A strategy for developing high-resolution DNA barcodes for species discrimination of wood specimens using the complete chloroplast genome of three Pterocarpus species

Main conclusionA method for extraction of wood DNA and a strategy for designing high-resolution barcodes for wood were developed. Ycf1b was the prioritized barcode to resolve the Pterocarpus wood species studied.DNA barcoding, an effective tool for wood species identification, mainly focuses on universal barcodes and often lacks high resolution to differentiate species, especially for closely related taxa within the same genus. Therefore, more highly informative DNA barcodes need to be identified. This study is the first to report a strategy for developing specific DNA barcodes of wood tissues. The complete chloroplast genomes of leaf samples of three Pterocarpus species, i.e., P. indicus, P. santalinus, and P. tinctorius, were sequenced, and thereafter, the most variable DNA regions were identified on the scale of the complete chloroplast genomes. Finally, wood DNA was extracted from 30 wood specimens of the three Pterocarpus species, and DNA recovery rates of the selected regions were tested for applicability to verification on the wood specimens studied. The seven regions with the most variation (rpl32-ccsA, rpl20-clpP, trnC-rpoB, ycf1b, accD-ycf4, ycf1a, and psbK-accD) were identified from the chloroplast genome by quantifying nucleotide diversity (Pi>0.02), which was remarkably higher than that of the plant universal barcodes (rbcL, matK, and trnH-psbA) and the previously reported barcodes (ndhF-rpl32 and trnL-F) used for phylogenetic analysis in Pterocarpus. After comprehensive evaluation of species discrimination ability and applicability, the ycf1b region performed well in terms of the recovery success rate (76.7%) and species identification (100%) for wood specimens of the three Pterocarpus species, and was identified as the preferred high-resolution chloroplast barcode for selected Pterocarpus species. It will offer technical support for curbing illegal timber harvesting activities and for conserving endangered and valuable wood species.