Cost-effective multiplex PCR assay for simultaneous detection of bacterial leaf blight, blast and brown planthopper resistance genes in rice

Rice production is drastically affected by biotic stresses, namely, bacterial leaf blight (BB), blast (BL) and brown planthopper (BPH). As marker-assisted selection for uniplex gene screening in breeding population is often tedious, time-consuming and expensive, multiplex PCR is practically a reasonable choice for the testing of genes in the pyramided lines as it offers significant time- and cost-saving advantages. Therefore, a cost-effective multiplex PCR-based screening for simultaneous detection of three BB ( Xa21 , xa13 , and Xa4 ), two BL ( Pi1 and Pi54 ) and two BPH resistance genes ( Bph20 and Bph21 ) has been developed. This multiplex system can detect different combinations of BB, BL and BPH resistance. In addition to this most cost-effective method, a five-gene ( Xa21 , xa13 , Pi54 , Bph20 and Bph21 ) multiplex assay in a single PCR tube for three biotic stresses, BB, BL and BPH, has been developed. These standardized multiplex assays have been incorporated into our breeding programs for accelerated results. These assays have displayed no deviation and are in perfect correlation with the individual marker screenings. As one of the most efficient and cost-saving technologies, this technology will have a great impact in the seed industry.