Development of SNP markers and Genome-Wide Association mapping of virulence factors of Xanthomonas oryzae pv. oryzae

Successful control of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight (BB), requires a specific and reliable diagnostic tool. Single nucleotide polymorphism (SNP) genotyping needs to be attempted to establish association between the identified mutations and bacterial virulence traits. This study was conducted to develop robust, lineage and race specific SNP markers using 100 Xoo strains from IRRI [International Rice Research Institute] collection of different rice growing regions in the Philippines, and to identify quantitative trait loci highly associated to important virulence traits of Xoo populations using Genome-Wide Association Study (GWAS). Allelic-specific PCR primer sets were developed for the identification of lineage and races of Xoo. Primer were designed based on the SNPs specific to three major lineages (PX-A, PX-B, and PX-C) and four subgroups of PX-A and PX-B of Philippines Xoo strains using modified allele-specific PCR methods. Two primer sets for each Philippine Xoo lineage (PX-A, PX-B, and PX-C) were found to be very specific. One primer sets for PX-Aa4, five for PXAb9x, five for PX-Ac139, four for PXAd139, one for PX-Ba8, two for PX-Ba278 and PX-Bb23b, one for PX-Bc5 were also found to be very specific. Specific and rapid detection assays (bacterial oozing out detection assay and single colony detection assay) were developed to detect Xoo lineages and races from the bacterial blight infected rice leaves. In the present study, a total of 26 isolates in 2014 collection from bacterial blight hot spot locations of Victoria, Laguna Province in the Philippines were classified as lineage A and as subgroup PX-Ab9x. Out of the 87 isolates for 2015 collection tested, 81 isolates fell into lineage A and were classified as PX-Ab9x subgroup. Six isolates fell into lineage B and these six isolates did not belong to any PX-A and PX-B subgroups and are probably new strains. Genome-Wide Association Studies (GWASs) have emerged as a popular tool for genetic research in recent decades. The virulence pattern of 91 bacterial strains of Xoo was assessed on IRBB4 and IRBB14 rice varieties. A significant differential interaction observed among the bacterial strains, the host-genotypes and in their interaction suggested that the host genotypes differed in resistance and bacterial strains differed in virulence. The result clearly revealed that most all the strains were able to overcome IRBB14 which harboured a Xa-14 resistant gene for BB resistance. Moreover, it was found that the strains in race 2, 3b and 9 were able to overcome IRBB4 possessing the Xa-4 gene. The linear mixed model (LMM) analysis detected 32 significant SNPs at P value 0.02 for IRBB14. The four most significant SNPs (SUNKNOWN_587, SUNKNOWN_9075, SUNKNOWN_11229 and SUNKNOWN_11402) were associated with hemagglutinin-like protein (PXO_03554), lysR (PXO_03773: transcriptional regulator LysR family), cas (PXO_02701: crispr-associated helicase Cas3 domain protein) and wxoA (PXO_03160: sugar nucleotide epimerase/dehydratase). LMM analysis generated 39 significantly virulence associated SNP markers based on P value 0.0001 for IRBB4 rice variety. The most significant SNP (SUNKNOWN_3696) was found to be associated with RND efflux membrane fusion protein (PXO_04839) that is important for the virulence of Xoo.