Table_4_VertU: universal multilocus primer sets for eDNA metabarcoding of vertebrate diversity, evaluated by both artificial and natural cases.xlsx

Environmental DNA (eDNA) metabarcoding is a powerful tool for monitoring biodiversity in natural ecosystems. The accuracy of eDNA metabarcoding relies heavily on the PCR primers that amplify target sequences. For vertebrates, most available primers are designed for particular groups. There have been inadequate attempts to design “universal” primers applicable to all vertebrates. Here, we developed three new universal primer sets (V12S-U, V16S-U, and VCOI-U) targeting mitochondrial 12S, 16S, and COI genes, respectively. They work for all vertebrate groups, with amplification length of 200-250 bp, facilitating the use of short-read sequencing platforms. We evaluated and compared the species detection ability of our primer sets and seven previously published ones through both in-silico PCR and experiment tests with mock DNA and zoo eDNA. The species detection success of the new primer sets is over 90%, showing better performance than previously published primer sets. We also tested our new primers with water eDNA samples of natural environments, and detected a total of 895 vertebrate OTUs (BLAST identity ≥ 0.95), comprising 182 species, 195 genera, and 94 families. Our results indicated that utilizing multiple markers can achieve better species detection than using only a single marker, which is especially important for monitoring large biological communities like vertebrates. The new primer sets enable researchers to detect the presence of species of different vertebrate groups in one eDNA metabarcoding survey, which simplifies the workflow and reduces the cost. It has the potential to serve as an alternative/complementary tool for future eDNA metabarcoding studies targeting vertebrates.

环境DNA（Environmental DNA，eDNA）宏条形码技术是监测自然生态系统生物多样性的有力工具。eDNA宏条形码技术的准确性高度依赖于用于扩增目标序列的聚合酶链式反应（Polymerase Chain Reaction，PCR）引物。对于脊椎动物而言，现有多数引物均针对特定类群设计，目前针对适用于所有脊椎动物的“通用”引物的开发尝试仍显不足。本研究开发了三套全新的通用引物组（V12S-U、V16S-U及VCOI-U），分别靶向线粒体12S、16S及细胞色素c氧化酶亚基I（COI）基因。该三套引物可覆盖所有脊椎动物类群，扩增产物长度为200~250 bp，适配短读长测序平台的使用需求。本研究通过电子PCR（in silico PCR）以及模拟DNA与动物园eDNA样本的实验测试，对本研究开发的引物组与7套已发表引物组的物种检测能力进行了评估与比较。新型引物组的物种检测成功率超过90%，性能优于已发表的引物组。本研究还利用自然环境中的水体eDNA样本对新型引物进行了测试，共检测到895个脊椎动物操作分类单元（Operational Taxonomic Units，OTUs）（BLAST比对相似度≥0.95），涵盖182个物种、195个属及94个科。研究结果表明，相较于仅使用单一标记基因，采用多标记基因可实现更优的物种检测效果，这对于脊椎动物这类大型生物群落的监测尤为重要。新型引物组可使研究人员在单次eDNA宏条形码检测中即可实现不同脊椎动物类群的物种检出，简化了实验流程并降低了检测成本。该引物组有望成为未来脊椎动物靶向eDNA宏条形码研究的替代/补充工具。