Identification of Important Variants of ATPase and Cytochrome b Coding Genes of the Mitochondrial Genome in Holstein and Cholistani Cows

Background: 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.It is necessary to implement breeding programs to quickly adapt to environmental changes.In the past studies that were conducted to investigate the differential analysis of genes in the Holstein and Cholistani breeds of the Punjab province in 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 were investigated in the mitochondrial genome of these two breeds.Methods: Transcriptome data (RNA-Seq) with free access to 40 samples of dairy cows from the University of Wisconsin, USA, and 45 female cows of Cholistani from the Gujatipir dairy cow unit of Bahawalpur City, located in the Punjab state of Pakistan, were used in the present study.Since the results of the differential gene expression of the two breeds showed different expression levels in several mitochondrial genes, and part of the differences were related to their different genetic structures in the two breeds, the present study aimed to investigate this topic.Using NCBI genome databases (https://www.ncbi.nlm.nih.gov/) and Ensemble (https://asia.ensembl.org/index.html), the sequences of three mitochondrial genes, including ATP6, ATP8, and CYTB, for Holstein cattle (Bos taurus) and Cholistani cattle (Bos indicus) were extracted and stored in FASTA format.IGB version 6 software was used to examine the level of transcriptomic coverage.MEGA6 software was used to calculate the polymorphic regions and to calculate the percentage of nucleotide substitution and alignment of the sequences.The types of polymorphisms occurring in the mitochondrial genomes of Holstein and Cholistani using nucleotide and amino acid sequences, as well as haplotype blocks, were specified by determining insert and deletion regions and protected regions in the genome using Dnasp5 software.Results: Deletion sites in the Holstein breed in the three ATP6, ATP8, and CYTB gene loci were 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 was 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 was 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 and ATP8 showed the highest and the lowest levels of coverage, respectively, and CYTB was in the middle of the other two loci.The comparison of the nucleotide