基于海甘蓝RNA-Seq序列开发EST-SSR分子标记  

Development and characterization of EST-SSR markers derived from RNA-Seq in Crambe abyssinica

作　　者：戚维聪[1,2] 程计华[2,3] 黄邦全[4] 李坦[1] 林峰[1] 

机构地区：[1]江苏省农业科学院农业生物技术研究所,江苏南京210014 [2]荷兰瓦赫宁根大学植物育种系,荷兰瓦赫宁根6708PB [3]中国科学院武汉植物园,湖北武汉430074 [4]湖北大学生命科学院,湖北武汉430062

出　　处：《江苏农业学报》2014年第5期997-1002,共6页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省农业科技自主创新基金[CX(13)3060]

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186 778条cDNA重叠群(Contigs)序列,通过MISA和Primer 3程序设计了6 639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1 206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-nbsp;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.

关 键 词：海甘蓝 分子标记 

分 类 号：S513[农业科学—作物学]