Optimized DNA extraction from a large plant multi-species volume of below- and aboveground samples for NGS-based analysis

1.             The application of NGS-based approaches has great practical potential for modern vegetation ecological surveys as an efficient and cost-effective means for detailed taxonomic identification in environmental samples but with a set of problems and pitfalls associated with eDNA extraction that can affect subsequent analyses and sequencing results. In this context, the widespread use of NGS is mainly limited by the lack of a standardized DNA extraction method for assessing plant communities.

2.      Here, we describe a highly efficient standardized protocol of DNA extraction that overcomes the limitations posed by poor quality and low yield of DNA derived from a large volume of dried plant multi-species sample collected from both above (green) and belowground (soil) parts of grasslands.

3.      The primary advantage of this optimized protocol is its reliance on readily available, cost-effective reagents and standard laboratory equipment, ensuring both accessibility and reproducibility. The critical innovations driving the extraction of high-quality DNA include the efficient precipitation of soluble compounds following lysis and the standardization of pH across all samples, resulting in consistent and uniform further purification processing.

4.      These critical steps ensure uniform sample processing, effectively compensating for variability in environmental properties and significantly enhancing the consistency and reliability of the extracted DNA for downstream applications. The effectiveness of the protocol was validated through high-throughput analysis, demonstrated by the successful construction of metabarcoding libraries targeting the ITS2 region for species detection across 36 grassland plots.

5.      The proposed eDNA extraction protocol is designed for implementation in laboratories equipped with basic equipment, making it highly accessible. It holds significant value for conservationists, researchers, and biodiversity managers as a tool for assessing ecosystem health and stability.