Genome-Wide Mitochondrial Heteroplasmy Reveals Predominantly Individual-Specific Variation in Indian Indigenous Cattle

Abstract

Mitochondrial heteroplasmy is a prominent source of intra-individual genetic variation. However, their genome-wide and population-level structure remains insufficiently characterized in livestock. This study considers complete mitochondrial genome sequences (16341 bp) from 46 indigenous cattle animals representing six Indian breeds. High-quality mitochondrial variants were identified using a standardized bioinformatics pipeline that included next-generation sequencing, quality filtering, de novo assembly, and heteroplasmy detection. Out of the total, 16 indigenous cattle mitochondrial sequences exhibited heteroplasmy variation and were further investigated to assess their distribution across individuals, populations, and functional gene categories. A total of 245 heteroplasmic events were found across 223 unique heteroplasmic sites, with 209 (93.72%) sites restricted to individual animals. Heteroplasmic profiles demonstrated very low similarity among individuals (mean Jaccard distance = 0.990 ± 0.020), indicating high inter-individual heterogeneity. A considerable variation in heteroplasmy burden was found among individuals and breeds (Kruskal–Wallis test: individuals χ² = 70.34, P = 3.89 × 10⁻⁹; breeds χ² = 34.76, P = 1.68 × 10⁻⁶). Although no significant clustering by breed identity was observed (PERMANOVA; F = 1.011, P = 0.303). Furthermore, 76 non-synonymous and 62 synonymous heteroplasmic substitutions were detected at the functional level. However, their allele frequency distributions were not noticeably different (Wilcoxon test, P = 0.881). Gene-wise analyses show no consistent enrichment of heteroplasmic variants after multiple-testing correction. Overall, mitochondrial heteroplasmy in cattle was characterized by highly individual variation, weak population structure, and a lack of detectable functional-class differentiation at the allele-frequency level.