Genetic Structure and Selection Signals in Hetian Gray Donkeys and Xinjiang Donkeys from Xinjiang, China

The Hetian Gray Donkeys (China_HTQD) and Xinjiang Donkeys (China_XJD) are representative local donkey breeds in Xinjiang, China, that show significant differences in production phenotypes, such as body size and coat color. However, their genetic background and the molecular mechanisms influenced by artificial selection have not been systematically elucidated. This study integrated whole-genome data from 60 newly sequenced China_HTQD and China_XJD individuals, along with 155 global reference datasets from the NCBI database, to construct a modern donkey whole-genome variation set covering domestic and overseas populations (including Africa, West and Central Asia, and Europe). The results of genetic diversity analysis indicated that China_XJD has slightly higher genetic diversity than China_HTQD, while the China reference population (China_Ref) and European population (Europe) have the lowest genetic diversity. China_HTQD and China_XJD have greater genetic variation. Both PCA and NJ phylogenetic analyses showed that China_HTQD and China_XJD are genetically very close, clustering within the Chinese donkey population background, and exhibit clear geographic genetic isolation from overseas reference populations. ADMIXTURE analysis (with K = 2 showing the lowest cross-validation error) revealed that the two breeds share a highly similar genetic structure, with only minor differences in the proportions of specific ancestral components, while both are distinctly different from the other four reference populations. A whole-genome differentiation scan (Fst) showed limited overall genetic differentiation between China_HTQD and China_XJD populations, but significant differentiation peaks were observed in some genomic regions. Based on the joint screening of windowed Fst (Fixation Index) and XP-CLR (Cross-Population Composite Likelihood Ratio), this study identified two candidate genomic regions containing the TBX3 and ZFAT genes. Among these, TBX3 emerged as a strong candidate with a robust selection signal and a well-established role in body size regulation. In contrast, ZFAT showed a weaker signal, and its potential involvement in coat color remains speculative and requires further validation. In summary, this study not only clarified the close genetic relationship and the level of interpopulation differentiation between China_HTQD and China_XJD at the whole-genome level but also identified candidate genes potentially associated with phenotypic divergence, providing gene-level confirmation of TBX3 and the novel identification of ZFAT as a candidate for coat-color variation, thereby providing a scientific foundation for elucidating the genetic mechanisms underlying phenotypic differences in Xinjiang’s local donkey genetic resources.