Screening Of Resistant Soybean Sources And Identification Of Causative Genes Against Soybean Cyst Nematode In Huang-Huai Valleys

Soybean cyst nematode(SCN)is one of the soybean diseases worldwide,the economic losses caused by it could reach hundreds of millions of dollars global annual.The methods of control SCN usually include some measures of crop rotation,resistant varieties planting,chemical control,biological control et al.As crop rotation needs the higher interval of the years,chemical control is prone to pesticide residues,biological control could protect the environment,but not in large scale,so resistant varieties planting become one of the most effective methods in all traditional control works.In recent years,with the rapid development of biotechnology,soybean transgenic technology through the expression of lethal gene ds RNA provides a new approach to combat the SCN.So researchers need adopt a comprehensive management to control SCN.This study was carried out screening of soybean germplasm resistant to SCN and identification of SCN pathogenicity-related genes according to resistant varieties and transgenic technology.The main results of this research were listed as follows:1.Screening for soybean germplasm resistant to SCN:Resistance of 350 soybean accessions from 7 provinces and cities to race 1 of SCN were evaluated by Suspension inoculation.Among the material evaluated,three accessions,Zhongpin 03-5368,Han 6192,Cangdou 6,showed high resistance;six accessions,Zhongzuo 50365-1,Zhou 01-14,Zhou 01015-1,Ji 01S56,Cangdou 7,Fen 9877-10,showed medium resistance.These varieties with yellow seed coats,good agronomic traits and high yield could meet production and breeding applications.2.Resistant identification for SCN causative genes(1)With A.rhizogenes-mediated soybean composite plants transformation system,thirty-five soybean genotypes had been evaluated under the conditions of 5-day seedlings,bacterial inoculating concentration OD600=0.8,23℃constant temperature,16 h/d illumination and moisture.Significant differences were found among genotypes in hairy roots inducing rate,the number of hairy roots and the transformation frequency.Twenty-one soybean genotypes were chosen to detect GUS histochemical staining whose hairy roots inducing rate over 60% and number of roots per plant longer 1.6,the higher transformation frequency of soybean genotypes were Y091,KF1,Z184,P108 and L010 with the transformation frequency of 96.2%,91.4%,91.7%,87.5% and 86.4%,respectively.Further RT–PCR analysis to the five soybean genotypes,GUS gene were transcribed correctly in the end.It was indicated that soybean genotypes Y091,KF1,Z184,P108 and L010 were ideal receptor materials to A.rhizogenes transformation.(2)With A.rhizogenes-mediated soybean composite plants transformation system,RNAi fragments of cgh-1、cpl-1、npp-17、vha-14 genes of SCN were transferred into soybean genotypes KF1 individualy in order to identify their function resistant to SCN Race 4.RT-PCR analysis of transgenic roots showed that four RNAi fragments of SCN were transcribed correctly in soybean hairy roots,respectively.Bioassays performed on transgenic composite plants expressing double-stranded RNA fragments of cgh-1,cpl-1,npp-17 and vha-14 genes resulted significant reduction for Cysts g-1 root and eggs g-1 root,respectively.The gene vha-14 could reduce significantly Eggs cyst-1.Parasitic index analysis of transgenic composite plants expressing double-stranded RNA fragments of cgh-1,cpl-1,npp-17 and vha-14 genes showed that four RNAi fragments of SCN made parasitism index(IP)from medium suscept to medium resistance.Bioassays and parasitism index indicated that four RNAi fragments of SCN were able to reduce a certain number of cysts,but due to the parasitism index did not reach the level of high resistance,these genes do not yet have the production value.In order to further detect expression of RNAi in vivo when SCN feeding transgenic soybean roots,RT-PCR and real-time PCR were performanced respectively to decect RNAi effect about cysts on transgenic soybean roots,the result ultimately showed that no RNAi effect could fetect,this can be speculated that the cyst nematodes by RNAi have died.In order to further detect lethal phenotypes of SCN,we will need to make some slices of different periods when SCN feeding transgenic roots.