Development and Validation of Tetranucleotide Repeat Microsatellite Markers at the Whole-Genome Level in the Yangtze Finless Porpoise

The Yangtze finless porpoise (<i>Neophocaena asiaeorientalis asiaeorientalis</i>, YFP) is the only freshwater cetacean species currently found in China’s Yangtze River. To accurately evaluate its genetic diversity and provide reliable molecular markers for population genetic studies, this study developed a highly efficient and reproducible method for identifying polymorphic microsatellite loci using whole-genome sequencing data. Using this method, we identified and validated a set of highly polymorphic microsatellite markers, which were then used to analyze the genetic diversity of the YFP populations in Poyang Lake to evaluate their effectiveness. Our results demonstrated that the screening pipeline successfully identified 220 tetranucleotide repeat microsatellite loci. Based on the principle of uniform chromosomal distribution, 190 loci were randomly selected for experimental validation, of which 19 exhibited stable amplification, high polymorphism, and a low genotyping error rate. Genetic diversity analysis based on these markers revealed significant genetic variation among YFP populations in Poyang Lake, confirming the effectiveness of the developed markers. The polymorphic microsatellite molecular marker system developed in this study demonstrates high reliability and applicability for assessing YFP genetic diversity. This system provides a critical technical foundation for future research in conservation genetics, genetic resource preservation, and the development of genetic management strategies for the species.