Genetic evidence of an Asian background in heteroplasmic Iberian cattle (Bos taurus): effect on food authentication studies based on polymerase chain reaction-restriction fragment length polymorphism analysis.
This work was aimed at identifying nucleotide polymorphic sites in a 359 bp region of the cytochrome b (cytb) mitochondrial gene of Iberian cattle (Bos taurus). This region is widely used as target in polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) species identification studies in foodstuffs destined for human and animal consumption. Two different coexisting restriction patterns were observed in four of the six animals studied when the 359 bp DNA fragment was cleaved with PalI, HinfI, MvaI, RsaI, or MboI. The amplification of both genotypes with the mitochondrial-specific primers L14735 and H15149 revealed the absence of nuclear pseudo-cytb genes, confirming the existence of mitochondrial heteroplasmy. The two coexisting mtDNA fragments were selectively sequenced in PCR extracts in which one genotype predominated over the other, both exhibiting a sequence variation of 10.4%. From the 37 nucleotide mismatches observed between genotypes, 32 were transitions and five were transversions. While 31 of the nucleotide mismatches between genotypes resulted to be conservative at the amino acid level, six changes implied amino acid substitutions, five of them being located in the variable transmembrane region. Genetic analysis suggests the presence of an Asian background in the mitochondria of Iberian cattle: while one of the genotypes matched the published sequence for Bos taurus, the other genotype clustered with a B. primigenius indicus animal and close to an Asian Bos taurus animal. These results also suggest that a number of current PCR-RFLP species identification methods based on cytb sequences may not be reliable for the accurate detection and identification of bovine material: an alternative battery of enzymes consisting of MmeI, NlaIV, and AluI is proposed.
Prado
Marta
M
Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences/College of Biotechnology, University of Santiago de Compostela, Lugo, Spain.
Calo
Pilar
P
Cepeda
Alberto
A
Barros-Velázquez
Jorge
J
D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D002417:Cattle / Q000235:genetics*; D045303:Cytochromes b / Q000235:genetics*; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics; D005838:Genotype; D008928:Mitochondria / Q000235:genetics; D018482:Muscle, Skeletal / Q000201:enzymology; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D013045:Species Specificity
D017931:DNA Primers; D004272:DNA, Mitochondrial; D045303:Cytochromes b