基于海甘蓝RNA-Seq序列开发EST-SSR分子标记

摘要

利用油料作物海甘蓝(十字花科)发育时期种子的RNA-Seq测序数据组装获得186778条cDNA重叠群( Contigs)序列，通过MISA 和Primer 3程序设计了6639个EST-SSR分子标记。在这些标记中，除了单核苷酸重复(45%)外，三核苷酸重复的 SSR 是最常见的碱基重复类型(29%)，其次是双核苷酸型(10%)、五核苷酸型(7%)、六核苷酸型(5%)和四核苷酸型(2%)型。采用电子定位的方法将1206个EST-SSR标记定位到近缘种白菜( Brassica napa)的基因组上。依据引物在白菜基因组中的分布，挑选了20条EST-SSR引物在海甘蓝中进行PCR验证，其结果显示所有引物均能够扩增出符合预期大小的PCR片段。这些新开发的EST-SSR引物可以作为功能标记应用于海甘蓝的分类鉴定、遗传图谱构建、种质资源鉴定以及分子标记辅助育种工作中。%Crambe abyssinica of Brassicaceae family is a new crop and cultivated for its erucic concentration as high as 60%. Although as a high-value commercial crop, the germplasm collection and breeding achievements of C. abyssinica have lagged behind other crops due to its limited molecular markers. In this study, 6 639 EST-SSR primers according to 186 778 cDNA contigs derived from 86 224 256 RNA-Seq tags of the developing seeds were designed with the aid of MISA and Primer 3 programs. In addition to mononucleotide repeats ( 45%) , trinucleotides SSR is the dominant repeat type (29%) followed by 10% dinucleotide, 7% pentanucleotide, 5% hexanucleotide, and 2% tetranucleotide. The 1 206 EST-SSR markers were in silico mapped onto the Brassica napa genome. Based on their distribution on Brassic napa ge-nome, 20 primers were selected and validated in Crambe abyssinica. All the primers successfully amplified the expected PCR band. The newly developed EST-SSR primers, so-called functional markers, will be powerful resource for further applications such as taxonomy, linkage map con-struction and molecular assisted breeding.