Data_Sheet_2_A Multi-Gene Region Targeted Capture Approach to Detect Plant DNA in Environmental Samples: A Case Study From Coastal Environments.docx

Metabarcoding of plant DNA recovered from environmental samples, termed environmental DNA (eDNA), has been used to detect invasive species, track biodiversity changes, and reconstruct past ecosystems. The P6 loop of the trnL intron is the most widely utilised gene region for metabarcoding plants due to the short fragment length and subsequent ease of recovery from degraded DNA, which is characteristic of environmental samples. However, the taxonomic resolution for this gene region is limited, often precluding species level identification. Additionally, targeting gene regions using universal primers can bias results as some taxa will amplify more effectively than others. To increase the ability of DNA metabarcoding to better resolve flowering plant species (angiosperms) within environmental samples, and reduce bias in amplification, we developed a multi-gene targeted capture method that simultaneously targets 20 chloroplast gene regions in a single assay across all flowering plant species. Using this approach, we effectively recovered multiple chloroplast gene regions for three species within artificial DNA mixtures down to 0.001 ng/μL of DNA. We tested the detection level of this approach, successfully recovering target genes for 10 flowering plant species. Finally, we applied this approach to sediment samples containing unknown compositions of eDNA and confidently detected plant species that were later verified with observation data. Targeting multiple chloroplast gene regions in environmental samples, enabled species-level information to be recovered from complex DNA mixtures. Thus, the method developed here, confers an improved level of data on community composition, which can be used to better understand flowering plant assemblages in environmental samples.

植物DNA环境样本的宏条形码分析，被称为环境DNA（eDNA），已被广泛应用于检测入侵物种、追踪生物多样性变化以及重建历史生态系统。由于片段长度较短且易于从降解DNA中恢复，trnL内含子P6环成为植物宏条形码分析中应用最为广泛的基因区域。然而，该基因区域的分类分辨率有限，往往无法实现物种水平的鉴定。此外，使用通用引物靶向基因区域可能会产生结果偏差，因为某些类群可能比其他类群更有效地扩增。为了提高DNA宏条形码在环境样本中解析开花植物物种（被子植物）的能力，并减少扩增偏差，我们开发了一种多基因靶向捕获方法，该方法在一次检测中同时对所有被子植物的20个叶绿体基因区域进行靶向。采用此方法，我们有效地从人工DNA混合物中恢复出三个物种的多个叶绿体基因区域，其DNA浓度低至0.001 ng/μL。我们测试了该方法的检测水平，成功恢复出10种开花植物的目标基因。最后，我们将此方法应用于含有未知eDNA组成的沉积物样本，并成功检测出植物物种，这些物种随后通过观察数据得到验证。在环境样本中靶向多个叶绿体基因区域，使得从复杂的DNA混合物中恢复物种水平信息成为可能。因此，此处开发的方法为群落组成提供了更高级别的数据，可用于更好地理解环境样本中的开花植物群落。