Struggle with over- and underestimatation of soil biodiversity in metabarcoding technology

2022 Fiscal Year Final Research Report

• Project/Area Number: 20K06101
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 39060:Conservation of biological resources-related
• Research Institution: Okinawa International University
• Principal Investigator: Seikoh Saitoh 沖縄国際大学, 経済学部, 准教授 (10623754)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Keywords: トビムシ / ミトゲノム / メタバーコーディング

Outline of Final Research Achievements

We established a laboratory protocol for sequencing mitochondrial genomes from individual Collembola specimens via whole-genome shotgun (WGS) sequencing. Contigs derived from WGS data provide sequencing depth information, reflecting their abundance in the total DNA. Consequently, it becomes possible to distinguish mitochondrial sequences from pseudogenes present in the nuclear genome.
We analyzed 5 newly sequenced mitogenomes in combination with 31 previously published mitogenomes. This analysis led to the identification of two conserved regions within the 16S ribosomal RNA gene. These regions matched the previously published metabarcoding primers described by Saitoh et al. (2016, Genome). Upon aligning the sequences, we observed primer sequence mismatches in certain taxa. These mismatches may explain the previously observed low PCR amplification of these taxa. To improve the metabarcoding method, we suggest utilizing degenerate PCR primers designed based on the consensus sequences.

Free Research Field

土壌動物学

Academic Significance and Societal Importance of the Research Achievements

本研究は高速並列DNAシーケンサー(NGS)による生物群集調査手法、特にその土壌動物における技術的改善を企図し、種によらず偏りなく増幅できるPCRプライマーを設計した。今後これを用いた群集調査を実施し、有効性を引き続き検証する。
本研究の派生的な成果として、土壌の微小な動物 1個体からの全ゲノムショットガン法の確立が挙げられる。NGSによる群集調査においてデータベースの蓄積が重要であるが、本手法では、容易にミトコンドリアゲノム全長配列が得られ、対象遺伝子が異なる複数のマーカーのために一度にデータベース蓄積が行える。また、トビムシ類の系統進化の解明にも貢献できる。