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三疣梭子蟹线粒体基因组SNP在增殖放流家系识别中的应用
赵 莲1,2,3, 李志辉1,2,3, 张 培1,2,3, 薛 蓓1,2,3, 赖晓芳1,2,3, 张庆起4, 高 焕1,2,3, 阎斌伦1,2,3
1.淮海工学院海洋生命与水产学院 江苏省海洋生物技术重点实验室 连云港 222005;2.江苏省海洋生物产业技术协同创新中心 连云港 222005;3.江苏省农业种质资源保护与利用平台南京 210014;4.连云港赣榆佳信水产开发有限公司 连云港 222005
摘要:
为开发三疣梭子蟹(Portunus trituberculatus)增殖放流分子标志技术,本研究在前期获得三疣梭子蟹线粒体基因组24个SNP位点的基础上,采用高分辨率熔解曲线(HRM)检测技术对4个用于增殖放流的家系(A、B、C和D) (代表约80~120万个放流个体)进行了鉴别工作。结果显示,含有SNP位点的22条PCR扩增序列中,有9条SNP位点扩增产物在亲代(母本)及其子代的28个个体之间具有基本一致的熔解峰,且子代个体间Tm的均一性较好,无明显差异;进一步以序列已知的野生型及其突变体作为同一SNP引物扩增片段,在各家系间分析HRM标准曲线,这9个SNP可以成功用于三疣梭子蟹4个放流家系的鉴别。在这9个SNP位点中,可鉴定C家系的有5个,可鉴定B家系的有2个,可鉴定A、D家系的各1个。该研究结果为三疣梭子蟹种质资源的鉴定及标志放流工作的开展提供了技术支持。
关键词:  三疣梭子蟹  增殖放流  分子标志  SNP  HRM
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基金项目:
Identification on Stock Enhancement Pedigrees of the Swimming Blue Crab Portunus trituberculatus Using mtDNA SNPs
ZHAO Lian1,2,3, LI ZhiHui1,2,3, ZHANG Pei1,2,3, XUE Bei1,2,3, LAI Xiaofang1,2,3, ZHANG Qingqi4, GAO Huan1,2,3, YAN Binlun1,2,3
1.College of Fisheries and Life Science, Huaihai Institute of Technology, Jiangsu Provincial Key Laboratory of Marine Biotechnology, Lianyungang 222005;2.Co-Innovation Center of Jiangsu Marine Bio-Industry Technology, Lianyungang 222005;3.Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing 210014;4.Lianyungang Ganyu Jiaxin Aquaculture Exploitation Ltd , Lianyungang 222005
Abstract:
The wild population of Portunus trituberculatus, an important economical species in China, has been dramatically decreased in past decades due to over capturing. To recover wild germplasm resources of P. trituberculatus, the activities of stock enhancement were performed, and the released individuals were about six millions each year in Haizhou Bay of Jiangsu Province. To evaluate the effects of stock enhancement, we utilized molecular markers (mtDNA SNPs) to track the released pedigrees of P. trituberculatus. Previous studies identified 22 SNPs of mtDNA from P. trituberculatus, which were used to trace four released families (on behalf of 0.8~1.2 million released individuals) (A, B, C and D) by HRM (High resolution melt) technology. Nine of 22 SNPs were usable, which resolution melt curve were coincident between female parentages and their offspring. Furthermore, the nine SNPs could be used to distinguish the four families, in which the parentage C, B, A and D could be differentiated by 5, 2, 1 and 1 SNPs, respectively. Our results indicate that the mtDNA SNPs, combined HRM technology, is a wonderful technology to evaluate the effects of stock enhancement of P. trituberculatus.
Key words:  Portunus trituberculatus  Stock enhancement  Molecular marker  SNP  HRM