Abstract:

The mitochondrial genes of animals are frequently utilized as molecular markers in phylogenetic studies， environmental DNA （eDNA） analysis， and species identification， including COX1 and 12S rRNA. However， the variability in locus selection among researchers has contributed to a lack of consistency in reference databases. Adopting complete mitochondrial genome sequences as references can efficiently solve this problem. For environmental DNA research， establishing a localized mitochondrial genome database for various species is particularly imperative. In our study， we designed bird-specific mitochondrial genome gene capture probes based on 10 conserved loci within the avian mitochondrial genome. Employing these probes， we captured， assembled， and annotated the mitochondrial genomes of 17 species， encompassing 19 bird samples collected in Shanghai， China. Despite the absence of a reference mitochondrial genome for the Pale Thrush， we successfully assembled its complete mitochondrial genome. A comparative analysis of sequencing outcomes utilizing both methods， namely direct library construction without enrichment and enrichment preceding library construction， for Pale Thrush samples revealed that gene capture significantly enhanced the proportion of mitochondrial DNA in the overall genome library. A subsequent random sampling of the sequencing outcomes from both methodologies revealed that， under stringent conditions involving roughly 40 000 reads （equivalent to approximately 0.012 gigabytes of sequencing data）， the captured results achieved a coverage of approximately 75%， whereas the non-captured samples exhibited only a 25% coverage. Notably， with an increased sequencing data of approximately 0.24 gigabytes， the gene-captured samples attained 100% coverage for the first time. In comparison to the conventional approach of segmentally amplifying mitochondrial genomes using primers， the probes introduced in this study offer a more efficient and expedited method for acquiring bird mitochondrial genomes.

Key words: mitochondrial genome, aves, gene capture

摘要：

动物线粒体基因常常被作为系统发育研究、环境DNA、物种鉴定的分子标记，如COX1、12S rRNA等，而不同学者使用的位点各不相同，导致参考数据库难以统一，而使用完整的线粒体基因组序列作为参考，可以很好地解决这个问题。建立本地物种的线粒体基因组数据库，对环境DNA研究尤为重要。根据鸟类线粒体基因组内10个保守位点开发了针对鸟类的线粒体基因组基因富集探针，利用该探针富集并组装、注释了17 种共19只采集于上海地区的鸟类样本的线粒体基因组，在没有白腹鸫（Turdus pallidus）参考线粒体基因组的条件下组装获得了该物种的完整线粒体基因组。同时，针对白腹鸫样本“不富集，直接建库”和“先富集，后建库”两种方法的测序结果进行了对比，结果表明基因富集可以显著提高全基因组文库中线粒体DNA的占比；随后对两种方法的测序结果进行随机抽样，结果表明在约40 000条序列（约0.012 G测序量）的极端情况下，富集后结果可达到约75%的覆盖度，而未富集的样本仅有约25%的覆盖度；在测序量约0.24 GB时，富集后的样本首次达到100%覆盖度。相对于传统使用引物逐段扩增获取线粒体基因组的方法，研究提出的RNA探针为获取鸟类线粒体基因组提供了更加高效、快捷的方法。

关键词: 线粒体基因组, 鸟类, 基因富集