Mapping And Cloning Of A Rice Blast Resistance Gene Pik～m

Rice blast (caused by Magnaporthe grisea) is one of the serious diseases of rice worldwide. It decreases rice yield and affects the quality of rice. Ultimately, utilization of resistance (R ) genes in rice breeding, which is friendly to the environment, has been the most effective and economical way to control this disease. However, many cultivars with one or few R genes are short-lived in environments conducive to the disease. One way to maintain the durability of the blast resistance is to pyramid several R genes into an elite cultivar (cv.) to provide multigenic resistance against a wide spectrum of blast races. Molecular marker-assisted selection (MAS) provides a rapid and effective method for genes detecting and pyramiding. On the other hand, R genes are crucial in plant-pathogen system. Isolating R genes will provide the bases for revealing mechanisms of plant resistance, plant-pathogen interaction, and host-pathogen co-evolution. Therefore, molecular mapping and developing linkage markers and cloning of the R genes will be important. The Pik^m gene in rice confers a stable resistance to many isolates of Magnaporthe grisea collected from Japan, Thailand and China. To effectively utilize this gene, we would like to map and clone this gene in this study.1. Primary localization of the Pik^m geneThis major dominant Pik^m gene was roughly mapped to the long arm of rice chromosome 11 with restriction fragment length polymorphic (RFLP) markers previously. In this chapter, through bulked-segregant analysis (BSA) in combination with recessive-class analysis (RCA), a linkage analysis using the publicly available PCR-based markers (STS, CAPS and SSR) mapped on the long arm was performed in a mapping population collected from one F₂ populations, which were derived from crosses between the japonica donor cv. Tsuyuake and a highly susceptible indica cv. AS20-1. The result showed that the Pik^m" gene was linked to the six SSR markers, RM5926, RM7443, RM2136, RM5766, RM144, RM254, and flanked by RM254 and RM144.2. Fine mapping of the Pik^m geneFor fine mapping of the Pik^m gene, an electronically physical map spanning the target region was constructed based on the sequences of BAC clones derived from cv. Nipponbare.Additional 10 PCR-based markers (STS and CAPS) were developed in the region defined by RM254 and RM144. Furthermore, the mapping population was enlarged through crossing the donor cv. Tsuyuake and three susceptible cvs. The Pild" gene was delimited to a 0.3 cM region flanked by K10 and K34, in which three markers, K27, K28, and K33, co-segregated with this gene. A high-resolution map of this gene was constructed.3. Gene prediction of the target region and making sure the candidate of the Pitt" gene To identify candidates of the Pild" gene, gene prediction was performed in the targetregion by 3 gene prediction systems, which were ORF Finder (http://www.ncbi.nlm.nih.gov), RiceGAAS (Autopredgeneset, http://ricegaas.dna.affrc.go.jp) and Soft Berry (FGENESH, http://www.softberry.com) , respectively. RT-PCR analysis was performed in the predicted candidate genes, which had conserved domain of R genes. The result showed that PK1 was the candidate of the Pi Id" gene.4. Obtaining the sequence of cDNA derived from PK1 and the structure and function prediction of the cDNATo obtain the whole sequence of the cDNA, the product of RT-PCR from PK1 was firstly cloned to vector pGEM-T and sequenced. Then, gene-special primers were developed based on this sequence, and 3'-RACE and 5'-RACE were performed with GeneRacer?kit, respectively. The products of the 3'-RACE and 5'-RACE were also cloned to vector pGEM-T and sequenced. At last, the sequences of them were assembled with a soft ware, DNAStar.Structure and function prediction of the cDNA with RiceGAAS showed that PK1 encoded a new type protein with three domains: one transmembrane helix, one calcium-binding EGF-like domain and one protein kinase.5. Analysis of the sequence and gene structure of PK1To fully characterized the difference in DNA sequence between the resistance and susceptible parents, primer set, which was designed based on the sequence of cDNA of PK1, was used to amplify the target region flanking the PK1 from the resistant parent Tsuyuake and three susceptible parents AS20-1 -. M142^ Kasalath and another susceptible cv. LTH, respectively. No amplicons were obtained from the three susceptible parents. It showed that no allele might exist in the three cvs. DNA sequencing was performed between the PK.1 in Tsuyuake and the allele pkl in LTH. Sequence alignment showed that the DNA sequence discrepancy was significant: Many nucleotide insertions or deletions were observed in pkl, and nucleotide substitutions were also found through out the pkl gene. To reveal the gene structure of PK1, sequence alignment was performed between the genomic DNA and the cDNA of PK1 gene. The result showed that the PK1 gene included 4 exons and 3 introns.6. Cloning and transformation of the candidate cDNATo confirm the candidate gene PK1, using long-rang PCR (LR-PCR), the cDNA of PK1was firstly amplified from the donor cv. Tsuyuake with the primer set newly developed, then the cDNA was cloned to the binary vector pCAMBIA1300-35S. The recombinants were transformed into the highly susceptible cv. Q1063 through Agrobacterium-mediatcd transformation.