Insights into pedigree- and genome-based inbreeding patterns in Martina Franca donkey breed

Conserving small, endangered equine populations demands tools that capture both recent and historical inbreeding more accurately than pedigree alone. The Martina Franca donkey, a large indigenous breed from southern Italy that approached extinction in the 1980s, offers a relevant model for conservation genetics in equids. Here, we combine pedigree- and genome-based approaches to quantify inbreeding, disentangle its temporal components via runs of homozygosity (ROH), and highlight putative selection targets to inform management. We sampled 101 studbook-registered animals (70 females, 31 males) and generated genome-wide data by the double-digest restriction-site associated DNA sequencing, retaining 21 280 high-quality Single Nucleotide Polymorphisms after filtering. We estimated pedigree inbreeding (F_PED1) and genomic indices based on excess homozygosity (F_HET, F_HAT1-3) and ROH-derived genomic inbreeding (F_ROH). F_PED1 ranged from 0.029 to 0.245 (mean 0.114), whereas F_ROH ranged from 0.006 to 0.316 (mean 0.147), with weak concordance between pedigree and genomic metrics, consistent with incomplete pedigrees or founder effects. Across individuals, we detected 4 433 ROH segments; medium-to-long segments (4-16 Mb) predominated, indicating substantial recent inbreeding, while very short ROH (< 2 Mb) were rare (about 2%), suggesting limited ancient autozygosity. ROH were unevenly distributed across the genome: chromosomes 2 and 3 harboured the most segments, whereas chromosome 18 had the fewest. We identified ROH islands on chromosomes 2, 6, 8, 12, 13, and 19; candidate genes therein included SHH (development), EPAS1 (hypoxia response), OPRK1 (stress response), and BIRC5 (apoptosis/cell cycle), pointing to historical pressures on development, resilience, and reproduction. Overall, genomic measures-particularly F_ROH-provided a finer-grained portrait of autozygosity than pedigree alone and revealed focal regions likely shaped by selection. These results deliver actionable guidance for breeding schemes seeking to limit further inbreeding while preserving adaptive variation, and they illustrate how genomic surveillance can bolster conservation strategies for endangered donkey breeds and other small equid populations worldwide.