Comparative study of three simple molecular approaches in search of mtDNA haplogroup identification of domestic sheep

There are five (A–E) identified mitochondrial DNA (mtDNA) haplogroups (HPGs) in domestic sheep. In search of a quick and reliable HPG classification, three different molecular approaches were tested: the restriction fragment length polymorphism (RFLP) method applied to mtDNA control region (CR), the single strand conformational polymorphism (SSCP) method applied to NADH dehydrogenase subunit 2 (MT-ND2) and to NADH dehydrogenase subunit 4 (MT-ND4) regions. The results were evaluated comparatively to elucidate their power in discriminating the sheep mtDNA HPGs. For this purpose, the HPGs of 622 domestic sheep all from Turkey, hence from the genetic diversity hot spot of domestic sheep distribution were examined using these three approaches. The HPGs for some of the individuals (n = 240) were also obtained based on CR sequences. The SSCP analysis of the MT-ND2 region (by identifying HPGs as A, B, C/E and D) exhibited higher discrimination power, sensitivity and specificity in HPG classification when compared with the SSCP analysis of MT-ND4 region and RFLP analysis of CR. In conclusion, to identify the mtDNA HPGs of sheep, and especially to identify the rare HPGs (D and E), first screening the individuals by MT-ND2 SSCP and then sequencing the CR of only those individuals revealing C/E HPGs (mixed strategy of genotyping) may be preferred when sequencing is not easily available.