Genetic Diversity in Coppice Chestnut Forests in Central Italy and Potential Use of SSR-Based Timber Traceability

Sweet chestnut (Castanea sativa Mill.) coppice forests are important Mediterranean resources, but the genetic structure of local coppice stands remains insufficiently characterized, limiting their use as reference systems for wood provenance studies. This study integrated population genetic analysis, optimization of wood-derived DNA recovery, and reference-based assignment to evaluate Lazio coppice stands and test the declared origin of timber samples. Four Lazio populations were genotyped at 12 SSR loci, screened for Hardy-Weinberg disequilibrium, null alleles, and kin structure, and compared with eight European/Mediterranean populations using five shared loci. DNA extraction from dried wood was optimized through a CTAB-PEG workflow, and 40 timber samples from four sawmills, including two declared from Lazio (MCt and RPt), one from Calabria (CALt) and one from France (FRAt), were assigned using supervised DAPC, STRUCTURE with population information and GDA_NT Bayesian assignment and exclusion testing. Lazio stands retained high SSR diversity but showed heterozygote deficiency and fine-scale family structure; therefore, a conservative post-COLONY dataset was used for downstream analyses. Differentiation was weak among Lazio stands, but they showed a coherent Central Italian affinity within the available European/Mediterranean reference set. The optimized protocol yielded reproducible SSR profiles from all timber samples, with an 80.0% successful wood extraction rate. Within the available reference panel, assignment analyses indicated compatibility of MCt and RPt with a Lazio origin, whereas CALt and FRAt were incompatible with the Lazio references and should be interpreted as extra-regional or insufficiently represented by the current baseline. SSRs provide a practical first-line tool for regional chestnut timber screening, although broader reference panels and complementary high-resolution markers are needed to strengthen fine-scale provenance inference.