Genetic Monitoring of the Endangered Acipenser dabryanus Using a High-Resolution MNP System

Acipenser dabryanus, once abundant in China’s freshwater ecosystems, is now extinct in the wild. Effective genetic tools are urgently needed to support conservation efforts under the Yangtze River Protection Law and the 10-year fishing ban. Traditional molecular markers (e.g., COI, SSR, SNP) often lack sufficient resolution for fine-scale population assessment. Here, we developed a high-resolution Multiple-Nucleotide Polymorphism (MNP) system for A. dabryanus, comprising 424 newly developed, highly polymorphic markers optimized for multiplex PCR and high-throughput sequencing. The MNP system demonstrated excellent performance in individual fin tissue samples, successfully distinguishing Acipenser sinensis and Acipenser ruthenus individuals from the A. dabryanus population. In addition, 41 characteristic alleles specific to A. dabryanus were further identified. Across samples, it achieved >90% MNP locus detection rate, with an average of 7.48 alleles per locus, 66.5% heterozygosity, >98% reproducibility, and 99% accuracy. A strong correlation was observed between DNA concentration and spike-in-based copy numbers (R2 > 0.99), and sensitivity analysis confirmed reliable detection at ~1 copy/reaction. Application of the system across 97 samples, including 51 A. dabryanus tissue samples and 46 water environmental samples, revealed clear population structure with an average genetic differentiation of 70.45%, highlighting substantial genetic diversity within the sampled populations. Based on the above experimental results, the high-resolution MNP system has the potential to enable construction of population-specific allelic genotypes to distinguish wild individuals from released ones and, when applied to tissue and eDNA samples, to facilitate monitoring of migration pathways and habitat connectivity. Such applications could provide essential genetic information to evaluate release programs, guide conservation strategies, and inform habitat restoration for the recovery of A. dabryanus.