Development of stem- and leaf rust resistant near-isogenic lines of spring triticale TOBIE and...

8/9/2019 Development of stem- and leaf rust resistant near-isogenic lines of spring triticale TOBIE and advanced line 98T376-

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UNIVERSITY OF STELLENBOSCH

DEPARTMENT OF GENETICS

Plant Breeding Laboratory (SU-PBL)

Honours Project Final Article

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Student:Yuriy Tsupko [US#14997665]

Lecturers: W. Botes / G.F. Marais / K. Pakendorf

Date: 20 October 2006


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Abstract

In triticale (Triticosecale spp.) the most widespread diseases are now those caused by wind-borne

obligate pathogens, such as rusts ( Puccinia spp.). During the 2005 season new pathotypes of leaf

rust (race UVPt19) and stem rust (race UVPgt57) appeared on triticale in the Western and Southern

Cape regions of South Africa. A population of first backcross (BC1) and doubled haploid (DH) lines

containing a source of rust resistance into an existing cultivar Tobie and an advanced breeders line

98T376-A-A-3 were established. The wide hybridization method of DH creation was evaluated on

local triticale material in comparison with near-isogenic lines (NILs) creation. Obtained rust-

resistant lines could be used in subsequent selection and testing as new advanced lines. They also

can be used as possible sources of resistance in a pre-breeding programme. Segregation analysis of

F1 showed that resistance to leaf rust race UVPt19 in the donor line 95T159-A-A-3-PL1 is

controlled by a dominant gene(s), and resistance to stem rust race UVPgt57 controlled by a

recessive gene(s).

Keywords: triticale, stem rust, leaf rust,Puccinia, doubled haploids.

Introduction

In triticale (Triticosecale spp.), as in other cereals such as wheat (Triticum aestivum L.) or

barley ( Hordeum vulgare L.), the most widespread diseases are now those caused by wind-borne

obligate pathogens, such as rusts (Puccinia spp.). Globally rust diseases of small grain cereals cause

significant economic losses every year by reducing grain yield and quality. Leaf rust can cause yield

losses of up to 20-40% (SOLODUKHINA, 1997; KOVALENKO et al., 2004) and stem rust up to

50-80% (LEONARD & SZABO, 2005). Since the establishment of triticale it had been considered

relatively resistant to rusts (SCHLEGEL, 1996). However, due to the increase in cultivation of

triticale the past two decades pathogens had a better chance adaptating to the crop (SCHINKEL,

2002), therefore of great significance is research committed to plant protection, and in particular to

breeding for resistance against this up and coming biotic threats.

A long-term breeding strategy must put emphasis in the reduction of crop losses and the

lowering of pathogen inoculum levels, which could be done by the breeding and deployment of

resistant cultivars (KOLMER, 2005). Such strategies are primarily aimed at achieving durability of

resistance combined with the deployment of genotypic diversity to buffer potential losses ofresistance (OELKE & KOLMER, 2004). Development of genetic resistance to rust is the most


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efficient, cost-effective and environmentally-friendly approach to prevent the losses caused by rust

epiphytoties. Breeding small grain crops for rust resistance is, however not like breeding for better

grain quality, higher yield, or even tolerance to physical stresses such as drought or heat. Breeding

gains made in quality and abiotic traits usually remains effective for as long as a variety is grown.

This is not true for gains against biotic stresses, i.e. rust resistance (USDA-ARS, 2005). Resistance

built into a crop variety over a number of years of breeding can be negated by a shift in pathogenic

races in the rust fungus population, which is exactly the current problem in triticale breeding

programmes (W.C. BOTES & H.S. ROUX, 2006, SU-PBL, personal communication).

The cereal breeders work is therefore never-ending, because new rust races can and do arise.

Usually breeders try to identify a source of resistance and to transfer that resistance to locally

adapted germplasm by applying such techniques as backcrosses, resistance tests, and molecular

marker-assisted selection.Since the early 1970s the Department of Genetics has been instrumental in developing

superior triticale cultivars for use within the Winter Rainfall Region of South Africa. Stellenbosch

University (SU) is the only institution in South Africa with a triticale programme of this extent. The

breeding programme main aims include: improving overall yield, grain quality, protein content and

disease resistance of triticale grown as a feed grain, and to select lines that can be used for grazing,

hay and/or silage.

During the 2005 season new pathotypes of leaf rust ( Puccinia triticina Eriks., race UVPt19)

and stem rust ( Puccinia graminis f. sp. tritici, race UVPgt57) appeared on triticale in the Western

and Southern Cape provinces of the Republic of South Africa (L.E. SNYMAN, 2006, SU-PBL,

personal communication). These proved to be virulent on many of the advanced breeding lines and

necessitated the urgent identification of diverse and alternative resistance genes, and their

deployment in the crossing block. Furthermore, the successful variety Tobie has become susceptible

to both leaf and stem rust pathotypes, whereas a very promising advanced breeding line, 98T376-A-

A-3 (pedigrees are presented in Table 1), has become susceptible to stem rust (H.S. ROUX & W.C.

BOTES, 2006, SU-PBL, personal communication). As a short-term response to the new threat it

was decided to use these two genotypes in a backcross breeding programme. The primary aim is to

improve their resistance by introducing gene(s) of hypersensitive response to rusts from resistant

breeding line 95T159-A-A-3-PL1. As a secondary objective, and longer term strategy it is

necessary to acquire and utilize as many diverse sources of resistance (from international

programmes) as possible in a pre-breeding effort, with different types of resistance (hypersensitive

and durable and their combination) controlled by diverse pool of genes. This makes it necessary to

identify a core group of local varieties/advanced lines with superior breeding value that can be used

in the initial crosses with the introductions and then in subsequent backcrosses.


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Material and Methods

The research consisted of three parts: creation of a DH population, backcrosses and creation of

NILs, and a segregation analysis of the identified resistance.

During the course of this research the following cultivars and lines of triticale were used: the

South African spring triticale segregating lines (F2) 05T32 and 05T92, cultivar Tobie and advanced

line 98T376-A-A-3. The pedigrees of these lines and some of their ancestors are given in Table 1. A

half of the available plant material of 05T32 and 05T92 was involved in backcrossing, and another

half was used in DH production (Table 3). Maize ( Zea mays L.) sweetcorn line HTB08173

(imported by HYGROTECH) was used as pollinator for the production of doubled haploids.

Testing for leaf and stem rust resistance was carried out at seedling stage (about 2 weeks after

planting) for all F2 seedlings of segregating lines 05T32 and 05T92, subsequent BC1 lines, and for

segregation analysis test in F1 (Table 4). Plants were inoculated with the virulent fungal races found

in the Western Cape: UVPgt57for stem rust and UVPt19 for leaf rust.

Rust spores were supplied by Ms. L.E. SNYMAN (SU-PBL). Rust spores were collected in a

growth chamber from susceptible cultivars Bacchus for leaf rust and Tobie for stem rust. Spores

were collected to a plastic cup with distilled water, surface-active agent was added and plants weresprayed thoroughly. Inoculated pots covered with wet plastic bags and left in a darkroom for 24

hours at 20C, brought to a growth chamber and the plastic bags removed. The plants were left in a

growth room for a 7-10 day period, at 25/20C day/night, for rust to develop. The temperature range

was chosen as a compromise between optimum temperatures for stem (day 25-30C, night 15-20C;

germination optimum 18C) and leaf (day 20-25C, night 15-20C; germination optimum 15-20C)

rusts development because of combined inoculation. After complete development of the pustules

plants were scored according to the scale (Table 2). Susceptible F2 and BC1 plants were removed;

resistant and partially resistant plants were replanted and used in DH production and first

backcrossing.

Creation of the DH populations. Maize was planted twice per week (3 seeds per pot) in a growth

chamber with controlled environment (CONVIRON PGV36) in order to have enough pollen for DH

production during all the period of research. Seeds were planted 15 mm deep in plastic pots (height

150 mm, diameter 150 mm) with coarse river sand. In the CONVIRON PGV36 combination of

sodium and lithium lamps are used with 16/8 h day/night light regime. Temperature is controlled

between 23/25C, but not relative humidity.


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Breeding lines 05T32 and 05T92 were obtained in 2005 from crosses between Tobie, 98T376-

A-A-3 and 95T159-A-A-3-PL1 (see pedigrees in Table 1). F1 seeds from both lines were planted in

order to obtain a F2 segregating population for rust resistance testing. The seedlings were scored and

resistant plants held back for use in haploid production. The selected resistant seedlings were

planted in pots (height 150 mm, diameter 150 mm) with coarse river sand and placed in a growth

chamber (CONVIRON E7H) with elevated growing temperatures (25/20C day/night) and long

light regime (16/8 h day/night). As soon as tillers started to develop they were moved to an outside

glasshouse in order to facilitate better flower development.

Spikelets were subsequently emasculated and pollinated with pollen from maize line

HTB08173 and this was followed by an application of growth regulators some 30 h later to induce

embryo formation (by injection into a spike). The following growth regulator combination was

applied: 50 mg 2,4-D + 100 mg GA3 /litre.Some 16-20 days after pollination, spikelets were harvested and embryos cultured on modified

MS medium containing 165 mg NH4NO3 + 20 g sucrose + 100 mg inositol + 0.4 mg t hiaminHCl

/litre.

For chromosome doubling, the roots of haploid plants were soaked in an aqueous solution of

0.05% colchicine and subsequently re-potted after rinsing. Nearly all colchicine-treated plants are

expected to produce a few spikes with fertile DH sectors which, after selfing, will give rise to

homozygous DH lines (PIENAAR, HORN & LESCH, 1997).

The development of the NILs. For the backcrosses with Tobie and 98T376-A-A-3, F2s of the

segregating lines 05T32 and 05T92 were planted in three portions, 2-3 weeks apart from each other

in a growth chamber CONVIRON E7H. For pots in the CONVIRON E7H mixture of of sand and

of peat mix was used. Plants were propagated using hand watering with a nutrient feeding

solution for hydroponics. Using the growth chamber CONVIRON E7H, generations of segregating

lines were forced making use of elevated temperatures (25/20C day/night) and long light regime

(16/8 h day/night). A combination of 8 fluorescent and 4 incandescent lamps are used. Humidity is

uncontrolled. When plants in the CONVIRON reached tube developmental stage they were moved

to the greenhouse for emasculation and subsequent pollination with their recurrent parents.

Recurrent parents Tobie and 98T376-A-A-3 were planted twice per week (4 seeds of each in

separate pots) in the greenhouse in order to have enough pollen for making backcrosses. Seeds were

planted 10 mm deep in plastic pots (height 150 mm, diameter 150 mm) with coarse river sand.

Plants were propagated using hydroponics. Watering was applied 1-3 times per day, 30 ml/pot,

depending on the development stage and environmental conditions in the greenhouse. Plants were


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grown under natural light conditions. A chimney cooling system was used in order to keep the

temperature not higher than +25C. Plants were sprayed against aphids, thrips and powdery mildew.

The following crosses were performed for NILs production (BC1):

05T32(F2) Tobie => 95T159-A-A-3-PL1/2*TOBIE

05T92(F2) 98T376-A-A-3 => 98T376-A-A-3*2/95T159-A-A-3-PL1

Segregation analysis. F1 from crosses between resistant 95T159-A-A-3-PL1 and susceptible Tobie

and 98T376-A-A-3 was studied in an attempt to determine whether the resistance in the donor line

95T159-A-A-3-PL1 is based on a single gene, dominant or recessive.

The following crosses were performed for segregation analysis:

95T159-A-A-3-PL1 Tobie => identical to 05T32

Tobie

95T159-A-A-3-PL1 => 05T32 98T376-A-A-3 95T159-A-A-3-PL1 => identical to 05T92

95T159-A-A-3-PL1 98T376-A-A-3 => 05T92

Test for resistance to stem and leaf rusts were performed at the seedling stage (about 2 weeks

after planting) in F1 (Table 4). Plants were scored for rust resistance according to Table 2.

Results and Discussion

Creation of a DH population. In order to create population of DH lines total number of 30 heads of

05T32 and 23 heads of 05T92 lines were emasculated, pollinated with maize and injected with

hormones. Immature seeds were examined under a microscope for embryo rescue (Figure 1). Four

haploid embryos were rescued, but only one of them developed both sprout and roots. Obtained

plant was propagated till growth stage 29 on the Zadoks scale (ZADOKS, CHANG & KONZAK,

1974) and treated with colchicine for chromosome doubling (Figure 2).

Such a discouraging result with haploid creation in triticale through pollination with maize

suggests that the protocol used for this purpose, initially developed for wheat (PIENAAR, HORN &

LESCH, 1997), has to be modified in order to be successfully implemented in triticale breeding.

Most likely the problem in triticale recalcitrance lies in incompatibility of tested genotypes with

used maize genotype, and/or timing of pollination/hormones injection. Due to the limited time span

of this project it was however not possible to explore such options.

Backcrosses and creation of NILs. In order to create near-isogenic lines of Tobie and 98T376-A-A-

3, resistant to leaf and stem rust, F2 plants obtained from their crosses with resistant line 95T159-A-


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A-3-PL1 were tested for rust resistance (Figure 3). Plants resistant to both pathogens were selected

and were used for backcrossing. In total 108 heads of 05T32 and 97 heads of 05T92 F2 lines were

crossed with their susceptible recurrent parents (Table 3). Obtained BC1 lines will be used in

consequent backcrossing as well as in field testing, during the latter part of 2006, as advanced

breeding lines resistant to rust pathogens.

Segregation analysis. A second, and almost more important, objective of the project is a

preliminary study of the resistance sources by means of a segregation analysis which can give the

breeding programme a better understanding of the real value of the resistance source that we are

using in this project (W.C. BOTES, 2006, SU-PBL, personal communication). F1 from crosses

between resistant 95T159-A-A-3-PL1 and susceptible Tobie and 98T376-A-A-3 were studied in an

attempt to determine whether the resistance in the donor line 95T159-A-A-3-PL1 is based on asingle gene, dominant or recessive.

According to the results obtained (Table 4), in all combinations all of the F1 plants inoculated

with leaf rust race UVPt19 shows highly resistant hypersensitive reaction (;). This can be explained

by presence of a dominant gene(s) for leaf rust resistance to this specific race in the donor line. The

exact number of resistance genes involved and their behaviour in new germplasm has to be further

investigated in F2.

In a contrast, all plants inoculated with stem rust race UVPgt57appear to be susceptible to the

pathogen (chlorotic susceptible reaction 3c). It can be concluded that no dominant gene of

resistance to the stem rust pathogen is involved and supposedly resistance to the pathogen in the

donor line is controlled by a recessive gene(s), which has to be also studied in F2 generation.

Identification of gene(s) of resistance and determination of the components of resistance, i.e.

their phenotypic and genotypic characteristics will give a basis for decisions regarding advisability

of, and the optimal strategy for, their further commercial exploitation, taking in account the

perceived risks of resistance breakdown (McINTOSH et al., 1995).

Conclusions

The aims of this project dictated that we establish a population of NILs and a DH population

containing a source of rust resistance into an existing cultivar Tobie and a very promising advanced

breeders line 98T376-A-A-3 (W.C. BOTES & H.S. ROUX, 2006, SU-PBL, personal

communication). Due to time constraints this was not achievable by the end of October 2006. The

wide hybridization method of DH creation was evaluated on local triticale material in comparison


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with NILs creation. Obtained rust-resistant BC1 lines of Tobie and 98T376-A-A-3, as well as DH

true-breeding line could be used in subsequent selection and testing as new advanced lines. These

rust-resistant advanced lines also can be used as possible sources of resistance in a pre-breeding

programme.

Segregation analysis of F1 showed that resistance to leaf rust race UVPt19 in the donor line

95T159-A-A-3-PL1 is controlled by a dominant gene(s), and resistance to stem rust race UVPgt57

controlled by a recessive gene(s).

Acknowledgements

I wish to thank the Stellenbosch University Plant Breeding Laboratory, Department of Genetics, for

a scholarship. Personal thanks to Willem C. Botes for constant support, open-mindness and

patience; to Herman S. Roux and Lisle E. Snyman for helpful advices and rust supply; to Stanley

Pretorius, Charles Toutie, Andr Julius and Samuel Pietersen for casual help and friendly attitude.


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References

KOLMER, J.A., 2005. Tracking wheat rust on a continental scale. Cur. Op. in Pl. Bio. 8, 441449.

KOVALENKO, E.D., KISELEVA, M.I., SOLOMATIN, D.A., ZHEMCHUZHINA, A.I. &

LAPOCHKINA, I.F., 2004. The main parameters of durable resistance to leaf rust in wheat.

Cereal Rusts and Powdery Mildews Bulletin. [cited 27 February 2006]. Available from World

Wide Web:

LEONARD, K.J. & SZABO, L.J., 2005. Stem rust of small grains and grasses caused byPuccinia

graminis. Mol. Pl. Path. 6(2), 99-111.

McINTOSH, R.A., WELLINGS, C.R. & PARK, R.F., 1995. Wheat rusts. An atlas of resistance

genes. Australia: CSIRO.

OELKE, L.M. & KOLMER, J.A., 2004. Characterization of leaf rust resistance in hard red spring

wheat cultivars.Plant Disease. 88, 1127-1133.

PIENAAR, R.V.DE, HORN, M. & LESCH, A.J.G., 1997. A reliable protocol for doubled haploid

accelerated wheat breeding. Wheat Information Service. 85, 49-51.

SCHINKEL, B., 2002. Triticalestill a healthy crop?Proc. 5th Int. Triticale Symp., June 30-July 5,

2002, Radzikow, Poland. 1, 8188.

SCHLEGEL, R., 1996. Triticaletoday and tomorrow. In: Guedes-Pinto, H., Darvey, N. &

Carnide, V.P. (eds.). Triticale: Today and Tomorrow. Kluwer Academic Publishers,

Netherlands. 21-31.

SOLODUKHINA, O., 1997. Genetics and identification of rust resistance in rye.J. Appl. Genet.

38B, 111-116.

SU-PBL., 2004. Annual report. Stellenbosch University, Plant Breeding Laboratory.

USDA-ARS., 2005. Cereal rusts. [cited 22 February 2006]. Available from World Wide Web:

ZADOKS, J.C., CHANG, T.T. & KONZAK C.F., 1974. A decimal code for the growth stages of

cereals. Weed Research. 14, 415-421.


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Table 1. Cultivars and advanced breeding lines involved in NILs and DH

creation, and some of their ancestors

(SU-PBL, 2004; H.S. ROUX, 2006, SU-PBL, personal communication).

Line / Cultivar Pedigree

05T32 95T159-A-A-3-PL1/TOBIE

05T92 98T376-A-A-3/95T159-A-A-3-PL1

TOBIE W.TCL 83/HOHI//RHINO 4/3/ARDI 1/4/KIEWIET

98T376-A-A-3 150.83/4/FABA/DWF RYE GOOD

SEED//DGO_4/3/BAER_1/5/LT1478.82/FARAS_1//NIMIR

95T159-A-A-3-PL1

(resistant to stem andleaf rust)

IBIS/7/HARE 212/3/CHAMPLAIN/ARONDE

68//VPM/MOISSON/4/JUANILLO 100/5/ANDASS/6/DURUMWHEAT/BALBO//BOKS/3/ANDALASS//TJ/BGLS

KIEWIET D7069//P1243741/SPY/3/ANZA/P1243741/6/CIT/UC90 C3

TRIPLE DWF/5/TOB/8156//CC/3/INIA/4SPY

IBIS FLORIDA 201/3/DURUM WHEAT/BALBO//BOKS


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Table 2. Major infection type classes for stem rust and leaf rust

(McINTOSH, WELLINGS & PARK, 1995).

Infectiontype*

Host response Symptoms

0 Immune No visible uredia

; Very resistant Hypersensitive flecks

1 Resistant Small uredia with necrosis

2 Resistant to moderately

resistant

Small to medium sized uredia with green islands

and surrounded by necrosis or chlorosis

3 Moderately resistant /

moderately susceptible

Medium sized uredia with or without chlorosis

4 Susceptible Large uredia without chlorosis

X Resistant Heterogeneous uredia, similarly distributed over

the leaves

Y ? Variable size with larger uredia towards the tip

Z ? Variable size with larger uredia towards the leaf

base

* Variations are indicated by the use of

(less than average for the class) and+

(more), as well as C

and N to indicate more than usual degrees of chlorosis and necrosis. Heterogeneity on a leaf not

adequately described with X, Y or Z may be written as a sequence, for example 12. A comma is

used to indicate heterogeneity between plants in a single test, for example 1+,X.


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Table 3. Average seed set and total number of heads used for the first backcross (BC1), creation of haploids (H) and

segregation analysis (SA).Line 05T32 Line 05T92

SA Totalx Tobie (BC1) x maize (H) Sub-total x 98T376 (BC1) x maize (H) Sub-total

Heads 108 30 138 97 23 120 22 280

incl. spikelets emasculated 3326 940 4266 3088 670 3758 638 8662

Seeds obtained 2505 n/a n/a 2199 n/a n/a 473 5177

Avg. seed set 75.3% n/a n/a 71.2% n/a n/a 74.1% 73.4%

Avg. spikelets/head 30.8 32.4 31.1 31.8 31.9 31.8 29.0 31.3Avg. seeds/head 23.2 n/a n/a 22.7 n/a n/a 21.5 22.8


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List of figures

Figure 1. Undeveloped seeds of triticale plants pollinated with maize, 2 weeks after pollination.

Figure 2. Chromosomes of a haploid triticale plant (1n=3x=21, line 05T92, F2).

Figure 3. Stem and leaf rust on plants inoculated in seedling stage.

Figure 1. Undeveloped seeds of triticale plants pollinated with maize, 2 weeks

after pollination.

Figure 2. Chromosomes of a haploid triticale plant (1n=3x=21, line 05T92, F2).


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Figure 3. Stem (left) and leaf (right) rust on BC1 plants inoculated in seedling

stage.


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Addendum

Table 1. The recipe of the tissue culture medium preparation.

Final solution 1000 ml(1) Weight sucrose and dissolve it in dH2O.

(2) Add BAC1, BAC2, BAC3, AV4, V1 and V4 (adjust in volumetricflack to 1000 ml).

(3) Adjust the pH to 5.8 (by adding NaOH or HCl).

(4) Add the Agar and the solution from the flack in a Scott bottle.

(5) Shake this vigorously and pour into tissue culture bottles (about 40 ml

per bottle).

(6) Fix tops (but do not fasten too tightly) and autoclave (wet).

BAC1

BAC2

BAC3

AV4

V1

V4

Sucrose

Agar

100 ml

10 ml

1 ml

10 ml

10 ml

4 ml

20 g

8 g

BAC1

(powder of each and dissolve into the final solution)BAC2

(powder of each and dissolve into the final solution)

KNO3

NH4NO3

KH2PO4

MgSO4 7H2O

CaCl2 2H2O

500 ml

9.500 g

0.825 g

0.850 g

1.850 g

2.200 g

MnSO4 4H2O

ZnSO4 7H2O

H3BO3

500 ml

0.825 g

0.430 g

0.310 g

BAC3

(powder of each and dissolve into the final solution)AV4

(heat to dissolve and stabilize)

KI

CuSO4 5H2O

Na2MoO4 2H2O

500 ml

0.4150 g

0.0125 g

0.1250 g

Na2EDTA

FeSO4 7H2O

200 ml

0.746 g

0.556 g

V1 V4

Inositol

100 ml

1 g Tiamin HCl

100 ml

0.01 g


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Table 2. Rust resistance testing of F2 of lines 05T32 and 05T92.

F2 (SSD lines)

Inoculation A Inoculation B Inoculation C

SusceptibleSelected clean

plants ( ; )Susceptible

Partially

resistant

Resistant

SusceptibleSelected clean

plants ( ; )Incl. selected cleanplants ( ; )

05T32-1 10 0 7 0 7 2 15 0

05T32-2 10 0 4 6 5 2 19 0

05T32-3 10 0 2 3 10 2 13 0

05T32-4 10 0 7 3 10 2 4 0

05T32-5 8 1 5 6 15 2 10 0

05T32-6 9 1 2 8 17 2 12 0

05T32-7 8 2 10 0 9 2 3 4

05T32-8 10 0 12 0 12 2 8 2

05T32-9 9 1 14 0 9 2 4 1

05T32-10 9 1 6 6 15 2 4 3

05T32-11 10 0 6 7 13 2 0 1

05T32-12 10 0 7 3 6 2 0 0

05T32-13 9 1 7 8 17 2 2 0

05T32-14 9 1 14 7 14 2 7 0

05T92-2 9 1 8 0 14 2 2 4

05T92-3 9 1 6 4 16 2 11 3

05T92-4 8 2 9 2 12 2 11 0

05T92-5 8 2 12 2 12 2 20 5

05T92-6 10 0 8 7 16 2 8 0

05T92-7 7 3 9 3 14 2 5 0

05T92-8 6 4 5 1 17 2 8 2

05T92-9 5 5 5 0 16 2 6 405T92-10 5 5 6 4 14 2 9 3


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Table 4. Rust resistance testing of BC1 of lines 05T32 and 05T92.

Line no.

(BC1)

No. of

plantsHead no.

No. of

spikelets

emasculated

No. of seeds

Leaf rust Stem rustIndistinguish-

able rust (mix)

Selected clean

plants ( ; )Infection

type

No. of

plantsInfection type No. of plants

05T32-1-B 2

3 34 31 3-4 12 3-4 16 2

4 30 30 3-4 18 3-4 10 2

5 30 18 3-4 9 3-4 8

6 30 19 4 7 3-4 7

05T32-2-B 2

2 30 20 ? ? ? ? [1-4] 14

3 36 26 4 12 3-4 12

4 28 26 ? ? ? ? [3-4] 194 36 14 3-4 5 2-4 6

05T32-3-B 2

1 32 20 3-4 9 3-4 10 1

3 32 28 4 10 3-4 11

4 32 22 3-4 6 2-3n 2 [3-4n] 9

5 28 18 3-4 6 1-4 10 1

6 36 29 3-4 19 3c 9

05T32-4-B 2

2 32 20 3-4 6 2-4 14

3 22 14 3-4 4 2-4 8 1

4 30 23 3-4 10 3-4 13

5 28 20 ? ? ? ? [3-4c] 20

6 30 21 4 13 3-4 7

05T32-5-A 1

2 30 17 3-4 7 3-4n, 2c 5, 5

3 28 22 3-4 14 3-4 4

5 28 22 3-4 7 2-4 15

05T32-5-B 2

3 32 31 ? ? ? ? [3-4] 31

4 32 27 ? ? ? ? [3-4c] 235 34 23 ? ? ? ? [3-4] 15

6 30 26 ? ? ? ? [3-4] 25

7 30 29 ? ? ? ? [3-4c] 28


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Line no.

(BC1)

No. of

plantsHead no.

No. of

spikelets

emasculated

No. of seeds


able rust (mix)

Selected clean


typeNo. ofplants

Infection type No. of plants

05T32-6-A 1

1 34 24 3-4 13 2-4 9 1

2 32 17 N/A N/A N/A N/A

3 32 29 3-4 15 3-4 13

4 30 26 3-4 12 1-4 14

05T32-6-B 2

2 34 19 ? ? ? ? [3-4c] 16

3 32 25 3-4 10 3-4 12

4 32 29 0 0 1-4n,c 26 1

5 32 29 ? ? ? ? [3-4] 28

6 28 24 ? ? ? ? [3-4n] 207 30 22 ? ? ? ? [3-4] 22

05T32-7-A 22 28 21 4 1 3-4 18

3 36 19 4 6 3-4 11 1

05T32-7-B 2

1 32 17 4 11 ? ? [3-4] 5 1

2 30 18 ? ? ? ? [3-4] 12

3 30 23 4 9 3-4 11

4 32 24 3-4 7 2-4 12

5 30 22 ? ? ? ? [2-3c,n] 20 1

05T32-7-C 4

1 26 21 4 9 3-4 11

2 22 19 3-4 16 3-4 2

3 30 29 ? ? ? ? [3-4] 25

4 32 30 ? ? ? ? [3-4] 26

05T32-8-B 2

2 28 1 ? ? ? ? [1n] 1

3 26 21 4 10 3-4 11

4 30 24 4 12 1-4n,c 5 [1-3n,c] 5

05T32-8-C 21 30 27 ? ? ? ? [3-4c] 242 26 21 4 10 3-4 8

3 28 22 ? ? ? ? [2-4] 22


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20

Line no.

(BC1)

No. of

plantsHead no.

No. of

spikelets

emasculated

No. of seeds


able rust (mix)

Selected clean


typeNo. ofplants


05T32-9-A 1

1 30 12 4 3 3-4 9

2 28 20 4 10 3-4 7

3 38 23 ? ? ? ? [1] 23

4 32 20 4 11 3-4 9

05T32-9-B 2

1 36 28 4 1 1-4 21 6

3 32 15 0 0 3-4 14

5 34 24 0 0 1-4 17 3

6 40 33 3-4 12 1-3 11 6

05T32-9-C 1 1 28 24 3-4 9 3-4 13

05T32-10-A 1

1 32 25 4 12 3-4 13

2 38 23 4 10 3-4 9 2, [1-2] 2

3 36 31 3-4 13 3-4 17

05T32-10-B 2

2 36 33 4 14 2-3n 10 [3-4] 6 [1c] 2

3 36 31 3-4 12 2-4 13

6 34 33 3-4 14 3-4 18

8 36 31 3-4 19 1-3 7 4

05T32-10-C 3

1 16 14 4 2 4 1 [3-4] 8 1

2 30 22 3-4 13 3-4 8

3 32 25 4 12 3-4 11

4 30 21 4 11 3-4 10

5 28 18 4c 14 4 1 [3-4] 2

6 30 21 3-4 9 2-4 11

7 26 22 4 6 3-4 7

8 24 16 ? ? ? ? [3-4] 13


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21

Line no.

(BC1)

No. of

plantsHead no.

No. of

spikelets

emasculated

No. of seeds


able rust (mix)

Selected clean


typeNo. ofplants


05T32-11-B 2

1 36 34 3-4 14 1-4 18 1

2 28 22 ? ? ? ? [3-4] 22

3 30 27 3-4 13 3-4 12

4 34 34 3-4 18 3-4 14 1

5 34 26 4 16 3-4 10

6 32 27 ? ? ? ? [3-4] 26

7 32 30 ? ? ? ? [3-4] 30

8 30 14 ? ? ? ? [3-4] 13

05T32-11-C 11 32 28 4 9 3-4 19 [mix?]2 28 26 3-4 15 2-4c 8

3 26 26 3-4 8 3-4 11

05T32-12-B 2

3 30 21 3-4 11 3-4 9

4 30 9 3-4 3 2-4 6

5 32 28 3-4 10 1-4 8 4

6 30 28 3-4 15 3-4 12

7 30 21 3-4 8 3-4 9 2

8 34 29 3-4 17 3-4 11

05T32-13-A 1 1 30 22 4 11 4 11

05T32-13-B 2

3 28 20 4 7 3-4 11

4 32 27 ? ? ? ? [3-4c] 27

5 26 22 ? ? ? ? [3-4] 17

6 28 20 4 9 1-2 4 3

7 30 24 3-4 7 3-4 7

05T32-14-A 1

1 30 10 0 0 3-4 10

2 34 23 0 0 3-4 233 32 25 0 0 1n, 3-4 3, 21 1


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22

Line no.

(BC1)

No. of

plantsHead no.

No. of

spikelets

emasculated

No. of seeds


able rust (mix)

Selected clean


typeNo. ofplants


05T32-14-B 2

1 30 23 4 13 3-4 9

2 32 24 3-4 3 3c 13 [4] 2

3 32 30 4 17 3-4 12

4 30 23 3-4 9 2-4 11

5 30 18 2-3 8 0 [missed?] 0 1

05T92-2-A 12 36 23 ? ? ? ? [3-4] 23

3 36 35 3-4 2 3-4 29 3

05T92-2-B 2

2 30 27 4 11 1-3 12 1

3 30 13 4 9 2-3 3 14 30 14 3-4 8 3-4 4

5 32 26 3-4 9 1-3n,c 8

6 34 22 3-4 10 3 10

05T92-2-C 4

1 30 15 N/A N/A 3c 15

2 32 25 N/A N/A 3c 25

3 24 13 N/A N/A 3c 13

05T92-3-A 12 38 27 4 15 3-4 10

3 34 25 3-4 16 2-4 9

05T92-3-B 2

2 34 24 0 0 1-3n,c 21

3 30 24 0 0 1-4 22

4 36 29 0 0 3-4 26

5 34 23 0 0 3-4 21 [1 kept] (1)

6 34 24 4 1 3-4 22

7 30 19 0 0 1-3 17 1

05T92-3-C 3

1 34 24 3-4 7 3-4 9 2

2 28 22 N/A N/A 3c 223 30 26 ? ? ? ? [3-4] 23 1

4 28 24 N/A N/A 3c 24

5 28 23 N/A N/A 3c 23


24/29


25/29

24

Line no.

(BC1)

No. of

plantsHead no.

No. of

spikelets

emasculated

No. of seeds


able rust (mix)

Selected clean


typeNo. ofplants


05T92-7-B 2

1 24 15 3-4 7 3-4 6

2 36 28 3-4 16 3-4 9 1

3 38 27 4 13 1-3 11

4 26 23 4 13 3-4 9

5 34 26 4 10 3-4 13

05T92-8-A 4

5 30 18 3-4 6 1-4 10

6 34 18 ? ? ? ? [1-4] 18

7 28 8 4 6 3n 1 1

8 30 27 3-4 6 3-4 9 5

05T92-8-B 2

2 36 23 0 0 1-4n,c 15

3 34 31 ? ? ? ?[3-4n,c] 28

2 [slowrusting]

4 38 29 ? ? ? ? [1-4] 23

5 36 32 ? ? ? ? [1-4] 30

6 34 30 0 0 1-3c 263 [slowrusting]

7 32 29 3-4 18 1-4c 10

8 30 22 ? ? ? ? [3-4] 20

05T92-8-C 2

1 32 7 N/A N/A 3c 7

2 32 19 N/A N/A 3c 19

4 26 20 N/A N/A 3c 20

5 26 19 N/A N/A 3c 19

05T92-9-A 5

1 32 14 3-4 9 3-4 4

2 24 1 N/A N/A N/A N/A

3 34 9 3-4 2 1 1 54 34 11 3-4 4 3-4 7

5 38 19 3-4 10 2-4 6 2

6 32 23 4 12 1-4 11

7 38 33 0 0 1-4 31 2

8 30 23 1-4 10 1-4 4


26/29

25

Line no.

(BC1)

No. of

plantsHead no.

No. of

spikelets

emasculated

No. of seeds


able rust (mix)

Selected clean


typeNo. ofplants


05T92-9-B 2

1 28 23 3-4 12 1-4 11

2 28 20 4 7 1-3 7 2

3 40 28 4 13 3-4 14

4 36 28 3-4 17 3-4 7

05T92-9-C 4

1 30 26 ? ? ? ? [3-4] 25

2 26 17 N/A N/A N/A N/A

3 24 13 N/A N/A 3c 13

4 32 28 N/A N/A 3c 28

05T92-10-B 2

1 28 12 4 3 1-4 93 34 26 4 14 1-4 11 1

4 32 25 ? ? ? ? [3-4] 21

5 30 27 3-4 6 2-4 9

05T92-10-C 3 1 32 21 ? ? ? ? [3-4] 20

05T92-11-C 2

1 30 25 ? ? ? ? [3-4] 23

2 26 20 ? ? ? ? [3-4] 19

3 32 30 ? ? ? ? [3-4] 30


27/29

26

Table 5. F1 segregation analysis for stem and leaf rust resistance.

Combination Cross codeNo. of spikelets

emasculated

No. of seeds

obtained

Leaf rust Stem rust

Infection type No. of plants Infection type No. of plants

95T159 x 98T376

95T159-SA1 28 26 ; 26 n/a n/a

95T159-SA2 28 25 n/a n/a 3c 25

95T159-SA4 28 18 ; 18 n/a n/a

95T159-SA5 34 26 n/a n/a 3c 26

98T376 x 95T159

98T376-SA2 24 16 n/a n/a n/a n/a

98T376-SA3 30 23 ; 23 n/a n/a

98T376-SA5 32 26 n/a n/a 3c 2698T376-SA6 28 26 n/a n/a 3c 26

98T376-SA7 30 25 n/a n/a n/a n/a

98T376-SA8 30 16 n/a n/a n/a n/a

98T376-SA9 32 29 ; 29 n/a n/a

Tobie x 95T159

Tobie-SA1 24 21 ; 21 n/a n/a

Tobie-SA2 26 20 ; 20 n/a n/a

Tobie-SA3 24 20 n/a n/a 3c 20



95T159 x Tobie

95T159-SA3 30 17 n/a n/a 3c 17

95T159-SA6 34 14 n/a n/a n/a n/a

95T159-SA7 28 27 ; 27 n/a n/a

95T159-SA8 28 20 ; 20 n/a n/a

95T159-SA9 30 25 n/a n/a n/a n/a95T159-SA10 32 30 n/a n/a 3c 30


28/29

27

Figure 1. The scheme of doubled haploids population creation through wide

hybridization with maize.

Resistant cultivar / lineSusceptible cultivar / line X

F1 (selfing)

F2Rust inoculation and

selection of resistant plants

Resistant plants crossed with maize

Application of growth regulators(30 h later) to induce embryo formation

Embryos are rescued (16-20 days after

pollination) and cultured in vitro

Chromosome doubling regenerated plantletsare treated with colchicine

(for 24 h) and repotted

After selfing, population of homozygous DH lines is

created. DH seeds harvested and planted

Final rust inoculation and selection of resistant

plants among DH population

Resistant true-breeding line of the

susceptible parent is created


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Figure 2. The scheme of NILs creation through backcrosses (BC).

Figure 3. The scheme of segregation analysis of rust resistance.

Resistant cultivar / lineSusceptible cultivar / line X

F1 (selfing)Rust inoculation Counting ofsegregation ratio

Rust inoculation F2 (selfing) Counting ofsegregation ratio

Resistant cultivar / lineSusceptible cultivar / line

(recurrent parent) X

F1 (selfing)

F2Rust inoculation and


Resistant plants backcrossed with

recurrent parent

BC line (1...4)Rust inoculation and


Resistant near-isogenic line (NIL) of the

recurrent parent is created

Date post:	30-May-2018
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Development of stem- and leaf rust resistant near-isogenic lines of spring triticale TOBIE and...

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