Laboratory of Marine Genetics and Breeding (MGB), College of Marine Life Sciences, Ocean University of China (OUC), Qingdao 266003, China.
Mol Ecol Resour. 2008 Jul;8(4):790-5. doi: 10.1111/j.1755-0998.2007.02066.x.
Marine scallops are sessile as adult but have a long planktonic larval phase showing great possibility to migrate in marine realm lacking of obvious barriers. Genetic analysis of scallop embryos/larvae based on molecular markers is very essential to clarify the spatial and temporal gene flow and the unique population and community structure. However, the technical challenges, such as single embryos/larvae isolation and low quantity and poor quality of DNA extracted, make genotyping for a single embryo/larva long preserved in ethanol to be a really difficult task. In this study, we analysed the factors that might affect the DNA quantity and quality for simple sequence repeat-based genotyping for single embryos/larvae. Based on the factors analysed, we developed a LoTEPA buffer-based method, of which the accuracy, stability and reproducibility were evaluated by controlled inter- and intraspecies and self-fertilized scallop families. The genotyping results showed the high success rate of more than 90% in total for embryos/larvae preserved in ethanol for 1-5 years. Furthermore, the successful genotyping for the larvae sampled from a natural habitat well demonstrated the potential use of this method in practical ecological analysis.
海洋扇贝在成年后是固着的，但有一个很长的浮游幼体阶段，在缺乏明显障碍的海洋环境中，有很大的迁移可能性。基于分子标记的扇贝胚胎/幼虫的遗传分析对于阐明时空基因流以及独特的种群和群落结构非常重要。然而，技术挑战，如单个胚胎/幼虫的分离以及提取的 DNA 数量少且质量差，使得对保存在乙醇中的单个胚胎/幼虫进行基因分型成为一项非常困难的任务。在这项研究中，我们分析了可能影响基于简单序列重复的单个胚胎/幼虫基因分型的 DNA 数量和质量的因素。基于分析的因素，我们开发了一种基于 LoTEPA 缓冲液的方法，通过控制种间和种内以及自交扇贝家族对其准确性、稳定性和重现性进行了评估。基因分型结果表明，对于保存在乙醇中的胚胎/幼虫，保存 1-5 年的成功率超过 90%。此外，从自然栖息地采集的幼虫的成功基因分型充分证明了该方法在实际生态分析中的潜在用途。
