INTROGRESSION OF LEAF RUST AND STRIPE RUSTRESISTANCE GENES FROM AEGILOPS UMBELLULATATO HEXAPLOID WHEAT THROUGH INDUCEDHOMOEOLOGOUS PAIRING

P. CHHUNEJA1�∗, S. KAUR1, R.K. GOEL1, M. AGHAEE-SARBARZEHAND H.S. DHALIWAL2

1Dept. Plant Breeding, Genetics and Biotechnology, Punjab Agricultural University,Ludhiana 141 004, India2Indian Institute of Technology, Roorkee, India∗E-mail: pchhuneja@rediffmail.com

Abstract: First alien leaf rust resistance gene Lr9 was transferred from Ae. umbellulata intohexaploid wheat in the year 1956 through irradiation induced translocation. A number ofother genes were subsequently transferred from non-progenitor and progenitor Aegilopsspecies and exploited commercially. However, the appearance of new virulences neces-sitates the search of novel sources of rust resistance. An Ae. umbellulata acc. 3732 wasfound to be resistant to several wheat diseases such as leaf rust, stripe rust, Karnal bunt,powdery mildew and cereal cyst nematode. An amphiploid (AABBUU) synthesizedfrom the cross of Ae. umbellulata acc. 3732 and T. durum cv. WH890 was crossed toCS PhI for inducing homoeologous pairing between Ae. umbellulata and wheat chromo-somes. The F1 (AABBDU) was crossed to a susceptible hexaploid wheat cv. WL711 andintrogression lines carrying resistance to leaf and stripe rust were selected in backcrossprogenies. The BC2F1 plants with leaf and stripe rust resistance genes were selfed,and homozygous lines with least linkage drag were selected and screened against fiveleaf rust and three stripe rust pathotypes. The introgression lines were resistant to allthe leaf rust pathotypes and stripe rust pathotypes. Transfer of Lr9 was ruled out byscreening the introgression line with 77-7, virulent on Lr9. For studying the inheri-tance of transferred rust resistance genes, an F2 population was generated by crossingthe introgression line with the recipient parent WL711. Screening of F2 populationat the seedling stage against leaf rust pathotype 77-5 revealed that a single dominantgene governs the resistance at seedling stage. The F2 plants screened at the seedlingstage were grown in the field and screened at adult plant stage against a mixture ofpathotypes under artificial epiphytotic conditions. All plants that were resistant at theseedling stage maintained resistance at adult plant stage. Out of a total of 48 susceptibleseedlings, 28 showed resistance at adult plant stage indicating the presence of adultplant resistance (APR) gene as well. The population segregated for two genes for leafrust, one seedling and one APR, with a �2 (15:1) of 12.5. Thus from Ae. umbellulata,two novel leaf rust resistance genes(one seedling resistance and one APR) and stripe

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84 Chhuneja et al.

rust resistance has been transferred to hexaploid wheat. The introgression lines wereanalysed with SSR markers for identifying the introgressed regions. The introgressionswere detected for chromosomes of homoeologous group 2, 4 and 5. Bulk segregantanalysis is in progress to identify the SSR markers segregating with resistance

Keywords: leaf rust resistance, stripe rust resistance, Aegilops umbellulata, homeologous pairing

INTRODUCTION

Leaf rust (Puccinia triticina) and yellow rust (Puccinia striformis) are the twomajor rust diseases of wheat. Leaf rust or brown rust caused by the fungal pathogenPuccinia triticina is one of the most common diseases affecting wheat productionworldwide. The impact of leaf rust on yield reduction in wheat ranges from 10percent under moderate conditions to 65 percent under intense epidemics (Saari andPrescott 1985). An effective, economical and ecologically safe method to controlleaf and stripe rust epidemics is the cultivation of resistant cultivars. More than 50leaf rust resistance genes have been designated (Knott 1989, McIntosh et al. 1995)and of these 46 genes have been mapped to specific chromosomes. Out of these 50genes, 20 are of alien origin having been introgressed into the wheat genome fromwild relatives like Agropyron and Aegilops spp. (Kolmer 1996). However, a numberof genes transferred from related species of wheat viz. Lr9, Lr19, Lr24, Lr26, Yr 9and Pm8 etc. and exploited commercially, have been overcome by the emergence ofvirulent pathotypes, therefore, necessitating the search for new sources of resistance.Less closely related Aegilops species especially non-progenitor C, U and M genomespecies are excellent sources of resistance to various wheat diseases (Dhaliwal et al.1991, 1993, Harjit-Singh et al. 2000).

However, only a few genes for resistance to diseases and other traits transferredfrom non progenitor species have been commercially exploited due to substantialamount of undesirable genetic information (linkage drag) associated with usefulgenes and yield reduction (Jiang et al. 1994). Chen et al. (1994) transferred thedominant homoeologous pairing inducer PhI gene from Aegilops speltoides highpairing line to hexaploid wheat cultivar Chinese Spring (CS). This gene suppressesthe effect of the Ph1 locus and induces homoeologous pairing between wheatand alien chromosomes in F1 hybrids (Chen et al. 1994, Aghaee-Sarbarzeh et al.2000). Leaf and stripe rust resistance genes has been transferred from Ae. ovatachromosome 5M to wheat chromosome 5D through induction of homoeologouspairing with CS�PhI� (Aghaee-Sarbarzeh et al. 2002).

Evaluation of different accessions of wild Triticum and Aegilops speciesmaintained at the Punjab Agricultural University, Ludhiana, Punjab (India) has ledto the identification of a number of novel sources of resistance to leaf rust, stripe rust,Karnal bunt, powdery mildew and cereal cyst nematode (Dhaliwal et al. 1993, Gillet al. 1995, Dhaliwal and Harjit-Singh 1997, Harjit-Singh et al. 1998, Harjit-Singhand Dhaliwal 2000). Aegilops umbellulata, a diploid species with UU genome, was

Introgression of Leaf Rust and Stripe Rust 85

found to be highly resistant to different wheat diseases such as leaf rust, striperust, Karnal bunt, powdery mildew and cereal cyst nematode. An accession ofAe. umbellulata with multiple disease resistance was selected and leaf and striperust resistance has been transferred from this accession to cultivated wheat throughinduction of homoeologous pairing using CS�PhI�. In the present communication,the transfer of leaf rust and stripe rust resistance from Ae. umbellulata throughinduction of homoeologous pairing and molecular characterization of introgressionlines using SSR markers is being reported.

MATERIAL AND METHODS

An amphiploid was synthesized between T. durum cv. WH890 and Ae. umbellulataaccession 3732 and crossed with T. aestivum cv Chinese spring carrying PhI geneof Ae. speltoides to induce homoeologous pairing. The F1 plants from this crosswere selfed and also crossed to a rust susceptible T. aestivum cv. WL711 havingnon-necrotic gene WL711(NN). All BC1 plants were screened at the seedling stagefor leaf and stripe rust. The resistant plants were backcrossed to WL711(NN) torecover the recurrent genotype. Homozygous BC1F4 and BC2F4 introgression lineswith leaf and stripe rust resistance transferred from Ae. umbellulata have beenselected.

Leaf and Stripe Rust

The rust resistant genes were followed in the segregating generations by screeningagainst leaf rust pathotypes 77-5 and 104-2 and stripe rust pathotype 46S119 atthe seedling stage. The resistant plants were transplanted in the field and terminaldisease severity recorded for leaf and stripe rust. The homozygous leaf and/or striperust resistant introgression lines were tested with five most virulent and prevalentpathotypes of leaf rust 12-2, 77-2, 77-5, 77-7, and 104-2, and three pathotypes ofstripe rust 46S119, 46S102 and 46S103 at the seedling stage and under artificialepiphytotic conditions in the rust screening nursery in the field. Seedling reactionwas recorded in parents and introgression lines using the standard inoculationprocedure (Nayar et al. 1997). Infection type was recorded 14 days after inoculationsfollowing the modified 0; -4 scale of Stakman et al. (1962). The disease severityunder field conditions was recorded as percentage of leaf area covered by rustfollowing modified Cobb’s scale (Peterson et al. 1948).

Molecular Studies

SSR markers were used to detect alien transfer, in the homozygous wheat-umbellulata introgression lines. The PCR was performed as described by Röder et al.(1998) with some modifications. PCR products were resolved by electrophoresis in2.5% agarose gels. Gels were visualized by staining with ethidium bromide usingUVP Gel Documentation System.

86 Chhuneja et al.

RESULTS AND DISCUSSION

Leaf and Stripe Rust Screening

All the selected ILs showed resistance to the four test leaf rust pathotypes at theseedling stage and remained free from leaf rust in the rust screening nursery atPAU, Ludhiana (Table 1) and off season nursery at Wellington, Neelgiri Hills,India (data not given). A leaf rust resistance gene transferred from U genome ofAe. umbellulata (Sears 1956) is present on chromosome 6BL as a compensatingtransfer. To establish that the leaf rust resistance gene present in these introgressionlines is different from Lr9, some of the lines were tested with Lr9 virulence 77-7. Three of the selected ILs 329-1, 333-4 & 367-4, however, showed susceptibleinfection type to 77-7 at the seedling stage indicating that these ILs might be carryingLr 9. All the introgression lines except 329-1, 333-4, 367-4, 380-3 were found tobe resistant to stripe rust at the seedling stage and under field conditions (Fig. 1).

Inheritance Studies

One of the ILs 403-1 was crossed with recurrent parent WL711 and leaf and striperust resistant F1 plants were selfed to develop an F2 for studying the inheritanceof leaf rust resistance in this IL. The F2 was screened at the seedling stage againstleaf rust pathotype 77-5 and all the plants were then transplanted in the field andstudied for leaf rust reaction at the adult plant stage. The population segregated forsingle gene for leaf rust at the seedling stage with a �2 of 3.5 and for two genesfor leaf rust at the adult plant stage with a �2 (15:1) of 12.5. However, out of 48susceptible plants at the seedling stage, 28 plants were observed to be resistant atthe adult plant stage indicating that an APR gene for leaf rust is present in additionto the seedling resistance gene. The progeny of another introgression line 351-1was also found to be segregating for one seedling and one APR gene for leaf rustresistance (Table 2).

SSR Analysis

A total of 100 SSR markers were amplified in the parents and 19 primers werefound to be polymorphic. 13 primers did not amplify in umbellulata and hencewere not used for genotyping of introgression lines. The analysis of the wheat-Ae.umbellulata introgression lines using Xwmc and Xgwm SSR markers led to theidentification of introgressed segments of umbellulata chromatin in homoeologousgroup 2 in different introgression lines (Fig. 2). The 2AL/2DL mapped SSR markerXwmc181 detected the introgression in the progenies of 315-5, 351-1, 388-5 and403-1. The Xwmc181 was amplified in the two leaf and stripe rust resistant andone susceptible progeny derived from IL 351-1. The introgression was detected inthe progeny carrying seedling resistance gene for leaf rust (lane 16) and wheat typeallele was observed in the progeny with APR gene and in the susceptible progeny.The five leaf and stripe rust resistant and one susceptible F3 progeny of IL 403-1

Tab

le1.

Lea

fan

dst

ripe

rust

reac

tion

ofth

ese

lect

edin

trog

ress

ion

lines

from

the

cros

sT

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rum

c.W

H89

0-A

e.um

bell

ulat

aac

c.PA

U#3

732/

CS

(PhI

)//W

L71

1(N

N)

S.N

o.IL

IDG

ener

atio

nL

eaf

rust

-see

dlin

gSt

ripe

rust

-see

dlin

gR

ust

reac

tion-

Fiel

d

12-2

77-2

77-5

77-7

104-

246

S119

46S1

0246

S103

Lea

fSt

ripe

1W

L71

13

33

33

33

360

S80

S2

Ae.

umbe

llul

ata

;;

;;

;0;

;;

00

3C

S�P

hI�

33+

33

33

33

320

S20

S4

WH

890

33+

33

-3

33

310

S10

S5

T.

duru

m-A

e.um

bell

ulat

aam

phip

loid

--

--

--

--

40S

40S

631

5-5

F 7;

-;

;-;

3;

-0

07

329-

1F 7

--

0;3+

;3

--

080

S8

333-

4-

1;

3+1

3-

-0

40S

933

9-4

BC

1F 7

--

;11

1;

;0;

00

1035

1-1

BC

1F 6

--

3-

3-

--

05M

R11

351-

5B

C1F 6

;10

;;1

-1

0;

;0;

00

1235

3-2

BC

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--

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00

1335

4-4

BC

1F 6

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;;

;;

00

1435

7-1

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;1-

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;;

00

1535

9-1

BC

1F 6

--

;;

;;

;;

00

1636

7-4

BC

2F 5

--

0;

33+

;3

--

040

S17

380-

3B

C3F 4

;0

;0

;-

;3

33

040

S18

388-

5B

C1F 6

;1;1

;1;1

;10;

--

00

1939

3-4

BC

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;;1

;;1

-;

;-

00

2040

3-1

BC

2F 5

--

32

-0;

--

00

88 Chhuneja et al.

Figure 1. Rust reaction of wheat-Ae umbellulata introgression lines under artificial epiphytoticconditions in the field

Table 2. Segregation for leaf rust resistance in the F2 developed from the cross of IL403-1 with recipientparent WL711

Resistant plants Susceptible plants Segregation ratio �2

Seedling 104 48 3:1 3.5Adult plant 132 20 15:1 12.5

Figure 2. PCR amplification profile of SSR marker Xwmc181 in the homozygous resistant progeniesof some selected leaf and/or Stripe rust resistant Introgression lines developed from the cross T. durum

cv. WH890-Ae. Umbellulata acc. PAU#3738/CS (Ph)//WL711(NN) and segregating F3 progenies of351-1 and 403-1. Lane 1. T. durum WH890; Lane 2. WL711; Lane 3. CS(Ph); Lane 4. Ae.

Umbellulata Lane 5-14. Progeny of 315-5, 353-2, 357-1, water, 367-4, 380-3, 393-4, 388-5, 403-1 and403-1 (repeated); Lane 15-17. 351-1(R), 351-1(R), 351-1(S); Lane 18-22 resistant F3 progenies of

403-1; Lane 26. susceptible F3 progeny of 403-1

Introgression of Leaf Rust and Stripe Rust 89

were also analysed with Wmc181. The introgression was detected in all the resistantprogenies and only wheat type allele was amplified in susceptible progeny. Theamplification of wheat as well as umbellulata specific alleles of the 2A/2D mappedXwmc181 in resistant introgression lines and progenies indicates the presence ofintrogressed segment from 2U of Ae. umbellulata on the either chromosome 2A or2D as a compensating transfer. The work to define the introgressed regions and totag the genes using F2 population is in progress.

Results indicated the precise transfer of one seedling and one APR gene forleaf rust and one seedling resistance gene for stripe rust from Ae. umbellulata acc.3732 to hexaploid wheat cultivar WL711 and the introgression of Ae. umbellulatachromatin, carrying these genes, has been detected on wheat homoeologous group 2.

The leaf and stripe rust resistance genes transferred from Ae. umbellulata arenovel genes which can be exploited for broadening the genetic base of cultivatedwheats for rust resistance. The identification of the closely linked molecular markersmay lead to marker-assisted mobilization of these genes in elite wheat backgrounds.

ACKNOWLEDGEMENTS

The financial assistance of USDA-ARS under Project IN-ARS-842, and GrantNumber FG-In-792 to carry out the research work is gratefully acknowledged.The rust inoculum was provided by Director, Regional Research Station, Directorateof Wheat Research, Flowerdale, Shimla, India.

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