PCR-SSCP: a simple method for the authentication of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets

A method of DNA analysis has been developed to verify the authenticity of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets. A short fragment (208 bp) of the mitochondrial 12S rRNA gene was amplified by the polymerase chain reaction and analyzed by single-strand conformation polymorphism to get species-specific patterns of single-stranded DNA (ssDNA). DNA strands were separated by native polyacrylamide gel electrophoresis and visualized by silver staining. Discrimination among the three fish species studied was possible, because each one expressed a specific ssDNA pattern.     

Identification of whale species by thin-layer isoelectric focusing of sarcoplasmic proteins.

Thin-layer isoelectric focusing was applied to the identification of whale (Cetacea) species by using water-soluble sarcoplasmic proteins of skeletal muscles. Twenty-eight samples consisting of 4 species (10 samples) of baleen whales (Mysticeti) and 8 species (18 samples) of toothed whales (Odontoceti) were analyzed. Each sample (approximately 1 g) was electrophoresed with Ampholine PAGplate, pH 3.5-9.5. The electrophoretic profiles were species-specific on the 4 toothed whale species that did not have a marked intra-species difference, and all 4 baleen whale species. However, the profiles were not specific on the 4 other dolphin species, even though they were discriminable from the other 4 toothed whale species. Numerical values of pIs and relative peak heights were obtained by densitometric analysis of the isoelectro-focused protein bands. The bands were also species-specific for the 8 toothed whale species mentioned. The values may be applicable to species identification without the need for a standard sample, which may not be readily obtainable. Experiments on test samples of minke and sel whales showed that bloodletting with ice water made the densities of isoelectro-focused bands thinner, although species identification was still possible by using the inside part of muscles. Heat treatment at below 60 degrees C for 10 min caused little denaturation; at higher temperatures the protein bands were diminished in a temperature-dependent fashion. Therefore, the present isoelectric focusing analysis should be applicable to small samples of whale meat, excluding several species of dolphins.     

Identification of goose (Anser anser) and mule duck (Anasplatyrhynchos x Cairina moschata) foie gras by multiplex polymerase chain reaction amplification of the 5S RDNA gene.

Polymerase chain reaction (PCR) amplification of the nuclear 5S rDNA gene has been used for the identification of goose and mule duck foie gras. Two species-specific reverse primers were designed and used in a multiplex reaction, together with a forward universal primer, to amplify specific fragments of the 5S rDNA in each species. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose and mule duck foie gras samples. This genetic marker can be useful for detecting fraudulent substitution of the duck liver for the more expensive goose liver.     

Identification of sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides) by PCR amplification of the 5S rDNA gene.

Polymerase chain reaction (PCR) amplification of the nuclear 5S rDNA gene, has been used for the identification of sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides). Two species-specific primers were designed to amplify specific fragments of the 5S rDNA gene in each species. The remarkably different size of the amplicons obtained gives, by simple agarose gel electrophoresis, two distinguishable band patterns for both flatfish species. This genetic marker can be very useful for the accurate identification of S. solea and Greenland halibut, to enforce labeling regulations.     

实时荧光定量聚合酶链式反应快速鉴定三种鳕鱼

为鉴定常见的3种鳕科鳕鱼(大西洋鳕鱼、太平洋鳕鱼和黑线鳕),选用16S rDNA基因设计区分鳕科和非鳕科鱼类的引物,选用线粒体Cytb基因设计区分3种鳕鱼的物种特异性引物,建立实时荧光定量聚合酶链式反应(qPCR)体系,对其进行物种分析。结果表明,16S rDNA qPCR体系及3种鳕鱼品种特异性qPCR体系均具有良好的性能,结合标准曲线,可以对单一或混合样品中的目标鳕鱼进行定量检测。在模拟混合样品中目标鳕鱼的相对检测灵敏度可达0.01%。通过对13种市售样品的检测,本研究设计的qPCR体系可检测出原料及深加工产品中的鳕鱼成分。综上,所建立的qPCR体系具有很强的实用性,能满足日常检测的要求,并有望作为未来鳕鱼市场管理的检测方法。     

One-step triplex-polymerase chain reaction assay for the authentication of yellowfin (Thunnus albacares), bigeye (Thunnus obesus), and skipjack (Katsuwonus pelamis) tuna DNA from fresh, frozen, and canned tuna samples

A one-step triplex-polymerase chain reaction (PCR)-based assay was developed to discriminate between three tuna species, Thunnus albacares, Thunnus obesus, and Katsuwonus pelamis, even in highly processed food samples such as canned or cooked tuna. Diagnostic nucleotides were identified by direct sequencing and alignment of part of the mitochondrial cytochrome b gene of 30 authenticated exemplars, which allowed us to evaluate intraspecific variation and the genetic distance between three tuna species. The assay relies on a one-step triplex-PCR reaction in which in a single tube species-specific amplification products are generated only in the presence of the correct template nucleic acid and the species of origin of the DNA is indicated by the distinctive size of the PCR product. The identification of tuna species can be performed with a good accuracy, low cost, and with potential automation for large-scale high-throughput screenings in small in-house laboratories.     

根瘤菌中的nodD基因及其与弗兰克氏菌（Frankia）中的类nodD基因的功能互补

根瘤菌可在几种豆科植物上成瘤,结瘤基因（ｎｏｄ基因）赋予这些细菌以寄主专一性的方式诱导瘤的形成。目前,已被鉴定的三类主要结瘤基因是：＂公共＂结瘤基因,如ｎｏｄＡＢＣ,这类基因是结瘤不可缺少的,并具有高度的结构和功能上的保守性;另一类是寄主专一性基因,这类基因具有种属或菌株的专一性,它们决定了细菌的寄主范围;最后一类是结瘤基因Ｄ（ｎｏｄＤ）,这类基因编码一类调节蛋白,并和植物分泌的酚类化合物一道起动其他结瘤操纵子的转录。一些根瘤菌种属只有一个拷贝的ｎｏｄＤ基因,而另一些则可拥有三个拷贝之多。苜蓿根瘤菌（Ｒ．ｍｅｌｉｌｏｔｉ）中存在三个拷贝的ｎｏｄＤ基因,其中的任何一个发生突变都以依赖于寄主的方式影响结瘤效果。在慢生型大豆根瘤菌（Ｂ．ｊａｐｏｎｉｃｕｍ）中,ｎｏｄＤ１突变菌株还可在大豆或其他豆科植物寄主上结瘤,只是稍往后推迟而已,ｎｏｄＤ２基因对ｎｏｄＹＡＢＣＳＵＩＪ操纵子诱导的影响却很小,两个ｎｏｄＤ拷贝突变后尚表现出结瘤活性。在只有一个ｎｏｄＤ基因拷贝的根瘤菌种属中,如豌豆根瘤菌（Ｒ．ｌｅｇｕｍｉｎｏｓａｒｕｍ）和茎瘤固氮菌（Ａ．ｃａｕｌｉｎｏｄａｎｓ）中,ｎｏｄＤ突变后会导致菌株丧失结瘤能力。一般说来,不     

Identification of the clam species Ruditapes decussatus (Grooved carpet shell), Venerupis pullastra (Pullet carpet shell), and Ruditapes philippinarum (Japanese carpet shell)by PCR-RFLP

PCR-RFLP analysis has been applied to the identification of three clam species: Ruditapes decussatus (grooved carpet shell), Venerupis pullastra (pullet carpet shell), and Ruditapes philippinarum (Japanese carpet shell). PCR amplification was carried out using a set of primers designed from the DNA nucleotide sequences reported for alpha-actins from humans and various animals. Restriction endonuclease analysis based on sequence data of the PCR products of each clam species revealed the presence of species-specific polymorphic sites for MaeIII and RsaI endonucleases. Electrophoretic analysis of the amplicons digested with MaeIII and RsaI produced species-specific profiles that allowed the genetic identification of the three clam species.     

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.     

Assessment of EST-microsatellites markers for discrimination and genetic diversity in bread and durum wheat landraces from Afghanistan

Accurate and reliable means for identification are necessary to assess the discrimination between landraces of tetraploid wheat [T.␣turgidum L. subsp. durum (Desf.) Husn.] and hexaploid wheat (T. aestivum L. em. Thell.). In Afghanistan, farmers usually cultivate mixed landraces, and thus distinction between bread and durum is difficult. A set of 18 microsatellites derived from the DuPont EST-database were used to describe genetic diversity in a sample of 82 Afghan wheat landraces. A total of 101 alleles were detected, with allele number per locus ranging from 2 to 13, and a mean allele number of 6.31. The percentage of polymorphic loci was 89%. The EST-SSRs markers showed different level of gene diversity: the highest Polymorphism Information Content value (0.921) was observed with DuPw 221. Our results demonstrated that with a reasonable number of expressed sequences target microsatellites (EST-SSRs) it is possible to discriminate between T. durum and T. aestivum species of wheat germplasm. Our results showed that EST-databases could be a useful source for species-specific markers and have the potential for new genic microsatellites markers that could enhance screening germplasm in gene banks.     

Identification of goose,mule duck,chicken, turkey,and swine in foie gras by species-specific polymerase chain reaction

A specific Polymerase Chain Reaction (PCR) has been developed for the identification of goose (Anser anser), mule duck (Anas platyrhynchos x Cairina moschata), chicken (Gallus gallus), turkey (Meleagris gallopavo), and swine (Sus scrofa domesticus) in foie gras. A forward common primer was designed on a conserved DNA sequence in the mitochondrial 12S ribosomal RNA gene (rRNA), and reverse primers were designed to hybridize on species-specific DNA sequences of each species considered. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose, mule duck, chicken, turkey, and swine in foie gras. Analysis of experimental mixtures demonstrated that the detection limit of the assay was approximately 1% for each species analyzed. This genetic marker can be very useful for the accurate identification of these species, avoiding mislabeling or fraudulent species substitution in foie gras.     

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.     

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).     

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.     

Detecting fish parvalbumin with commercial mouse monoclonal anti-frog parvalbumin IgG

Parvalbumin is a calcium-binding muscle protein that is highly conserved across fish species and amphibians. It is the major cross-reactive allergen associated with both fish and frog allergy. We used two-dimensional electrophoretic and immunoblotting techniques to investigate the utility of a commercial monoclonal anti-frog parvalbumin IgG for detecting parvalbumin present in some commonly consumed fish species. The 2D electrophoresis and immunoblots revealed species-specific differences in proteins that appear to represent various numbers of isoforms of parvalbumin in carp (5), catfish (3), cod (1) and tilapia (2). No parvalbumin was detected in yellowfin tuna. Based on minor differences in relative intensities of protein staining and immunodetection, parvalbumin isoforms may have slight differences in the epitope region recognized by the anti-frog parvalbumin antibody. These results suggest that the frog anti-parvalbumin antibody can be used as a valuable tool to detect parvalbumins from the fish tested in this study, except yellowfin tuna.     

Identification of anchovy (Engraulis encrasicholus L.) and gilt sardine (Sardinella aurita) by polymerase chain reaction, sequence of their mitochondrial cytochrome b gene, and restriction analysis of polymerase chain reaction products in semipreserves

A method of authenticating anchovy (Engraulis encrasicholus L.) and gilt sardine (Sardinella aurita) semipreserves (salt-cured and fillets in oil) has been developed by polymerase chain reaction (PCR) followed by sequence and restriction site analysis. The amplification of a fragment of the cytochrome b gene by universal primers produced a 376 base pairs (bp) fragment in all samples analyzed. Digestion of PCR products with XhoI, TaqI, AluI, and HinfI endonucleases yielded species-specific profiles distinguishing anchovy from gilt sardine. Therefore, the restriction length fragment polymorphism (RLFP) technique can be used to determine the species identity of anchovy and gilt sardine in semipreserves.     

Electrophoretic analysis of some <Emphasis Type="Italic">Lathyrus</Emphasis> L. species based on seed storage proteins

Present study reported the variation in electrophoretic seed subfraction (albumins, globulin A, globulin B, glutelins) patterns in some Lathyrus species collected from their natural habitats different localities in Turkey. Electrophoretic data were documented by using a gel documentation system (Bio-Rad, USA) and analysed by using Quantity 1-D analysis software and also the dendograms were formed with 4.0% tolerance in UPGAMA (Unweighed Pair-Group Arithmetic Mean). The differences among species were observed and all seven species were clearly identifiable from the protein patterns. The dendograms obtained from cluster analysis of seed storage proteins data showed examined species have displayed a number of distinct relationships. According to our results, it seems justified to recommend a wider use of electrophoretic analysis of seed storage proteins in taxonomic investigations of leguminous plants.     

Molecular phylogeny of three subfamilies of the Neanuridae (Insecta, Collembola) and the position of the antarctic species Friesea grisea Schäffer.

The nuclear 28S rRNA and the mitochondrial COII gene were used to establish phylogenetic relationships among species of the family Neanuridae, with special emphasis on species of the subfamily Neanurinae. Phylogenetic analysis was conducted using genetic distances, parsimony and likelihood methods. The D3-D5 fragment of the rRNA gene was very conserved, both in sequence and in secondary structure features. This fragment supplied little information on relationships at this level. The phylogenetic reconstruction based on 1st and 2nd codon positions of the COII gene was partly in accordance with morphological data, but it was discordant for the placement of some species. Relationships among the subfamilies Frieseinae, represented by the Antarctic species Friesea grisea, Pseudachorutinae and Neanurinae were uncertain. The subfamily Neanurinae and its tribes Neanurini and Paleonurini were shown as monophyletic taxa. Relationships between three species of the genus Bilobella were in accordance with morphological and biochemical data. Relationships between genera within the Neanurini were more controversial. In accordance with morphological hypotheses, a basal position of Thaumanura was suggested, but the molecular data placed Neanura muscorum in a derived position, in sharp contrast with morphological evidence. A close relationship was suggested between Deutonura conjuncta, Cansilianura malatestai and Lathriopyga longiseta. The disagreement between molecular and morphological data suggests that one or both data sets might be affected by a certain degree of homoplasy and that these data should be interpreted with caution in phylogenetic reconstructions.     

Spatial and interspecific variability in phenological responses to warming temperatures

A comprehensive understanding of species phenological responses to global warming will require observations that are both long-term and spatially extensive. Here we present an analysis of the spring phenological response to climate variation of twelve taxa: six plants, three birds, a frog, and two insects. Phenology was monitored using standardized protocols at 176 meteorological stations in Japan and South Korea from 1953 to 2005, and in some cases even longer. We developed a hierarchical Bayesian model to examine the complex interactions of temperature, site effects, and latitude on phenology. Results show species-specific variation in the magnitude and even in the direction of their responses to increasing temperature, which also differ from site-to-site. At most sites the differences in phenology among species are forecast to become greater with warmer temperatures. Our results challenge the assertion that trends in one geographic region can be extrapolated to others, and emphasize the idiosyncratic nature of the species response to global warming. Field studies are needed to determine how these patterns of variation in species response to climate change affect species interactions and the ability to persist in a changing climate.     

Genetic variation of five species of Trifolium L. from south-west Turkey

Following a legume collection mission to south-west Turkey in 1996, five species of Trifolium were analysed for genetic variation within and between species in eleven morphological and flowering characters. The five species included two outcrossing species, T. michelianum and T. resupinatum, and three inbreeding species, T. clypeatum, T. glomeratum and T. tomentosum. The genetic diversity found was related to climate and edaphic factors. All five species showed significant amounts of genetic differentiation between sites and the species could be separated morphologically by principal components analysis and cluster analysis. The most significant source of genetic variation was found to be related to geographical distribution with those species which were widely distributed across south-west Turkey exhibiting much greater amounts of genetic variation between sites, than those which had a narrow distribution. The breeding system was found to be less important, but only the morphology of the outbreeding species showed any environmental clines in relation to climate. A multiple regression analysis was computed to estimate the effect of growing season on the days to flowering of each of the species.