Costa Adriana, Giraldo Giovanny, Bishell Amy, He Tuo, Kirker Grant, Wiedenhoeft Alex C
Department of Sustainable Bioproducts, Mississippi State University, Starkville, MS, USA.
Forest Products Laboratory, Madison, WI, USA.
Plant Methods. 2022 Apr 20;18(1):51. doi: 10.1186/s13007-022-00885-z.
Illegal logging is a global crisis with significant environmental, economic, and social consequences. Efforts to combat it call for forensic methods to determine species identity, provenance, and individual identification of wood specimens throughout the forest products supply chain. DNA-based methodologies are the only tools with the potential to answer all three questions and the only ones that can be calibrated "non-destructively" by using leaves or other plant tissue and take advantage of publicly available DNA sequence databases. Despite the potential that DNA-based methods represent for wood forensics, low DNA yield from wood remains a limiting factor because, when compared to other plant tissues, wood has few living DNA-containing cells at functional maturity, it often has PCR-inhibiting extractives, and industrial processing of wood degrades DNA. To overcome these limitations, we developed a technique-organellar microcapture-to mechanically isolate intact nuclei and plastids from wood for subsequent DNA extraction, amplification, and sequencing.
Here we demonstrate organellar microcapture wherein we remove individual nuclei from parenchyma cells in wood (fresh and aged) and leaves of Carya ovata and Tilia americana, amyloplasts from Carya wood, and chloroplasts from kale (Brassica sp.) leaf midribs. ITS (773 bp), ITS1 (350 bp), ITS2 (450 bp), and rbcL (620 bp) were amplified via polymerase chain reaction, sequenced, and heuristic searches against the NCBI database were used to confirm that recovered DNA corresponded to each taxon.
Organellar microcapture, while too labor-intensive for routine extraction of many specimens, successfully recovered intact nuclei from wood samples collected more than sixty-five years ago, plastids from fresh sapwood and leaves, and presents great potential for DNA extraction from recalcitrant plant samples such as tissues rich in secondary metabolites, old specimens (archaeological, herbarium, and xylarium specimens), or trace evidence previously considered too small for analysis.
非法采伐是一场全球性危机，会造成严重的环境、经济和社会后果。打击非法采伐的行动需要运用法医鉴定方法，以确定整个林产品供应链中木材标本的物种身份、来源和个体识别。基于DNA的方法是唯一有可能回答这三个问题的工具，也是唯一可以通过使用树叶或其他植物组织进行“非破坏性”校准并利用公开可用的DNA序列数据库的工具。尽管基于DNA的方法在木材法医鉴定方面具有潜力，但木材中DNA产量低仍然是一个限制因素，因为与其他植物组织相比，木材在功能成熟时含活DNA的细胞很少，通常含有抑制PCR的提取物，并且木材的工业加工会使DNA降解。为了克服这些限制，我们开发了一种技术——细胞器微捕获技术，用于从木材中机械分离完整的细胞核和质体，以便后续进行DNA提取、扩增和测序。
在这里，我们展示了细胞器微捕获技术，即从山核桃（Carya ovata）和美洲椴（Tilia americana）木材（新鲜和陈旧）以及叶片的薄壁细胞中分离单个细胞核，从山核桃木材中分离造粉体，从羽衣甘蓝（Brassica sp.）叶中脉分离叶绿体。通过聚合酶链反应扩增内部转录间隔区（ITS，773 bp）、ITS1（350 bp）、ITS2（450 bp）和核糖体大亚基叶绿体基因（rbcL，620 bp），进行测序，并利用对NCBI数据库的启发式搜索来确认回收的DNA与每个分类单元相对应。
细胞器微捕获技术虽然对于许多标本的常规提取来说劳动强度过大，但成功地从65年多前采集的木材样本中回收了完整的细胞核，从新鲜边材和叶片中回收了质体，并且在从富含次生代谢物的组织、旧标本（考古标本、标本馆标本和木材标本馆标本）或以前认为太小而无法分析的微量证据等难处理的植物样本中提取DNA方面具有巨大潜力。
