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Identifying the Genus origin of historic leather using DNA mini-barcoding and DNA meta-barcoding technologies

Qian Wu,Hailiang Yang,Yang Zhou,Xiaojing Kang,Liping Bao,Zvezdana Dode,Bingjian Zhang,Yulan Hu

The utilization of leather products dates back to the Upper Paleolithic era, serving diverse aspects of human life and production. Species identification of archaeological leather artifacts provides material evidence for understanding the development of ancient pastoralism and technological levels, while also informing critical decisions in conservation material selection and the reconstruction of historical craftsmanship. Archaeological leathers uncovered worldwide exhibit considerable diversity. However, due to complex burial environments, the epidermal structures of these artifacts are often damaged or entirely lost, making them unidentifiable through traditional morphological methods. Contamination and complex sample matrices impair the biomolecular specificity of existing techniques, limiting taxonomic resolution. DNA analysis has thus provided a novel pathway for species identification of leather artifacts. DNA barcoding represents a prevalent methodology within this domain. Unlike previous DNA barcoding studies that required preliminary identification of relic sources to design specific primers—often leading to sample waste—this study introduces a novel technique that uses only one pair of universal primers to identify unknown species in a single experiment. Moreover, it enables the identification of mixed samples via second-generation sequencing. Specifically, we designed a pair of universal primers based on DNA mini-barcoding and meta-barcoding concepts, allowing accurate species-level identification of 11 historical leather specimens from different periods and preservation conditions. Notably, this method requires only minimal sample input, as little as 1 mg per artifact. This study attempts to identify historical leathers from five different species using a single pair of primers in one experiment. The proposed "DNA mini-barcode and DNA meta-barcode technologies" enable highly efficient analysis, reducing sample consumption compared to traditional DNA techniques that rely on screening multiple primers for unknown samples. Furthermore, it enhances taxonomic resolution from the genus to the species level. This approach holds great promise for the species identification of historical leather artifacts.