Distribution and Variation of PWL Gene Family in Rice Magnaporthe oryzae from Heilongjiang Province and Hainan Province

The objective of this study is to understand the distribution and variation characteristics of PWL gene family in different Magnaporthe oryzae strains, and to provide a basis for studying the genetic diversity and specificity of different M. oryzae strains.

A total of 8 pairs of specific primers were designed for the promoter region and CDS region of the PWL gene family by referring to the avirulence gene sequence published in NCBI. DNA was extracted from 397 single spore isolates of M. oryzae collected from different regions of Heilongjiang Province and Hainan Province in 2020. PCR amplification of avirulence genes was conducted and the genes were detected by agarose gel electrophoresis. Representative strains from different regions were selected from the test results to sequence and analyze the amplified fragments. The sequencing results were compared with the base and amino acid sequences of the corresponding avirulence gene promoter region and CDS region in NCBI.

In the results of PCR electrophoresis, PWL1 was not detected in all strains. The specific fragments of PWL2, PWL3 and PWL4 were amplified in Heilongjiang Province and Hainan Province, indicating that these three genes were distributed in both provinces and had different distribution frequencies and variation types. Among them, PWL2 had the highest distribution frequency in Heilongjiang Province and Hainan Province, which were 98.14% and 100%, respectively. The distribution frequencies of PWL3 and PWL4 in the two provinces were significantly different. The frequencies of these two genes in Heilongjiang strains were 89.30% and 82.79%, respectively, while in Hainan strains were 5.49%. Through the analysis of avirulence gene combinations, the results showed that the combination types could be divided into six types, namely PWL(f), PWL2, PWL3, PWL2+PWL3, PWL2+PWL4, PWL2+PWL3+PWL4. Among them, Heilongjiang strains contained all combination types, and Hainan strains contained only two, indicating that Heilongjiang strains were more abundant in avirulent genotypes than Hainan strains. By sequencing the PCR products of the PWL gene family, it was found that the PWL gene family had abundant variation sites in the promoter region and CDS region, and was divided into nine types with point mutations and deletions as the main variation types, and the variation types of strains from different populations were specific and consistent. Among them, five variant types PWL2- (1, 2, 3, 4, 5) were detected in PWL2, base sequence changes led to amino acid sequence missense mutations; two mutation types were detected in PWL3 and PWL4, which were PWL3- (1, 2) and PWL4- (1, 2), respectively. Both of them had frameshift mutations that caused changes in the following amino acids.

The distribution and variation types of PWL gene family in M. oryzae strains from different populations have regional differences, and the variation sites are abundant.