Construction Of A SSH Library Of Wheat Leaves Induced By Puccinia Striifomis And Its ESTs Analysis

Stripe rust, caused by Puccnia striiformis Westend f.sp.tritici Eriks, is one of the most devastating wheat diseases worldwide. Development and utilization of resistant cultivars is the most economic, effective and ecological approach to control the disease. However, many cultivars with stripe rust resistance genes have lost resistance to new races because of the co-evolution of host resistance and pathogen virulence. T. aestivum-H. villosa 6VS/6AL translocation lines were highly resistant to stripe rust with resistance gene Yr26 located on chromosome 1B. Cloning of Yr26 gene will play an important role in future disease resistance breeding. In this research, a SSH (suppression subtractive hybridization) library using the leaves of resistant and susceptible plants from advanced generation of 92R137 backcrossed with Yangmai 5 inoculated by Puccinia striiformis f. sp. Tritici.had been constructed.3082 positive clones in the SSH library had been screen out, from which 70 non-redundant ESTs have been obtained after sequencing. Subsequently, similarity analysis based on BLASTx and BLASTn softwares in GenBank was taken. The results showed that 18 of them might be related to disease resistance,8 of them might be related to stress response,6 of them might be related to transcriptional regulation,9 of them had significant similarity with the metabolic and membrane protein genes,8 of them were related to electronic transformation and cell construction. The other 20 ESTs have been found no significant similar genes which may represent new genes or high variant non-coding regions of cDNAs.After analyzing by semi-quantitative RT-PCR using Yangmai 5 and 92R137 cDNA which inculated with Puccinia striiformis f. sp. Tritici at different hs as templates, the expressing patterns of 15 ESTs were revealed.Among which 8 ESTs were up-regulated,3 ESTs were down-regulated, and 4 ESTs were constitutive expressed. HSSH17, which has significant similarity with the heat shock protein gene, its expression was much higher in 92R137 than that in Yangmai 5 after inoculation.Nine ESTs'homologous genes were mapped on the chromosomes of homoeologous group 1,2,3,5. Using Chinese Spring nulli-tetrasomic and deletion lines.homologous genes of HSSH71 were mapped on 5A and 5D; Homologous genes of HSSH12 were mapped on the FL 0.71-1.0 of 5BS; Homologous genes of HSSH16 were mapped on the FL 0.77-1.0 of 2AL and FL 0.69-0.89 of 2BL; Homologous genes of HSSH17 were mapped on the FL 0.31-0.63 of 3BL; Homologous genes of HSSH19 were mapped on the FL 0.65-0.69 of 2BL and 0.49-0.76 of 2DL; Homologous genes of HSSH28 were mapped on the FL 0.42-0.78 of 3AL; Homologous genes of HSSH61 were mapped on the FL 0.63-1.0 of 3BL; Homologous genes of HSSH58 were mapped near to centromere.Six STS markers of Yr26, WE171, WE173, WE177, WE201, WE202 and WE210, which were derived from ESTs on chromosome 1B-6 bin, had been developed by Wang. BLASTing these ESTs to the genome sequence of Brachypodium distachyon,we found Super₈ of Brachypodium distachyon had high similarity to 1BL-6. Many primers of RGAs in Super₈ were designed, and primer HJ-9 could amplify polymorphic fragments between 92R137 and Yangmai 5. Then,HJ-9 was used to amplify the DNA of individuals in Yangmai 5/92R137 F2 population. Most of the susceptible plants amplified the bands as same as that from the susceptible parent and most of the resistant plants amplified the bands as same as that from the resistant parents.The result shows that marker HJ-9 was closely linked to Yr26, and was located between WE173 and WE210.