空心李RNA-seq SSR信息分析及分子标记开发

RNA-seq SSR Information Analysis and Development of Molecular Markers in Kongxinli

为开发适用于空心李的目的基因SSR分子标记。基于空心李果实中果皮转录组测序数据,利用Trinity软件组装45 389条Unigene,其中32 822条获得功能注释。通过KEGG代谢通路分析发现817条Unigene与碳水化合物代谢相关。利用MISA软件在所有Unigene中共检测到9 797个SSR位点,分布于7 963条Unigene中,其占总Unigene数的17.54%。优势重复基序为二核苷酸(占总SSR位点数的40.57%)、单核苷酸(39.34%)和三核苷酸(18.58%)。AG/CT是二核苷酸中最多的重复类型(32.40%),三核苷酸重复类型以AAG/CTT为主(5.87%)。根据已注释的与果实品质(大小,糖和酸合成等)相关Uningene序列,挖掘到47个目的基因SSR分子标记,以‘李子王30号’空心李种质叶片基因组DNA为模板对这些SSR标记进行有效性验证,39对引物能扩增出清晰条带,其中11对引物在5份不同空心李种质中表现出多态性。开发的空心李目的基因SSR标记为空心李种质遗传多样性分析、指纹图谱构建和果实品质性状关联分析提供有效标记。

In order to develop the SSR molecular markers for the target gene of Kongxinli, 45 389 Unigenes were assembled by Trinity software based on the transcriptome sequencing data of pericarp in Kongxinli fruit, of which32 822 were functional annotations. Analysis of KEGG metabolic pathway revealed that 817 Unigenes were related to carbohydrate metabolism. A total of 9 797 SSR loci were detected in 7 963 U nigenes using MISA software,accounting for 17.54% of the total number of Unigenes. The dominant repeat motifs were dinucleotides(40.57% of total SSR loci), single nucleotides(39.34%) and trinucleotides(18.58%). AG/CT was the most common duplicate type of dinucleotides(32.40%) and AAG/CTT was the main duplicate type of trinucleotides(5.87%). According to the annotated Unigene sequence related to fruit quality(size, sugar and acid synthesis), 47 target gene SSR markers were identified. The validity of these SSR markers was validated by using genomic DNA of leaves of ‘Liziwang No. 30’ Kongxinli germplasm as template. 39 pairs of primers could amplify clear bands, and 11 pairs of primers showed polymorphism in 5 different hollow plum germplasms. The SSR markers for target gene developed in this study could provide effective markers for genetic diversity analysis, fingerprint map construction and traits association of Kongxinli.