舟山近海水样环境DNA获取方法的建立

以曼氏无针乌贼(Sepiella japonica)为研究对象, 通过绝对定量技术建立和优化了舟山近海高浊度水样环境DNA(Environmental DNA, eDNA)的获取方法。研究结果如下: (1)同体积水样采用乙醇沉淀法获得的eDNA产量是滤膜抽滤法的1.76—2.53 倍, 但在实际应用中由于受到采样体积、处理方式、配套设备的限制, 乙醇沉淀法难以发挥出优势; (2)滤网对泥沙等杂质无过滤作用, 添加滤网并不能过滤掉泥沙及增加抽滤体积; (3)滤膜孔径的大小对少量水样的eDNA产量有很大影响, 但对大体积水样eDNA的产量无影响; (4)水样静置处理有可能会增大eDNA产量, 但也有可能会增大eDNA结果的波动性, 使生物量评估结果误差较大; (5)阳离子表面活性剂对eDNA降解有明显的抑制作用; (6)去膜法效果优于碎膜法, 建议使用去膜法进行eDNA消化, 使用时增加离心时间; (7)酚抽除沙法虽然不能提高eDNA产量, 但能明显提高产物纯度。研究首次建立了舟山近海水样大生物eDNA最适获取方法, 为相似水域的水样采集及eDNA提取提供了借鉴参考。

THE EDNA COLLECTION METHOD OF ZHOUSHAN COASTAL WATERS

This study used Sepiella japonica as the research object to establish and optimize the acquisition method of high-turbidity water DNA (environmental DNA, eDNA) in Zhoushan offshore by absolute quantitative technique. The results indicated that the eDNA yield by ethanol precipitation method is 1.76—2.53 times higher than that of filtrating method, but the limitations of the collection volume, treatment requirement and supporting equipment make the ethanol precipitation method difficult to employ in practice. Filter screen with small aperture have no filtering effect on sediment. The size of filter aperture has a great effect on eDNA yield only when small volume water samples were collected. Precipitation treatment to water sample enhanced the yield of eDNA, but it also increased the variation of eDNA yield. Cationic surfactant significantly inhibited eDNA degradation. The effect of the membrane removal method is better than the membrane method, and it is recommended to increase the centrifugation time when the membrane removal method was used. Although the phenol extraction method can not improve the eDNA yield, it can significantly improve the purity of the product. This study is the first to establish an optimal method for obtaining eDNA of macro-organisms from Zhoushan offshore water, which provides reference for water sample collection and eDNA extraction in similar waters.