| Soybean [Glycine max(L.)Merr] originated in China,is one of the most widely cultivated economic crops in the world,and also an important source of animal protein feed and vegetable oil in China.Soybean mosaic virus(SMV)disease is one of the most common diseases in soybean production,which seriously harms the yield and quality of soybean.Breeding SMV resistance cultivars is the most effective and direct measure to solve soybean mosaic virus.Therefore,it is of great significance to search for molecular markers closely linked to SMV resistance genes and promote the mining of SMV resistance genes for soybean breeding.SMV strain SC3 is an endemic strain in soybean production in the Huanghuai-Hai and Yangtze River Valley of China.There are many reports on the resistance location of SC3 at China and abroad.In this study,we used the reported molecular markers and the developed molecular marker near the Rsc3 resistance genes to identify the new soybean cultivars(lines)in China and combined with the results of phenotype,genotype and serological identification.The molecular markers with more than 80% consistent with phenotypic identification of disease resistance were screened,and the function of the linkage genes near the markers were analyzed.The main contents and results of the study are as follows:1.Screening and verification of molecular markers for resistance genes of soybean mosaic virus SC3 strain30 newly developed SSR markers near SMV resistance locus and 50 SSR markers linked to Rsc3 were screened from 96 soybean cultivars(lines)by combining phenotypic,genotype and serological identification.The selected markers were used to detect and analyze the remaining 192 cultivars(lines)(288 in total).The results showed that a newly developed SSR marker CH0211 was screened,and the coincidence rate with soybean variety resistance phenotype selection was 87.8%.In addition,the remaining 192 materials were further verified by using the marker.Combined with artificial inoculation phenotype and serological identification,the coincidence rate of disease resistance phenotype and genotype was 80.56%.158 soybean cultivars showing resistance to SC3 were selected from288 soybean cultivars(lines),accounting for 54.86% of the total number of tested materials.116 disease resistant cultivars with the same inoculation phenotype and molecular band type were identified,such as PI96983,Qihuang 34,Fendou 104,Shanning 28,and the like.2.Identification of resistance genes linked to SSR marker CH0211Based on previous mapping results,9 genes were selected near the Rsc3 resistance marker CH0211,which were Glyma.13g185000、Glyma.13g184900、Glyma.13g184800、Glyma.13g184700 、 Glyma.13g184600 、 Glyma.13g184500 、 Glyma.13g184400 、Glyma.13g184300、Glyma.13g184200.Then these genes were analyzed for SMV induced expression between resistance and susceptible cultivars.After inoculation with SMV,the expression of gene Glyma.13g184800 were significantly different between resistance and susceptible cultivars at 3 hpi.The expression level of PI96983 was 12 times higher than that of Nannong 1138-2,indicating that Glyma.13g184800 in PI96983 could quickly respond to SMV infection.While the other genes had little difference between resistance and susceptible cultivars.The results of tissue-specific expression analyses of Glyma.13g184800 showed that it was highly expressed in the roots of PI96983,but only a few in the stems,leaves,flowers and pods;In Nannong 1138-2,it had the highest expression in the flower organs.It was suggested that although the expression level of this gene was low in leaves,it could quickly respond to SMV infection in resistant cultivars,so it may be an induced expression gene.Therefore,the gene Glyma.13g184800 were selected as the target gene of this study.The complete CDS of the gene Glyma.13g184800 in PI96983 and Nannong 1138-2 were cloned.The CDS of Glyma.13g184800 is 3786 bp in length and encode 1261 amino acids,with an isoelectric point of 4.6 and a relative molecular mass of 313.7k Da.Sequence alignment showed that there were a large number of differences in Glyma.13g184800 CDS sequences among resistant and susceptible cultivars(to be supplemented)with 86 SNPs,resulting in 58 amino acid mutations.The amino acid sequence encoded by Glyma.13g184800 has a typical NBS-LRR resistance domain.Subcellular localization showed that the gene was expressed in the nucleus.3.VIGS verifies the function of SC3 resistance candidate gene Glyma.13g184800The silencing vector VIGS-Gm800 based on BPMV(Bean Pod Mosaic Virus)was constructed.One week after inoculation,the plants appeared chlorosis,curling and dwarfing.The transcription level of Glyma.13g184800 was detected by q RT-PCR.The results showed that Glyma.13g184800 in the VIGS-Gm800 plant(Si Gm800)inoculated with the silent vector was silenced by 60% compared with the control plant(p BPMV-V2),indicating that the gene is silenced.SMV(SC3)was inoculated on the diseased leaves of BPMV.Two weeks after inoculation,the upper leaves of Nannong 1138-2 Si Gm800 plants were observed,and it was found that Nannong 1138-2 Si Gm800 plants had more serious symptoms than p BPMV-V2 plants.The SMV virus accumulation in silent plants was detected 20 days after SC3 inoculation.The results of q RT-PCR showed that there was no significant difference between the Si Gm800 plants of PI96983 and the control;but the expression of SMV CP in Si Gm800 plants of Nannong 1138-2 was 2.5 times of that in p BPMV-V2 plants.The results of DAS-ELISA was the same as q RT-PCR.This result indicates that Glyma.13g184800 may be involved in soybean resistance to SMV. |
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