Construction Of Genomic Library And Characterization Of Resistant Gene Pihk1(T) Locus In A Rice Blast-resistant Cultivar Hekezijing

Blast, caused by the filamentous ascomycete Mangnaporthe grisea (Hebert) Barr, is one of the most devastating rice diseases worldwide. Using the transgenic and Marker Assist Selection (MAS) technology in rice breeding program, specific resistance genes, ordinary resistance genes and quantity trait resistance genes can be transferred into landraces which possess better agricultural traits, thus speed up the breeding process, enhance landraces resistance and yield stability.Heikezijing is a rice cultivar in Taihu area with broad-spectrum and high resistance to blast fungus. According to previous study, the blast resistant gene from Heikezijing was mapped on chromosome11between flanking SSR marker RM7654and In5, and was named Pihkl (t).Based on the method of bacterial artificial chromosome libraries construction by Danial G. Peterson, some modifications were made to meet the need of BAC library construction of rice and then we established a high-efficient technology system of BAC libraries construction. Utilizing improved methods, taking pIndigoBAC5as vector, the BAC library contained39936clones with a mean insert size of about70Kb. Based on the rice haploid genome size of430Mb, the BAC library was estimated to have6.5folds genome coverage.The BAC library was consisted of104384-well plates and we made a replicate of the master for long-term storage. All the clones were used to develop a3-dimensional PCR screening system. For facilitating BAC library screening, the whole library was arranged in two levels of pools(column pools and the super pools). Screening the super pools with markers RM7654and in5, which tightly linked to the resistant gene loci,4positive clones were detected. By sequencing the end of the positive clones, we constructed the physical map cover the gene locus with totally6clones. The BAC clone No.242and No.66P14were selected as the candidate gene clone for sequencing. The picked clones were sequenced by shotgun and a contig sequence of140Kb was obtained. Compare to published rice genome sequence, a5-kb insertion was found in Heikezijing between markers RM7654and In5, which contained a retrotransposon-like sequence. Two NB-ARC gene were predicted in this region, which were considered as the candidate genes for next cloning. Comparison of the sequences BAC clone N0.66P14with the Nipponbare sequence of the equivalent region revealed that Heikezijing genome lacked a30-kb segment present in the Nipponbare genome and also existed a10-kb segment. Another NB-ARC gene was predicted in this region, which was different from the allele in Nipponbare.