A low-cost DNA extraction method from pine sawdust reveals novel ITS sequences for forensic wood identification.

The illegal timber trade threatens biodiversity, highlighting the need for forensic identification tools. Extracting DNA from processed wood like sawdust is challenging due to inhibitory substances, and commercial kits are often cost-prohibitive. This study developed and validated RUIZTECMA, a cost-effective DNA extraction protocol for pine sawdust, using eight endemic Pinus species from Michoacán, Mexico, as proof-of-concept. DNA yields ranged from 210.7 to 1662.5 ng/µL, with A260/280 ratios of 1.40-1.91, confirming effective inhibitor removal. ITS amplification was successful for all species. Remarkably, BLAST analysis revealed no significant matches with existing GenBank entries, indicating previously unreported ITS variants. This study provides a cost-effective forensic tool and adds new sequence data to improve reference databases against illegal logging of Mexican pines.