Introduction and Objective: Selection over the past years has caused commercially modified livestock to have less genetic resistance and adaptability compared to native livestock, which is the reason for the reduction of genetic diversity. Implementing any breeding programs to adapt to environmental changes quickly is necessary. In the past studies that were conducted to investigate the differential analysis of genes in the Holstein and Cholistani breeds of the Punjab province of Pakistan, a very significant difference in expression was observed in some genes, including mitochondrial genes, which became the basis of the present study. Therefore, this research aims to investigate the reasons for the difference in gene expression between Holstein and Cholistani cattle breeds in mitochondrial genes, including ATP6, ATP8 and CYTB genes, which are involved in important processes such as energy metabolism in dealing with biotic and abiotic stresses. and also play a role in disease resistance, using transcript data (RNA-Seq). For this purpose, transcriptome coverage, nucleotide and protein regions and mutations, and deletion and addition genetic differences in the mitochondrial genome of these two breeds were investigated.

  Results: The number of deletion sites in the Holstein breed in the three ATP6, ATP8, and CYTB gene loci was 96, 28, and 91, respectively, which were more than the number of deletion points in the Cholistani breed (vs. 84, 9, and 57). In the Cholistani breed, there is an insertion of 64 bp in the ATP6 gene locus at position 8733 bp, and two insertion regions in the CYTB gene locus at positions 15846 and 14779 bp, 17 and 24 bp in length, respectively. No insertion was observed in the ATP8 locus in the Cholistani breed. In the Holstein breed, there is an insertion region at position 8185 with a length of 16 bp in the ATP8 locus, an insertion region at position 8733 bp with a length of 20 bp in the ATP6 gene locus, and in the CYTB gene, three insertion regions were observed at positions 14779, 15355, and 15356, respectively, 27, 42, and 16 base pairs in length. Among the three gene loci, ATP6 had the highest level of coverage and ATP8 had the lowest level of coverage, and CYTB was in the middle of the other two loci. The comparison of the nucleotide sequences of the ATP6 gene locus with a length of 678 base pairs in two Holstein and Cholistani breeds showed 8 polymorphic regions and 3 polymorphic regions based on the amino acid sequence. They included two threonine-to-alanine conversions and one isoleucine-to-valine conversion position. Comparison of the nucleotide sequences of the ATP8 gene locus with a length of 198 base pairs in Holstein and Cholistani breeds containing 6 polymorphic regions and based on the amino acid sequence, they contained 2 polymorphic regions. Amino acid changes included valine to isoleucine and threonine to alanine. Comparison of the nucleotide sequences of the CYTB gene locus with a length of 1137 bp in the Holstein and Cholistani breeds containing 19 polymorphic regions and based on the amino acid sequence, only two polymorphic regions were found where the amino acid valine was converted to valine-isoleucine and isoleucine-valine. Finally, the results of analyzing the conserved regions in the aligned fragment of ATP6, ATP8 and CYTB genes showed that from the sequence of the mentioned genes, the ATP6 gene has a 217 bp conserved fragment, the ATP8 gene without the conserved region. Non-protected genes are susceptible to nucleotide changes and mutations, which have caused the coming into being of new proteins and their new functions. and most of its regions of the CYTB gene were protected. CYTB gene had the most conserved regions in DNA with a length of 605 bp. Also, the results showed that transitional substitution in all genes is more than transversional substitution.