文献类型：期刊文献

英文题名：Organellar microcapture to extract nuclear and plastid DNA from recalcitrant wood specimens and trace evidence

作者：Costa, Adriana[1,3] Giraldo, Giovanny[2,3] Bishell, Amy[3] He, Tuo[4,5] Kirker, Grant[1,3] Wiedenhoeft, Alex C.[1,2,3,6,7]

第一作者：Costa, Adriana

通信作者：Wiedenhoeft, AC[1];Wiedenhoeft, AC[2];Wiedenhoeft, AC[3];Wiedenhoeft, AC[4];Wiedenhoeft, AC[5]

机构：[1]Mississippi State Univ, Dept Sustainable Bioprod, Mississippi State, MS 39762 USA;[2]Univ Wisconsin, Dept Bot, Madison, WI 53706 USA;[3]Forest Prod Lab, Madison, WI 53726 USA;[4]Chinese Res Inst Wood Ind, Chinese Acad Forestry, Dept Wood Anat & Utilizat, Beijing, Peoples R China;[5]Chinese Acad Forestry, Wood Collect WOODPEDIA, Beijing, Peoples R China;[6]Purdue Univ, Dept Forestry & Nat Resources, W Lafayette, IN 47907 USA;[7]Univ Estadual Paulista Botucatu, Dept Ciencias Biol Botanica, Sao Paulo, Brazil

年份：2022

卷号：18

期号：1

外文期刊名：PLANT METHODS

收录：;Scopus(收录号：2-s2.0-85128465408);WOS:【SCI-EXPANDED(收录号:WOS:000784136300002)】；

基金：This work was supported in part by a grant from the US Department of State via Interagency Agreement number 19318814Y0010.

语种：英文

外文关键词：Organellar microcapture; Nucleus isolation; Plastid isolation; Single-cell sequencing; Wood identification

摘要：Background 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. Results 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. Conclusion 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.