Klinbunga Sirawut, Amparyup Piti, Leelatanawit Rungnapa, Tassanakajon Anchalee, Hirono Ikuo, Aoki Takashi, Jarayabhand Padermsak, Menasveta Piamsak
Marine Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120 Thailand.
J Biochem Mol Biol. 2004 Mar 31;37(2):213-22. doi: 10.5483/bmbrep.2004.37.2.213.
A randomly amplified polymorphic DNA (RAPD) analysis was used to identify the species- and population-specific markers of abalone; Haliotis asinina, H. ovina, and H. varia in Thai waters. Fifteen species-specific and six population-specific RAPD markers were identified. In addition, an 1650 bp band (UBC195) that was restricted to H. ovina from the Gulf of Thailand (east) was also found. All of the specific RAPD markers were cloned and sequenced. Twenty pairs of primers were designed and specificity-tested (N = 12 and 4 for target and non-target species, respectively). Seven primer pairs (CUHA1, 2, 4, 11, 12, 13, and 14) were specifically amplified by H. asinina DNA, whereas a single pair of primers showed specificity with H. ovina (CUHO3) and H. varia (CUHV1), respectively. Four primer pairs, including CUHA2, CUHA12, CUHO3, and CUHV1, were further examined against 216 individuals of abalone (N = 111, 73, and 32, respectively). Results indicated the species-specific nature of all of them, except CUHO3, with the sensitivity of detection of 100 pg and 20 pg of the target DNA template for CUHA2 and CUHA12 and CUHV1, respectively. The species-origin of the frozen, ethanol-preserved, dried, and boiled H. asinina specimens could also be successfully identified by CUHA2.
采用随机扩增多态性DNA（RAPD）分析来鉴定泰国海域鲍鱼物种和种群特异性标记；即皱纹盘鲍、羊鲍和多变鲍。鉴定出15个物种特异性和6个种群特异性RAPD标记。此外，还发现了一条1650bp的条带（UBC195），该条带仅在泰国湾（东部）的羊鲍中出现。所有特异性RAPD标记均进行了克隆和测序。设计了20对引物并进行特异性测试（分别针对目标物种和非目标物种各12对和4对）。七对引物（CUHA1、2、4、11、12、13和14）能特异性扩增皱纹盘鲍的DNA，而一对引物分别对羊鲍（CUHO3）和多变鲍（CUHV1）具有特异性。包括CUHA2、CUHA12、CUHO3和CUHV1在内的四对引物，进一步对216只鲍鱼个体进行检测（分别为111只、73只和32只）。结果表明，除CUHO3外，其他引物均具有物种特异性，其中CUHA2、CUHA12和CUHV1对目标DNA模板的检测灵敏度分别为100 pg和20 pg。利用CUHA2也能成功鉴定冷冻、乙醇保存、干燥和煮熟的皱纹盘鲍标本的物种来源。
