Development of a High‐Resolution <scp>MNP</scp> Marker System for Aquatic Biodiversity Monitoring: A Case Study With <scp> <i>Schizothorax prenanti</i> </scp> in the Yangtze River

Effective monitoring of aquatic biodiversity is critical for conservation, yet current approaches such as mitochondrial COI barcoding and microsatellite markers exhibit limitations in resolution, sensitivity, and scalability, particularly for detecting low-abundance or degraded DNA in mixed aquatic samples. To address these challenges, we developed a novel Multiple Nucleotide Polymorphism (MNP) marker system tailored to S. prenanti, an endangered endemic fish species emblematic of biodiversity crises in the Yangtze River. Through restriction-site associated DNA sequencing, we identified 115 genome-wide MNP markers. These markers demonstrated ultrasensitive detection (~1 DNA copy/reaction) and high specificity (mean discriminative power = 0.77, calculated as the probability that two random samples differ at a locus). When applied to environmental DNA from the Yangtze River, the MNP system revealed substantial genetic diversity among 86 samples (84% average differentiation rate) and quantified the contribution of artificially stocked fish to natural populations, identified 567 shared alleles between stocked and wild populations. By outperforming traditional methods in analysing fragmented DNA and enabling high-throughput applications, this MNP framework provides a transformative approach for conservation genetics. Our scalable solution bridges the gap between genetic research and conservation action, offering global applicability for aquatic biodiversity monitoring.