Mitochondrial 16S rRNA Gene Sequences Research Articles

Molecular phylogeny of Barbin fishes of North - East Indiabased on mitochondrial 16SrRNA gene sequences

The Barbin fishes (Cypriniformes: Barbinae) have achieved great diversity in the world and cosmopolitan in distribution but their phyletic classification is ambiguous. Molecular phylogeny of barbin fishes of North-East India was studied by using mitochondrial 16S rRNA sequences. Partial sequences (540-615 bp) of mit. 16S rRNA genes of four species of barbin fishes – Pethia atra, Puntius chola, P. javanicus, and P. sophore were generated and sequences of 7 species were downloaded from GenBank for present study. The aim of our present study is to resolve the taxonomic relationship and to establish the molecular phylogeny of Barbin fishes of North-East India based on mitochondrial 16S rRNA genes sequences. The Maximum Parsimony Tree shows three clusters with high bootstrap value and present data indicates that all fishes in this group had a common ancestor but now they have separated into distinct evolutionary lineages.

Sclerotised spines in the female bursa associated with male’s spermatophore production in cantharidin-producing false blister beetles

Cantharidin is a defence chemical synthesised in only two beetle families Meloidae and Oedemeridae. In Meloidae, cantharidin is used as a defence chemical in eggs. However, in Oedemeridae the function of cantharidin remains unclear. Based on morphological comparison of female internal reproductive organs in 39 species of Oedemeridae, we found that some species have sclerotised spines in the bursa copulatrix (bursal spines), while others have no such spines. Molecular phylogenetic trees inferred from mitochondrial 16S and nuclear 28S rRNA gene sequences suggested multiple evolutionary origins of bursal spines from an ancestor without spines. In the species which lacked spines, males transferred small amounts of ejaculates to females; however, in species with spines, males transferred large spermatophores. Deposited spermatophores gradually disappeared in the bursa, probably owing to absorption. To compare the amounts of cantharidin in eggs laid by species with and without bursal spines, we constructed a new bioassay system using the small beetle Mecynotarsus tenuipes from the family Anthicidae. M. tenuipes individuals were attracted to droplets of cantharidin/acetone solution, and the level of attraction increased with cantharidin concentration. This bioassay demonstrated that the eggs of Nacerdes caudata and N. katoi, both of which species have conspicuous bursal spines, contain more cantharidin than the eggs of N. waterhousei, which lacks spines. In the former species, males transfer large spermatophores to the female, and spermatophores are eventually broken down and digested within the female’s spiny bursa. Thus, females with bursal spines may be able to provide more cantharidin to their eggs.

Identification of Megaselia scalaris (Diptera : Phoridae) based on morphology and mitochondrial 16S rRNA and COI gene sequences

Megaselia scalaris (Diptera: Phoridae), commonly known as scuttle fly, is widely distributed all over the world. It is easily cultured in the laboratory condition making it a potential model organism. Besides, it has forensic importance. However, no report from Bangladesh could be retrieved about this fly. So, in the present study, identification of this species was attempted using both morphological and molecular approaches. Characteristics of male hypopygium and legs played key roles in morphological identification. To strengthen identification, mitochondrial COI and 16S rRNA gene fragments were amplified and sequenced. Blast search at NCBI provided highest hits to available COI and 16S rRNA sequences of M. scalaris. A neighbor joining phylogenetic tree was built using sequences of respective COI gene region to show its relationship among other closely related dipteran flies.
 Dhaka Univ. J. Biol. Sci. 25(2): 149-159, 2016 (July)

Estimating diversity of crabs (Decapoda: Brachyura) in a no-take marine protected area of the SW Atlantic coast through DNA barcoding of larvae

DNA barcoding was used to identify crab larvae from the Marine Biological Reserve of Arvoredo, encompassing a coastal archipelago off the SW Atlantic coast (27°S, 48°W). Partial mitochondrial COI or 16S rRNA gene sequences were obtained for 488 larvae, leading to the identification of 20 species. The COI sequences generated 13 barcode index numbers (BINs) within Barcode of Life Data Systems (BOLD), among which 11 were concordant with single species. DNA from ∼ 6% of the larvae did not amplify using the primers tested; based on external morphological characteristics, these larvae represented four possible additional operational taxonomic units (OTUs) at the family level. Intraspecific variation for the COI and 16S rRNA genes was found to be < 2.6% and < 2.1% respectively (Kimura 2-parameter distance), whereas interspecific divergence ranged from 7.9% to 21.5% and 6.4% to 14.5%, respectively. These results imply that both genes are suitable for use in species identification of brachyuran crabs of this area. Molecular identification of this group successfully enabled the diagnosis of larvae of closely related species, including congeners in Mithrax, Achelous and Callinectes. In addition, eight out of 20 species recognized represent new records for the reserve suggesting that the brachyuran fauna in the area has been underestimated based on traditional biodiversity measures. The availability of primers suited to the targeted species, and the development of a taxonomically comprehensive DNA barcoding database are the major recommendations to improve the accuracy and feasibility of using DNA barcoding for species identification of SW Atlantic brachyuran crabs.

The shark panel: An InDel multiplex for shark species identification

The Elasmobranchii comprises the diverse and important group of sharks and rays. The Selachii or sharks clade includes some of the ocean's largest predatory fishes, being commercially overexploited due to unsustainable fishing activities for their meat and fins. Overfishing has resulted in significant population declines and several Selachii species are now considered under high threat and facing extinction, with about 93% of its nominal species included in the IUCN Red List. Molecular data has provided important information about these species, allowing the management of natural stocks and their decline. Population genetics, connectivity data, and their population genetics knowledge are now available and play an important role on establishing conservation policies. However, despite the ecological, commercial and conservation importance, no molecular method is available to identify sharks in a forensic analytic context. As a first step in the construction of a reliable method for shark identification, we carried out a molecular systematic analysis using 85 previously published mitochondrial 16S rRNA gene sequences obtained from the NCBI database. We found that indels in the 16S rRNA gene can be used to distinguish almost all the analyzed shark species, including some of the most threatened according to IUCN Red List. The regions selected in this study can be used for the construction of molecular assays for shark identification in a forensic context.

Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture.

Aim:This study was aimed to detect Mannheimia haemolytica in lung tissues of sheep and from a bacterial culture.Introduction:M. haemolytica is one of the most important and well-established etiological agents of pneumonia in sheep and other ruminants throughout the world. Accurate diagnosis of M. haemolytica primarily relies on bacteriological examination, biochemical characteristics and, biotyping and serotyping of the isolates. In an effort to facilitate rapid M. haemolytica detection, polymerase chain reaction assay targeting Pasteurella haemolytica serotype-1 specific antigens (PHSSA), Rpt2 and 12S ribosomal RNA (rRNA) genes were used to detect M. haemolytica directly from lung tissues and from bacterial culture.Materials and Methods:A total of 12 archived lung tissues from sheep that died of pneumonia on an organized farm were used. A multiplex polymerase chain reaction (mPCR) based on two-amplicons targeted PHSSA and Rpt2 genes of M. haemolytica were used for identification of M. haemolytica isolates in culture from the lung samples. All the 12 lung tissue samples were tested for the presence M. haemolytica by PHSSA and Rpt2 genes based PCR and its confirmation by sequencing of the amplicons.Results:All the 12 lung tissue samples tested for the presence of PHSSA and Rpt2 genes of M. haemolytica by mPCR were found to be positive. Amplification of 12S rRNA gene fragment as internal amplification control was obtained with each mPCR reaction performed from DNA extracted directly from lung tissue samples. All the M. haemolytica were also positive for mPCR. No amplified DNA bands were observed for negative control reactions. All the three nucleotide sequences were deposited in NCBI GenBank (Accession No. KJ534629, KJ534630 and KJ534631). Sequencing of the amplified products revealed the identity of 99-100%, with published sequence of PHSSA and Rpt2 genes of M. haemolytica available in the NCBI database. Sheep specific mitochondrial 12S rRNA gene sequence also revealed the identity of 98% with published sequences in the NCBI database.Conclusion:The present study emphasized the PCR as a valuable tool for rapid detection of M. haemolytica in clinical samples from animals. In addition, it offers the opportunity to perform large-scale epidemiological studies regarding the role of M. haemolytica in clinical cases of pneumonia and other disease manifestations in sheep and other ruminants, thereby providing the basis for effective preventive strategies.

Genetic similarity of the Hainan medaka populations collected from hyper- and hypo-osmotic environments in northern Vietnam

Ricefishes of the genus Oryzias, including Japanese medaka (O. latipes), are known as excellent model organisms for studies in various fields of science. Some species of the genus inhabit brackish water, and such species are recognized to be useful to investigate physiological phenomena in seawater. However, only a limited number of species have been recorded from brackish waters. In addition, there is no information about the genetic relationship among populations inhabiting sites with different salinities. Here we report the discovery of Oryzias fish in two locations near Haiphong, northern Vietnam, a brackish mangrove planting area and a freshwater pond. A phylogenetic analysis using mitochondrial 12S and 16S ribosomal RNA (rRNA) gene sequences indicated that the fish from the two localities are the same species, Hainan medaka, O. curvinotus. Population genetic analysis using the mitochondrial 12S and 16S rRNA gene sequences revealed a close genetic relationship between the two populations. These results suggest that O. curvinotus is adaptable to both hyperosmotic and hypoosmotic environments. Due to its osmotic adaptability and ease of rearing in the laboratory, this species is expected to become a model for marine environmental and toxicological studies, as well as for studies of osmotic adaptation mechanisms.

A new species of Indian caecilian highlights challenges for species delimitation within Gegeneophis Peters, 1879 (Amphibia: Gymnophiona: Indotyphlidae).

A new species of indotyphlid caecilian amphibian, Gegeneophis tejaswini sp. nov., is described based on eight specimens from lowlands of the most northerly district of the state of Kerala in the southern part of the Western Ghats region, India. This species is distinguished from all other Gegeneophis in annulation characters and genetics (&gt; 6% different from most similar nominal species for 883 base pairs of mitochondrial 12S and 16S rRNA gene sequence data). The high degree of morphological similarity of G. krishni, G. mhadeiensis and the new species underlines that, for some Gegeneophis, larger samples and/or new characters will be needed to further advance the taxonomy of this genus.

New insights into the taxonomy of the skittering frog Euphlyctis cyanophlyctis complex (Schneider, 1799) (Amphibia: Dicroglossidae) based on mitochondrial 16S rRNA gene sequences in southern Asia

The Skittering frog ( Euphlyctis cyanophlyctis ) is considered to be a species complex distributed in southern and southeastern Asia. Genetic diversity and taxonomic status of populations across their ranges is unclear and existence of several cryptic species is expected. In this study we used sequence variation in the mitochondrial 16S ribosomal RNA gene to elucidate the taxonomic status of Iranian populations of E. cyanophlyctis and compare their genetic diversity and divergence to populations from the Indian subcontinent. Phylogenetic analysis indicated that the populations of E. cyanophlyctis from Iran, Bangladesh-Assam (northeastern India), southern India, and Sri Lanka are partitioned into different clades. At least four different haplogroups were detected which are here proposed to be considered as allopatric cryptic species. The sedimentation of the Helmand River into the Sistan depression during the Neogene and subsequent formation of dry land barriers are proposed to have caused the Iranian populations of skittering frogs to be disconnected from those of the Indian subcontinent, resulting in differentiation of these lineages. In addition, some populations from southern India and those from Sri Lanka that were previously recognized as E. cyanophlyctis belong to E. mudigere . A preliminary investigation on the genetic diversity of the populations from southeastern Iran highlights the low genetic diversity among these populations.

A Preliminary Study of Phylogenetic Relationships and Taxonomic Problems of Vietnamese Rhacophorus (Anura: Rhacophoridae)

We investigated the phylogenetic relationships among species of Vietnamese tree frogs in the genus Rhacophorus and their allies from China and Malaysia based on 434-bp partial sequences of the mitochondrial 16S rRNA gene. Rhacophorus forms a monophyletic group containing two clades, within each of which the phylogenetic relationships are unresolved. Rhacophorus robertingeri is closer to R. orlovi than to R. calcaneus . Rhacophorus exechopygus and R. annamensis are sister species, while R. vampyrus is very remote from all other species studied. Rhacophorus duboisi forms a substantially supported clade with R. puerensis and is considered a member of the R. dugritei group. Rhacophorus pingbianensis is very close to R. omeimontis , whereas R. rhodopus is not monophyletic, with Yunnan samples not conspecific with R. rhodopus from Vietnam, but are possibly conspecific with Malaysian R. bipunctatus . Further studies both from morphology and molecular phylogeny based on many samples from their whole distribution range are necessary.

Single-Gene Versus Double-Gene Tree Analyses in Molecular Classification of Saudi Venomous Snakes

Aiming to use DNA sequence for proper identification of venomous snakes in Saudi Arabia, mitochondrial 12S rRNA gene sequences of Bitis arietans, Cerastes cerastes, C. gasperettii, and Echis coloratus were investigated. Concatenated analysis of 16S and 12S rRNA gene sequences (as mosaic) was also performed to validate the usefulness of using more than one gene marker in molecular taxonomy of venomous snakes. DNA extracted from whole blood samples and 12S rRNA was amplified using PCR. Sequences submitted to GenBank were analyzed for similarity using BLAST and aligned using Clustal X followed by manual editing with BioEdit software. Phylogenetic analysis was generated using PAUP version 4, and the evolutionary distance among different groups was calculated using MEGA5 software for both 12S and 16S rRNA genes. Contrary to the morphological classification, phylogenetic analysis revealed that the collected isolates formed three different statistically supported clusters (B. arietans, C. cerastes, and E. coloratus). Although the combined analysis of 16S and 12S rRNA sequences improved the signal, the C. cerastes and C. gasperettii intermixing have not been resolved. Both single and combined genetic distance analyses showed that Echis is more distant from Cerastes than from Bitis. Further studies are mandatory to evaluate other gene markers in molecular classification.

Phylogeny of the freshwater crabs of the Western Ghats (Brachyura, Gecarcinucidae)

The Western Ghats mountain range in India is a biodiversity hotspot for a variety of organisms including a large number of endemic freshwater crab species and genera of the family Gecarcinucidae. The phylogenetic relationships of these taxa, however, have remained poorly understood. Here, we present a phylogeny that includes 90% of peninsular Indian genera based on mitochondrial 16S rRNA and nuclear histone H3 gene sequences. The subfamily Gecarcinucinae was found to be paraphyletic with members of two other subfamilies, Liotelphusinae and Parathelphusinae, nesting within. We identify a well‐supported clade consisting of north Indian species and one clade comprising mostly south Indian species that inhabit the southern ‘sky islands’ of the Western Ghats. Relationships of early diverging genera, however, were resolved with low support. This study also includes newly sampled material from an isolated mountain plateau in the northern part of the Western Ghats, representing a new species of Gubernatoriana, which we describe here as Gubernatoriana basalticola sp. n. The new species is immediately distinguished from its congeners and the related genera Ghatiana and Inglethelphusa by its carapace and cheliped morphology, which are unique among Indian freshwater crabs. This study highlights the urgent need for continued faunistic studies to assess the true diversity of gecarcinucid crabs on the Indian subcontinent, to fully understand the basal phylogenetic relationships within the freshwater crab family Gecarcinucidae, and to evaluate the conservation threat status and biogeography of the montane freshwater crabs of the Western Ghats.

Genetic divergences of South and Southeast Asian frogs: a case study of several taxa based on 16S ribosomal RNA gene data with notes on the generic name Fejervarya

To elucidate the genetic divergences of several Asian frog taxa, the mitochondrial 16S rRNA gene (16S) sequences of 81 populations across 6 Asian countries were analyzed. In total, 109 haplotypes were found, and the concept of a 3% difference in 16S sequence corresponding to species threshold was applied to define candidate amphibian species, for which corroborating evidence, such as morphology, ecological characteristics, and/or nuclear gene data, is required. Polypedates leucomystax, Hylarana chalconota, and Hylarana sp. from Chantaburi, Maelippet Siberut, and Langkawi Island, respectively, correspond to 3 candidate species and H. erythraea from Malaysia/Thailand represents a possible candidate species. Hylarana cf. nicobariensis from Muara Siberut, Amolops larutensis from Gombak, and Microhyla okinavensis from Ishigaki Island showed divergences from the topotypic specimen, all suggesting a relevant candidate species. Microhyla heymonsi from Malaysia and M. ornata from 2 regions (Mudigere and Talapu) in India did not fit any congeneric species based on available 16S data, suggesting 3 possible candidate species in total. Two lineages of Duttaphrynus melanostictus from Malaysia denote 2 possible candidate species. Consequently, this study indicates the occurrence of 6 candidate and 6 possible candidate species, and argues that the generic allocation of the Fejervarya-Minervarya-Zakerana complex needs to be studied in detail.

Sequence Characterization of Mitochondrial 12S rRNA Gene in Mouse Deer (Moschiola indica) for PCR-RFLP Based Species Identification

Mitochondrial 12S rRNA has proven to be a useful molecular marker for better conservation and management of the endangered species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the mitochondrial 12S rRNA gene has proven to be a reliable and efficient tool for the identification of different Indian deer species of family cervidae. In the present study, mitochondrial 12S rRNA gene sequence of mouse deer (Moschiola indica) belonging to the family Tragulidae was characterized and analysed in silico for its use in species identification. Genomic DNA was isolated from the hair follicles and mitochondrial 12S rRNA gene was amplified using universal primers. PCR product was cloned and sequenced for the first time. The sequence of mouse deer showed 90.04, 90.08, 90.04, 91.2, 90.04, and 90.08% identities with sika deer, sambar, hog deer, musk deer, chital, and barking deer, respectively. Restriction mapping in Lasergene (DNAstar Inc., Madison, WI, USA) revealed that mouse deer mitochondrial 12S rRNA gene sequence can be differentiated from the other deer species in PCR-RFLP using RsaI, DdeI, BsrI, and BstSFI. With the help of predicted pattern, mouse deer can be identified using genomic DNA from a variety of biomaterials, thereby providing molecular aid in wildlife forensics and conservation of the species.

Mitochondrial DNA variation in wild and hatchery populations of northern pike, Esox lucius L.

Esox lucius is an economically important freshwater species. Mitochondrial cytb, 12SrRNA, and 16SrRNA gene sequences were used in order to clarify the genetic variation and population structure in three E. Lucius populations, i.e., one Wild population (W) and two hatchery populations (Hatchery Population I-HPI and Hatchery Population II-HPII). A total of 55 individuals, with 19 from wild and 18 from each hatchery population, were sequenced. The results are as follows: 1) cytb. Eleven variable sites defined eight distinct haplotypes, with 1137 base pairs (bp) complete cytb sequences. The nucleotide diversity (π) values were W 0.009%, HPI 0.075% and HPII 0.068%, respectively. Genetic distances within and between populations were both between 0 and 0.1%. Analysis of molecular variance (AMOVA) analysis revealed that variation within populations accounts for 98.49% of total variation. 2) 12SrRNA. We obtained 380 bp consensus 12SrRNA sequence and found little variation among the 55 sequences. 3) 16SrRNA. Twelve variable sites defined seven haplotypes, with 519 bp consensus 16SrRNA sequence. The π values were W 0.041%, HPI 0.310% and HPII 0.021%, respectively. Genetic distances were between 0 and 0.3% within populations and from 0 to 0.2% between populations. AMOVA disclosed that variation within populations accounts for 91.79% of total variation. Variation within populations is the main source of total variation. The results suggest that low genetic variation can be found in both wild and hatchery populations of E. lucius, and the effect of genetic drift and adaptation to culture conditions gradually emerged under only two generations’ artificial cultivation.

Molecular phylogenetic relationship between Trichiurus margarites Li, 1992 and Trichiurus sp.2 (sensu Nakabo, 2002) (Perciformes: Trichi-uridae) based on mitochondrial 16S rRNA sequence analysis

The taxonomic identification of the genus Trichiurus(Trichiuridae) has historically been challenging because of the morphological similarities between the species.As a result,Trichiurus lepturus Linnaeus,1758 was recognized as a monotypic cosmopolitan species of Trichiurus prior to 1990.In 1992,Li proposed the new species T.margarites Li,1992,based on morphological differences in the skull patterns and dorsal fin coloration,i.e.su-praoccipital bone with a hard knob and yellow dorsal fin respectively.Subsequently,Yamada et al.described a species of Trichiurus with a yellow dorsal fin from the Ryukyu Islands and East China Sea coast of Kyushu,Japan.This species is very similar to T.margarites,but shows some variation in the size of a pair of knobs on the su-praoccipital crest.Accordingly,the yellow dorsal fin species of Trichiurus from Japan was provisionally called Trichiurus sp.2 by Nakabo.There remains no molecular evidence that elucidates the genetic differences between T.margarites and T.sp.2(sensu Nakabo,2002).Thus,the phylogenetic relationship and taxonomic status of these two species remain unresolved. In this study we examined the mitochondrial 16S rRNA gene sequence from 11 T.margarites individuals collected from the coast of western Hainan Island(Baimajing,Yinggehai and Sanya),South China Sea,China which is the type locality of T.margarites.The 16S rRNA genes were amplified using PCR techniques.Homolo-gous sequences from T.sp.2 and closely-related species from previous studies were also included in the compara-tive analysis.Genetic information indexes,including base composition,sequence variation,and Kimura-2 pa-rameter net genetic distance were examined.Phylogenetic analysis using neighbor-joining(NJ) and maxi-mum-likelihood(ML) methods were conducted using Eupleurogrammus muticus as the outgroup. There was evidence of insertion or deletion of base pairs in the 435 bp sequence alignment,with 63 variable sites,48 parsimony informative sites,and 15 singleton sites.The net genetic distances between T.margarites(from western Hainan Island) and T.sp.2(from Japan,eastern Hainan Island,and Indian Ocean) ranged from 0.000 to 0.006.These values were much lower than those found for interspecific 16S rRNA comparisons in some species of Trichiurus(0.037–0.061),and for intraspecies comparisons between populations of T.lepturus(0.010–0.016).Moreover,the haplotypes of T.margarites and T.sp.2 did not form reciprocal monophyletic clades in the phylogenetic trees(NJ,ML),and the close relationship of the haplotypes of these two species was supported by high confidence levels(NJ 100%,ML 99%).These results indicated that the genetic distance between T.mar-garites and T.sp.2 was at the intraspecific level,and the species T.sp.2(sensu Nakabo,2002) from Japan should be classified as T.margarites Li,1992.It has been suggested that T.margarites is widely distributed from the In-dian Ocean to the western Pacific,and not merely limited to the South China Sea,Taiwan Strait,and Taiwan coastal waters.Based on coalescent estimates of divergence times among species,the origin and evolution of T.margarites are primarily discussed in this study.

A new montane species of Philautus (Amphibia: Anura: Rhacophoridae) from western Sarawak, Malaysia, Borneo.

A new species of Philautus is described from western Sarawak. The new species was collected in lower montane forest in two national parks in Sarawak and recorded from another park. It differs from its congeners by a unique combination of morphological characters, including a long, acuminate snout, long legs, and comparatively extensive toe webbing. The advertisement call of the new species differs from all calls of other species that have been analyzed so far. Comparison of the mitochondrial 16S rRNA gene sequence corroborates its distinct specific status.

Assessing the genetic diversity and population structure of Culter alburnus in China based on mitochondrial 16S rRNA and COI gene sequences

The genetic diversity and population genetic structure of Culter alburnus were investigated using mitochondrial cytochrome c oxidase subunit I (COI) and ribosomal 16S subunit (16S rRNA) gene sequences. A total of 89 individuals from four localities were included in the analysis. Overall, 12 polymorphic sites were observed and 10 haplotypes were defined. The C. alburnus populations were characterized by high haplotype diversity (0.587 ± 0.047) and low nucleotide diversity (0.00197 ± 0.00073). Pairwise fixation index (FST) analysis indicated significant genetic differentiation among different populations. The hierarchical analysis of molecular variance (AMOVA) analysis also showed significant genetic divergence (φST = 0.3792, P < 0.01) among these populations. The present results suggest that subdivisions exist among four C. alburnus populations, and should be considered as different management unit for effective conservation and management purposes. This study provides new information for genetic assessment and will be crucial for establishing fisheries management and strategies for this species.

Sequence analysis of mitochondrial 16S rRNA gene to identify meat species

In the present study, mitochondrial 16S rRNA gene sequence analysis was used for identification of Cattle (Bos taurus), buffalo (Bubalus bubalis), sheep (Ovis aries), goat (Capra hircus) and pig (Sus scrofa) species in fresh and processed meat. The DNA was extracted from fresh and processed meat including autoclaved meat and meat emulsion followed by polymerase chain reaction (PCR) amplification of about 497 bp DNA fragments of mitochondrial 16S rRNA gene. Then the amplified PCR fragments were sequenced and analysed to differentiate the species. No adverse effect of ingredients and processing conditions was observed on PCR amplification of DNA extracted from heat-treated meat and meat emulsion. The closely related species such as cattle and buffalo, sheep and goat were differentiated from each other by sequence analysis. Thus, PCR amplification and sequence analysis of mitochondrial 16S rRNA gene can be used as a tool for authentic identification of meat species.

A novel approach for simultaneous differentiation of closely related meat species by PCR assay using single primer pair

The aim of the present study was to design the primer pair for differentiation of closely related meat species i.e. cattle from buffalo, and sheep from goat employing PCR assay. The primer pair was designed based on the mitochondrial 12S rRNA gene sequences of buffalo and then designed primer pair matched with other species available 12S rRNA gene sequences. The designed primer pair was successfully amplified to the desired DNA fragments of 316 bp from the buffalo and sheep in single PCR reaction assay without any cross reaction with cattle, goat, pig and chicken species. The developed PCR assay was able to differentiate the closely related meat species; however, this assay was not suitable for differentiation of buffalo from sheep species meat. This PCR assay was also effective in heat treated meat and meat emulsion without any adverse effect on amplification of DNA. It can be concluded that the developed PCR assay has potential application for the purpose of differentiation of closely related meat species.