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enetic diversity and structure in a collection of tulip cultivarsssessed by SNP markers
an Tanga,b, Arwa Shahinb, Paul Bijmanb, Jianjun Liua, Jaap van Tuylb, Paul Arensb,∗
Northwest A&F University, Taicheng Road 3, Yangling, Shaanxi, ChinaWageningen University and Research Centre, Plant Breeding, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
r t i c l e i n f o
rticle history:eceived 17 May 2013eceived in revised form 11 July 2013ccepted 12 July 2013
eywords:ulipulb crop
a b s t r a c t
Although tulip is one of the most important bulbous crops worldwide, the genetic background of mostcultivars is unclear at present. The purposes of this study are to investigate genetic diversity and toidentify the genetic structure and relationships among tulip cultivars. A total of 236 polymorphic singlenucleotide polymorphisms (SNPs) were obtained from ‘Kees Nelis’ and ‘Cantata’, from which 121 SNPswith a minor allele frequency above 0.1 were selected for genetic analysis. The total observed heterozy-gosity (Ho) among the 72 accessions was 0.35, Ho of cultivar groups ranged from 0.22 (Tulipa fosteriana)to 0.43 (Tulipa gesneriana × T. fosteriana hybrids). Rather small genetic distances were found among T.
gesneriana cultivar groups which are defined according to flowering time and morphology. In both PCoA(principle coordinate analysis) and STRUCTURE analysis, the 72 accessions were separated into threeclusters (FST = 0.208, P < 0.0001). A significant difference could be detected between early and late flower-ing cultivars (FST = 0.072, P = 0.02). T. gesneriana × T. fosteriana hybrid breeding lines were easily identified
STR
as admixed individuals inalso inferred as hybrids.
. Introduction
Tulip is one of the most important ornamental crops in theorld. The primary gene diversity centre of tulips is in the Pamirlai and Tian Shan mountains in central Asia (Hoog, 1973), whereas
he whole range extends from southern Europe, north Africa, Ana-olia and Iran to the northwest of China. The genus has more thanne hundred species. Taxonomy and species relationships wereainly described using morphological characters, geographical
istribution, cytogenetic characteristics and biochemical markeruch as esterase isozyme polymorphisms (Van Raamsdonk et al.,997; Van Raamsdonk and Vries, 1995). Recently, a molecularharacterization of Tulipa L. species using ISSR markers providedore genetic information about the taxonomy of this genus (Kiani
t al., 2012).Tulips have been classified into two major groups: Garden tulips
Tulipa gesneriana) and Species tulips. The original species involvedn the current garden and cut flower tulip, T. gesneriana, haveot been determined. Species tulips are composed of Tulipa spp.,uch as Kaufmaniana, Fosteriana, Greigii and their hybrids (Hall,
940). Several species can be crossed with cultivars of T. gesner-
ana. For example, interspecific crosses between Tulipa fosterianand T. gesneriana Darwin tulips created the widely grown group
of Darwin hybrid tulips (Bryan, 2002). The first Darwin hybridwas developed in 1943 and about 180 Darwin hybrid cultivarshave been introduced since then. Thirty seven cultivars were reg-istered during 1940–1960, 46 were registered during 1960–1980,55 were registered during 1980 and 2000. After 2000, there areabout 33 cultivars were introduced. At present, more than 2600T. gesneriana cultivars are available (Botschantzeva, 1982) andthese cultivars have been divided into 15 divisions by the KAVB(Koninklijke Algemeene Vereniging voor Bloembollencultuur,http://www.kavb.nl/index.cfm?act=home.tonen), the Royal DutchBulbgrowers Association in their 1996 Classified List and Inter-national Register of Tulip Names (Van Scheepen, 1996). Most ofthe divisions were defined according to blooming time and flowershape except for Kaufmanniana, Fosteriana and Greigii divisions,which were classified by their species of origin (Richard, 2006).
Genetic relationships among cultivars and information on theirparentage are useful to geneticists, breeders and public policy mak-ers. Morphological traits and biochemical and molecular markerscan be used to investigate genetic relationships and parentageinformation. However, morphological traits are often limitingin numbers and are easily influenced by environment. Althoughbiochemical markers are independent of environment influenceand have shown potential for cultivar identification, they are
dependent on the developmental stage of plant tissues. As a usefulcomplement to previous characterization, molecular markersprovide large amounts of information and can be used to detectvariation among individuals, populations and species reliably.
andom amplified polymorphic DNA (RAPD) were used to analyseenetic diversity of 10 tulip cultivars and 4 wild species in Xinjiangrovince of China (Luan et al., 2008). However, RAPD markers areominant makers and comparison of data can be limited due to its
ow reproducibility especially between labs (Jones et al., 1997). Sin-le nucleotide polymorphisms (SNPs) are ideal molecular markerso perform genetic studies because they are co-dominant markers,bundant in both coding and non-coding regions and amendableor high throughput genotyping. Next Generation Sequencingechnologies have enabled SNP identification at increasing lowerosts. This makes it possible to develop SNP markers in non-modelpecies, for which little or no genome sequence information isvailable (Osman et al., 2003). Although SNPs have limited amountf information per locus (each locus usually has only two alleles),he sheer number of putative markers as well as the possibilityor high genotyping throughput contribute to overcome thishortcoming (Helyar et al., 2011). Recent studies have shownhat even small subsets of SNPs (∼30) could also be sufficient toetect moderate (FST = 0.01) levels of differentiation (Morin et al.,009). A subset of 14 selected structure informative SNPs wasufficient in differentiating intercontinental human populationtructure (Paschou et al., 2007) and a core set of 49 SNPs was usedn distinguishing barley lines and varieties (Hayden et al., 2010).
Since the relationships among majority of tulip cultivars arenknown at present, the aims of this paper are (i) to investigate theenetic diversity and structure of a collection of tulip accessions;ii) to study the usefulness for identification of tulip hybridsetween T. gesneriana and T. fosteriana using SNP markers; (iii)o study whether the selection for forcing has led to geneticrosion and whether there are differences between tulip cultivarypes.
. Materials and methods
.1. Plant material
A total of 72 tulip accessions were used, including 55 widelyrown cultivars and 17 breeding lines which were produced atlant Breeding, Wageningen UR. Cultivars represented 2 superroups of tulips: T. gesneriana and T. fosteriana. Three aspectsere considered when selecting cultivars available from the tulip
ollection of Wageningen UR Plant Breeding. Firstly, the selectedultivars were in any time point popular in the tulip cut flowerroduction. Secondly, cultivars from different cultivar groups wereelected to investigate the genetic relationship among groups.hirdly, cultivars were selected differed in introduction timeo study the changes in levels of genetic diversity over time.reeding lines are interspecific hybrids between T. gesneriana and. fosteriana (short for GF hybrids). They were selected becausehey can be used as reference for hybrid identification since theenetic background of them is known. Description of cultivarroup, year of registration, country of origin and background of thereeding lines are presented in (Table 1). As representatives of T.esneriana and T. fosteriana tulips, ‘Kees Nelis’ and ‘Cantata’ wereelected to develop SNP markers. An F1 population made from annterspecific cross between ‘Kees Nelis’ and ‘Cantata’ was used toheck marker segregation. All plants were obtained from the tulipollection of Plant Breeding, Wageningen UR. A single young leafor each cultivar was collected in the field, put into liquid nitrogenmmediately and then kept in −80 ◦Cuntil DNA solation.
.2. SNP marker development
SNP markers were developed from cultivars ‘Kees Nelis’ andCantata’ (Shahin et al., 2012). In brief, RNA was isolated usinghe Trizol protocol (Invitrogen, Carlsbad, CA, USA) and purified
urae 161 (2013) 286–292 287
by RNeasy MinElute kit (Qiagen, Hilden, Germany). cDNA syn-thesis, normalization and adaptor ligation were performed byVertis Biotechnologie AG (Freising, Germany). cDNA libraries weresequenced on a GS-FLX Titanium at Greenomics Wageningen UR(Wageningen, the Netherlands) according to standard 454 LifeSciences procedures. Adapter sequences and low quality baseswere removed using CLC genomics workbench (CLC bio, Denmark,http://www.clcbio.com/. Fragments between 100 and 800 bp werekept for further analysis. PCR duplicates were removed by CD-HIT(Li and Godzik, 2006) with a threshold of 98% similarity (Shahinet al., 2012). SNPs were detected from the contigs assembled fromCLC by QualitySNP (Tang et al., 2006). A total of 316 SNPs wereselected for genotyping, 151 SNPs identified from ‘Kees Nelis’ and165 SNPs identified from ‘Cantata’.
2.3. SNP genotyping and marker filtration
Putative SNP markers were genotyped across 72 tulipaccessions using KASPar genotyping technology (KBioscience,http://www.kbioscience.co.uk/). Genotyping data were visualizedgraphically using the software program SNPViewer2 (KBioscience,http://www.kbioscience.co.uk/). Marker filtration was performedbefore doing genetic diversity analysis. Firstly, monomorphicmarkers were removed. Secondly, segregation ratio of SNP markersin the ‘Kees Nelis’ x ‘Cantata’ F1 population was checked. Theoreti-cally, the expected segregation ratio of markers is 1:1 (polymorphicin only one parent) or 1:2:1 (polymorphic in both parents). Markersthat showed skewed segregation were not used for further analysisbecause null-allele(s) might exist in these markers. Null-allele(s)would remain undetectable within cultivars and could influenceanalysis of genetic distance between cultivars. Thirdly, markersthat have more than 10% missing data were also excluded. As alast step, SNP markers with a minor allele frequency below 10%were excluded because they may not be informative for diversityanalysis and may lead to discriminating bias.
2.4. Data analysis
Allele frequency, observed and expected heterozygosity(Nei, 1973) of SNP markers were calculated using Popgene32(Quardokus, 2000). Total genetic diversity among 72 accessions anddiversity in different cultivar groups were estimated with percent-age of polymorphic loci, expected heterozygosity (Nei’s 1973) andobserved heterozygosity.
To investigate the genetic structure of the selected tulip geno-types, a principal coordinate analysis (PCoA) were used to clusterthe samples. PCoA used simple matching coefficient to plot n indi-viduals in (n − 1) dimensional space and was performed usingNTSYSpc 2.10 (Jensen, 1989). Beyond that, a Bayesian model-basedprogram STRUCTURE 2.3.3 (Pritchard et al., 2000) was used to inves-tigate the genetic structure among the tulip accessions. The best Kvalue (number of groups) was estimated through two approaches:the likelihood of the probability of data L(K) (=Ln P(D)) (Rosenberget al., 2001) and an ad hoc quantity �K (Evanno et al., 2005). Tworuns of analysis using the admixture model were performed. Ini-tial runs were performed with a burnin length of 1000 and 10,000MCMC (Markov Chain Monte Carlo) replicates for 20 times at eachK from1 to 10. The probable number of groups (K = 2 or 3) was esti-mated by Ln P(D). The second run was 10,000 for burnin length and100,000 for MCMC replicates, 20 times for each K between 1 and5. When the results suggested that the K groups could be furtherstructured in sub-groups, a second level (nested) STRUCTURE anal-
ysis was performed for each K group (Jacobs et al., 2011). The resultswere summarized in a bar plot using Distruct (Rosenberg, 2004).
The partitioning of the molecular variance at different levelswas tested by hierarchical analysis of molecular variance (AMOVA)
288 N. Tang et al. / Scientia Horticulturae 161 (2013) 286–292
Table 1List of tulip cultivars and breeding lines used in this study and their phenotypic division, introduced year, raiser’s name, country/city and flower colour.
NO Cultivar Cultivar group (KAVBa) Introduced year Country/city of originb Genetic background
1 Alliance Double early 1999 NL/Lisse T. gesneriana2 Largo Double early 1994 NL/St Pancras T. gesneriana3 Monte Carlo Double early 1955 Unknown T. gesneriana4 Double Princess Double late 1999 NL/Schagerbrug T. gesneriana5 Freeman Double late 2000 NL/Breezand T. gesneriana6 Barbados Fringed 1999 NL/Oude Niedorp T. gesneriana7 Mon Amour Fringed 2001 NL/Breezand T. gesneriana8 Target Fringed 2002 NL/Breezand T. gesneriana9 Seattle Lily-flowered 2007 NL/Breezand T. gesneriana
10 Amethyst Parrot 1975 NL/Wageningen T. gesneriana11 Bird of Paradise Parrot 1962 Unknown T. gesneriana12 Blue Parrot Parrot Before 1927 Unknown T. gesneriana13 Bellona Single early 1944 NL/Lisse T. gesneriana14 Christmas Marvel Single early 1954 Unknown T. gesneriana15 Duc van Tol Red and Yellow Single early 1620 Unknown T. gesneriana16 Generaal de Wet Single early 1904 Unknown T. gesneriana17 Baronne de la Tocnaye Single late 1891 NL/haarlem T. gesneriana18 Queen of Night Single late Before 1936 Unknown T. gesneriana19 Rhodos Single late 1985 NL/Wageningen T. gesneriana20 Wisley Single late 1987 NL/Wageningen T. gesneriana21 Bolroyal Silver Triumph 2002 Czech T. gesneriana22 Captivio Triumph 1930 Unknown T. gesneriana23 Caruso Triumph Before 1927 NL/Haarlem T. gesneriana24 Cheers Triumph 1990 NL/Nieuwe Niedorp T. gesneriana25 Debutante Triumph 1984 NL/Wageningen T. gesneriana26 Escape Triumph 1999 NL/Lisse T. gesneriana27 Fusor Triumph 1996 NL/Wageningen T. gesneriana28 Holland Sun Triumph 2000 NL/Heiloo T. gesneriana29 Ile de france Triumph 1968 NL/Beverwijk T. gesneriana30 Indus Triumph Before 1927 NL/Haarlem T. gesneriana31 Kees Nelis Triumph 1951 Unknown T. gesneriana32 Laura Fygi Triumph 2000 NL/Metslawier T. gesneriana33 Leen v/Mark Triumph 1968 NL/Noordwijk T. gesneriana34 Lucky Strike Triumph 1954 Unknown T. gesneriana35 Lustige Witwe Triumph 1942 Unknown T. gesneriana36 Orange Monarch Triumph 1962 Unknown T. gesneriana37 Pax Triumph 1942 Unknown T. gesneriana38 Purple Flag Triumph 1983 NL/Lisse T. gesneriana39 Recreado Triumph 1979 NL/St. Pancras T. gesneriana40 Ronaldo Triumph 1997 NL/De Goorn T. gesneriana41 Sachi Triumph 2000 NL/Breezand T. gesneriana42 Snow Lady Triumph 1992 NL/St Pancras T. gesneriana43 Snowboard Triumph 2006 NL/Metslawier T. gesneriana44 Strong Gold Triumph 1989 NL/Obdam T. gesneriana45 Tibet Triumph 1999 NL/Metslawier T. gesneriana46 Yellow Crown Triumph 2003 NL/Metslawier T. gesneriana47 Yellow Flight Triumph 1994 NL/Lisse T. gesneriana48 Yokohama Triumph 1961 Unknown T. gesneriana49 Zorro Triumph 1998 NL/Wageningen T. gesneriana50 Cantata Fosteriana 1942 NL/Heemstede T. fosteriana51 Juan Fosteriana 1961 NL/Heemstede T. fosteriana52 Madame Lefeber Fosteriana 1931 NL/Heemstede T. fosteriana53 Princeps Fosteriana Unknown Unknown T. fosteriana54 1560-1 Breeding line 2001 NL/Wageningen GF hybrid55 20185-1 Breeding line 2000 NL/Wageningen GF hybrid56 20185-4 Breeding line 2000 NL/Wageningen GF hybrid57 20185-5 Breeding line 2000 NL/Wageningen GF hybrid58 20208-2 Breeding line 2000 NL/Wageningen GF hybrid59 20241-2 Breeding line 2000 NL/Wageningen GF hybrid60 20241-3 Breeding line 2000 NL/Wageningen GF hybrid61 20255-4 Breeding line 2000 NL/Wageningen GF hybrid62 20259-11 Breeding line 2000 NL/Wageningen GF hybrid63 20259-12 Breeding line 2000 NL/Wageningen GF hybrid64 20622-36 Breeding line 2000 NL/Wageningen GF hybrid65 89183-105 Breeding line 1989 NL/Wageningen GF hybrid66 89183-53 Breeding line 1989 NL/Wageningen GF hybrid67 89183-55 Breeding line 1989 NL/Wageningen GF hybrid68 89183-73 Breeding line 1989 NL/Wageningen GF hybrid69 89183-80 Breeding line 1989 NL/Wageningen GF hybrid70 S-20253-1 Breeding line 2000 NL/Wageningen GF hybrid71 Purissima Fosteriana 1943 NL/Heemstede GF hybrid72 Flair Single early 1978 Unknown GF hybrid
a KAVB: Koninklijke Algemeene Vereniging voor Bloembollencultuur.b NL: the Netherlands.
N. Tang et al. / Scientia Horticulturae 161 (2013) 286–292 289
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Table 2Genetic diversity parameters of tulip cultivar groups.
ig. 1. Distribution of minor allele frequencies KN SNPs in T. gesneriana and CA SNPsn T. fosteriana.
sing Arlequin (Excoffier et al., 2005). A total of 1000 permutationsere performed to test the significance of variance.
. Results
.1. Description of SNP marker
In total, 316 SNP markers (151 from ‘Kees Nelis’, 165 fromCantata’) were genotyped across 72 tulips. Two hundred andighty markers (88.6%) were polymorphic. Eight SNP markers werexcluded due to more than 10% missing data. SNP markers wereenotyped across the offspring of ‘Kees Nelis’ × ‘Cantata’ and seg-egation ratio of markers was checked. As a result, 36 SNP markershowed inconsistence with Mendelian segregation. Therefore, 236NPs (111 from ‘Kees Nelis’, 125 from ‘Cantata’) were used forurther analysis. Considering the origin of markers and their infor-
ativeness, it is observed that 99 (89.2%) SNP markers derived fromKees Nelis’ (KN SNPs) were only polymorphic in T. gesneriana cul-ivars and 12 SNP markers (10.8%) were polymorphic in both T.esneriana and T. fosteriana cultivars. Similarly, for SNPs from ‘Can-ata’ (CA SNPs), 107 (85.6%) markers were only polymorphic in T.osteriana while 18 (14.4%) SNP markers were polymorphic in T.esneriana as well.
Minor allele frequency (MAF) is an important factor for SNP locivaluation. In this study, many SNPs showed very low MAF valuesnd were almost monomorphic. There were 25 KN SNPs (22.5%)nd 17 CA SNPs (13.6%) which showed an MAF < 0.10 in T. gesneri-na and T. fosteriana, respectively (Fig. 1). Considering all selected36 SNPs in 72 accessions, a total of 115 SNPs (83 CA SNPs; 32N SNPs) presented MAF < 0.10. These SNPs were removed from
urther analysis to increase statistical power and avoid possible
iscriminating bias. MAF value of the remaining 121 SNPs rangedrom 0.10 to 0.47 and about half of these markers have a MAF valuearger than 0.30.
able 3enetic distance among tulip cultivar groups.
Cultivar groups Double early Double late Fringed Lily-flowered
Double late 0.04Fringed 0.04 0.02Lily-flowered 0.12 0.06 0.07Parrot 0.09 0.03 0.02 0.07Single early 0.04 0.02 0.04 0.07
Single late 0.07 0.03 0.02 0.06
Triumph 0.04 0.02 0.01 0.05
Fosteriana 0.27 0.26 0.28 0.35
GF hybrids 0.11 0.09 0.11 0.16
a Percentage of polymorphic loci.b Observed heterozygosity.c Nei’s 1973 expected heterozygosity.
3.2. Genetic diversity analysis
Of the 121 selected SNPs, the highest frequency of polymor-phic loci (96.7%) was observed in the group of GF hybrids asexpected and the lowest (28.1%) was found in the Lily-floweredcultivar group (Table 2). The total observed heterozygosity amongthe 72 accessions was 0.35. When comparing the genetic diver-sity among cultivar groups, the Lily-flowered group was excludedfrom comparisons with other groups as it contained only one cul-tivar. Ho values ranged between 0.22 (T. fosteriana) and 0.43 (GFhybrids). The genetic distances (Nei’s 1978) among T. gesnerianacultivar groups (Double early, Double late, Fringed, Lily-flowered,Parrot, Single early, Single late and Triumph) were relatively low(Table 3). T. fosteriana group showed the largest genetic distances(0.26–0.35) to other groups except for GF hybrids (only 0.07). Thedistance between GF hybrids and other groups were intermediate(0.09–0.16).
The differentiation of all individuals was investigated usingPCoA (principal coordinate analysis) (Fig. 2a). The first and sec-ond coordinates of PCoA accounted for 19.6% and 6.5% of the totalvariance. All 72 tulips were separated into three clusters, whichwas in agreement with the unweighted pair group method forarithmetic averages (result not shown). Cluster 1 included all T. ges-neriana cultivars, while T. fosteriana cultivars ‘Cantata’, ‘Madamelefeber’, ‘Juan’ and ‘Princeps’ were included in cluster 2. Cultivar‘Purissima’ and ‘Flair’ together with all GF hybrids were groupedin cluster 3. Clusters were separated from each other in the firstcoordinate. The differentiation of the three clusters was furtherconfirmed by AMOVA which showed that the difference amongclusters was significant (P < 0.0001) and explained 20.8% of the totalgenetic variance (Table 4a).
A subsequent PCoA was performed for 49 T. gesneriana cultivarsto zoom in on the relationships among them (Fig. 2b). In total, 17.6%of the total variance can be explained by the first two coordinates.
There was no clear group division of these cultivars as describedby the KAVB (Van Scheepen, 1996). However, it was observed thatcultivars can be separated on the second principle coordinate by
Parrot Single early Single late Triumph Fosteriana
Fig. 2. Principal coordinates of PCoA based on genetic similarity of genetic dis-tances obtained from SNP markers. (a) Distribution of 72 tulip accessions basedon 121 SNPs. (b) Distribution of 49 T. gesneriana cultivars based on KN SNP mark-ers. Cultivar groups were indicated by different markers: Double early (open circle);Dr(
flao(psstfl
TA
ouble late (open square); Fringed (up triangle); lily-flowered (down triangle); Par-ot (cross); single early (plus); single late (star); Triumph (closed circle); Fosterianaopen diamond); GF (closed square). Individual numbers of (b) conform to Table 1.
owering time. Double early and Single early cultivars (Nos. 1–3nd 13–16) are early season flowers and located in the upper sidef the PCoA plot. Double late (4 and 5), Fringed (6–8), Lily-flowered9) and Single late (18–20) cultivars located in the lower part of thelot are late season flowers. Cultivars in the Triumph group were
cattering in the centre of the plot, in accordance with being middleeason flowering cultivars. Results of AMOVA (Table 4) showed thathere was significant variation between Early flowering and Lateowering groups (FST = 0.072, P = 0.02).
able 4nalysis of molecular variance (AMOVA): (a) for three clusters of the 72 tulips obtained fr
Source of variation df Sum of squares
(a)Among clusters 2 262.091
Within clusters 141 1887.7
Total 143 2149.792
Fixation index: FST: 0.208 (P < 0.0001)
(b)Among groups 1 24.145
Within groups 38 380.505
Total 39 404.65
Fixation index FST: 0.072 (P = 0.02)
urae 161 (2013) 286–292
The structure of genetic differentiation among cultivar groupsand the ancestry of individuals were assessed using SNP data by theprogram STRUCTURE. The L (K) value reached its maximum at K = 2and the variance of L (K) at larger K were increasing. The maximum�K also presented at K = 2, suggesting that there are two distinctclusters in the 72 accessions. One cluster was the T. gesneriana cul-tivars, the other one was T. fosteriana. In the barplot (Fig. 3), 49T. gesneriana cultivars in 8 cultivar groups (Triumph, Single early,Single late, Double early, Double late, Fringed, Lily-flowered andParrot) were assigned to one cluster (red). T. fosteriana cultivarswere assigned to the other cluster (green), while GF hybrids, ‘Puris-sima’ and ‘Flair’ showed almost equal percentages of admixture toboth clusters (red and green). Thus the genetic background of GFhybrids was visualized directly and their hybrid origin was clearlyshown. Therefore, SNP markers used in this study can be used foridentification of interspecific hybrids of T. gesneriana and T. foste-riana. Subsequent STRUCTURE analysis showed no within-groupsubstructure in the T. gesneriana group as could be expected.
4. Discussion
4.1. SNP markers
Single nucleotide polymorphisms are the most abundant type ofpolymorphism in organisms’ genomes. Recent studies have shownthat SNPs can perform as highly informative markers in investigat-ing genetic relationships among cultivars and natural populations(Ebana et al., 2010; Jiang et al., 2010; Lao et al., 2006; McCallumet al., 2008; Paschou et al., 2007). All SNP loci used in this studywere developed from expressed sequence tags (ESTs), which is anuseful method to identify SNPs in non-model plants (Shahin et al.,2012). A total of 316 SNP markers were identified from cultivars‘Kees Nelis’ and ‘Cantata’ representing T. gesneriana and T. fosteri-ana, respectively. Due to the origin of markers, SNPs which weredeveloped from T. gesneriana ‘Kees Nelis’ (KN SNPs) were mainlypolymorphic in T. gesneriana cultivars and have limited power indifferentiating T. fosteriana tulips. Similarly, CA SNPs which comefrom T. fosteriana ‘Cantata’ have limited power in differentiating T.gesneriana cultivars. Both KN SNPs and CA SNPs are polymorphicin GF hybrids which make it useful for hybrids identification.
The informativeness of markers is very important for efficientestablishment of genetic relationships among individuals. It hasbeen suggested that the statistical power of markers for individ-ual identification and population structure studies is closely linkedto allele frequency and SNPs with higher minor allele frequencies(MAF) are preferred (Krawczak, 1999; Morin et al., 2004; Rymanet al., 2006). Mean MAF value of KN SNPs in T. gesneriana cultivarswas 0.21, while mean MAF of CA SNPs in T. fosteriana was 0.23. The
SNP dataset used in this study has a similar level of informativenesswith crops such as rice (MAF = 0.27, Chen et al., 2011) and grapevine(MAF = 0.24) (Lijavetzky et al., 2007), but lower than commonbean (MAF = 0.36) (Gupta et al., 2012). To increase the power of
om PCoA; (b) for early flowering and late flowering group of T. gesneriana cultivars.
Variance components Percentage of variance
3.52093 Va 20.813.38795 Vb 79.216.90887
0.77644 Va 7.210.01330 Vb 92.810.78974
N. Tang et al. / Scientia Horticulturae 161 (2013) 286–292 291
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the analysis, for each individual its admixture for these two groupsis quantified. Therefore, the third group (GF hybrids) which wasseparated in PCoA was identified as admixed individuals instead
Table 5Genetic diversity of T. gesneriana cultivars at different periods.
ig. 3. Inferred genetic structure of tulips based on posterior probability by STRUCT, Fringed; 4, Lily-flowered; 5, Parrot; 6, Single early; 7, Single late; 8, Triumph; 9, Fdark) shows background from T. gesneriana tulips. Green (light) shows background
iscriminating hybrid individuals and to minimize possible false-ositives, KN SNPs and CA SNPs were combined into one dataset.oncerning about genotyping accuracy, SNPs with low MAF (<0.10)ere removed from further analysis.
.2. Genetic differentiation and diversity
The results of PCoA and STRUCTURE analyses showed that the2 tulip accessions were clearly divided into 3 clusters. FST valuef 0.21 (P < 0.0001) verified the significant large genetic varia-ion among the 3 clusters (Wright, 1978), indicating that the SNPataset is efficient in distinguishing tulip species T. gesnerianand T. fosteriana. However, the genetic distances among cultivarroups of T. gesneriana (Single early, Single late, Double early, Dou-le late, Fringed, Lily-flowered, Parrot and Triumph) were rather
ow and without significant differentiation. Classification of tulipultivars were discussed previously and lack of clear grouping ofccessions according to cultivar groups were noticed previouslyVan Raamsdonk and DeVries, 1996). Selection for common traitsn forcing might be responsible for this lack of differentiationmong these widely grown tulip cultivars. It was reported thatulip cultivars can be divided into Early, Middle and Late flower-ng types (Van Raamsdonk and Wijnker, 2000). In accordance theresent study showed that Early flowering cultivars (Double early,ingle early) were genetically clearly separated from Late flower-ng cultivars (Double late, Single late, Fringed and Lily-flowered)FST = 0.072, P = 0.02). This significant division shows the two groupsave formed separate breeding pools with limited interaction.
Tulips have a long and fabled breeding history. In theetherlands, the first tulip was planted in the Botanical Garden
n Leiden in 1593 and the first cultivation was established in Southf Haarlem in 1600. Although limited numbers of cultivars werencluded in this study, it was noticed that T. gesneriana cultivarsntroduced after 1960 roughly grouped in the centre of the PCoAlot. Older cultivars (before 1960) were found to be scattered acrosshe whole plot and especially along the edges. Concerning thehanges in levels of genetic diversity over time, heterozygosity ofroups of T. gesneriana cultivars at different periods (before 1940,940–1960, 1960–1980, 1980–2000, after 2000) were calculatedTable 5). It was found that genetic diversity of cultivars introducedefore 1940 was lower than others and diversity was increasingntil 1960. During the twenty years from 1940 to 1960, the diver-ity of tulip cultivars was higher than that of cultivars before 1940Mann Whitney U tests at p 0.05). This might because tulips were
ore commonly used as garden flowers before 1940. However,fter the World War II, tulip cut flower industry developed rapidly
nd new groups of tulips such as Darwin hybrids emerged, tradef bulbs widened and crossings diversified. The leading goal ofost tulip breeders shifted from selection based on ornamental
raits to cut-flower production with forcing ability. The acreage of
umbers below the figure represent cultivar groups. 1, Double early; 2, Double late;iana; 10, GF hybrids. Different colours represent different genetic background. Red
T. fosteriana tulips. P, Purissima; F, Flair.
cultivars with good forcing ability was increasing obviously, whilesome cultivars become less important due to poor forcing abil-ity (Van Tuyl et al., 2012). Continuous selection among crosses ofgenetically related cultivars could narrow the genetic base againand may lead to the reduction of genetic diversity found in recentcultivars. The disadvantage of intensive selection on forcing traitsin tulips is that other traits (e.g. resistance to diseases or abioticstress) in modern cultivars might have been lost in the process.
Genetic diversity of tulip cultivar groups was described pre-viously based on morphological characters such as first date offlowering, height of flower, number of tepals, presence/absenceof blotch, length and width of tepals and pollen colour (VanRaamsdonk and DeVries, 1996; Van Raamsdonk and Wijnker,2000). This study is the first to assess the genetic diversity of abroad set of tulip cultivars using SNP markers. The level of geneticdiversity that we found in the studied species is similar with Liliumtsingtauense (He = 0.23) (Baldwin et al., 2012) and lower than Heobserved in onion (0.30) (Guo et al., 2011). The heterozygosity ofGF hybrids (Ho = 0.43) is higher than that of the other two groupsas expected. Due to the small sample size of T. fosteriana that couldbe analysed, we cannot compare the genetic diversity of T. foste-riana and T. gesneriana since sample size is an important factor ininfluencing genetic diversity. Considering cultivar groups within T.gesneriana, heterozygosity in Triumph group was higher than inother groups. Records show that Triumph tulips were raised fromcrosses of Single early tulips with Cottage, Old Darwin and Dutchbreeder tulips (Van Raamsdonk and DeVries, 1996). This could bethe reason of the relatively high heterozygosity in Triumph group.
Bayesian inference is an effective method to analyse human dis-turbed materials (e.g. cultivars) because the assemblage of thesematerials cannot be strictly regarded as biological populations.One important application of the Bayesian model based soft-ware STRUCTURE is to identify distinct genetic groups, admixedindividuals and present the ancestry of individuals. In this study,two distinct groups (T. gesneriana and T. fosteriana) were found,showing the clear separation of the two genomes involved. During
1980–2000 17 0.47 0.37After 2000 10 0.44 0.37
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f a distinct new group. Apart from the GF hybrid breeding lines,ultivar ‘Purissima’ and ‘Flair’ were also identified as admixed indi-iduals. For a long time, ‘Purissima’ was classified as a member ofosteriana group (Van Scheepen, 1996). In this study, based on SNParkers, ‘Purissima’ was identified as a GF hybrid. This result agreesith the study of Marasek and Okazaki (2008) in which ‘Purissima’as found as an interspecific hybrid of T. gesneriana and T. foste-
iana by southern hybridization and genomic in situ hybridizationGISH). Cultivar ‘Flair’ was supposed to be a Single early cultivarelonging to T. gesneriana, which is also found to be a GF hybrid
n this study. Both ‘Purissima’ and ‘Flair’ clustered together withF breeding lines in the PCoA plot. This means that SNP markerssed in this study are efficient in identifying hybrids of T. gesnerianand T. fosteriana and thus can be used to screen tulip cultivars forybrids that have not been identified previously.
In conclusion, the selected 121 SNP markers are efficient in eval-ation of genetic diversity, and inference of genetic relationshipsnd hybrid distinction in tulips. Genetic differentiation among T.esneriana cultivars was quite low. It is concluded that only someeparation is appearing among cultivar groups due to floweringime and breeding of tulip is using material from among cultivarroups. Given that T. fosteriana is marked as donor for introducingulip Breaking Virus resistance into T. gesneriana, the set of SNParkers used are well suited for recurrent parent selection.
cknowledgements
We are thankful for the support from the Foundation Techno-ogical Top Institute Green Genetics (TTI-GG), Land-en Tuinbouwrganisatie (LTO) Noord Fondsen and ECO Tulips BV.
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