Genome-wide Association Studies Of Bacterial Blight Resistance Genes In Rice And Detection Of Bacterial Blight From Rice Seeds Using Loop-Mediated Isothermal Amplification Assay

Rice is one of the most important food crops in the world.Rice bacterial blight,caused by Xanthomonas oryzae,is a worldwide rice disease.Infected rice generally reduces its yield by 20%-30%,and it reaches 50%in severe cases.Meanwhile,the rice seeds is crisp and the seed germination rate is reduced,resulting in huge losses.Therefore,Xanthomonas oryzae is classified as a harmful quarantine pathogen in China.It’s important to detect this pathogens on seeds and exploring rice bacterial blight resistance genes for controlling bacterial blight disease.Up to date,39 bacterial blight resistance genes have been reported,and 10 bacterial blight resistance genes have been cloned(Xa1,Xa5,Xa8,Xa10,Xa13,Xa21,Xa23,Xa25,Xa3/Xa26 and Xa27).In this study,five different races of rice bacterial blight were used to inoculate 240 rice accessions collected from worldwide.It was found the Philippines strain PXO99 from has the strongest pathogenicity.Among the 240 accessions,34 resistance accessions are high resistant or medium resistant to three or more races.According to the origin and subspecies,15 accessions from these 34 resistant accessions are originated from Asia,and 26 accessions are japonica rice.In total,277 SNPs related to rice blight resistance were obtained using GWAS assay,and 12 resistance sites have been reported.141 candidate genes were obtained in the 200 kb interval associate with these sites.Loop-mediated isothermal amplification(LAMP)technology is a new rapid detection method.The LAMP assay is carried out at a constant temperature(60℃-65℃),and it has higher specificity and sensitivity than conventional PCR,which can amplify 109-1010orders of magnitude during 30-60 minutes.In this study,a set of six specific primers were designed according to the specific region of a epimerase gene(AF169031)in the Xanthomonas oryzae pv.Oryzae genome.Then the versatility,specificity,and sensitivity of the LAMP assay were tested and all of them met the expected requirements.We also tested this LAMP detection system through detecting Xanthomonas oryzae pv.oryzae in rice seeds.The results showed that this LAMP system could successfully detect X.oryzae in rice seeds.This LAMP detection system is convenient,rapid and low requirements on instruments and it provides a reliable method for detecting seed-borne rice bacterial blight.