UNIVERSITY OF STELLENBOSCH

DEPARTMENT OF GENETICS

Plant Breeding Laboratory (SU-PBL)

Honours Project Final Article

Development of stem- and

leaf rust resistant
near-isogenic lines
of spring triticale TOBIE and
advanced line 98T376-A-A-3

Student: Yuriy Tsupko [US#14997665]

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

Date: 20 October 2006

 1

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 breeder’s 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 of
resistance (OELKE & KOLMER, 2004). Development of genetic resistance to rust is the most
 2

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 breeder’s 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 1970’s 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: UVPgt57 for 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 were
sprayed thoroughly. Inoculated pots covered with wet plastic bags and left in a darkroom for 24
hours at 20ºC, 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 25/20ºC day/night, for rust to develop. The temperature range
was chosen as a compromise between optimum temperatures for stem (day 25-30ºC, night 15-20ºC;
germination optimum 18ºC) and leaf (day 20-25ºC, night 15-20ºC; germination optimum 15-20ºC)
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 23/25ºC, but not relative humidity.
 4

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 (25/20ºC 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 thiamin·HCl
/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, F2’s 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 ¾ of sand and
¼ 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 (25/20ºC 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
 5

grown under natural light conditions. A chimney cooling system was used in order to keep the
temperature not higher than +25ºC. 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-
 6

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 a
single 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 UVPgt57 appear 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
breeder’s 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
 7

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, André Julius and Samuel Pietersen for casual help and friendly attitude.
 8

References

KOLMER, J.A., 2005. Tracking wheat rust on a continental scale. Cur. Op. in Pl. Bio. 8, 441–449.

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: <http://www.crpmb.org/2004/1029kovalenko>

LEONARD, K.J. & SZABO, L.J., 2005. Stem rust of small grains and grasses caused by Puccinia
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. Triticale – still a healthy crop? Proc. 5th Int. Triticale Symp., June 30-July 5,
2002, Radzikow, Poland. 1, 81–88.

SCHLEGEL, R., 1996. Triticale – today 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:
<http://www.ars.usda.gov/Main/docs.htm?docid=9854&pf=1&cg_id=0>

ZADOKS, J.C., CHANG, T.T. & KONZAK C.F., 1974. A decimal code for the growth stages of
cereals. Weed Research. 14, 415-421.
 9

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

68//VPM/MOISSON/4/JUANILLO 100/5/ANDAS”S”/6/DURUM
(resistant to stem and
WHEAT/BALBO//BOK”S”/3/ANDALAS”S”//TJ/BGL”S”
leaf rust)

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//BOK”S”

 10

Table 2. Major infection type classes for stem rust and leaf rust
(McINTOSH, WELLINGS & PARK, 1995).

Infection Host response Symptoms

type*
0 Immune No visible uredia

; Very resistant Hypersensitive flecks

1 Resistant Small uredia with necrosis

2 Resistant to moderately Small to medium sized uredia with green islands

resistant and surrounded by necrosis or chlorosis

3 Moderately resistant / Medium sized uredia with or without chlorosis

moderately susceptible

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.
 11

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 Total
x 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 31.3
Avg. seeds/head 23.2 n/a n/a 22.7 n/a n/a 21.5 22.8
 12

Table 4. Segregation analysis of F1 for leaf and stem rust resistance.

Leaf rust inoculation
Combination Cross code No. of resistant plants
No. of susceptible plants
(infection type ; )
95T159-SA1 0 26
95T159 x 98T376
95T159-SA4 0 18
98T376-SA3 0 23
98T376 x 95T159
98T376-SA9 0 29
Tobie-SA1 0 21
Tobie x 95T159
Tobie-SA2 0 20
95T159-SA7 0 27
95T159 x Tobie
95T159-SA8 0 20
Stem rust inoculation
No. of susceptible plants No. of resistant plants
(infection type 3c ) (infection type ; )
95T159-SA2 25 0
95T159 x 98T376
95T159-SA5 26 0
98T376-SA5 26 0
98T376 x 95T159
98T376-SA6 26 0
Tobie-SA3 20 0
Tobie x 95T159 Tobie-SA4 15 0
Tobie-SA5 8 0
95T159-SA3 17 0
95T159 x Tobie
95T159-SA10 30 0
 13

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, F 2).
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).

 14

Figure 3. Stem (left) and leaf (right) rust on BC1 plants inoculated in seedling
stage.
 15

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 volumetric
BAC1 100 ml
flack to 1000 ml).
BAC2 10 ml
(3) Adjust the pH to 5.8 (by adding NaOH or HCl).
BAC3 1 ml
(4) Add the Agar and the solution from the flack in a Scott bottle.
AV4 10 ml
(5) Shake this vigorously and pour into tissue culture bottles (about 40 ml
V1 10 ml
per bottle).
V4 4 ml
(6) Fix tops (but do not fasten too tightly) and autoclave (wet).
Sucrose 20 g
Agar 8g

BAC1 BAC2
(powder of each and dissolve into the final solution) (powder of each and dissolve into the final solution)
500 ml 500 ml
KNO3 9.500 g MnSO4 · 4H2O 0.825 g
NH4NO3 0.825 g ZnSO4 · 7H2O 0.430 g
KH2PO4 0.850 g H3BO3 0.310 g
MgSO4 · 7H2O 1.850 g
CaCl2 · 2H2O 2.200 g

BAC3 AV4
(powder of each and dissolve into the final solution) (heat to dissolve and stabilize)
500 ml 200 ml
KI 0.4150 g Na2EDTA 0.746 g
CuSO4 · 5H2O 0.0125 g FeSO4 · 7H2O 0.556 g
Na2MoO4 · 2H2O 0.1250 g

V1 V4
100 ml 100 ml
Inositol 1g Tiamin · HCl 0.01 g
 16

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

Inoculation A Inoculation B Inoculation C
Resistant
F2 (SSD lines) Selected clean Partially Selected clean
Susceptible Susceptible Incl. selected clean Susceptible
plants ( ; ) resistant plants ( ; )
plants ( ; )
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 4
05T92-10 5 5 6 4 14 2 9 3
 17
Table 3. List of heads used for creation of haploids through maize pollination.
Line No. Head no. No. of spikelets emasculated No. of seeds No. of embryos rescued
05T32-1-B 1 28 1 0
2 32 0 0
05T32-2-B 1 38 0 0
05T32-3-B 2 30 0 0
05T32-4-B 1 30 0 0
05T32-5-A 1 30 0 0
4 30 0 0
05T32-5-B 1 32 26 0
2 36 24 0
05T32-6-B 1 30 21 0
05T32-7-A 1 n/a n/a 0
4 34 0 0
5 28 0 0
05T32-8-B 1 30 25 0
05T32-9-A 5 36 0 0
6 36 0 0
05T32-9-B 2 38 17 0
4 38 0 0
05T32-10-A 4 34 0 0
05T32-10-B 1 30 0 0
4 32 10 0
5 30 5 0
7 36 4 0
05T32-12-B 1 28 0 0
2 28 0 0
05T32-13-A 2 34 0 0
3 34 0 0
05T32-13-B 1 30 11 0
2 34 23 0
05T32-14-A 4 34 1 0
05T92-2-A 1 40 36 1
05T92-2-B 1 26 26 0
05T92-3-A 1 n/a n/a 0
05T92-3-B 1 36 34 0
05T92-4-A 1 n/a n/a 0
05T92-4-B 5 34 9 0
05T92-5-A 1 36 25 0
2 30 29 0
3 36 1 1 [died]
05T92-6-B 3 28 26 0
05T92-7-A 2 34 0 0
6 42 36 1 [roots only]
05T92-8-A 1 22 16 0
2 32 0 0
3 30 0 0
4 32 0 0
05T92-8-B 1 38 31 0
05T92-9-A 8 38 7 0
05T92-10-A 1 n/a n/a 0
2 32 0 0
3 30 7 0
4 42 15 0
05T92-10-B 2 32 32 1 [died]
Tobie 1 n/a n/a 0
2 n/a n/a 0
Total: 55 1610 498 1
 18

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

No. of Leaf rust Stem rust
Line no. No. of Indistinguish- Selected clean
Head no. spikelets No. of seeds Infection No. of
(BC1) plants Infection type No. of plants able rust (mix) plants ( ; )
emasculated type plants
3 34 31 3-4 12 3-4 16 2
4 30 30 3-4 18 3-4 10 2
05T32-1-B 2
5 30 18 3-4 9 3-4 8
6 30 19 4 7 3-4 7
2 30 20 ? ? ? ? [1-4] 14
3 36 26 4 12 3-4 12
05T32-2-B 2
4 28 26 ? ? ? ? [3-4] 19
4’ 36 14 3-4 5 2-4 6
1 32 20 3-4 9 3-4 10 1
3 32 28 4 10 3-4 11
05T32-3-B 2 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
2 32 20 3-4 6 2-4 14
3 22 14 3-4 4 2-4 8 1
05T32-4-B 2 4 30 23 3-4 10 3-4 13
5 28 20 ? ? ? ? [3-4c] 20
6 30 21 4 13 3-4 7
2 30 17 3-4 7 3-4n, 2c 5, 5
05T32-5-A 1 3 28 22 3-4 14 3-4 4
5 28 22 3-4 7 2-4 15
3 32 31 ? ? ? ? [3-4] 31
4 32 27 ? ? ? ? [3-4c] 23
05T32-5-B 2 5 34 23 ? ? ? ? [3-4] 15
6 30 26 ? ? ? ? [3-4] 25
7 30 29 ? ? ? ? [3-4c] 28
 19

No. of Leaf rust Stem rust

Line no. No. of Indistinguish- Selected clean
Head no. spikelets No. of seeds Infection No. of
(BC1) plants Infection type No. of plants able rust (mix) plants ( ; )
emasculated type plants
1 34 24 3-4 13 2-4 9 1
2 32 17 N/A N/A N/A N/A
05T32-6-A 1
3 32 29 3-4 15 3-4 13
4 30 26 3-4 12 1-4 14
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
05T32-6-B 2
5 32 29 ? ? ? ? [3-4] 28
6 28 24 ? ? ? ? [3-4n] 20
7 30 22 ? ? ? ? [3-4] 22
2 28 21 4 1 3-4 18
05T32-7-A 2
3 36 19 4 6 3-4 11 1
1 32 17 4 11 ? ? [3-4] 5 1
2 30 18 ? ? ? ? [3-4] 12
05T32-7-B 2 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
1 26 21 4 9 3-4 11
2 22 19 3-4 16 3-4 2
05T32-7-C 4
3 30 29 ? ? ? ? [3-4] 25
4 32 30 ? ? ? ? [3-4] 26
2 28 1 ? ? ? ? [1n] 1
05T32-8-B 2 3 26 21 4 10 3-4 11
4 30 24 4 12 1-4n,c 5 [1-3n,c] 5
1 30 27 ? ? ? ? [3-4c] 24
05T32-8-C 2 2 26 21 4 10 3-4 8
3 28 22 ? ? ? ? [2-4] 22
 20

No. of Leaf rust Stem rust

Line no. No. of Indistinguish- Selected clean
Head no. spikelets No. of seeds Infection No. of
(BC1) plants Infection type No. of plants able rust (mix) plants ( ; )
emasculated type plants
1 30 12 4 3 3-4 9
2 28 20 4 10 3-4 7
05T32-9-A 1
3 38 23 ? ? ? ? [1] 23
4 32 20 4 11 3-4 9
1 36 28 4 1 1-4 21 6
3 32 15 0 0 3-4 14
05T32-9-B 2
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
1 32 25 4 12 3-4 13
05T32-10-A 1 2 38 23 4 10 3-4 9 2, [1-2] 2
3 36 31 3-4 13 3-4 17
2 36 33 4 14 2-3n 10 [3-4] 6 [1c] 2
3 36 31 3-4 12 2-4 13
05T32-10-B 2
6 34 33 3-4 14 3-4 18
8 36 31 3-4 19 1-3 7 4
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
05T32-10-C 3
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
 21

No. of Leaf rust Stem rust

Line no. No. of Indistinguish- Selected clean
Head no. spikelets No. of seeds Infection No. of
(BC1) plants Infection type No. of plants able rust (mix) plants ( ; )
emasculated type plants
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
05T32-11-B 2
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
1 32 28 4 9 3-4 19 [mix?]
05T32-11-C 1 2 28 26 3-4 15 2-4c 8
3 26 26 3-4 8 3-4 11
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
05T32-12-B 2
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
3 28 20 4 7 3-4 11
4 32 27 ? ? ? ? [3-4c] 27
05T32-13-B 2 5 26 22 ? ? ? ? [3-4] 17
6 28 20 4 9 1-2 4 3
7 30 24 3-4 7 3-4 7
1 30 10 0 0 3-4 10
05T32-14-A 1 2 34 23 0 0 3-4 23
3 32 25 0 0 1n, 3-4 3, 21 1
 22

No. of Leaf rust Stem rust

Line no. No. of Indistinguish- Selected clean
Head no. spikelets No. of seeds Infection No. of
(BC1) plants Infection type No. of plants able rust (mix) plants ( ; )
emasculated type plants
1 30 23 4 13 3-4 9
2 32 24 3-4 3 3c 13 [4] 2
05T32-14-B 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
2 36 23 ? ? ? ? [3-4] 23
05T92-2-A 1
3 36 35 3-4 2 3-4 29 3
2 30 27 4 11 1-3 12 1
3 30 13 4 9 2-3 3 1
05T92-2-B 2 4 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
1 30 15 N/A N/A 3c 15
05T92-2-C 4 2 32 25 N/A N/A 3c 25
3 24 13 N/A N/A 3c 13
2 38 27 4 15 3-4 10
05T92-3-A 1
3 34 25 3-4 16 2-4 9
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
05T92-3-B 2
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
1 34 24 3-4 7 3-4 9 2
2 28 22 N/A N/A 3c 22
05T92-3-C 3 3 30 26 ? ? ? ? [3-4] 23 1
4 28 24 N/A N/A 3c 24
5 28 23 N/A N/A 3c 23
 23

No. of Leaf rust Stem rust

Line no. No. of Indistinguish- Selected clean
Head no. spikelets No. of seeds Infection No. of
(BC1) plants Infection type No. of plants able rust (mix) plants ( ; )
emasculated type plants
2 34 24 4 12 2-4 12
3 42 38 3-4 21 3-4 17
05T92-4-A 2
4 42 30 4 9 3-4 17
5 38 26 3-4 13 3-4 11
1 32 30 3-4 20 1-4 10
2 32 26 3-4 12 3-4 14
05T92-4-B 2 3 32 21 4 8 1-4 9 3
4 36 27 3-4 16 2-4 9 2
6 34 29 3-4 17 2-4 9 2
05T92-5-A 2 4 34 29 4 16 1-4 12
1 26 14 0 0 1c, 3-4 5, 9
2 30 16 4 10 2 6 1
3 28 22 0 0 2-4 15 5
05T92-5-B 2 4 34 30 ? ? ? ? [3-4] 28 2
5 36 32 4 20 2-4 11
6 34 30 3-4 14 3-4 13 2
7 32 21 3-4 12 3-4 8 1
1 28 23 ? ? ? ? [3-4] 20
2 24 12 N/A N/A 3c 12
05T92-5-C 5 3 26 21 N/A N/A 3c 21
4 26 23 N/A N/A 3c 23
5 30 25 N/A N/A 3c 25
1 32 24 4 10 3-4 11 3
2 32 18 3-4 10 1-3 7 1
05T92-6-B 2 4 32 28 3-4 18 3-4 9
5 20 12 4 6 3-4 6
6 28 10 4 6 3-4 4
3 32 13 3-4 3 2-4 7 3
05T92-7-A 3 4 46 34 4 18 3-4 9 3
5 38 31 3-4 12 3-4 17
 24

No. of Leaf rust Stem rust

Line no. No. of Indistinguish- Selected clean
Head no. spikelets No. of seeds Infection No. of
(BC1) plants Infection type No. of plants able rust (mix) plants ( ; )
emasculated type plants
1 24 15 3-4 7 3-4 6
2 36 28 3-4 16 3-4 9 1
05T92-7-B 2 3 38 27 4 13 1-3 11
4 26 23 4 13 3-4 9
5 34 26 4 10 3-4 13
5 30 18 3-4 6 1-4 10
6 34 18 ? ? ? ? [1-4] 18
05T92-8-A 4
7 28 8 4 6 3n 1 1
8 30 27 3-4 6 3-4 9 5
2 36 23 0 0 1-4n,c 15
2 [“slow
[3-4n,c] 28
3 34 31 ? ? ? ? rusting”]
4 38 29 ? ? ? ? [1-4] 23
05T92-8-B 2 5 36 32 ? ? ? ? [1-4] 30
3 [“slow
6 34 30 0 0 1-3c 26 rusting”]
7 32 29 3-4 18 1-4c 10
8 30 22 ? ? ? ? [3-4] 20
1 32 7 N/A N/A 3c 7
2 32 19 N/A N/A 3c 19
05T92-8-C 2
4 26 20 N/A N/A 3c 20
5 26 19 N/A N/A 3c 19
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 5
4 34 11 3-4 4 3-4 7
05T92-9-A 5
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
 25

No. of Leaf rust Stem rust

Line no. No. of Indistinguish- Selected clean
Head no. spikelets No. of seeds Infection No. of
(BC1) plants Infection type No. of plants able rust (mix) plants ( ; )
emasculated type plants
1 28 23 3-4 12 1-4 11
2 28 20 4 7 1-3 7 2
05T92-9-B 2
3 40 28 4 13 3-4 14
4 36 28 3-4 17 3-4 7
1 30 26 ? ? ? ? [3-4] 25
2 26 17 N/A N/A N/A N/A
05T92-9-C 4
3 24 13 N/A N/A 3c 13
4 32 28 N/A N/A 3c 28
1 28 12 4 3 1-4 9
3 34 26 4 14 1-4 11 1
05T92-10-B 2
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
1 30 25 ? ? ? ? [3-4] 23
05T92-11-C 2 2 26 20 ? ? ? ? [3-4] 19
3 32 30 ? ? ? ? [3-4] 30
 26

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

No. of spikelets No. of seeds Leaf rust Stem rust
Combination Cross code
emasculated obtained Infection type No. of plants Infection type No. of plants
95T159-SA1 28 26 ; 26 n/a n/a
95T159-SA2 28 25 n/a n/a 3c 25
95T159 x 98T376
95T159-SA4 28 18 ; 18 n/a n/a
95T159-SA5 34 26 n/a n/a 3c 26
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 26
98T376 x 95T159 98T376-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-SA1 24 21 ; 21 n/a n/a
Tobie-SA2 26 20 ; 20 n/a n/a
Tobie x 95T159 Tobie-SA3 24 20 n/a n/a 3c 20
Tobie-SA4 30 15 n/a n/a 3c 15
Tobie-SA5 28 8 n/a n/a 3c 8
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 x Tobie
95T159-SA8 28 20 ; 20 n/a n/a
95T159-SA9 30 25 n/a n/a n/a n/a
95T159-SA10 32 30 n/a n/a 3c 30
 27

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

hybridization with maize.

Susceptible cultivar / line X Resistant cultivar / line

F1 (selfing)

Rust inoculation and

selection of resistant plants F2

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 plantlets

are 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
 28

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

Susceptible cultivar / line

X Resistant cultivar / line
(recurrent parent)

F1 (selfing)

Rust inoculation and

selection of resistant plants F2

Resistant plants backcrossed with

recurrent parent

Rust inoculation and

selection of resistant plants BC line (1...4)

Resistant near-isogenic line (NIL) of the

recurrent parent is created

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

Susceptible cultivar / line X Resistant cultivar / line

F1 (selfing) Counting of
Rust inoculation
segregation ratio

F2 (selfing) Counting of
Rust inoculation
segregation ratio