以川陕哲罗鲑为目标物种的水样环境DNA分析流程的优化

水样环境DNA分析包括水样采集、DNA提取和分析等流程,已成为监测濒危水生生物种群分布调查的重要手段.为减少在监测目标物种尤其濒危物种中的不确定性,对水环境DNA分析流程的优化至关重要.本研究以川陕哲罗鲑为目标物种,采用滤膜法采集养殖池中的水样,设计了 250 mL、500 mL、1 L和2 L等4种水样采集量,分别采用 PoweWater DNA Isolation kit和DNeasy Tissue and Blood DNA extraction kit 提取水样环境DNA(eDNA),使用物种mtDNA D_loop区特异性引物进行PCR扩增,通过研究滤膜法、水样采集量和水样DNA提取方法对水样eDNA中目标基因检出率的影响,探索适宜的eDNA分析操作方案.结果表明: 使用DNeasy Tissue and Blood DNA extraction kit提取的水样DNA中目的基因的检出率为100%,效果明显优于PoweWater DNA Isolation kit(目标基因的检出率为0);目标基因扩增条带的亮度随水样采样量的增加而增加,其中2 L水样目标基因的扩增效果较理想;序列比对结果显示,本试验从水样DNA中成功扩增得到了川陕哲罗鲑mtDNA Dloop区部分序列.表明DNA提取方法和水样采集量对目标物种的检出率有显著的影响,滤膜法、2 L水样采集量、DNeasy Tissue and Blood DNA extraction kit更适宜进行水样的DNA分析,mtDNA D-loop区可作为川陕哲罗鲑识别的特异性分子标记.

Protocol optimization of eDNA analysis workflow for detecting Hucho bleekeri.

Environmental DNA (eDNA) analysis in water, consisting of water sample collection, DNA extraction and analysis, is increasingly important in detecting rare aquatic species and determining their distribution. Optimization of protocols used in an eDNA molecular workflow is critical to reduce its uncertainty in detecting target species, especially rare species. In this study, 250 mL, 500 mL, 1 L and 2 L water samples were collected by filtering method from a pond for Sichuan taimen (Hucho bleekeri) culture, followed by extraction and purification of eDNA using DNeasy Tissue and Blood DNA extraction kit or MoBio Power Water DNA extraction kit. A PCR procedure was then applied using specific primers from the D-loop region of mtDNA in Sichuan taimen. To explore an appropriate eDNA analysis protocol, the effect of filtering method, sample size and extraction protocols on the detection rate of target gene in water eDNA was studied. The results showed that the target gene was 100% detected from the water sample by using DNeasy Tissue and Blood DNA extraction kit, which was much better that the MoBio Power Water DNA extraction kit with a detection rate of 0%. Larger volume of water samples yielded better PCR products, with 2 L being the most appropriate volume. Sequence alignment results showed that target sequence from the D-loop region of mtDNA in Sichuan taimen was successfully amplified. The results indicated that DNA extracting method and water sample volume could strongly affect the detection rate. The protocol combining filtration, 2 L water sample, DNeasy Tissue and Blood DNA extraction kit was appropriate for eDNA analysis, and mtDNA D-loop region was recommended for detecting DNA of Sichuan taimen from water samples.