Primer effect in the detection of mtDNA heteroplasmy: insights from horse Cytochrome b gene.

Heteroplasmy, the presence of bi-allelic mtDNA types within an individual, has been previously detected in the D-loop region and Cytochrome b gene (Cytb) of mitochondrial DNA using PCR-RFLP. However, heteroplasmy was absent in thousands of equine mtDNA sequences deposited in GenBank. To address whether heteroplasmy widely exists in mitochondria of Chinese indigenous horses, we generated the data set of the target sites in Cytb region with Sanger sequencing and PCR-RFLP method as well. In this study, 23 heteroplasmic individuals were detected in 430 Chinese local horses. Both site and length heteroplasmy were identified in horse Cytb, especially in Xinihe and Ujumqin breeds. Our data provide evidence that the forward and reverse primers seem to produce a similar approximation to the proportion of mutation base call. However, locations of primers affected the proper detection of mtDNA heteroplasmy. The data obtained in this study highlight the importance of the primers in the accurate detection of heteroplasmy.