Development of a multiplex microsatellite genotyping system and its application in parentage assignment of hybrid snakehead (Channa argus ♀ × C. maculata ♂)

Microsatellite markers are important tools for genetic analysis and parentage testing in aquaculture. The all-male hybrid snakehead ( Channa argus ♀ × Channa maculata ♂) is characterized by rapid growth and strong disease resistance and is an aquaculture strain of considerable economic value. In this study, we developed a high-throughput multiplex microsatellite genotyping system suitable for this hybrid. Genome-wide screening identified 170,124 and 174,062 perfect microsatellite loci in C. argus and C. maculata , respectively. On this basis, 19 highly polymorphic loci were selected and assembled into six optimized multiplex PCR systems. Two hybrid groups were produced by crossing YY supermale C. maculata with female C. argus from Shandong (SD) and Hunan (HN), respectively, and the performance of the system was evaluated. Both groups exhibited high genetic diversity, with 297 alleles detected in the SD group (mean Na = 15.6; PIC = 0.602–0.879) and 317 alleles detected in the HN group (mean Na = 16.7; PIC = 0.465–0.879). Under the candidate-parent sets and analytical conditions used in this study, all 334 offspring in the SD group and all 343 offspring in the HN group achieved two-parent assignment, with cumulative exclusion probabilities (CE-2P) exceeding 99.99%. In addition, descriptive statistics stratified by sire indicated that offspring body weight was generally higher in the SD group than in the HN group; based on the microsatellite-derived two-parent assignment results, the preliminary heritability estimate for body weight was 0.275 in the SD group and 0.180 in the HN group. Overall, the multiplex microsatellite genotyping system established here provides effective technical support for parentage assignment, pedigree reconstruction, and population genetic analysis in hybrid snakehead, and lays a methodological foundation for subsequent broodstock management and molecular-assisted breeding research.