Chhuneja P

Dhaliwal HS

Kaur A

Kaur S

Singh K

English

Sydney eScholarship

 1A novel leaf rust resistance gene transferred from Aegilops caudata L. to Triticum aestivum L. maps on chromosome 5D  Chhuneja P1, Kaur A1, Kaur S1, Dhaliwal HS2 and Singh K1 1School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana 141 004, India, 2Indian Institute of Technology Roorkee, Roorkee, India  ABSTRACT Leaf rust and stripe rust are the two most important foliar diseases of wheat worldwide. Host genetic resistance is the most economical and environmentally friendly approach to reduce the losses due to rust diseases in wheat.  The germplasm of non-progenitor Aegilops species have been found to be an invaluable source for new resistance genes. The transfer and mapping of a new leaf rust resistance gene from Ae. caudata L. is reported here. A leaf rust resistant accession PAU #3556 of Aegilops caudata was crossed with T. durum and an amphiploid synthesized for transferring leaf rust resistance to T. aestivum through induced homoeologous pairing. Leaf rust resistant introgression lines (ILs) were developed through backcrossing with a leaf rust susceptible cultivar WL711. An F2 population derived from one of the introgression lines segregated for a single leaf rust resistance gene at the seedling (208R:78S; χ2 =0.79) and adult plant stage (219R:67S; χ2 =0.37). The testing of F3 progenies confirmed the transfer of a single gene for leaf rust resistance. Bulk segregant analysis was conducted using 101 polymorphic D-genome specific SSR markers.  The SSR markers Xcfd18, Xcfd189, Xcfd78 and Xfd81 detected Ae. caudata  specific alleles in the parental introgression line and the resistant bulks. All these markers mapped on distal region of 5DS indicated a terminal translocation.  Analysis of the F2 population mapped Ae. caudata leaf rust resistance gene on chromosome 5DS with Xcfd18 as the closest  linked marker.  INTRODUCTION  Wild species of wheat are a reservoir of variability for several biotic and abiotic stresses. Genes especially for disease resistance transferred from wild species have provided an insurance cover for sustaining wheat production globally. Among the wheat diseases, leaf rust, stripe rust and stem rust are the most important worldwide. A total of 60 genes for leaf rust, 40 for stripe rust and 45 for stem rust have been designated so far and out of these 30 genes for leaf rust, 12 for stripe rust and 18 for stem rust resistance have been introgressed from wild progenitor and non-progenitor species (McIntosh et al. 2007). Although a number of genes have been introgressed from wild Triticum and Aegilops species, only a few have been commercially exploited due to substantial linkage drag. The T1BL.1RS translocation carrying complete IRS arm from Secale cereale, is the most successful example of utilization of the alien variability in wheat (Hoisington et al. 1999).   Most of the commercially exploited genes condition hypersensitive reaction and interact with the pathogens in gene-for-gene fashion. Deployment of such genes over large areas leads to the development of new virulence in the pathogen. A large number of these genes have been rendered ineffective due to the emergence of virulent races of the pathogens necessitating the constant search and transfer of new and effective sources of rust resistance to counter balance the continuous evolution of rust populations. Wild progenitor and non-progenitor species of wheat are highly valuable source of additional resistance genes (Gale and Miller 1987; Jiang et al 1994; Friebe et al. 1996; Singh et al 1998; Singh et al 2007). In our laboratory leaf rust and stripe rust resistance genes have been transferred from Aegilops geniculata (Lr57 and Yr40), Ae. triuncialis (Lr58) and Ae. umbellulata (LrU1, LrU2, YrU1) (Kuraparthy et al 2007a,b; Chhuneja et al. 2007).  Two new stripe rust resistance genes from T. monococcum and T. boeoticum have been mapped and transferred to cultivated wheat (Chhuneja et al. 2008).  Ae. caudata, a non-progenitor diploid species with the CC genome, was also found to be an excellent source of resistance for various diseases. In the present study we report the transfer of a leaf rust resistance gene from Ae. caudata accession pau#3556 to hexaploid wheat and its inheritance and mapping in hexaploid wheat background.  TRANSFER OF LEAF RUST RESISTANCE FROM AE. CAUDATA A leaf rust and stripe rust resistant Ae. caudata acc. pau3556, was used for developing a synthetic amphiploid with T. durum cv. WH868. The amphiploid was crossed with Chinese Spring {CS(PhI)} to induce homoeologous pairing. The F1 plants were crossed with a leaf rust susceptible genotype, WL711(NN). The resistant F1 plants from the cross T. durum WH868-Ae. caudata//CSPh1/3/WL711(NN) were then selfed and homozygous resistant introgression lines were selected in advanced self generations. In the segregating generations, selection for leaf rust resistance was carried out by screening at the seedling stage against two most virulent and prevalent leaf rust pathotypes (121R63-1 and 21R5) using the standard inoculation procedure (Nayar et al., 1997). The adult plant screening was conducted against a mixture of pathotypes and data was  2 recorded according to modified Cobb’s scale (Peterson et al. 1948).  Recipient parent WL711 (NN) and CS (PhI) were susceptible to the leaf rust pathotypes 121R63-1 and 21R5 at the seedling stage. WL711 developed 60S-80S leaf rust at the adult plant stage while CS (PhI) developed 20S leaf rust.  Hexaploid and cytologically stable homozygous WL711-Ae. caudata introgression lines, however, were resistant at the seedling as well as adult plant stage (Fig. 1).               Fig. 1.  Leaf rust reaction of the WL711-Ae. caudata introgression lines and F2 plants derived from the cross WL711-Ae. caudata/PBW343  INHERITANCE STUDIES FOR LEAF RUST RESISTANCE  An F2 population was developed by crossing a selected WL711-Ae. caudata introgression line with a widely grown wheat cv. PBW343 for studying the inheritance of leaf rust resistance transferred from Ae. caudata. The  F1 of WL711-Ae. caudata/PBW343 was resistant at both seedling and adult plant stage indicating that the resistance was controlled by a dominant gene. The F2 population segregated into 208R:78S (χ2 3:1=0.79) and 219R:67S (χ2 3:1=0.37) at the seedling and adult plant stage, respectively (Table 1). Progeny testing in F3 also showed 1:2:1 segregation, thereby confirming that single dominant gene transferred from Ae. caudata conditioned leaf rust resistance.  Table 1. Segregation of leaf rust (P. triticina) resistance in an F2 from the cross of WL711-Ae. caudata  introgression line with T. aestivum cv. PBW343 1Seedling screening was done with leaf rust pathotype 121R63-1 2Adult plant screening was done with a mixture of pathotypes 109R31-1, 121R63-1 and 21R5  MOLECULAR CHARACTERIZATION OF AE. CAUDATA INTROGRESSION LINES For characterizing the introgression carrying leaf rust resistance gene of Ae. caudata transferred to bread wheat, bulk segregant analysis based on the F2 population was conducted. The DNA of the parents, WL711-Ae. caudata introgression line and F2 plants was isolated using CTAB method. The DNA of the 10 resistant and 10 susceptible F2 plants was bulked to generate resistant ‘R’ and susceptible ‘S’ bulks. For molecular analysis only D-genome specific SSR markers were used because in the F1 derived from the cross of T. durum-Ae. caudata amphiploid with CS (PhI) only C-genome chromosomes of Ae. caudata and D-genome chromosomes of CS were present as univalents and hence had higher probability of pairing.  A total of 168 SSR markers were tested on the parents and 101 markers were polymorphic which were amplified on the parents, wheat-Ae. caudata introgression line, ‘R’ and ‘S’ bulks. Ae. caudata specific introgressions were detected on chromosome 4D, 5D, 6D and 7D in the wheat-Ae. caudata introgression line. However, only 5D mapped SSR markers Xcfd18, Xgwm190, Xcfa2104, Xcfd189,  Xgwm205, Xcfd78 and Xfd81 showed Ae. caudata specific amplification in the introgression line as well as the resistant bulks indicating that the leaf rust resistance gene of Ae. caudata has been transferred to wheat chromosome 5D.  Graphical genotyping of the introgression line showed that the almost complete short arm of chromosome 5D has been transferred from Ae. caudata with translocation break point near the centromere  (Fig. 2).      Figure 2. Graphical genotyping of a WL711-Ae. caudata introgression line for chromosome 5D based on SSR markers.  Black areas indicate Ae. caudata specific introgression and grey areas indicate wheat specific alleles. Map distances are according to the Somers et al. 2004.              Resistant Susceptible Total χ2 Leaf rust-seedling1 208 78 286 0.79 Leaf rust-Adult plant2 219 67 286 0.37 Xcfd81 WL711 IL F2 plants WL711 IL F2 plants  3 MAPPING OF AE. CAUDATA LEAF RUST RESISTANCE   SSR markers Xcfd18, Xcfd189, Xcfd78 and Xfd81 showing introgression in the resistant bulks were amplified on the F2 population for studying the co-segregation of these markers with the leaf rust resistance gene transferred from Ae. caudata (Fig. 3). A total of 254 F2 plants were analysed with these four markers. PCR products for Xcfd18, Xcfd78 and Xcfd189 were resolved on  Agarose gel while Xcfd81 was resolved on 8% PAGE. All the markers co-segregated with the leaf rust resistance.  Ae. caudata is a non-progenitor species and hence no recombination was expected within the introgressed segment.         Fig. 3. PCR amplification of the F2 population derived from the cross WL711-Ae. caudata IL/PBW343 for SSR marker Xcfd81. PCR products were resolved in 8% PAGE and visualized on the LICOR sequencer. Lane 1- WL711; Lane 2-PBW343; Lane 4-WH868, Lane 5-  WL711-Ae. caudata IL and Lane 6-64: F2 plants.     During the present investigation a leaf rust resistance gene has been transferred from Ae. caudata pau3556 to cultivated wheat and the introgression carrying this resistance has been mapped to wheat chromosome 5D.   REFERENCES  Chhuneja P,  Kaur S,  Goel RK,  Aghaee-Sarbarzeh M,  Prashar M, Dhaliwal HS, (2007) Transfer of leaf rust and stripe rust resistance from Aegilops umbellulata Zhuk. to bread wheat (Triticum aestivum L.). Genet. Resour. Crop Evol. DOI 10.1007/s10722-007-9289-3. Chhuneja P,  Kaur S,  Garg T, Ghai M,  Kaur S, Prashar M,  Bains NS, Goel RK, Keller B, Dhaliwal HS, Singh K. (2008)  Mapping of adult plant stripe rust resistance genes in diploid A genome wheat species and their transfer to bread wheat. Theor Appl Genet. 116:313–324 Friebe B, Jiang J, Raupp WJ, McIntosh RA, Gill BS (1996) Characterization of wheat-alien translocations conferring resistance to diseases and pests: current status. Euphytica 91:59–87 Gale MD, Miller TE (1987) The introduction of alien genetic variation into wheat. In: Lupton FGH (ed) Wheat Breeding: its Scientific Basis, Chapman and Hall, UK, pp 173–210  Hoisington D, Khairallah M, Reeves T, Ribaut JM, Skovmand B, Taba S, Warburton M (1999) Plant genetic resources: What can they contribute toward increased crop productivity? Proc. Natl. Acad. Sci. USA. 96: 5937–5943 Jiang J, Friebe B, Gill BS (1994) Recent advances in alien gene transfer in wheat. Euphytica 73: 199–212 Kuraparthy V, Chhuneja P, Dhaliwal HS, Kaur S, Bowden RL, Gill BS (2007a) Characterization and mapping of cryptic alien introgression from Aegilops geniculata with novel leaf rust and stripe rust resistance genes Lr57 and Yr40 in wheat. Theor Appl Genet 114:1379–1389 Kuraparthy V, Sood S, Chhuneja P, Dhaliwal HS,  Kaur S, Bowden RL, Gill BS (2007b) A cryptic wheat-Aegilops triuncialis translocation with leaf rust resistance gene Lr58. Crop Science 47:1995-2003 McIntosh RA, Devos KM, Dubcovsky J, Rogers WJ, Morris CF, Appels R, Somers DJ, Anderson OA (2007) Catalogue of gene symbols for wheat: 2007 supplement. In: KOMUGI–Integrated Wheat Science Database. (http://www.grs.nig.ac.jp/wheat/komugi/ genes/macgene/supplement2007.pdf) Nayar SK, Prashar M, Bhardwaj SC (1997) Manual of current techniques in wheat rusts. Research Bull No.2, 32pp Regional Station, Flowerdale, Shimla 171002, India Peterson RF, Campbell AB, Hannah AE (1948) A diagrammatic scale for rust intensity on leaves and stems of cereals. Can J Res 26: 496–500 Singh H, Grewal TS, Dhaliwal HS, Pannu PPS, Bagga PS (1998) Sources of leaf rust and stripe rust resistance in wild relatives of wheat. Crop Improvement 25:26–33 Singh K, Chhuneja P, Ghai M, Kaur S, Goel RK, Bains NS, Keller B, Dhaliwal HS (2007) Molecular mapping of leaf and stripe rust resistance genes in Triticum monococcum and their transfer to hexaploid wheat. In: Buck H, Nisi JE and Solomon N (eds) Wheat production in stressed environments. Developments in Plant Breeding, Vol. 12. Springer, Netherlands, pp 779–786  Somers DJ, Isaac P, Edwards K (2004) A high-density microsatellite consensus map for bread wheat (Triticum aestivum L.). Theor Appl Genet 109:1105–1114  

A novel leaf rust resistance gene transferred from Aegilops caudata L. to Triticum aestivum L. maps on chromosome 5D

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A novel leaf rust resistance gene transferred from Aegilops caudata L. to Triticum aestivum L. maps on chromosome 5D

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