基于转录组测序的辅助鉴定南方大豆皱叶症分子标记开发

Development of molecular markers based on transcriptome sequencing for assisting the identification of southern soybean crinkle leaf disease

  • 摘要: 【目的】基于转录组测序(RNA-Seq)开发辅助鉴定南方大豆皱叶症(SSCLD)的分子标记,为快速鉴定生产上遇到的大豆皱叶是否为SSCLD提供技术支撑。【方法】利用转录组测序技术分析遗传背景相近的皱叶大豆和正常叶大豆材料在皱叶症诱导环境下的差异表达基因(DEGs),从中去除核苷酸序列高度相似的同源序列,筛选出表达差异较大的DEGs进行分子标记开发,利用皱叶大豆材料和逆境处理试验评价分子标记的表达专一性,并利用不同类型的皱叶表型样本对分子标记进行表达专一性及可靠性验证。【结果】 7株皱叶植株样本和5株正常叶植株样本的测序碱基错误率为0.0226~0.0238,Q20和Q30分别在99.00%和99.90%以上的碱基数量占总碱基数量均大于95.00%,GC含量为45.60%~46.24%,表明转录组测序数据质量较好。皱叶大豆材料较正常叶大豆材料有1063个DEGs上调表达,85个DEGs下调表达。根据基因的表达量和同源性,共筛选8个DEGs用作开发分子标记的候选基因,均表现为明显上调表达。8个候选基因的实时荧光定量PCR检测结果与转录组数据基本相符。此外,所筛选基因中有6个基因表达在不同程度上受干旱、水渍、冷害、荫蔽和盐胁迫等逆境胁迫诱导,其中,种植于皱叶症土壤中的皱叶型株系叶片GLYMA_18G033400基因的相对表达量均高于其在正常土壤中受干旱、水渍、冷害、荫蔽和盐等逆境胁迫后的相对表达量。根据GLYMA_18G033400基因序列设计分子标记qCL-18G033400,并用该分子标记对19份不同皱叶类型的叶片样品进行实时荧光定量PCR检测,结果发现,以ΔCt值小于10.00作为SSCLD的判定标准,分子标记鉴定结果同田间鉴定结果一致。【结论】利用转录组测序技术开发的分子标记qCL-18G033400可辅助鉴定SSCLD。

     

    Abstract: 【Objective】Molecular markers based on transcriptome sequencing(RNA-Seq)for assisting the identification of southern soybean crinkle leaf disease(SSCLD)was developed to provide technical support for quickly identifying whether the soybean leaves encountered in production were SSCLD.【Method】Transcriptome sequencing technology was used to analyze differentially expressed genes between crinkle leaf and normal leaf soybean materials with similar genetic background in the crinkle leaf-induced soil environment. Homologous sequences with highly similar nucleotide sequences were removed,DEGs with large expression differences were selected for molecular marker development,the expression specificity of molecular markers was evaluated by using crinkle leaf soybean material and stress treatment test,and the expression specificity and reliability of molecular markers were verified by different types of crinkle leaf phenotype samples. 【Result】The sequencing base error rate of seven crinkle leaf plants and five normal leaf plants were 0.0226-0.0238,the numbers of bases in Q20 and Q30 were 99.00% and 99.90%,and the GC contents were 45.60% to 46.24%,indicating good quality of transcriptome sequencing data. Transcriptome data showed that 1063 genes were up-regulated and 85 genes were down-regulated in leaves from the crinkle leaf type material, compared to that from normal leaf type material. Based on the expression level and homology of the genes,eight genes were screened according to the expression level and the identification of the homology in soybean genome, and both of them were up-regulated genes. Real-time fluorescence quantitative PCR analysis of the eight candidate genes generally agreed with the transcriptome data.The expression levels of six of these genes showed varying degrees of interference by diverse abiotic stress, including dry stress, water logging stress, cold stress, shade and salt stress, and the expression level of GLYMA_18G033400 in crinkle leaves from the crinkle leaf-induced environment was higher than that from normal soil under dry stress, waterlogging stress, cold stress, shade and salt stress. Molecular marker qCL-18G033400 was designed according to the GLYMA_18 G033400 gene sequence,and used to perform real-time fluorescence quantitative PCR on 19 leaf samples of different crinkle leaf types. The results showed that ΔCt value less than 10.00 was used as the determination criterion of SSCLD,and the identification results were consistent with the field identification results.【Conclusion】Molecular marker qCL-18G033400 developed using transcriptome sequencing can assist in the identification of SSCLD.

     

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