Mitochondrial DNA (mtDNA) is a useful genetic marker that can be used for species identification. The cytochrome b (Cyt b) gene is a suitable mtDNA candidate gene for use in phylogenetic analyses due to its sequence variability, which makes it appropriate for comparisons at the subspecies, species, and genus levels. This study was conducted to develop a rapid molecular method for species identification of Malayan gaur (Bos gaurus hubbacki), Kedah-Kelantan (KK) (Bos indicus), and Bali (Bos javanicus) cattle in Malaysia. DNA was extracted from blood samples of 8 Malayan gaurs, 30 KK, and 28 Bali cattle. A set of both specific and universal primers for the Cyt b gene were used in PCR amplification. DNA sequences obtained were then analyzed using BioEdit and Restriction Mapper softwares. The PCR products obtained from Cyt b gene amplification were then subjected to restriction enzyme digestion. The amplification, using both specific and universal primers, produced a 154- and a 603-bp fragment, respectively, in all three species. Two restriction enzymes, NlaIV and SspI, were used to obtain specific restriction profiles that allowed direct identification of Malayan gaur, KK, and Bali cattle. Our findings indicate that all three species can be identified separately using a combination of universal primers and the restriction enzyme SspI.
The Malayan gaur (Bos gaurus hubbacki) is one of the three subspecies of gaurs that can be found in Malaysia. We examined the phylogenetic relationships of this subspecies with other species of the genus Bos (B. javanicus, B. indicus, B. taurus, and B. grunniens). The sequence of a key gene, cytochrome b, was compared among 20 Bos species and the bongo antelope, used as an outgroup. Phylogenetic reconstruction was employed using neighbor joining and maximum parsimony in PAUP and Bayesian inference in MrBayes 3.1. All tree topologies indicated that the Malayan gaur is in its own monophyletic clade, distinct from other species of the genus Bos. We also found significant branching differences in the tree topologies between wild and domestic cattle.
The chromosomes of five gaur (Bos gaurus hubbacki) domestic cattle (B indicus cross B taurus) hybrids (three females, two males) were studied using the leucocyte culture method and centromeric (C) banding technique. All the hybrids had a diploid chromosome number of 2n = 58, made up of two submetacentric autosomes (different in size) and 54 acrocentric autosomes, most of which could be arranged in pairs in descending order of size. The sex (X) chromosomes in females were a pair of submetacentric chromosomes smaller than the submetacentric autosomes. The Y chromosome in males was a small submetacentric chromosome. The C banding patterns were useful in identifying the X and Y chromosomes and the inherited submetacentric autosomes from the gaur sire. Phenotypically, the hybrids resembled normal B indicus cross B taurus calves except for the presence of a distinct hump-like dorsal ridge containing the spinous processes of the third to 11th thoracic vertebrae, upright 'deer-like' ears and long lean legs. The potential of these hybrids as important genetic resources for meat production is stressed.
Interspecific hybridization has been reported for a wide variety of vertebrate species either spontaneous or by organized crossing of bovine species. The hybrids were often carrying intermediate characters genetically and phenotypically of the parents. Thus, status information of both aspects is valuable in animal production for selection and breeding management. The Gaur-cattle hybrids was reported to be superior in production value compared to their parent cattle but fertility status was still questionable. The project was abandoned due to their fertility issue and the hybrids were kept within the cattle in a dairy farm. Cytogenetic status and breeding record of the remaining herd were unavailable since then. The herd was then translocated to a deer farm (PTH Lenggong) and kept freely in the paddock. Recently, two female calves were born via inter se mating. Peripheral blood cultures of Malayan Gaur, Sahiwal-Friesian cattle and Gaur x cattle hybrid backcrosses were analyzed via Giemsa stained metaphase. The Gaur and cattle were having diploid chromosome number (2n) of 56 and 60, respectively. Interestingly, the backcrosses from the hybrids by cattle bulls were found to have two chromosome arrangements, which are 2n = 58 and 2n = 60.
Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.
Beef, buffalo, and pork adulteration in the food chain is an emerging and sensitive issue. Current molecular techniques to authenticate these species depend on polymerase chain reaction (PCR) assays involving long and single targets which break down under natural decomposition and/or processing treatments. This novel multiplex polymerase chain reaction-restriction fragment length polymorphism assay targeted two different gene sites for each of the bovine, buffalo, and porcine materials. This authentication ensured better security, first through a complementation approach because it is highly unlikely that both sites will be missing under compromised states, and second through molecular fingerprints. Mitochondrial cytochrome b and ND5 genes were targeted, and all targets (73, 90, 106, 120, 138, and 146 bp) were stable under extreme boiling and autoclaving treatments. Target specificity and authenticity were ensured through cross-amplification reaction and restriction digestion of PCR products with AluI, EciI, FatI, and CviKI-1 enzymes. A survey of Malaysian frankfurter products revealed rampant substitution of beef with buffalo but purity in porcine materials.
Bali cattle is a domestic cattle breed that can be found in Malaysia. It is a domestic cattle that was purely derived from a domestication event in Banteng (Bos javanicus) around 3,500 BC in Indonesia. This research was conducted to portray the phylogenetic relationships of the Bali cattle with other cattle species in Malaysia based on maternal and paternal lineage. We analyzed the cytochrome c oxidase I (COI) mitochondrial gene and SRY of Y chromosome obtained from five species of the Bos genus (B. javanicus, Bos gaurus, Bos indicus, Bos taurus, and Bos grunniens). The water buffalo (Bubalus bubalis) was used as an outgroup. The phylogenetic relationships were observed by employing several algorithms: Neighbor-Joining (PAUP version 4.0), Maximum parsimony (PAUP version 4.0) and Bayesian inference (MrBayes 3.1). Results from the maternal data showed that the Bali cattle formed a monophyletic clade, and together with the B. gaurus clade formed a wild cattle clade. Results were supported by high bootstrap and posterior probability values together with genetic distance data. For the paternal lineage, the sequence variation is low (with parsimony informative characters: 2/660) resulting an unresolved Neighbor-Joining tree. However, Bali cattle and other domestic cattle appear in two monophyletic clades distinct from yak, gaur and selembu. This study expresses the potential of the COI gene in portraying the phylogenetic relationships between several Bos species which is important for conservation efforts especially in decision making since cattle is highly bred and hybrid breeds are often formed. Genetic conservation for this high quality beef cattle breed is important by maintaining its genetic characters to prevent extinction or even decreased the genetic quality.