基于Illumina测序技术的枸杞微卫星引物的开发  

Discovering the microsatellite sites in L. ruthenicumand L. barbarum using transcriptome analysis

作　　者：赵鑫[1] 李建民[1] 朱雪冰[1] 宗渊 魏乐[1] 刘宝龙 

机构地区：[1]青海师范大学生地学院,青海西宁810008 [2]青海大学,青海西宁810016 [3]青海省作物分子育种重点实验室,青海西宁810001

出　　处：《青海师范大学学报（自然科学版）》2017年第3期61-66,共6页Journal of Qinghai Normal University(Natural Science Edition)

基　　金：青海省应用基础研究项目(2015-ZJ-701);中国科学院西部之光项目

摘　　要：目的:分离和鉴定红、黑枸杞基因组中的简单重复序列(SSR)位点.方法:Illumina Hiseq 2000进行红、黑枸杞的高通量转录组测序后,生物信息分析SSR的分布频率和重复基元的类型特征,PCR扩增和聚丙烯酰胺电泳验证SSR位点及引物.结果表明:转录组测序共得到了192,869条reads,总长度205,220,696bp.拼接后序列的数量为35,572条,序列平均长度约为1064bp.从中共预测到44,828个SSR位点,其中单、二、三、四、五、六个碱基重复单元的微卫星位点数分别有25,800、9186、8479、363、464和536个.从中随机选择30个位点,设计引物进行验证.23对引物在红黑枸杞中能扩增出产物,其中5对仅一个材料中有扩增产物,另有5个位点扩增产物能明显区分红黑枸杞.结论:利用Illumina测序技术得到的枸杞转录组中SSR位点出现频率高,类型丰富,对今后枸杞遗传多样性的研究以及种质资源鉴定打下了良好的理论基础.Objective： discovering and proving the microsatellite sites in L. ruthenicum and L. barba- rum. Methods： The transcriptome analysis was carried out in the fruits of L. ruthenicurn and L. barbarum with the Illumina Hiseq 2000. Msat commander was employed to predict the miscrosatellite sites and de- sign the correspondence primers. The PCR and electrophoresis were performed to prove the existences of the miscrosatellite site. Results： A total of 192,869 reads were obtained with 205,220,696 bp. The assembly analysis produced 35,572 contigs with the average length 1064bp. A total of 44828 SSR--contained contigs were found. The number of microsatellite loci were 25,800, 9186, 8479, 363, 464 and 536, for the repeat units containing single, two, three, four, five and six bases respectively. 30 microsatellite sites were selected randomly to design the primers for proving their existences in the genomes of L. ruthenicum and L. barbarurn. 23 primers can produce amplification in the genome DNA of L. ruthenicurn and L. bar- barum. Five primers can produce amplifications in one Species, and five primers can differentiate the two species. Conclusion： The Illumina transcriptome sequence discovered so many miscrosatellite sites, which benefit the genetic variation study of L. ruthenicum and L. barbarurn, and differentiating the two species.

关 键 词：转录组分析 红果枸杞 黑果枸杞 微卫星位点 

分 类 号：S435.622.1[农业科学—农业昆虫与害虫防治]