Microdissection Of H. Villosa 6VS Conferring Resistance To Wheat Powdery Mildew And Cloning Of Disease Resistance Related Genes

Powdery mildew caused by Eiysiphe. graminis f. sp. tritici is one of the most important diseases in China. Breeding for resistant cultivars has been proved the most effective way to control this disease, and study of disease resistance genes is the foundation to carry on the resistance-breeding program successfully. At present, 29 loci with 45 main effective genes were named. In the present study, we report the construction and screening of 6VS-specific library by microdissection and microcloning, cloning the resistance gene analogs (RGA) from 6VS, construction and screening of cDNA library of Taestivum-H villosa translocation line. The main results are followings:1.The monotelosomic substitution line H 1-2 and ditelosomic line H 1-2-6 were regenerated from young embryo of RW1 5/CA92 11 hybrids. The 6VS-telosome lines was screened and identified by resistance test, cytogenetics analysis and genomic in situ hybridization.2.The 6VS was microdissected with needle and transferred into a 0.5 ml Ep tube. In the 搒ingle tube?, all the subsequence steps were conducted. After two round of LA-PCR amplification, the size of PCR band ranged from 100 to 3000bp, with predominate bands 600?500 bp. The products were confirmed that originated from the H J>2llosa(L.) Schur by Southern blot using H T扽llosa (L.) Schur genomic DNA labeled with 32P as probe. The PCR products were purified and ligated with clone vector-pGEM-T easy Vector. Then the plasmids were transformed into competence B. coli JM1O9 with cool CaC12.3.It was estimated that there were more than 17000 white clones in the library. The size of insert fragments distributed from 100?500 bp, with average of 600 bp. Using H villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It demonstrated that there were about 37% repeat sequence clones and 63% single/unique sequence clones in the library. Five H villosaspecific insert repeat sequence clones, pHVMK 134 .. pHVMK137pHVMK125~ pHVMKI3O and pHVMK67 were screened from the library. 3 Hvillosa-specific insert single/low copy sequence clones, pHVMK 22~IIIpHVMK15 and pHVMK129 were also screened from the library.Among them, pHVMK22 may be applied to detect the exist of Pm2 1.4.The degenerate oligonucleotide primers were designed according to the tobacco N, Arabidopsis RPS2, rice Xa2 1, tomato Pto, and L6 , PCR amplification was carried out from 6VS genomic DNA. 300 clones were isolated and 10 types of disease resistance gene analogs were characterized. They were designated as Hvrgakl-Hvrgakl0, and GenBank accession numbers are AF3871 13AF387121, AY040671- AY040672. Identity among RGAs was about 10-50%, and identity with cloned R gene from plants was 5-20%. Southern hybridization analysis showed three RGAs, Hvrgak2, Hvrgak4, and Hvrgak5 were linked to wheat powdery mildew resistance.5.cDNA library from the leaf of Taestivum-H.~villosa translocation line was constructed. It contained 3 X 1 o~ pfulml. The size of insert fragments distributed from 0.4--4.3kb, with average of 1.8kp.6.The library was screened by the RGA probe of Hvrgak4. Four positive clones were obtained among 1.2 X 106 clones in the library through two rounds southern hybridization. These four clones are kongl, kong28, kong32, and kong36 with insert fragments of 2.9kb, 1.3kb, 1.2kb, and 1.1kb. Sequencing of these four clones is being conducted.7.The different display with primer pairs of anchor primer and degenerate oligonucleotide primer designed according to the plant disease resistance genes was carried out. The 10 different eDNA fragments among leaves of Ohour, 24hours and 48hours after induced with Erysiphe. graminis f. sp. tritici were collected and cloned. Among them, two clones R3-1 and SC1 .3-6 showed positive signals in Northern blot. P2-i has 840bp and SC 1.3-6 587bp. Blast analysis showed R3-1 had no disease resistance sequences with high high identity in GenBank. 8C1 .3-6 had 10...