Panprommin Dutrudi, Soontornprasit Kanyanat, Pangeson Tanapat
a School of Agriculture and Natural Resources , University of Phayao , Phayao , Thailand.
b Center of Excellence on Agricultural Biotechnology (Ag-BIO/PERDO-CHE) , Bangkok , Thailand.
Mitochondrial DNA A DNA Mapp Seq Anal. 2019 Jan;30(1):184-190. doi: 10.1080/24701394.2018.1472248. Epub 2018 May 18.
The species diversity of cichlids was investigated in Kwan Phayao from August 2016 to May 2017. Four cichlid species were found, including Oreochromis niloticus, Oreochromis mossambicus, Coptodon rendalli and Coptodon zillii. Due to similar characterizations, it is very difficult to identify each species. Three molecular methods were used to distinguish these four species. DNA barcodes or partial cytochrome c oxidase I (COI) gene sequences were amplified by PCR and sequenced. In Oreochromis sp. and Coptodon sp., 707- and 704-bp fragments were amplified, respectively. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis showed clear differences between the four cichlid species after digestion with three restriction enzymes, ScaI, HindIII and PdiI. ScaI and HindIII separated Oreochromis sp. from Coptodon sp. due to different fragment sizes. PdiI distinguished each cichlid species in the same genus. Finally, high resolution melting (HRM) analysis showed the sensitivity of the primers for discriminating these species with small amplicons and melting curves. From the comparison, HRM analysis was the most efficient method because the primer was shown to be sensitive for discriminating the four cichlids. In addition, it was inexpensive and required a short time to detect large samples. However, direct sequencing or DNA barcodes were still necessary in the case of the COI sequences of organisms of interest, which have not been reported in any databases. These four cichlids are alien species in Thailand; thus, species identification is very important for fishery management.
2016年8月至2017年5月，对坤帕夭的丽鱼科鱼类物种多样性进行了调查。共发现4种丽鱼科鱼类，包括尼罗罗非鱼、莫桑比克罗非鱼、伦氏非鲫和齐氏非鲫。由于特征相似，很难区分每种鱼类。使用了三种分子方法来区分这4个物种。通过聚合酶链式反应（PCR）扩增并测序DNA条形码或细胞色素c氧化酶I（COI）基因的部分序列。在罗非鱼属和非鲫属中，分别扩增出707bp和704bp的片段。聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）分析表明，在用ScaI、HindIII和PdiI三种限制性内切酶消化后，这4种丽鱼科鱼类之间存在明显差异。由于片段大小不同，ScaI和HindIII将罗非鱼属与非鲫属区分开来。PdiI区分了同一属中的每种丽鱼科鱼类。最后，高分辨率熔解曲线分析（HRM）显示了引物对区分这些小扩增子和熔解曲线物种的敏感性。通过比较，HRM分析是最有效的方法，因为该引物对区分这4种丽鱼科鱼类具有敏感性。此外，它成本低廉，检测大量样本所需时间短。然而，对于感兴趣的生物的COI序列，如果在任何数据库中都没有报道，则仍然需要直接测序或DNA条形码。这4种丽鱼科鱼类在泰国是外来物种；因此，物种鉴定对渔业管理非常重要。
