福尔马林固定匙吻鲟样本线粒体基因组的测定

Determining mitochondrial genome sequences from formalin fixed paddlefish (Polyodon spathula) samples

摘要:

在鱼类分子系统学的研究中，常常会碰到样本难以采集而博物馆中保存的大量标本又因经过福尔马林处理而无法利用的难题，比如：鲟形目鱼类。为了研究鲟鱼的系统分类，获取福尔马林固定标本的线粒体基因组，我们将匙吻鲟（Polyodon spathula）样本用福尔马林进行不同时间的固定 （1 h、1 d、3 d、10 d、30 d 和150 d ），采用提取古 DNA 的方法获得样本的 DNA，以新鲜匙吻鲟线粒体基因组设计诱饵（Baits），通过靶基因富集“钓取”固定样本中的线粒体基因组序列，进行 Illumina 测序，获得线粒体基因组序列。结果表明处理 1 h、1 d、10 d、30 d、150 d 的匙吻鲟样本测得的线粒体基因组序列长度均为 16 524 bp，覆盖率为 100%，目标序列占总数据的比率均大于 45%，错误率均为 0。另外，采用 Q-ratio 方法检测固定时间对 DNA 片段化的影响：前端引物保持不变，设计 4 种右端引物分别进行 PCR 扩增，目标片段长度分别为 41、129、305和 650 bp。结果前3组引物在各样本 DNA 中均扩增成功，650 bp 片段引物仅在固定 1 h的样本 DNA 中扩增成功；固定时间越长，各组引物所得到的 Q-ratio 值（不同长度扩增片段和41 bp 扩增片段的比值）越低，DNA 片段化程度越大。此研究结果可为提取福尔马林固定标本 DNA 用于分子遗传学研究提供参考。

Abstract:

When studying molecular phylogenetics of fishes, we often encounter difficulty in collecting fresh samples of rare species; in the meaning time there are a large number of specimens stored in museum collections that could not be used due to formalin treatment on those specimens. For example, the Acipenseriformes. In order to study taxonomy and get the mitochondrial genome of formalin-fixed sturgeon specimens, we tested a new strategy of determining mitochondrial genome sequence from formalin-fixed samples. We treated muscle sample of paddlefish (Polyodon spathula) with formalin for different length of time, 1 hour, 1 day, 3 days, 10 days, 30 days and 150 days. We then extracted DNA from those samples using an ancient DNA method, captured and sequenced the mitochondrial genome of all samples using homemade biotinylated baits and Illumina sequencing. The ratio of on-target reads was above 45% for all samples. The genome sequences assembled are 16 524 bp in length, with 100% coverage and zero error rate. We also studied the relationship between DNA fragmentation and time used for formalin treatment. The results showed that when the length of the target amplicon was 650 bp, it could be amplified only in sample treated with formalin for 1-hour . When the length of the amplicons was 41 bp, 129 bp or 305 bp, a trace of product could be amplified for samples treated with formalin for up to 150 days. According to Q-ratio scores (the ratio between different size amplicon and the 41 bp amplicon), we found that the longer of formalin fixation time was, the lower Q-ratio score became. In other word, the longer of formalin-fixed time was, the greater degree of DNA fragmentation occurred. These results can augment usefulness of DNA extraction from formalin-fixed samples in molecular studies.