Analysis of Mitochondrial D-Loop Cloning Sequence Variations in Yak (Bos Grunniens)

Abstract Background: Yaks (Bos grunniens) are a livestock species that is highly adapted to the extremely conditions of the Qinghai-Tibet plateau. Heteroplasmy and nuclear-mitochondrial DNA have been reported in many species, but are still unknown in yaks. The diversity of D-loop sequences, derived from the sequencing of PCR products, provides widely-used maternal DNA markers for the study of yak population and biology. D-loop variations were examined by sequencing the mono clones of the PCR products from individuals, and a comparative analysis with other documented PCR-product based sequences was conducted.Results: Twenty-two and 27 variants were detected in 25 and 38 clones in two different yak individuals, respectively. Phylogenetic analysis in each demonstrated that variants could be grouped into two distinct major clades individual. Analysis of the D-loop sequences of domesticated yaks and wild yaks (Bos grunniens mutus) from the gene bank, which were originally obtained by sequencing of the PCR products, also revealed the two distinct clades. Integrative analysis of all the above variants indicated two major distinct clades, consistent with the respective analysis. One common variant was revealed to be shared by the differently sourced sequences. Furthermore, consensus comparison of all clone sequences revealed 13 differential sites between the two major clades, which are still strongly conserved in sequences from the gene bank. A few recombination events were detected between individual variants, and the supposed recombinants displayed distinct phylogenetic divergence from those in the same clade. Conclusion: Highly varied D-loop sequences were detected in the individual yaks. The detected variants were clustered into two deeply divergent clades as the haplotypes reported by other researchers employing PCR-product based D-loop sequences. This suggests either or both of heteroplasmy and nuclear-mitochondrial DNA in yaks, and also emphasizes the need for reconsidering D-loop sequences as haploid and matrilineal markers for phylogenetic analysis in yaks.