Molecular phylogeny and species identification of sardines

The DNA sequence diversity of Sardina pilchardus (Walbaum, 1792) and some closely related species of Clupeomorpha was investigated using the mitochondrial DNA gene encoding cytochrome b. The nucleotide sequences of complete and partial mtDNA cytochrome b were determined in numerous specimens. Sequence divergence between species and genera was evenly distributed in the cytochrome b gene but rather high compared to reports for other fish species. Phylogenetic analyses on complete cytochrome b were used to study the relationships among the considered species. S. pilchardus was easily differentiated, showing a genetic distance of 0.25 with respect to Clupeidae species and 0.26 with respect to the other species. A species-specific short fragment (<150 bp) was isolated by polymerase chain reaction (PCR) using primers designed for Clupeomorpha. A rapid and reliable PCR method using restriction fragment length polymorphism (RFLP) with two restriction enzymes (MnlI/HinfI) was optimized for unambiguous differentiation of S. pilchardus from the other species tested (raw and canned products).     

Development of a DNA-based method aimed at identifying the fish species present in food products

Analysis of restriction fragment length polymorphism (RFLP) profiles of a 464 bp amplicon obtained from the mitochondrial cytochrome b gene was used to differentiate between several different fish species. The method was tested by a collaborative study in which 12 European laboratories participated to ascertain whether the method was reproducible. Each laboratory was required to identify 10 unknown samples by comparison with RFLP profiles from authentic species. From a total of 120 tests performed, unknown samples were correctly identified in 96% of cases. Further work attempting to use the method to analyze mixed and processed fish samples was also performed. In all cases the species contained within mixed samples were correctly identified, indicating the efficacy of the method for detecting fraudulent substitution of fish species in food products.     

Applying DNA techniques to the identification of the species of dressed toasted eel products

To differentiate the species of processed eel products, the gene identification of four fresh eel species was first established and the species of eel products collected from markets were investigated. Polymerase Chain Reaction (PCR) and sequence analysis were used to determine the genetic variation in a 362-nucleotide region of the mitochondrial cytochrome b gene in four fresh eels including Anguilla japonica, Anguilla anguilla, Anguilla rostrata, and Muraenesox cinereus. It was found that each eel species had a unique genotype, which was no different among fresh, frozen, and sterilized meats. The restriction enzyme HinfI could differentiate the species of A. japonica and A. rostrata but could not differentiate A. anguilla and M. cinereus. Another restriction enzyme, Sau96 I, was valuable in the differentiation of M. cinereus from the other three species of Anguilla. By applying PCR and restriction enzymes, the species of 12 commercial eel products were identified as A. japonica (9 samples), A. anguilla (2), and A. rostrata (1). This indicated that the sequence and restriction enzyme cutting site analyses were very usable to authenticate species of different processed eel products.     

Use of restriction fragment length polymorphism to distinguish between salmon species

Identification of 10 salmon species using DNA-based methodology was investigated. Amplification of DNA was carried out using a primer set which amplified a region of the mitochondrial cytochrome b gene. Sequences of PCR-amplified DNA from the salmon species were used to select six restriction enzymes allowing species to be uniquely classified. RFLP patterns generated following analysis with each enzyme were resolved using polyacrylamide gel electrophoresis and visualized by silver staining. Results indicate that it is possible to differentiate between all 10 salmon species and that the technique could be easily adopted by the food industry for analysis of processed salmon products.     

Identification of flatfish (Pleuronectiforme) species using DNA-based techniques

Identification of flatfish species using a DNA-based methodology was studied. The polymerase chain reaction was employed to obtain a 464 bp amplicon from mitochondrial cytochrome b gene. The sequences from this fragment belonging to 24 species were analyzed using a genetic distance method, and polymorphic sites were determined. The fragment was found to be highly polymorphic (231 sites), and this permitted the differentiation of most of the species. Phylogenetic tree construction was employed to allow the identification of flatfish species. As a result, each species was grouped in a well-differentiated clade, except for two pairs: Limanda ferruginea and L. limanda, and Solea impar and S. lascaris, which could not be differentiated. On the basis of the sequences obtained, restriction enzymes were selected to provide specific restriction profiles, which allow the differentiation of 21 species of flatfish in a faster and less expensive manner than sequencing. This polymerase chain reaction-restriction fragment length polymorphism methodology (PCR-RFLP) was tested using commercial samples.     

PCR-RFLP analysis of nuclear nontranscribed spacer for mackerel species identification

Scomber mackerel have been marketed in fresh and frozen forms and as processed seafood worldwide, and three species of Japanese mackerel S. japonicus, Pacific mackerel S. australasicus, and Atlantic mackerel S. scombrus have constituted a significant part of absolute Scombrid consumption in Japan. The present study was undertaken to develop a rapid and reliable method not only for differentiation of Scomber mackerel from related Scombrid fish by PCR amplification using Scomber genus-specific primers but also for identification of three Scomber mackerel species by PCR-RFLP analysis. Alignment of nucleotide sequences of the nuclear 5S ribosomal RNA gene (5S rDNA) among Scombrid fish allowed the selection of oligonucleotide primers specific for the Scomber genus. These primers enabled amplification of the nontranscribed spacer (NTS) of the 5S rDNA from S. japonicus, S. australasicus, and S. scombrus, whereas no amplification was demonstrated from other Scombrid fish. RFLP analysis of the PCR products with ScaI endonuclease generated unique restriction patterns for each Scomber species. This simple, robust, and reproducible PCR-RFLP technique using Scomber genus-specific primers can serve as a routine food inspection program to enforce labeling regulations of marketed Scombrid fish.     

Statistical validation of the identification of tuna species: bootstrap analysis of mitochondrial DNA sequences

Sequencing of the mitochondrial cytochrome b gene has been used to differentiate three tuna species: Thunnus albacares (yellowfin tuna), Thunnus obesus (bigeye tuna), and Katsuwonus pelamis (skipjack). A PCR amplified 528 bp fragment from 30 frozen samples and a 171 bp fragment from 26 canned samples of the three species were analyzed to determine the intraspecific variation and the positions with diagnostic value. Polymorphic sites between the species that did not present intraspecific variation were given a diagnostic value. The genetic distance between the sequences was calculated, and a phylogenetic tree was constructed, showing that the sequences belonging to the same species clustered together. The bootstrap test of confidence was used to determine the statistical validation of the species assignation, allowing for the first time a quantification of the certainty of the species assignation. The bootstrap values obtained from these results indicate that the sequencing of the cytochrome b fragments allows a correct species assignation with a probability > or =95%.     

Discrimination of three trachurus species using both mitochondrial- and nuclear-based DNA approaches

A double-DNA approach was developed to discriminate the three Trachurus species that abide in European waters: T. trachurus, T. mediterraneus, and T. picturatus. The analysis aimed at both mitochondrial and nuclear loci. Polymerase Chain Reaction (PCR) amplification of the cytochrome b gene of mtDNA was followed by restriction analysis with three species-specific enzymes: NlaIII, NciI, and BsmAI. Digestion with these endonucleases yielded species-specific electrophoretic profiles. The universality of the results was verified by screening a large number of individuals from 12 geographical regions covering most of the distribution of the species. Additionally, the nuclear multicopy 5S rRNA gene was selected as an alternative candidate for the discrimination of the three Trachurusspecies. A simple agarose gel electrophoretic analysis of the amplicons proved to be capable of leading to unambiguous identification of the three Trachurus species. Thus, the double-DNA methodology presented here allows the accurate discrimination of Trachurus fish species and the detection of commercial fraud.     

Preliminary study on puffer fish proteome-species identification of puffer fish by two-dimensional electrophoresis

The aims of this work were to determine the differential characterization of the urea soluble protein components of puffer fish species and to establish a preliminary proteomic database using an immobilized pH gradient two-dimensional electrophoresis (2DE) technique. The puffer fish muscle proteins resolved into 171-260 spots in the 2DE gels, with a pI range of 3-10 and molecular mass range of 7.4-205.0 kDa, following Comassie blue staining. Puffer fish muscle proteins fell in the region with pI values of 3.5-7.0, and molecular masses of 7.4-45.0 kDa were well-resolved and were good for species comparison. The more acidic proteins of lower molecular masses showed species specific characteristics. Therefore, the species of puffer fish can be differentiated from the comparison of the characteristic 2DE protein patterns.     

Fish species identification using PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis: application to detect white fish species in food products and an interlaboratory study

Identification of 10 white fish species associated with U.K. food products was achieved using PCR-RFLP of the mitochondrial cytochrome b gene. Use of lab-on-a-chip capillary electrophoresis for end-point analysis enabled accurate sizing of DNA fragments and identification of fish species at a level of 5% (w/w) in a fish admixture. One restriction enzyme, DdeI, allowed discrimination of eight species. When combined with NlaIII and HaeIII, specific profiles for all 10 species were generated. The method was applied to a range of products and subjected to an interlaboratory study carried out by five U.K. food control laboratories. One hundred percent correct identification of single species samples and six of nine admixture samples was achieved by all laboratories. The results indicated that fish species identification could be carried out using a database of PCR-RFLP profiles without the need for reference materials.     

Fish species identification in surimi-based products

Whole fish morphologically identified as belonging to Theragra chalcogramma, Merluccius merluccius, Merluccius hubbsi, and Merluccius capensis and 19 fish products commercialized as surimi with different commercial brands and labeled as T. chalcogramma were analyzed by direct sequence analysis of the cytochrome b gene. A phylogenetic analysis of surimi products was performed as well. Results demonstrated that mislabeling is a large-scale phenomenon, since 84.2% of surimi-based fish products sold as T. chalcogramma (16/19) were prepared with species different from the one declared. In fact, only three samples (samples 15-17) were found to belong to T. chalcogramma. In the remaining samples, Merluccidae (samples 4-14), Gadidae (samples 18 and 19), Sparidae (sample 1), and Pomacentridae (samples 2 and 3) families were detected. A phylogenetic tree was constructed, and the bootstrap value was calculated. According to this methodology, 11 samples were grouped in the same clade as Merluccius spp.     

Toward fish and seafood traceability: anchovy species determination in fish products by molecular markers and support through a public domain database

Traceability in the fish food sector plays an increasingly important role for consumer protection and confidence building. This is reflected by the introduction of legislation and rules covering traceability on national and international levels. Although traceability through labeling is well established and supported by respective regulations, monitoring and enforcement of these rules are still hampered by the lack of efficient diagnostic tools. We describe protocols using a direct sequencing method based on 212-274-bp diagnostic sequences derived from species-specific mitochondria DNA cytochrome b, 16S rRNA, and cytochrome oxidase subunit I sequences which can efficiently be applied to unambiguously determine even closely related fish species in processed food products labeled "anchovy". Traceability of anchovy-labeled products is supported by the public online database AnchovyID ( http://anchovyid.jrc.ec.europa.eu), which provided data obtained during our study and tools for analytical purposes.     

Genetic diversity and symbiotic efficiency of rhizobial isolates obtained from nodules of Arachis hypogaea in northwestern Morocco

The molecular characterization of 62 rhizobial isolates obtained from root-nodules of Arachis hypogaea growing in north-western Morocco was performed. Bacteria were firstly characterized by restriction of the 16S-rDNA region, and phylogeny was inferred from 16S gene sequences. Phylogenetically, isolates were grouped with species belonging to the Bradyrhizobium and Rhizobium genera. A high degree of variability was detected among isolates in terms of their nitrogen-fixing ability. This is, to our knowledge, the first study on genetic diversity and symbiotic effectiveness of rhizobia isolated from peanut nodules grown in Morocco. This characterization provides a basis for the selection of peanut-nodulating rhizobia which may have applications in the formulation of appropriate inocula to improve peanut crop yield on Moroccan soils.     

Identification of species in animal feedstuffs by polymerase chain reaction--restriction fragment length polymorphism analysis of mitochondrial DNA

Restriction site analysis of Polymerase Chain Reaction (PCR) products of cytochrome b mitochondrial DNA was applied to identify species in meat meal and animal feedstuffs. PCR was used to amplify a variable region of cytochrome b mitochondrial DNA gene. Species differentiation was determined by digestion of the obtained 359 bp amplicon with restriction enzymes, which generated species-specific electrophoresis patterns; the sequencing of PCR products was used as confirming analysis. PCR-RFLP analysis revealed the presence of meat meal in animal feedstuffs and distinguished species of interest. The results supported the application of the method in control measures which should be adopted for meat-meal-based animal feed, as suggested by EU law. As a technical improvement, to simplify the analysis, the number of enzymes presented in this study for the detection of different species was smaller than others described in the literature; discrimination between ruminant and nonruminant species and between mammalian and poultry species was possible with few digestions.     

Development of a DNA microarray for authentication of ginseng drugs based on 18S rRNA gene sequence

Ginseng drugs, derived from underground parts of Panax species (Araliaceae), are the most important group of herbal medicines in the Orient. Previously, the nucleotide sequences of the nuclear 18S rRNA gene of 13 Panax taxa were determined, as were the specific polymorphic nucleotides for identification of each species. On the basis of the nucleotide difference, a DNA microarray (PNX array) was developed for the identification of various Panax plants and drugs. Thirty-five kinds of specific oligonucleotide were designed and synthesized as probes spotting on a decorated glass slide, which included 33 probes corresponding to the species-specific nucleotide substitutions and 2 probes as positive and negative controls. The species-specific probes were of 23-26 bp in length, in which the substitution nucleotide was located at the central part. Triplicate probes were spotted to warrant accuracy by correcting variation of fluorescent intensity. Partial 18S rRNA gene sequences amplified from Panax plants and drugs as well as their derived health foods were fluorescently labeled as targets to hybridize to the PNX array. After hybridization under optimal condition, specific fluorescent patterns were detected for each Panax species, and the analyzed results could be indicated as barcode patterns for quick distinction. The developed PNX array provided an objective and reliable method for the authentication of Panax plants and drugs as well as their derived health foods.     

Phylogeographic analysis reveals multiple cryptic species of amphipods (Crustacea: Amphipoda) in Chihuahuan Desert springs

Biodiversity conservation and the identification of conservation units among invertebrates are complicated by low levels of morphological difference, particularly among aquatic taxa. Accordingly, biodiversity is often underestimated in communities of aquatic invertebrates, as revealed by high genetic divergence between cryptic species. We analyzed PCR-amplified portions of the mitochondrial cytochrome c oxidase I (COI) gene and 16S rRNA gene for amphipods in the Gammarus pecos species complex endemic to springs in the Chihuahuan Desert of southeast New Mexico and west Texas. Our analyses uncover the presence of seven separate species in this complex, of which only three nominal taxa are formally described. The distribution of these species is highly correlated with geography, with many present only in one spring or one spatially-restricted cluster of springs, indicating that each species likely merits protection under the US Endangered Species Act. We present evidence suggesting that habitat fragmentation, long-distance colonization, and isolation-by-distance have occurred at different temporal and spatial scales within this system to produce the lineages that we report. We show that patterns detected in the G. pecos species complex also correlate with endemic fishes (Gambusia spp., pupfish) and hydrobiid snails. Our results provide clues important for future biodiversity investigations in geographically isolated aquatic habitats, and shed light on the understudied and underestimated levels of biodiversity present in desert spring systems.     

Identification of shark species in seafood products by forensically informative nucleotide sequencing (FINS)

The identification of commercial shark species is a relevant issue to ensure the correct labeling of seafood products, to maintain consumer confidence in seafood, and to enhance the knowledge of the species and volumes that are at present being captured, thus improving the management of shark fisheries. The polymerase chain reaction was employed to obtain a 423 bp amplicon from the mitochondrial cytochrome b gene. The sequences from this fragment, belonging to 63 authentic individuals of 23 species, were analyzed using a genetic distance method. Nine different samples of commercial fresh, frozen, and convenience food were obtained in local and international markets to validate the methodology. These samples were analyzed, and sequences were employed for species identification, showing that forensically informative nucleotide sequencing (FINS) is a suitable technique for identification of processed seafood containing shark as an ingredient. The results also showed that incorrect labeling practices may occur regarding shark products, probably because of incorrect labeling at the production point.     

鲤鱼品系的部分线粒体序列的遗传变异

运用PCR测序法,以9个我国常见鲤鱼（Cyprinus carpio）养殖品种和2个野生群体为研究材料,分析线粒体DNA的16S rRNA、Cyt b和D-loop序列片段,用于分析的序列共有1 457 个位点,其中变异位点32个,简约信息位点21个,共有单倍型16个。分别利用以上3个基因片段以及合并后的序列, 构建NJ和MP进化树。由不同方法获得相似的进化树,由不同基因片段得到的进化树均为两支,其中贝尔湖野鲤（BE）单独成支,其它群体聚为1支,只是由D-loop得到的进化关系更为详细,而由合并后的序列构建的进化树也与利用不同基因序列所得到的进化树并不矛盾。各品系间的分化时间约为3.03×104～1.21×105年前。根据序列的特征,16S rRNA的1个变异位点、Cyt b基因的4个变异位点和D-loop的14个变异位点可以作为鉴定不同的养殖品系和野生群体的SNP,证明mtDNA序列分析可以应用于品系的鉴定。     

Microsatellite analysis of genetic population structure of the endangered cyprinid Anaecypris hispanica in Portugal: implications for conservation

The endangered fish species Anaecypris hispanica is restricted to eight disjunct populations in the Portuguese Guadiana drainage. The genetic structure of these populations was studied in order to determine levels of genetic variation within and among populations and suggest implications for conservation of the species. Based on five microsatellite loci, the null hypothesis of population homogeneity was tested. Tests for genetic differentiation revealed highly significant differences for pairwise comparisons between all populations, and substantial overall population subdivision (F_ST=0.112). All sampled populations contained unique alleles. Our findings indicate marked genetic structuring and emphasise limited dispersal ability. The high levels of genetic diversity detected within and among A. hispanica populations suggest, however, that the observed fragmentation and reduction in population size of some populations during the last two decades, has impacted little on levels of genetic variability. Data imply that most A. hispanica populations should be managed as distinct units and that each has a high conservation value containing unique genetic variation. It is argued that geographic patterns of genetic structuring indicate the existence of eight management units.     

Fast and efficient discrimination of the Isotoma viridis group (Insecta: Collembola) by PCR-RFLP

Identification of collembolan species is generally based on specific morphological characters, such as chaetotaxy and pigmentation pattern. However, some specimens do not match to described characters because these refer to adult specimens, often of one specific sex, or the characters are highly variable in adults (e.g. pigmentation, setae or furcal teeth). Isozymes have frequently assisted species discrimination, and also these may vary with developmental stage or environmental conditions. For identification of single species of the Isotoma viridis group, we present both direct sequencing of the cytochrome oxidase subunit II (COII) gene and a simple DNA-based molecular method.

Five PCR primers amplifying the COII region (717 bp) of the mitochondrial DNA were used. The sequences clearly separated the species I. viridis, I. riparia and I. anglicana, irrespective of colour varieties within the first species. DNA amplification products of different species can also be distinguished by digestion with restriction endonucleases, followed by gel electrophoresis for separation of fragments. This restriction fragment length polymorphism (RFLP), obtained after digestion with the endonucleases TaqI, VspI, MvaI and Bsp143I, revealed specific fragments that separated the three species from each other. Since restriction enzymes are sensitive to single base mutations, we suggest to use a combination of enzymes with at least two species-specific restriction sites when using the RFLP technique. For the I. viridis complex, VspI and Bsp143I appear to be an appropriate combination.