INTROGRESSION OF LEAF RUST AND STRIPE RUST RESISTANCE GENES FROM AEGILOPS UMBELLULATA TO HEXAPLOID WHEAT THROUGH INDUCED

HOMOEOLOGOUS PAIRING

P. CHHUNEJA1 , S. KAUR1 , R.K. GOEL1 , M. AGHAEE-SARBARZEH AND H.S. DHALIWAL2

1 Dept. Plant Breeding, Genetics and Biotechnology, Punjab Agricultural University, Ludhiana 141 004, India 2 Indian Institute of Technology, Roorkee, India E-mail: pchhuneja@rediffmail.com

Abstract:

First alien leaf rust resistance gene Lr9 was transferred from Ae. umbellulata into hexaploid wheat in the year 1956 through irradiation induced translocation. A number of other genes were subsequently transferred from non-progenitor and progenitor Aegilops species and exploited commercially. However, the appearance of new virulences necessitates the search of novel sources of rust resistance. An Ae. umbellulata acc. 3732 was found to be resistant to several wheat diseases such as leaf rust, stripe rust, Karnal bunt, powdery mildew and cereal cyst nematode. An amphiploid (AABBUU) synthesized from the cross of Ae. umbellulata acc. 3732 and T. durum cv. WH890 was crossed to CS PhI for inducing homoeologous pairing between Ae. umbellulata and wheat chromosomes. The F1 (AABBDU) was crossed to a susceptible hexaploid wheat cv. WL711 and introgression lines carrying resistance to leaf and stripe rust were selected in backcross progenies. The BC2F1 plants with leaf and stripe rust resistance genes were selfed, and homozygous lines with least linkage drag were selected and screened against five leaf rust and three stripe rust pathotypes. The introgression lines were resistant to all the leaf rust pathotypes and stripe rust pathotypes. Transfer of Lr9 was ruled out by screening the introgression line with 77-7, virulent on Lr9. For studying the inheritance of transferred rust resistance genes, an F2 population was generated by crossing the introgression line with the recipient parent WL711. Screening of F2 population at the seedling stage against leaf rust pathotype 77-5 revealed that a single dominant gene governs the resistance at seedling stage. The F2 plants screened at the seedling stage were grown in the field and screened at adult plant stage against a mixture of pathotypes under artificial epiphytotic conditions. All plants that were resistant at the seedling stage maintained resistance at adult plant stage. Out of a total of 48 susceptible seedlings, 28 showed resistance at adult plant stage indicating the presence of adult plant resistance (APR) gene as well. The population segregated for two genes for leaf rust, 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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rust resistance has been transferred to hexaploid wheat. The introgression lines were analysed with SSR markers for identifying the introgressed regions. The introgressions were detected for chromosomes of homoeologous group 2, 4 and 5. Bulk segregant analysis 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 two major rust diseases of wheat. Leaf rust or brown rust caused by the fungal pathogen Puccinia triticina is one of the most common diseases affecting wheat production worldwide. The impact of leaf rust on yield reduction in wheat ranges from 10 percent under moderate conditions to 65 percent under intense epidemics (Saari and Prescott 1985). An effective, economical and ecologically safe method to control leaf and stripe rust epidemics is the cultivation of resistant cultivars. More than 50 leaf 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 50 genes, 20 are of alien origin having been introgressed into the wheat genome from wild relatives like Agropyron and Aegilops spp. (Kolmer 1996). However, a number of genes transferred from related species of wheat viz. Lr9, Lr19, Lr24, Lr26, Yr 9 and Pm8 etc. and exploited commercially, have been overcome by the emergence of virulent pathotypes, therefore, necessitating the search for new sources of resistance. Less closely related Aegilops species especially non-progenitor C, U and M genome species 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 transferred from non progenitor species have been commercially exploited due to substantial amount of undesirable genetic information (linkage drag) associated with useful genes and yield reduction (Jiang et al. 1994). Chen et al. (1994) transferred the dominant homoeologous pairing inducer PhI gene from Aegilops speltoides high pairing line to hexaploid wheat cultivar Chinese Spring (CS). This gene suppresses the effect of the Ph 1 locus and induces homoeologous pairing between wheat and 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. ovata chromosome 5M to wheat chromosome 5D through induction of homoeologous pairing with CS PhI (Aghaee-Sarbarzeh et al. 2002). Evaluation of different accessions of wild Triticum and Aegilops species maintained at the Punjab Agricultural University, Ludhiana, Punjab (India) has led to 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, Gill et al. 1995, Dhaliwal and Harjit-Singh 1997, Harjit-Singh et al. 1998, Harjit-Singh and Dhaliwal 2000). Aegilops umbellulata, a diploid species with UU genome, was

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found to be highly resistant to different wheat diseases such as leaf rust, stripe rust, Karnal bunt, powdery mildew and cereal cyst nematode. An accession of Ae. umbellulata with multiple disease resistance was selected and leaf and stripe rust resistance has been transferred from this accession to cultivated wheat through induction of homoeologous pairing using CS PhI . In the present communication, the transfer of leaf rust and stripe rust resistance from Ae. umbellulata through induction of homoeologous pairing and molecular characterization of introgression lines using SSR markers is being reported. MATERIAL AND METHODS An amphiploid was synthesized between T. durum cv. WH890 and Ae. umbellulata accession 3732 and crossed with T. aestivum cv Chinese spring carrying PhI gene of Ae. speltoides to induce homoeologous pairing. The F1 plants from this cross were selfed and also crossed to a rust susceptible T. aestivum cv. WL711 having non-necrotic gene WL711(NN). All BC1 plants were screened at the seedling stage for leaf and stripe rust. The resistant plants were backcrossed to WL711(NN) to recover the recurrent genotype. Homozygous BC1 F4 and BC2 F4 introgression lines with leaf and stripe rust resistance transferred from Ae. umbellulata have been selected. Leaf and Stripe Rust The rust resistant genes were followed in the segregating generations by screening against leaf rust pathotypes 77-5 and 104-2 and stripe rust pathotype 46S119 at the seedling stage. The resistant plants were transplanted in the field and terminal disease severity recorded for leaf and stripe rust. The homozygous leaf and/or stripe rust resistant introgression lines were tested with five most virulent and prevalent pathotypes of leaf rust 12-2, 77-2, 77-5, 77-7, and 104-2, and three pathotypes of stripe rust 46S119, 46S102 and 46S103 at the seedling stage and under artificial epiphytotic conditions in the rust screening nursery in the field. Seedling reaction was recorded in parents and introgression lines using the standard inoculation procedure (Nayar et al. 1997). Infection type was recorded 14 days after inoculations following the modified 0; -4 scale of Stakman et al. (1962). The disease severity under field conditions was recorded as percentage of leaf area covered by rust following modified Cobbs scale (Peterson et al. 1948). Molecular Studies SSR markers were used to detect alien transfer, in the homozygous wheatumbellulata introgression lines. The PCR was performed as described by Rder et al. (1998) with some modifications. PCR products were resolved by electrophoresis in 2.5% agarose gels. Gels were visualized by staining with ethidium bromide using UVP Gel Documentation System.

86 RESULTS AND DISCUSSION Leaf and Stripe Rust Screening

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All the selected ILs showed resistance to the four test leaf rust pathotypes at the seedling stage and remained free from leaf rust in the rust screening nursery at PAU, Ludhiana (Table 1) and off season nursery at Wellington, Neelgiri Hills, India (data not given). A leaf rust resistance gene transferred from U genome of Ae. umbellulata (Sears 1956) is present on chromosome 6BL as a compensating transfer. To establish that the leaf rust resistance gene present in these introgression lines is different from Lr9, some of the lines were tested with Lr9 virulence 777. Three of the selected ILs 329-1, 333-4 & 367-4, however, showed susceptible infection type to 77-7 at the seedling stage indicating that these ILs might be carrying Lr 9. All the introgression lines except 329-1, 333-4, 367-4, 380-3 were found to be 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 stripe rust resistant F1 plants were selfed to develop an F2 for studying the inheritance of leaf rust resistance in this IL. The F2 was screened at the seedling stage against leaf rust pathotype 77-5 and all the plants were then transplanted in the field and studied for leaf rust reaction at the adult plant stage. The population segregated for single gene for leaf rust at the seedling stage with a 2 of 3.5 and for two genes for leaf rust at the adult plant stage with a 2 (15:1) of 12.5. However, out of 48 susceptible plants at the seedling stage, 28 plants were observed to be resistant at the adult plant stage indicating that an APR gene for leaf rust is present in addition to the seedling resistance gene. The progeny of another introgression line 351-1 was also found to be segregating for one seedling and one APR gene for leaf rust resistance (Table 2). SSR Analysis A total of 100 SSR markers were amplified in the parents and 19 primers were found to be polymorphic. 13 primers did not amplify in umbellulata and hence were not used for genotyping of introgression lines. The analysis of the wheat-Ae. umbellulata introgression lines using Xwmc and Xgwm SSR markers led to the identification of introgressed segments of umbellulata chromatin in homoeologous group 2 in different introgression lines (Fig. 2). The 2AL/2DL mapped SSR marker Xwmc181 detected the introgression in the progenies of 315-5, 351-1, 388-5 and 403-1. The Xwmc181 was amplified in the two leaf and stripe rust resistant and one susceptible progeny derived from IL 351-1. The introgression was detected in the progeny carrying seedling resistance gene for leaf rust (lane 16) and wheat type allele 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

Table 1. Leaf and stripe rust reaction of the selected introgression lines from the cross T. durum c. WH890-Ae. umbellulata acc. PAU#3732/CS (PhI )//WL711(NN) Leaf rust-seedling 12-2 3 ; 33+ 33+ 3 ; 3 3 3 ; 3 3 3 ; 3 3 ; 3 3 3 0; 3 3 3 ; 3 3 3 ; 3 3 60S 0 20S 10S 40S 77-2 77-5 77-7 104-2 46S119 46S102 46S103 Leaf Stripe 80S 0 20S 10S 40S Stripe rust-seedling Rust reaction-Field

S. No.

IL ID

Generation

1 2 3 4 5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 ; ;1 ;1 ; ;1 ;1 1 0; ; 0; ;1 0; ; 0; ; ;1 3 ;1 ; ; ;1 ; 0; 0; ;1 ;1 3 ;3+ 3+ 1 ; 33+ ;1 ; 2 ; ; 1 1 3 1 ; ; ;1 ; ; ; ;1 ;13 3 3 ; 0; ; ; ; ; 3 3 0; ; 0; ; ; ; ; ; ; 3 ; -

WL711 Ae. umbellulata CS PhI WH890 T. durum-Ae. umbellulata amphiploid 315-5 329-1 333-4 339-4 351-1 351-5 353-2 354-4 357-1 359-1 367-4 380-3 388-5 393-4 403-1 0; 0; ; ; ; 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

F7 F7

BC1 F7 BC1 F6 BC1 F6 BC1 F6 BC1 F6 BC1 F6 BC1 F6 BC2 F5 BC3 F4 BC1 F6 BC2 F5 BC2 F5

0 80S 40S 0 5MR 0 0 0 0 0 40S 40S 0 0 0

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

Table 2. Segregation for leaf rust resistance in the F2 developed from the cross of IL403-1 with recipient parent WL711 Resistant plants Seedling Adult plant 104 132 Susceptible plants 48 20 Segregation ratio 3:1 15:1
2

3.5 12.5

Figure 2. PCR amplification profile of SSR marker Xwmc181 in the homozygous resistant progenies of 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 of 351-1 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 and 403-1 (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

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were also analysed with Wmc181. The introgression was detected in all the resistant progenies and only wheat type allele was amplified in susceptible progeny. The amplification of wheat as well as umbellulata specific alleles of the 2A/2D mapped Xwmc181 in resistant introgression lines and progenies indicates the presence of introgressed segment from 2U of Ae. umbellulata on the either chromosome 2A or 2D as a compensating transfer. The work to define the introgressed regions and to tag the genes using F2 population is in progress. Results indicated the precise transfer of one seedling and one APR gene for leaf rust and one seedling resistance gene for stripe rust from Ae. umbellulata acc. 3732 to hexaploid wheat cultivar WL711 and the introgression of Ae. umbellulata chromatin, carrying these genes, has been detected on wheat homoeologous group 2. The leaf and stripe rust resistance genes transferred from Ae. umbellulata are novel genes which can be exploited for broadening the genetic base of cultivated wheats for rust resistance. The identification of the closely linked molecular markers may 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 Grant Number FG-In-792 to carry out the research work is gratefully acknowledged. The rust inoculum was provided by Director, Regional Research Station, Directorate of Wheat Research, Flowerdale, Shimla, India. REFERENCES
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