Development And Application Of Resistance Gene Chromosome Novel Molecular Markers To Powdery Mildew And Isolation Of Resistance Gene Fragments Containing NBS Domain In Dasypyrum Villosum

Dasypyrum villosum, wheat related species, including many excellentcharacteristics is an important material for wheat breeding and variety improvement.Research showed that, the resistant reaction in Dasypyrum villosum varied from originto origin, and existing DNA polymorphism. Dasypyrum villosum NO.1026is adiploid and origins of former Union of Soviet Socialist Republics. Triticumdurum-Dasypyrum villosum amphidiploid that derived from Dasypyrum villosumNO.1026showed outstanding grain quality, immuned to powdery mildew, and hadhigh resistance to take almost all disease characteristics in the filed, which was aexcellent gene resource to researchers. On the one hand, in this study, the probe ofSourtern Blot had been designed from the promoter region of Stpk, which was a keyPm21resistance gene to powdery mildew. And Southern Blot analysis had been usedto analysis two different origins of Dasypyrum villosum. On the other hand, in orderto further analysised and determined the number of Dasypyrum villosum NO.1026chromosome respectively into wheat, according to the EST sequence and SSR markerof Triticum durum, Hordeum vulgare,and Brachypodium distachyon, we develepedthe chromosome specific molecular marker of Dasypyrum villosum. At the same time,according to the conserved sequences of NBS-LRR resistance genes, we designedprimer pairs to screen and identify the chromosome specific molecular marker ofDasypyrum villosum in present study. And we used the chromosome arm6VS specificmarkers of Dasypyrum villosum to check the progeny population which transferedT6VS·6AL resistance gene to powdery mildew. We made progress as follows:(1) Sourtehrn Blot analysis showed that, Stpk-v gene had two copies in Dasypyrumvillosum NO.1026and a single copy in Haynaldia villosa RM3159, which proved thepolymorphism of DNA sequence and copies to exist in different sources ofDasypyrum villosum once again;(2) According to linear relationship of homologous chromosome in the wheatrelative species, we developed24pairs PCR markers of chromosome specific ofDasypyrum villosum by using their EST sequences and SSR markers, including1V, 2V,3V4V,5V,7V chromosome and6VS chromosome arm of Dasypyrum villosum. Asingle plant of BC1segregating population which deprived from Triticumdurum-Dasypyrum villosum amphidiploid had been detected to use those markers,combing with genmic in situ hybridization analysising and identifying the number ofchromosome from Dasypyrum villosum to them. As a result, Triticum durum-Dasypyrum villosum materials S8-1was carrying Dasypyrum villosum1VL and2VSchromosome arm, S2-1was carrying2VS and4V chromosome, S3-7was carrying6Vchromosome, and1V,2V,6V,3VS,4VS,7VS chromosomes were carried by S3-4.Those offered important information and were useful to marker assisted selection toidentify Triticum durum-Dasypyrum villosum materials which carried a single alienchromosome. What’s more, in this study, we also found some Triticum durum-Dasypyrum villosum materials which didn’t carry6VS chromosome arms showedresistance to powdery mildew in the filed. It means that new resistance genes maybeexist in Dasypyrum villosum NO.1026.(3) According to the conserved sequence of primers of R gene NBS domain, wedesigned primer pairs to amplify from Dasypyrum villosum gDNA and cDNA, and weacquired23Resistance Gene Analogs sequence, based on5sequences of them, wedeveloped some chromosome specific molecular marker of Dasypyrum villosum,which laid foundation for the further identification and isolation of resistance gene inDasypyrum villosum.(4) By using6VS chromosome specific molecular markers, we detected thebackcross progenies of Ningchun wheat which transferred from resistance gene fromCB037to powdery mildew. It showed that the ratio of separation were accorded with3:1in BC3F2population of Ningchun wheat, which was identical as resistancephenotype of them to powdery mildew in the filed. It provides the basis for theselection of disease resistant plants under the field conditions which are disandvage tothe occurance and prevalence of wheat powdery mildew.