Development Of Molecular Markers Of The Rice Blast Resistance Gene Pi-ta And Its Application

The rice blast disease caused by the fungal pathogen Magnaporthe grisea (Hebert) Brr is one of the most damaging diseases worldwide and leads to the significant loss of rice production. Currently, how to effectively control this disease is the central topic for many plant pathologists and rice breeders. For the long term, the major method for cotntrolling the blast disease is to develop the resistant cultivars. However, major challenge to rice breeders is to make the appropriate selection of resistance(R) genes. The quarantinee restrictions often prevent the exchanges of the races of the rice blast fungi, which causes problems to confirm the identity of R genes in rice germplasm.With the development of different DNA molecular markers, markers-assisted selection(MAS) becomes an effective approach for proper selection of R genes. Commonly, recombination can occur between R genes and linked molecular markers in different mapping populations. It is possible that molecular markers unlinked with target R genes in breeding populations, which will lead to the failure of the selection of R genes. Meanwhile, the mapping populations for constructing the molecular markers often differ from the breeding populations in different breeding programs. So these molecular markers have limited applications for breeding. Therefore, the best molecular marker is the R gene itself. Cloning R genes have made it possible to develop R gene markers. Rice Pi-ta gene is located on the centromeric region of chromosome 12 encoding a predicted 928-amino acid cytoplasmic receptor with a centrally localized nucleotide binding site and leucine-rich repeats at the carboxyl terminus and is an important rice blast resistance gene worldwide. The Pi-ta gene in rice prevents the infection of M.grisea strains containing the corresponding avirulence AVR-Pita gene. The objectives of current research were to develop Pi-ta gene markers based on R gene itself to distinguish the dominant indica Pi-ta allele from the japonica/j/-to allele and to utlize these markers for MAS in the southern USA. Accomplished specific objectives are described below:1. Four pairs of Pi-ta specific primers YL100 (5'-CAATGCCGAGTGTGCAAAGG-3')/YL102(5'-TCAGGTTGAAGATGCATAGC-3')(6259-6659), YL153(5'-CAACAATTTAATCATACACG-3')/YL154 (5'-ATGACACCCTGCGATGCAA-3')(2021-2442), YL155(5'-AGCAGGTTATAAGCTAGGCC-3')/YL87(5'-CTACCAACAAGTTCATCAAA-3') (4409-5450) and YL183(5'-AGCAGGTTATAAGCTAGCTAT-3') /YL87(4409-5450) were designed based on DNA polymorphisms of nucleotides between resistant indica Pi-ta allele and susceptiblejaponica pi-ta allele, respectively. The former three pairs of primers can amplify the dominant indica Pi-ta allele, while the fourth pair of primers can amplify the susceptible pi-ta allele. These four pairs of primers were used to amplify the genomic DNAs of US important rice cultivars Katy, Drew, Kaybonnet, M202 and Japanese rice cultivar Nipponbare. It was found that DNA fragments of 403, 440, 1042 base pairs (bp) were amplified by the former three pairs of primers from Katy, Drew and Kaybonnet, respectively, but not amplified from M202 and Nipponbare; Contrarily, DNA fragments of 1042 bp were amplified by the fourth pair of primers from M202 and Nipponbare, respectively, but not amplified from Katy, Drew and Kaybonnet. These results indicate that Katy, Drew and Kaybonnet contain Pi-ta gene, but M202 and Nipponbare don't contain the Pi-ta gene.To verify the amplification of the dominant indica Pi-ta allele, PCR products were purified, and cloned into pDrive vector, and sequenced. Searching the current GenBank database for sequences of all PCR products revealed 100% identity with DNA fragments from nucleotide positions 2021 to 2460(YL153/YL154), 4409 to 5450(YL155/YL87) and 6257 to 6659(YL100/YL102) of the Pi-ta gene. These results demonstrated that portions of the indica dominant Pi-ta alleles were amplified from Katy> Drew and Kaybonnet. Thus the dominant rice Pi-ta...