Identification of Mulki as a Distinct Native Indian Cattle Breed Using Microsatellite-Based Population Differentiation

India being the world’s largest cattle population has a significant proportion of non-registered cattle population. These non-registered cattle population plays an important role in local economy. Mulki cattle is a non-descript cattle found in the Betul district, Narmadapuram region of Madhya Pradesh, India. These cattle have unique characteristics such as heat tolerance, disease resilience, and agricultural utility. Thus, the characterization of such populations is important to enable effective improvement and conservation strategies. Thus, this study aims to decipher the genetic diversity of Mulki cattle using 25 FAO-recommended microsatellite markers. The blood samples from 75 unrelated Mulki cattle were analysed, with DNA amplified and genotyped on an ABI Genetic Analyzer 3500xL. A total of 271 alleles were detected across 25 loci, with a mean observed allele number of 10.84 ± 0.847 and effective allele number of 3.971 ± 0.38. Observed heterozygosity (mean 0.735 ± 0.05) exceeded expected heterozygosity (mean 0.678 ± 0.03), indicating outbreeding and high genetic variability. The average polymorphic information content (PIC) was 0.663 ± 0.02, with most loci highly informative (PIC > 0.5). Sixteen loci deviated from Hardy-Weinberg equilibrium (P < 0.05), suggesting evolutionary influences such as null alleles or genetic drift. The mean FIS value of -0.094 ± 0.06 indicated heterozygote excess, supporting outbreeding in the population. These findings highlight the genetic distinctiveness of Mulki cattle, providing a scientific basis for their recognition as a potential native Indian breed and informing conservation and breeding strategies for non-descript cattle populations.