on lettuce seed quality: IV. Individually measured embryo and seed charactereistics in relation to continuous plant growth (vigor) under controlled conditions. Journal of the American Society for Horticultural Science 99, 270-275.
Somers DJ, Rakow G, Prabhu V K , Friesen K R D (2001) Identification of a major gene and R A P D markers for yellow seed coat colour in Brassica napus. Genome 44, 1077-1082.
Song K, Tang K, Osborn TC, Lu P (1996) Genome variation and evolution of Brassica
amphidiploids. Acta Horticulturae 407, 35-44.
Stamp N E (1990) Production and effect of seed size in a grassland annual {Erodium brachycarpum, Geraniaceae). American Journal of Botany 11, 874-882.
Staub JE, Lopez-Sese Al, Fanourakis N (2004) Diversity among melon landraces {Cucumis melo L.) from Greece and their genetic relationships with other melon
germplasm of diverse origins. Euphytica 136, 151-166.
Stewart A V (2002) A review of Brassica species, cross-pollination and implications for pure seed production in N e w Zealand. Agronomy New Zealand 32, 63-82.
Stirling K, Lancaster R (2005) Alternative planting configurations influence cauliflower
development. Acta Horticulturae 694, 301-305
Susko DJ, Lovett-Doust L (2000) Patterns of seed mass variation and their effects on seedling traits in Alliaria petiolata {Brassicaceae). American Journal of Botany
87,56-66.
Swofford D L (1993) PAUP: Phylogenetic analysis using Parsimony, version 3.1 Illinois
Natural History Survey, Champaign, Illinois.
Tongue M , Griffiths P D (2004) Genetic relationships of Brassica vegetables determined using database derived simple sequence repeats. Euphytica 137, 193-201.
U N (1935) Genomic analysis of Brassica with special reference to the experimental formation of Brassica napus and its peculiar mode of fertilization. Japanese
Journal of Botany 1, 389-452.
U P O V - B M T (2002) BMT/36/10 Progress Report of the 36th session of the technical committee, the technical working parties and working group on biochemical and molecular techniques and DNA-profiling in particular, Geneva.
Van Molken T, Jorritsma-Wienk LD, Van Hoek P H W , de Kroon H (2005) Only seed size matters for germination in different populations of the dimorphic Tragopogon pratensis subsp. pratensis (Asteraceae). American Journal of Botany 92, 432-
437 Voss A, Snowdon RJ, Liihs W (2000) Intergeneric transfer of nematode resistance from
Raphanus sativus into the Brassica napus genome. Acta Horticulturae 539, 129-
134
Warren Cauliflower Group (2003) Priorities for research and development for the export cauliflower industry. Warren Cauliflower Group Incorporated, Manjimup.
Warwick SI, Soleimani V (2001) Genetic diversity in Brassical carinata, K juncea and B. nigra based on molecular A F L P markers. Cruciferae Newsletter 23, 15-16.
112
Watanabe M , Hinata K (1999) Self-Incompatibility. In Biology of Brassica Coenospecies. Gomez-Campo C (Ed.). Elsevier Science B.V. Amsterdam.
Webster R E (1964) Effect of size of seed on plant growth and yield of Fordhook 242 bush lima bean. Proceeding of the American Society of Horticultural Sciences 84, 327-331.
Wien HC, Wurr DCE (1997) Cauliflower, Broccoli, Cabbage and Brussel Sprouts. In The Physiology of Vegetable Crops. Wien H C (Ed.). CABI Publishing, Wallingford, U K .
William JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18, 6531-6535.
Williams CN, Uzo JO, Peregrine W T H (1991) Vegetable Production in the Tropics. Intermediate Tropical Agriculture Series. Longman Scientific and Technical, Essex.
Wills A B , Fyfes K, Wiseman E M (1980) Testing Fi hybrids of Brassica oleracea for sibs by seed isoenzyme analysis. Annals of Applied Biology 91, 263-270.
Wolfe A D , Liston A (1998) Contributions of PCR-based methods to plant systematics and evolutionary biology. In Molecular Systematics of Plants II, D N A Sequencing. Soltis DE, Soltis PS, Doyle JJ (Eds). Kluwer Academic Publishers, Dordrecht, The Netherlands.
Wurr D C E (1990) Prediction of the time of maturity in cauliflowers. Acta
Horticulturae 267, 387-391.
Yan G, Shan F, Plummer JA (2002) Genetic relationship within Boronia {Rutaceae) as revealed by karyotype analysis and R A P D molecular markers. Plant Systematics
and Evolution 233, 147-161
Yuan M , Zhou Y, Liu D (2004) Genetic diversity among populations and breeding lines from recurrent selection in Brassica napus as revealed by R A P D markers. Plant
Breeding 123, 9-12.
Zhang J (1993) Seed dimorphism in relation to germination and growth of Cakile edentula. Canadian Journal of Botany 71,1231-1235.
Zhao J, Wang X, Deng B, Lou P, W u J, Sun R, X u Z, Vromans J, Koornneef M, Bonnema G (2005). Genetic relationships within Brassica rapa as inferred from AFLP fingerprints. Theoretical and Applied Genetics 110, 1301-1314.
Zheng X Y , Liu Y (1994) Inbred testing of Chinese cabbage F! varieties by peroxidase and esterase isozyme analysis. Acta Horticulture Sinica 21, 65-70.
Zhuang FY, Chen JF, Staub JE, Qian C T (2004) Assessment of genetic relationships among Cucumis spp. by SSR and R A P D marker analysis. Plant Breeding 123,
167-172.
Zur I, Klein M , Dubert F, Samek L, Waligorska H, Zuradzka I, Zawislak E (2003) Environmental factors and genotypic variation of self-incompatibility in Brassica
oleracea L. var. capitata. Acta Biologica Cracoviensia 45,49-52.
113