A Single-Tube, Functional Marker-Based Multiplex PCR Assay for Simultaneous Detection of Major Bacterial Blight Resistance Genes Xa21, xa13 and xa5 in Rice

In marker-assisted breeding for bacterial blight (BB) resistance in rice, three major resistance genes, viz., Xa21, xa13 and xa5, are routinely deployed either singly or in combinations. As efficient and functional markers are yet to be developed for xa13 and xa5, we have developed simple PCR-based functional markers for both the genes. For xa13, we designed a functional PCR-based marker, xa13-prom targeting the InDel polymorphism in the promoter of candidate gene Os8N3 located on chromosome 8 of rice. With respect to xa5, a multiplex-PCR based functional marker system, named xa5FM, consisting of two sets of primer pairs targeting the 2-bp functional nucleotide polymorphism in the exon II of the gene TFIIA.5 (candidate for xa5), has been developed. Both xa13-prom and xa5FM can differentiate the resistant and susceptible alleles for xa13 and xa5, respectively, in a co-dominant fashion. Using these two functional markers along with the already reported functional PCR-based marker for Xa21 (pTA248), we designed a single-tube multiplex PCR based assay for simultaneous detection of all the three major resistance genes and demonstrated the utility of the multiplex marker system in a segregating population.