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Targeting stem rust resistance genes Sr32 and Sr1644 for cloning by mutagenesis and sequence capture

Resistance offers the best means of control of the cereal rusts, but must be strategically deployed so as to avoid exposure of single major genes, which have faltered so many times in the past. The pyramiding of multiple effective resistance genes is a strategy that has proven effective in a number of wheat production areas around the world. However, the process of incorporating multiple resistance genes into a single cultivar using standard breeding techniques is time consuming, laborious, and hampered by the problem of linkage drag. If a suite of effective resistance genes could be efficiently cloned and transferred into wheat as a cassette, it would accelerate the development of durably resistant varieties without the problem of linkage drag. Toward this end, we have developed a resistance gene cloning technology based on resistance gene enrichment sequencing (RenSeq) of EMS-derived mutant R gene alleles. As a proof of concept test, we successfully ‘re’-cloned the already characterized gene Sr33 and are now targeting the cloning of eight other effective resistance genes. For the identification of susceptible mutants for the cloning of Sr32 from Aegilops speltoides, we screened 1,109 M2 families with race TPMKC — as a surrogate for race TTKSK. Five susceptible M2 mutants were confirmed by progeny testing. These mutants were also susceptible to race TTKSK. For the population involving Sr1644 from Ae. sharonensis, 1,649 M2 families were screened, yielding 33 M2 families that appeared to segregate for susceptibility. Thirteen of 33 families were confirmed as bona fide susceptible mutants by progeny tests in the M3 generation. Identification of susceptible EMS mutants of Sr32 and Sr1644 suggests that the underlying resistance in these lines is conferred by single genes. We will report on progress to clone and characterize these genes using R gene exome capture and sequencing technology (RenSeq).

John Innes Centre, UK
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Pyramiding genes Sr33, Sr35, Sr36, SrCad, and Sr43 for resistance to Ug99

Most of the current stem rust resistance genes (Sr) in Canadian wheat varieties are ineffective against the Pgt race Ug99 lineage, which pose a major threat to wheat production worldwide. Several stem rust resistance genes, including Sr33, Sr35, Sr36, SrCad/Sr42 and Sr43, are effective against race TTKSK. Although Sr36 is ineffective against Ug99 race TTTSK, it is still potentially useful for pyramiding genes to develop germplasm with durable stem rust resistance. For this purpose, we made crosses among RL5405 (Sr33), RL6099 (Sr35), Lang (Sr36), AC Cadillac (SrCad/Sr42), and RWG34 (Sr43) containing the respective Sr genes. A total of 54 doubled haploid (DH) lines were produced from the F1 from AC Cadillac/Lang//RWG34/RL5405, and 82 DH lines were obtained from RWG34/RL5405//RL6099. The DH progeny were tested at the seedling stage with race TTKSK and susceptible lines were discarded. We putatively developed 12 genotypes with multiple Sr gene combinations, including Sr33+Sr36+SrCad/Sr42+Sr43, Sr33+Sr36+SrCad/Sr42, Sr33+Sr36+Sr43, Sr33+SrCad/Sr42+Sr43, Sr36+SrCad/Sr42+Sr43, Sr35+Sr33+Sr43, Sr33+Sr36, Sr33+Sr43, Sr36+SrCad/Sr42, Sr36+Sr43, Sr35+Sr33, and Sr35+Sr43, based on positive association with  linked PCR markers. Another population with 63 DH lines was derived from (Hoffman*2/RL6099)//(Hoffman*2/Lang) to combine the Fusarium head blight (FHB) resistance of Hoffman (Fhb1) with Sr35 and Sr36. We found 17 of 63 DH lines containing both Sr35 and Sr36 based also on linked PCR markers. This indicated that the combination Sr35+Sr36 was pyramided into the Canadian cultivar Hoffman; this derivative will be useful for development of cultivars resistant to Ug99 and FHB in Canada.

Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Centre, Canada
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