Marine Biotechnology Research Unit, Faculty of Marine Technology, Burapha University, Chanthaburi Campus, 57 Moo 1 Chonpratan Road, Kamong, Thamai, Chanthaburi, 22170, Thailand.
Faculty of Marine Technology, Burapha University, Chanthaburi Campus, 57 Moo 1 Chonpratan Road, Kamong, Thamai, Chanthaburi, 22170, Thailand.
Mol Biol Rep. 2020 Sep;47(9):6807-6816. doi: 10.1007/s11033-020-05738-3. Epub 2020 Aug 29.
Mantis shrimp has become commercially valuable in many countries, while the commercially aquaculture still unsuccessful. The stable supply of the species-specific markers for precise identification can play a key role of foods authentication as well as restoring/enhancing mantis shrimp stocks in future. The aim of this research was to identify species-specific markers for Squillid and Harpiosquillid mantis shrimp taxa using Amplified fragment length polymorphism-Single strand conformation polymorphism (AFLP-SSCP) approaches. Selective amplification would be substituted as a total of 40 primer combinations was performed using either three-base (i.e., EcoRI+3 and MseI+3 in 20 primer combinations) or two-base (i.e., EcoRI+2 and MseI+2 in 20 primer combinations) selective primers. These had been size-fractionated via 6% denaturing polyacrylamide gel electrophoresis, ten AFLP fragments exhibiting species or genus-specific characteristics were cloned, sequenced, and GenBank interrogated. A primer pair was designed and their specificity was tested versus the genomic DNA of various species. Results show that the primer E-13/M-13Hr generated PCR products for just H. harpax, while E-14/M-2HhHr and E-13/M-13Hh generated PCR products for both H. harpax and H. raphidea and not others (i.e., M. nepa, O. oratoria, and E. woodmasoni). SSCP was then applied in order to differentiate between H. harpax and H. raphidea. These SSCP results indicate that species can be differentiated based on polymorphic fragment nucleotides. Indeed, primers E-13/M-13Hr, E-14/M-2HhHr and E-13/M-13Hh were all successfully confirmed as present in processed mantis shrimp samples (i.e., saline-preserved and heat-dried). These results provide new species-specific markers for mantis shrimp identification.
螳螂虾在许多国家已经具有商业价值，而商业养殖仍然不成功。物种特异性标记物的稳定供应可以在食品鉴定以及未来恢复/增强螳螂虾种群方面发挥关键作用。本研究的目的是使用扩增片段长度多态性-单链构象多态性（AFLP-SSCP）方法鉴定 Squillid 和 Harpiosquillid 螳螂虾分类群的物种特异性标记物。选择扩增将取代总共进行了 40 个引物组合，这些引物组合使用三碱基（即 EcoRI+3 和 MseI+3 在 20 个引物组合中）或二碱基（即 EcoRI+2 和 MseI+2 在 20 个引物组合中）选择性引物进行。这些通过 6％变性聚丙烯酰胺凝胶电泳进行了大小分离，克隆了 10 个表现出物种或属特异性特征的 AFLP 片段，对其进行测序，并在 GenBank 中进行了查询。设计了一对引物，并针对各种物种的基因组 DNA 测试了其特异性。结果表明，引物 E-13/M-13Hr 仅产生 H. harpax 的 PCR 产物，而引物 E-14/M-2HhHr 和 E-13/M-13Hh 则产生 H. harpax 和 H. raphidea 的 PCR 产物，而不会产生其他物种（即 M. nepa、O. oratoria 和 E. woodmasoni）的产物。然后应用 SSCP 以区分 H. harpax 和 H. raphidea。这些 SSCP 结果表明，可以根据多态性片段核苷酸来区分物种。实际上，引物 E-13/M-13Hr、E-14/M-2HhHr 和 E-13/M-13Hh 都已成功确认为加工过的螳螂虾样品（即盐保存和热干燥）中存在。这些结果为螳螂虾鉴定提供了新的物种特异性标记物。
