Mapping,Cloning And Functional Analysis Of Soybean Mosaic Virus Resistance Gene R_SC3Q

Soybean mosaic virus（SMV）is one of the main pathogens that reduce soybean yield and soybean quality.Therefore,it is the most economic and safe method to study soybean disease resistance mechanism and cultivate disease-resistant varieties.High-throughput sequencing technology has been used in plant researches.But nobody ever used this technology to map SMV resistance gene in soybean.In this study,the resistance inheritance and resistance gene allelic analysis of Qihuang NO.1 to SC3（SMV）were studied;Near-isogenic lines from Qihuang NO.1× Nannong 1138-2 were used for whole genome resequencing and transcriptome sequencing;mapping of R_SC3Q and screen its candidate genes;the function of the candidate gene Glyma13g26380 and four GmNAC was identified using virus-induced gene silencing（VIGS）technology.The main experimental results are as follows:1.Genetic analysis and Allelic analysisPopulations F_2:3 derived from Qihuang NO.1× Nannong 1138-2 were inoculated with SC3.Results showed that Segregation of resistant（R）and susceptible（S）individuals in F_2:3 lines matched a 1:2:1 ratio.It is indicated that the R_SC3Q in Qihuang No.1 was controlled by a pair of dominant genes.Populations derived from Qihuang NO.1×Kefeng NO.1 and PI96983×Qihuang NO.1 were inoculated with SC3,seperately.The populations of F1 and F2 derived from PI96983×Qihuang No.1 showed resistance to SC3.The F1 of Qihuang No.1 × Kefeng No.1 showed resistance to SC3.And resistant（R）and susceptible（S）individuals in F2 lines matched a 15:1 ratio.It is indicated that SC3 resistance gene in Qihuang NO.1 and PI96983 was an allelic gene,and in Qihuang NO.1 and Kefeng 1 was independent inheritance.2.Mapping and screen Rsc3Q by high-throughput sequencingR line（Resistance SC3）and S line（Susceptible SC3）were Near isogenic lines from Qihuang No.NO.1×Nannong 1138-2.After whole genome resequencing,R line and S line obtained 153 million and 163 million clean reads,with an average sequencing depth of 29.51.87.64%and 87.74%reads after removal of repeated reads matched the soybean reference genome Williams 82 av1.1.The coverage rate of reads on the soybean reference genome was 96.17%and 96.25%.There were 47183 SNPs,1287 insertion fragments and 1559 deletion fragments between R line and S line.The number of SNPs on different chromosomes was 848-3952,except chromosome 13.Chromosome 13 contained the highest number of SNPs,8967.The distribution of the number of InDels almost the same as the SNP.The number of InDels on different chromosomes was 28-188,except chromosome 13.Chromosome 13 contained the highest number of InDels,1010.The number of SNPs and InDels on chromosome 13 was significantly higher than other chromosomes.Based on distribution of SNP and InDel,initial mapping result of disease resistance genes,we located R_SC3Q in the range of 27.4-29.9 Mbp on chromosome 13.After transcriptome sequencing of R line and S line at 0h,6h,20h and 48h after SC3 inoculation,a total of 42,969,016 to 67,068,306 clean reads were obtained.85.86%～92.57%of reads were compared to the soybean reference genome.About 40,000 genes were mapped on soybean reference genome,accounted for 30%～32%of the total soybean gene.After compared two samples of R line and S line at four time points,differentially expressed genes（DEG）were found.The results showed that after inoculation with SC3,an amount of DEG were induced to express in R line and S line.The number of DEGs in R line was more than S line.0hpi as a control,R line and S line had similar expression patterns at 48hpi and different expression patterns at 6hpi and 20hpi.;more DEGs in R line was involved in signaling pathways,secondary metabolism,and hormone pathways and cell wall metabolism-related pathways by GO enrichment analysis and Mapman analyzsis.Most of them was involved in mechanical injury responses and PTI responses.It is speculated that R line may increase its resistance to SMV by regulating basic physiological and biochemical reactions.In R_SC3Q location,there were 12 non-synonymous NBS-LRR genes for SNP and InDel:Glyma13g25440,Glyma13g25780,Glyma13g25920,Glyma13g25950,Glyma13g25970,Glyma13g26000,Glym a13g26141,Glyma13g26230,Glyma13g26250,Glyma13g26310,Glyma13g2 6380,Glymal3g26460.All genes were checked in the transcriptome results,except Glyma13g25780.However,they were not reached the threshold of the DEG due to the low expression level.It was speculated that the R_SC3Q gene can induce subsequent resistance responses just by a low expression level.Based on the heat map of these 12 genes,the expression level of Glyma13g25950,Glyma13g25970 and Glyma13g26380 in R line was higher than in S line.These 3 genes were selected to be R_SC3Q candidate genes.3.Functional analysis of resistance candidate genesThe expression of Glyma13g25950,Glyma13g25970 and Glyma13g26380 were detected in resistant parent（Qihuang NO.1）and susceptible parent（Nannong 1138-2）after SC3 inoculation at 0h,6h,24h and 48h.The results of qRT-PCR showed that expression levels of Glyma13g25950,Glymal3g25970 and Glymal3g26380 in Qihuang No.1 were higher than in Nannong 1138-2.N.benthamiana was used as experimental materials for subcellular localization experiment.Green fluorescence was observed in both the cell membrane and the nucleus,indicating that Glyma13g26380 protein expressed in both areas.Glyma13g26380 silencing plants of Qihuang No.1 had more virus content than control after inoculation with SC3.But over time,virus content of plants silenced Glyma13g26380 reduced.It was suggested that Glyma13g26380 may to be related to the SC3 resistance pathway in early time.4.Functional analysis of 4 soybean NAC genes and Arabidopsis Homologous GenesDuring the transcriptome analysis,expression of seven NAC genes were significantly different in R line and S line.After sequence homology alignment,four NAC genes with higher homology（GmNAC043,GmNAC085,GmNAC09 and GmNAC101）were selected for subsequent study.N.benthamiana was used as experimental materials for subcellular localization experiment.Green fluorescence was observed in the nucleus,indicating that 4 GmNAC protein expressed in nucleus.After 6 hours of SMV inoculation,the expression levels of four GmNAC were increased in all soybeans.However,no significant differences in GmNAC expression levels of Qihuang NO.1 and Nannong 1138-2.After SMV inoculation,SMV contents in silencing plants（GmNAC043,GmNAC085,GmNAC092 and GmNAC101）and control plants were similar.It indicated that these four GmNAC were not related to SC3 resistance pathway.After different stress treatments,it was found that the four GmNAC were induced by mechanical injury,Na+,K+and ABA.With NaCl treatment,silencing plants were more wilting than control plants.The relative water content（RWC）and relative conductivity（REC）results showed that the leaves of silencing plants lost more water than control plants,and the cell membrane damage was more serious than control plants.The expression level of NaCl marker gene GmASN1 in Sil1N2 was significantly higher than V after NaCl treatment.The above results indicated that silenced GmNAC043,GmNAC085,GmNAC09 and GmNAC101 can reduce the salt tolerance of soybean.