Factors Affecting SMV Transmission And Analysis Of Smv Resistance-related Genes

Soybean mosaic disease which was caused by soybean mosaic virus(SMV)belonged to potyviridae,is one of the most broadly distributed viral diseases worldwide in soybean[Glycine max(L.)Merr.].It resulted in yield losses and seed quality deficiency seriously.Understanding the factors of SMV epidemic and breeding resistance varieties is the most effective pathway to prevent SMV disease.However,the key to prevent SMV prevalent is to breed cultivars with SMV resistance.To understand the resistance mechanism of signaling pathways and related genes will lay the foundation for soybean resistant breeding.Recently the National Center for Soybean Improvement has completed the classification and identification of national SMV samples,and 21 SMV strains were distributed in China at present.In this research,50 soybean varieties were inoculated with SC3 and SC7 to screen the soybean cultivars with seed coat mottling resistance and seed transmitted resistance.By the way of two susceptible cultivars and two popular strains in Huang-Huai and Yangtze valleys,we aimed to understand the main factors affecting SMV disease epidemic to reduce the effect on soybean yield and seed quality.And also this paper describes SMV distribution of infected soybean plants in vegetative reproductive organs,and the virus carried incidence of the physiological and harvest mature seeds were also studied to understand the mechanism of seed transmission.To better understand the virus movement in soybean plants,we constructed SMV-based viral vector.At last,by gene expression,genome wide analysis and eQTL,we evaluate the potential application of GmRDR and GmNPR gene family in SMV resistance.And The main results were as followed:1.Screening soybean cultivars with seed coat mottling and seed transmitted resistance50 soybean cultivars were sown in the net house between 2012 and 2013 to observe SMV symptom,seed coat mottling incidences and seed transmitted incidences.Correlation analysis between the disease index and seed mottling incidence showed that correlation coefficient were 0.063(p>0.05)and 0.417(p<0.01)for SC3 and SC7 respectively.The average seed coat mottling rates were 44.98%and 49.42%of soybean cultivars inoculated with SC3 and SC7.And the average seed transmitted incidences were 1.47%and 1.22%of the soybean plants inoculated with SC3 and SC7.Several soybean cultivars were selected with different seed coat mottling incidence,while they were resistant to SMV seed transmission,e g.,Nongfeng12,Al,Nannong307-1 and so on.This shows that there was no direct relationship between seed coat mottling and seed transmission,and seed coat mottling could not be the "indicator" of vertical transmission.Among them,A3,SD1112 and SD1108 were resistant to seed coat mottling,but also resistant to seed transmission for SC3 and SC7 strains.But they have different SMV pathophysiology,high susceptible,middle susceptible and resistance.These species could provide the sources for the study of genetic mechanism of seed coat mottling and seed transmission resistance.2.Horizontal transmission of SMVTo understand well about disease epidemic,SC3 and SC7 strains with two soybean susceptible cultivars were used in this research.Four different seed transmitted incidence levels were constructed to compare SMV epidemic in 2009 and 2010.Results indicated that there were obvious differences on the infected incidences in different years,cultivars,strains and seed transmitted incidence levels.The infected incidence of the plots with SC7 was obviously more than the plots with SC3.Low temperature and more rain favored SMV epidemiology in 2009.The infected incidence of Nannnongcaidou 5 was remarkably more than Nannong 86-4.The infected incidence was increased as the seed transmitted incidence was high.The infected incidence of 2.0%level was obviously different with other treatments while the infected incidence of 1.0%and 0.5%plots resulted in higher infected incidences than 0.2%plots.CP and HC-Pro genes of SC3 and SC7 were sequenced and we found that there were 95.09%and 98.83%similarity of CP gene at the base and protein level separately while there were 99.71%and 99.54%similarity of HC-Pro gene for both strains.The motif related with aphid transmission of SC3 and SC7 in CP protein were DAG and DAD separately,while there were KLSC and PTK in two strains of HC-Pro.At last,seed coat mottling incidences of Nannnongcaidou 5 and Nannong 86-4 were studied,and there were difference among the infected soybean plants collected from different infected stages.The seed coat mottling incidence of infected plants in R3 stage was 2.50%,obviously lower than V2 and R1 stage(p<0.05).3.Distribution of SMV in infected soybean plantsTo research the vertical transmission of SMV,Nannnongcaidou5 and Nannong86-4 were inoculated at V4 stage.qPCR results showed that the virus content of the true leaves and the first trifoliolate leaves was lower than another two leaves at different part.By ELISA,the virus carried incidences of flowers and the seeds above or downward the inoculated leaves were almost the same.The physiology mature and harvest mature seeds were carefully dissected into three parts(seed coats,cotyledons and embryo)to detect the presence of SMV.Results showed that SMV virus was mainly distributed in seed coats of physiology mature seeds while only a few SMV samples were found in embryo and cotyledons.But we found that there was no SMV positive sample for embryo in Nannongcaidou5.The virus carried incidences of three parts in harvest mature seeds were lower than the physiology mature seeds especially in seed coats.For harvest mature seeds of Nannnong86-4,there was SMV positive sample in every part of the seeds,while positive samples were found only in the seed coats of Nannongcaidou5.By grown out experiment,the seed transmitted incidence of Nannong86-4 was 1.62%,and no seed transmitted plant was found for Nannongcaidou5 in repeated experiments.SMV symptom of the seed-transmitted plants was shown at the true leaf stage and V1 stage while most seed-transmitted seedlings had a weaker growth condition than the healthy plants.To research the influences of Nannong86-4 seed coats on the SMV seed transmitted,two methods were carried out.Results showed that disinfection of Nannnong86-4 seeds and seed coat-removing could not affect SMV seed transmission.Also SMV-based vector named pSC15 was constructed in this research and expressed in Nannong1138-2 plants.CP expression of pSC15 was lower than wild SMV strain SC 15,while no different on the symptom.pGFP was constructed with a weak CP expression and symptom in Nannong1138-2 plants.But green fluorescent could be observed under inverted microscope.4.The potential application of soybean RDR and NPR gene family in SMV resistanceSalicylic acid(SA)content in Kefeng No.1 is 235.3±11.8 ng/g.Fw which is two times of Nannong 1138-2.This indicates that metabolic activity in Kefeng No.1 is more vigorous than that in Nannong 1138-2.SA content reached up to 524.6±16.7 ng/g.Fw in Kefeng No.1 infected with SMV 1 hpi,and then gradually dropped.SA content reached up to the highest value,317.8±27.7 ng/g.Fw in Nannong 1138-2 infected with SMV 24 hpi.Results showed that SA content in Kefeng No.1 is more than that in Nannong 1138-2 no matter whether they were treated with SMV,and SA content change in Kefeng No.1 response rapidly than that in Nannong 1138-2By comparing the expression of Gm02g09470(NPR)and Gm19g31180(RDR)in soybean plants treated with SMV or SA,the expression of both genes were increased generally.The expression of RDR gene in Kefeng No.1 is rapid increased and higher than that in Nannong 1138-2.The expression of NPR gene in Kefeng No.1 is gradually increased while it was slowly down in Nannong 113 8-2.By linkage analysis using WinQTLCart software,we locate many quantitative trait loci.Four eQTLs(expression quantitative trait loci)of Gm02g09470(NPR)were detected on Gm2(Dlb)and five eQTLs for Gm19g31180(RDR)were detected on F and E linkage group.Two resistance QTLs were detected for SC13 was on D1b linkage group,which were near scaffold608.2386089 and Gm02sv068 and overlapped with the SC7 resistance location.For SC13,resistance locate was near scaffold1410.765801 on D1a linkage group.