Allele Mining Of Rice Blast Resistance Genes In Rice Germplasm Resources

Rice blast, one of the widespread fungal diseases of rice, causes tremendous yield lose in the rice production. Breeding of resistant varieties is considered as the most effective and environment-friendly way to control this kind of disease. Identification and utilization of the rice blast resistance genes are the foundation work for disease-resistant breeding of rice. It is meaningful that more rice blast resistance genes are isolated. More than 25 rice blast resistance genes have been cloned to date, most of them belong to NBS-LRR (nucleotide biding site-leucine rich repeat) class and some have been found to be allelic. In this study, we set out to identify new rice blast resistance genes in 502 rice landraces which were collected from different countries and/or regions, then we cloned the allelic genes of Pi2/9 and Pik genes from these resistant varieties using allele mining approach. The main results are summarized as follows:1.502 rice landraces were inoculated with 6 rice blast fungus strains from Philippines,58 lines showed high resistance and broad spectrum to all 6 isolates. And these resistant lines were not from the same country and/or region.2. Two positive primer pairs were designed according to the specific region of resistance locus of Pi2/9, then PCR was performed using these positive primer pairs to test all resistance lines which carried with Pi2/9 genes. Of total 58 resistance lines,29 lines were confirmed contain Pi2/9 gene.28 full length cDNA were successfully cloned from these resistant lines carrying Pi2/9 allleles using allele mining approach. Seven unique alleles of Pi2/9 were obtained including two known genes of Piz-t and Pi2, and other five novel allelic genes are Pi2-A15, Pi2-A35, Pi2-A16, Pi2-A56 and Pi2-A7, respectively. The function and resistance spectrum of these new alleles are subjected to be analyzed.3. Two primer pairs were designed according to the specific region of RGA4 and RGA5 which are required simultaneously for the function of Pik. PCR was performed using these positive primer pairs to detect the Pik alleles in all resistant lines.24 out of 58 resistant lines were found to carry Pik alleles. Then the entire genomic sequence of Pik-A43 was cloned and the deduced protein sequence was compared to known Pik alleles. Four and two amino acid changes were found in Pik-A43 at RGA4 and RGA5 region compared to protein sequences of known Pik alleles at these two regions, respectively. For genetic analysis, an F2 population derived from the cross between CO39 and A43 were inoculated with several isolates which contained different types of AVR-Pik genes. There is no segregation ratio of resistant versus susceptible plants showing 3:1. To validate the linkage relationship between phenotype and genotype, a Pik gene-pecific and an SSR marker RM224 were used for genetic analysis. Some of susceptible plants had the same genotype as resistant donor A43. We speculated that K type of Pik gene from A43 may not be the target recognized by the present four types ofAVR-Pik gene, in other words, a Pik allelic gene which belongs to K type, may perform its resistance function via recognition of a non-AVR Pik gene.