Abstract
To reveal mitochondrial DNA (mtDNA) diversity and indicus/taurus type maternal lineage of the three major Philippine native cattle breeds (Batangas cattle in Southern Luzon, Ilocos cattle in Northwest Luzon and Iloilo cattle in Panay Island ), we sequenced the D-loop region (436bp) and three protein coding regions (ND2, ND4 and ND5) of mtDNA in these breeds together with other nine cattle breeds (American Brahman cross, Holstein-Friesian, Holstein-Friesian×Sahiwal crossbred, Aberdeen-Angus, Hereford, Swiss Brown, Jersey, Japanese Black and Japanese Brown). The total cellular DNA from leukocyte was subjected to LA (Long and Accurate)-PCR to amplify the most part (15,393bp) of mtDNA including D-loop region in order to avoid the co-amplification of mtDNA fragments (numts) of nuclear genomic DNA. The LA-PCR primer sets and conditions were as follows : forward primer : 5prime;-CGGCTCAACTTTTTTTGTAGCCACAGGCTT-3prime;, reverse primer : 5prime;-CTATGCCTAGGTTTATTGATAGTTGTGTTGTTGGT-3prime;. Each 50-μl reaction for LA-PCR contained 500ηg of the total cellular DNA, 10pmol of each primer, 0.4mM of dNTPs, and 2.5U Takara LA Taq polymerase in the applied buffer. PCR conditions : 1 cycle of denaturation at 94°C for 1min and 30 cycles of denaturation at 98°C for 20sec and 68°C for 15min, and a final 10min elongation-step at 72°C. LA-PCR product was used for sequencing the D-loop region, the four protein-coding regions and for LA-PCR-RFLP analysis. Phylogenetic analyses of the D-loop sequence and LA-PCR-RFLP patterns were carried out using MEGA version 2.1 (UPGMA method) and the method described by Nei and Li (1979) respectively. Phylogenetic analyses of mtDNA confirmed that the Philippine native cattle breeds were divided into two distinct indicus and taurus types. There seems to still exist several unique maternal lineages in the three Philippine cattle breeds. In addition, both indicus and taurus mtDNA types were found in a herd of Jersey cattle in Okayama Prefecture (Japan). Furthermore, to distinguish between both mtDNA types, we developed a simple PCR-RFLP method based on a sequence difference of the ScaI site in the ND4 region between them (position 11,068 : G (taurus type : ScaI recognition site)↔A (indicus type : non-ScaI recognition site)). A total 19 substitutions in ND5 between the indicus type mtDNA (Iloilo cattle) and taurus type mtDNA were observed. Four of the 19 substitutions caused amino acid replacement. Bali cattle specific mtDNA types were not found in the three Philippine native cattle breeds. GenBank accession numbers reported here : AB079300∼AB079365, AB090957∼AB090996.
