Multiple haplotypes at the same rice blast R-gene locus share extremely high sequence similarity, which makes the gene diagnostic method using molecular markers less effective in differentiation from one another. The composition and distribution pattern of deployed R genes/haplotypes in elite rice varieties has not been extensively analyzed. In this study, we employed PCR amplification and sequencing approach for the diagnosis of R-gene haplotypes in 54 Chinese elite rice varieties. A varied number of functional and nonfunctional haplotypes of 4 target major R-gene loci, i.e., Pi2/9, Pi5, Pik, and Pib, were deduced by referring to the reference sequences of known R genes. Functional haplotypes accounted for relatively low frequencies for the Pi2/9 (15%) and Pik (9%) loci but for relatively high frequencies for the Pi5 (50%) and Pib (54%) loci. Intriguingly, significant frequencies of 33%, 39%, 46% of non-functional haplotypes at the Pi2/9, Pik, and Pib loci, respectively, with traceable original donors were identified, suggesting that they were most likely unintentionally spread by using undesirable donors in various breeding programs. In the case of Pi5 locus, only a single haplotype, i.e., Pi5 was identified. The reactions of 54 rice varieties to the differential isolates were evaluated, which showed a good correlation to the frequency of cognate avirulence (Avr) genes or haplotypes in the differential isolates. Four R genes, i.e., Pi2, Piz-t, Pi50, and Pikm were found to contribute significantly to the resistance of the elite rice varieties. Other two genes, Pi9 and Pikh, which were not utilized in rice varieties, showed promising values in breeding durable resistance due to their high resistance frequencies to the contemporary rice blast population. The sequence-based molecular diagnosis provided a promising approach for the identification and verification of haplotypes in different R-gene loci and effective R genes valuable for breeding durable rice resistance to rice blast.
Keywords: Avirulence gene; Resistance gene; Rice blast; Rice breeding; Sequence-based diagnosis.