Objective Tilia amurensis is an important economic tree species and a nationally protected endangered tree species. Based on SSR molecular markers, this study analyzed the genetic diversity of Tilia amurensis from different provenances and constructed DNA fingerprinting profiles, aiming to provide references for the conservation of Tilia amurensis populations, the nationwide collection and preservation of germplasm resources, and the establishment of germplasm resource banks.

Method A total of 173 Tilia amurensis samples from 16 provenances across 11 cities in 5 provinces in China were collected. SSR molecular markers were used for PCR amplification to analyze the genetic diversity of Tilia amurensis. Molecular variance analysis (AMOVA) and population clustering analysis were employed to investigate genetic differentiation among populations. Specific primers were selected to construct DNA fingerprinting profiles for Tilia amurensis.

Result (1) From 12 primer pairs, 8 highly polymorphic and reproducible primer pairs were selected, collectively identifying 101 alleles, with an average of 12.6 alleles per polymorphic locus. The polymorphism information content (PIC) across loci ranged from 0.506 to 0.897, with a mean value of 0.739. (2) Only 4 primer pairs (C110, C840, D150, TC5) were required to effectively distinguish all Tilia amurensis individuals and construct their DNA fingerprinting profiles. (3) The genetic diversity of Tilia amurensis provenances was relatively high, with an effective number of alleles (N_e) of 4.937, Shannon’s information index (I) of 1.777, and Nei’s gene diversity (H) of 0.771. (4) Molecular variance analysis (AMOVA) revealed that 91% of genetic variation originated from within-population differences. (5) A phylogenetic tree constructed based on genetic distances classified the 16 provenances into 5 subclusters, with geographically proximate provenances clustering together, indicating closer genetic relationship.

Conclusion The Tilia amurensis populations exhibit a relatively high overall level of genetic diversity, with moderate genetic differentiation observed. Intra-population genetic variation is predominant. Among the 16 populations, the seed sources from Wangqing, Jilin Province (JWQ3), Heilongjiang Province of northeastern China (HDN7, HDN8, HDN9), and Chengde City, Hebei Province of northern China (HCD15) show low genetic diversity levels and should be prioritized for in situ conservation and continuous monitoring. Populations JWQ2, JWQ4, JHL5, HNA10, LDD12, and LFX13 demonstrate higher genetic diversity and could serve as key targets for resource collection and conservation cultivation. This study provides a scientific basis for further research on population distribution, evolutionary patterns, and conservation strategies of Tilia amurensis in China.