SNV Analysis of CNDP1, ADCY8, and RYR3 Genes for Differentiation of Canis lupus and Canis lupus familiaris

Recent studies have identified distinct wolf population clusters in Russia, the Caucasus, Europe, and Scandinavia. The traditional method of differentiating wolves and dogs based on STR loci requires regular testing in specific regions, especially during intercountry migrations. Uncontrolled hunting and migration lead to sharp fluctuations in wolf populations, complicating the monitoring of genetic diversity. SNV analysis, which reveals only two alternative allele forms in 95% of cases, is more promising. Given current genomic data, it is advisable to develop a new test system for differentiating wolves and domestic dogs based on SNV analysis. Using modern bioinformatics methods to analyze the accumulated NGS dataset, it is possible to identify genetic markers that reliably distinguish between wolves and domestic dogs. In this study, 3551 genomes were analyzed using the SRA-BLAST and GENIS bioinformatics algorithms: 312 wolf (Canis lupus) and 3239 domestic dog (Canis lupus familiaris) genomes. The differentiating potential of the polymorphic variants under study was assessed, and a test system for classifying wolves and domestic dogs was proposed on the basis of three SNVs: rs21980477 (Chr.1:4882549A>G, CNDP1), rs22264087 (Chr.13:27748425T>C, ADCY8), and rs23658289 (Chr.30:1414373T>C, RYR3). The test system showed high values of accuracy (98.76%), specificity (99.165%), and sensitivity (98.41%). Successful testing was conducted on a sample of 105 wolves and 130 domestic dogs, with a classification accuracy of 97.45%. Various genotyping methods have been proposed on the basis of this test system, including those using fluorescent labels (KASP) and real-time PCR (ASPCR). The development of a molecular genetic test system for differentiating wolves and domestic dogs has several important applications: biodiversity conservation, population management, the study of evolutionary processes, reconstructing the history of domestication, determining the origins of various dog breeds, monitoring wolf reintroduction programs, and developing measures to prevent unwanted hybridization between species.