Abstract
This study was aimed to preserve mitochondria from liver tissues of Korean native cattle (KNC) as genetic resources and to analyze phylogenetic relationships among cattle breeds using the whole sequences of mtDNA. Mitochondria of KNC has been isolated with the alkaline lysis procedure using1 gof liver tissues, and measurements showed numbers of cells (2.64 × 1010), concentration (114.6 ug/ml), proteins (0.278 ug/ml), and absorbance (0.029) in 260 nm. Mitochondrial DNAs (mtDNA) were extracted from the isolated mitochondria to determine whole sequences that can be used to estimate genetic distances among cattle breeds. Designing of primers based on the bovine mtDNA sequence was resulted in 16 primer sets covering the whole mtDNA regions. The analysis of 40 KNC mtDNA sequences revealed 69 polymorphic sites that were less than the average number of SNP (single nucleotide polymorphism) for other populations (82 sites) in this study. As expected, the highest observation of SNPs was found in the D-loop region, and a total of 29 SNPs were shared between cattle populations. The haplotype analysis, which used 13 SNPs located in D-loop, COX2, ND5, CYTB, and non coding regions, revealed that KNC showed clearly different haplotypes that may be used to distinguish KNC from other breeds as well as to characterize individual identifications. The phylogenetic analysis revealed that KNC showed a relatively close genetic distance with Japanese black cattle (JBC) comparing with other breeds. Estimations of the average nucleotide diversity (0.0008) and the ratio of transition/ transversion (0.1050) of KNC were placed somewhere in the middle of genetic diversity among cattle breeds. The identified genetic variants and results of phylogenetic analysis will help understanding the phylogenetic relationships of KNC and provide useful genetic information for further studies regarding SNPs in mtDNA regions.
Highlights
Phylogenetic or population genetic studies have been focused on finding genetic variations in mitochondrial DNA because mutations in this region contained 5 times higher than other genetic materials [1,2]
Mitochondria has been successfully isolated using 1 g of liver tissues showing 2.64 × 1010 cells that were enough to preserve mitochondria as genetic resources and to isolate Mitochondrial DNAs (mtDNA) for further analysis of genetic variants
After verification of segments using direct sequencing analysis, alignments were conducted with the sequenced files for each individual, and complete contigs of mtDNA, which revealed approximately 16,388 bp showing similar sequence lengths compared with several cattle breeds from the GenBank, were obtained
Summary
Phylogenetic or population genetic studies have been focused on finding genetic variations in mitochondrial DNA (mtDNA) because mutations in this region contained 5 times higher than other genetic materials [1,2]. Genes in mtDNA are available to characterize breeds and individuals in phylogenetic studies [6,7], but a few reports are available for SNPs from the coding regions because studies believed that SNPs in coding regions may not be available to explain genetic diversity with low frequencies of genetic variations. It is still, required that uses of the whole mtDNA sequences are needed to estimate the genetic relationships among breeds, to characterize the breed specificity, and to identify individuals
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