Microsatellite-Based Evaluation of Genetic-Distance-Driven Crossbreeding in the Endangered Freshwater Fish Pseudopungtungia nigra

Artificial crossbreeding is a critical strategy in the restoration of endangered freshwater fishes with small, fragmented populations, where natural spawning alone cannot reverse genetic erosion. This study examined the genetic diversity and fitness traits of crosses between genetically distant (HGD) and closely related (LGD) broodstock individuals of Pseudopungtungia nigra, an endangered Korean freshwater fish. Using ten microsatellite loci, we evaluated genetic diversity, population structure, and early survival performance among crossbreeds and their broodstock. Both HGD and LGD progenies showed deviations from Hardy–Weinberg equilibrium and exhibited higher observed heterozygosity than expected, indicating the influence of artificial selection. The broodstock and both crossbred groups displayed bottleneck signals, while LD-based effective population size was infinite for the broodstock and HGD but finite for LGD, suggesting estimation bias because the parameter was undeterminable due to small sample size (each group, n = 28–30). STRUCTURE and DAPC analyses revealed that HGD_20 was most genetically similar to the broodstock population, while LGD and HGD_19 formed distinct clusters. Hatching rate was 1.5-fold higher in HGD compared with LGD (p < 0.05), although survival did not differ significantly (p > 0.05). These results highlight that crossbreeding based on genetic distance can enhance genetic diversity and hatching performance without causing excessive genetic divergence from the parental population, offering a practical model for the genetic management of endangered fish restoration.