Identification of Candidate Genes Controlling SSCLD by Utilizing High-Generation Segregating Populations RNA-seq
[Objective]Severe SSCLD can lead to about 40%yield reduction of soybean.In this study,candidate genes associated with controlling SSCLD were identified by HGRNA-seq technology to provide data support for revealing the molecular mechanisms of SSCLD.[Method]Two parents with SSCLD resistance difference and their derived F2:7 lines were used as the experimental materials.Two parents and 12 F2:7 lines(7 crinkled leaves and 5 normal leaves)were separately subjected to re-sequenced and transcriptome sequencing.Mixed pool method was carried out for gene localization by using SNP/InDel data from the parents and their progenies.GO and KEGG functional annotation and enrichment analysis was carried out by using differentially expressed genes(DEGs)from RNA-seq.7 KASP molecular markers were developed in the vicinity of localization interval,and 230 F2 populations were used to construct a localized linkage map to verify the localization results of the transcriptome data.Candidate genes controlling SSCLD were screened by combining gene mapping and transcriptome analysis.[Result]The gene controlling SSCLD named CL12 was located on chromosome 12 within a 1 473 464 bp interval by using mixed pool method,ranging from 39 231 651 bp to 40 705 115 bp.At the same time,the gene was localized in the 1 205 020 bp interval from 39 743 275 to 40 948 295 bp by using the F2 population,which was basically consistent with the results of the mixed-pool method.The GO annotation results showed that the metabolic processes included immune system processes and responses to stimuli,and cellular components were mainly related to membranes,etc.The KEGG annotation results showed that the biosystem pathways mainly included plant-pathogen interaction and environmental adaptation,etc.GO-enriched DEGs were mainly related to the activity of transmembrane receptor proteins,protein phosphorylation,and signaling receptors,etc.,KEGG enriched DEGs were mainly related to plant-pathogen interaction and MAPK signaling pathway.Combined with the characteristics of the causal factors of SSCLD,genes within the candidate interval associated with disease resistance which had non-synonymous mutations in the coding exons,or showed differential expression were selected as candidate genes for SSCLD resistance.GLYMA_12G223100,GLYMA_12G223900,GLYMA_12G224100,GLYMA_12G231800 and GLYMA_12G233000 were finally identified as the candidate genes controlling SSCLD by qRT-PCR.[Conclusion]HGRNA-seq realized the combination of RNA-seq and BSA-seq,and successfully mined the candidate genes controlling SSCLD.
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