Development of a method for the genetic identification of flatfish species on the basis of mitochondrial DNA sequences

In the present study a method for genetic identification of flatfish species was developed. The technique is based on DNA sequencing of amplified DNA by PCR and subsequent phylogenetic analysis ( FINS). A phylogenetic tree using the cytochrome oxidase subunit I (COI) was constructed and the bootstrap values calculated. The mentioned technique allows the genetic identification of more than 50 flatfish species in fresh, frozen, and precooked products. This analytical system was validated and subsequently applied to 30 commercial samples, obtaining 13 that were incorrectly labeled (43%). Four of the mislabeled samples were whole fish (31%), and nine were fillets (69%). The species with the higher rate of incorrect labeling were Pleuronectes platessa (17%) and Solea solea (10%). Other species incorrectly labeled were Hipoglossus hipoglossus (7%), Reinharditus hippoglossoides, Limanda ferruginea, and Microstomus kitt (3% each species). Therefore, this molecular tool is appropriate to clarify questions related with the correct labeling of commercial products, the traceability of raw materials, and the control of imported flatfish, and also can be applied to questions linked to the control of fisheries.     

Rapid identification of seaweeds in food products by PCR combined with ALF-RFLP and FINS methodologies

In the present study, two methods for the genetic identification of the most important seaweed species used for human consumption were developed. Both are carried out through PCR amplification of an 18S rRNA gene fragment. The first one is based on the phylogenetic analysis of DNA sequences (FINS), while the second is based on length polymorphism and RFLP visualized by means of an ALF system. The main novelty of this work lies in the fact that it allows genetic identification of the main commercial species of seaweed. Moreover, the developed systems can be applied to all kinds of processed products, including those that have undergone intensive transformation, as for instance canned foods. These methodologies also permit the detection of species in complex matrixes where more than one algal species is present. The methods were validated using products manufactured in a pilot plant showing correct functioning. Finally, the methods were applied to 23 commercial samples including some that had been subjected to intensive thermal treatment, allowing the detection of those that were incorrectly labeled (30%). Therefore, these molecular tools can be used for clarifying questions related to the correct labeling and traceability of commercial products that include some seaweeds in their composition.     

A rapid methodology for screening hake species (Merluccius spp.) by single-stranded conformation polymorphism analysis

An accurate screening method for hake species identification based in single-stranded conformation polymorphism analysis is presented. The differentiation of 11 species of the Merluccius genus and another five species of the Gadiformes order was studied. For this purpose, two fragments of the cytochrome b gene were sequenced; the first is the 5'-end, a fragment of 465 bp (Kocher fragment), and the second is the 3'-end of the cytochrome b, a 588 bp fragment (SB fragment). These two fragments were amplified, denatured, and submitted to native nondenaturing polyacrylamide gel electrophoresis. Results show that with this technique and both fragments, all of the species studied can be unequivocally identified. The validation of the methodology was carried out with 24 commercial hake products showing good performance of the technique for species identification in commercial products. Results show that all species were identified. This technique has advantages over other published methods, because only one polymerase chain reaction step is needed, saving time and money, and it decreases the time needed for hake species identification in food products, making it especially suitable as a screening methodology when a high number of samples should be analyzed in routine examinations.     

Genetic identification of horse mackerel and related species in seafood products by means of forensically informative nucleotide sequencing methodology

In the present study, a methodology based on the amplification of a fragment of mitochondrial cytochrome b and subsequent phylogenetic analysis (FINS: forensically informative nucleotide sequencing) to genetically identify horse mackerels have been developed. This methodology makes possible the identification of more than 20 species belonging to the families Carangidae, Mullidae, and Scombridae. The main novelty of this work lies in the longest number of different horse mackerel species included and in the applicability of the developed methods to all kinds of processed products that can be found by consumers in markets around the world, including those that have undergone intensive processes of transformation, as for instance canned foods. Finally, the methods were applied to 15 commercial samples, all of them canned products. Therefore, these methods are useful for checking the fulfillment of labeling regulations for horse mackerels and horse mackerel products, verifying the correct traceability in commercial trade, and fisheries control.     

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.     

Development of a method for the genetic identification of mussel species belonging to Mytilus, Perna, Aulacomya, and other genera

Legislation regarding the labeling of processed products is an important issue in the protection of consumer rights. This labeling is especially important in products that cannot be identified on the basis of their morphological characters, because these are removed from the animal in the transformation process. The goal of this study was the identification of mussel species using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Forensically Informative Nucleotide Sequencing (FINS) methodologies. The molecular marker selected was 18S rDNA (nuclear small-subunit rDNA gene), which allows identification at the genus level and at the species level in some cases. The genera included in this study were Mytilus, Perna, Aulacomya, Semimytilus, Brachidontes, Choromytilus, and Perumytilus. Different markers were used for genetic identification at the species level. To identify the species included in the genus Perna and Choromytilus, a fragment of ITS 1 (Internal Transcribed Spacer 1) was amplified by multiplex PCR and digested with restrictases. The species of Mytilus were identified by length polymorphism and RFLP of the polyphenolic adhesive protein gene. This methodology was validated with products manufactured in the authors' pilot plant and applied to commercial samples. Therefore, this sequential method can be completely or partially used to determine the mussel genus or species present in any food product.     

Analysis of 18S rRNA gene of Octostigma sinensis (Projapygoidea: Octostigmatidae) supports the monophyly of Diplura

The phylogenetic position of Diplura within Hexapoda has been controversial. There are three major lineages in Diplura: Campodeoidea, Projapygoidea, and Japygoidea. However, most of the previous studies were restricted to Campodeoidea and Japygoidea. Until now, only preliminary morphological study on Projapygoidea was reported, and no sequence data from Projapygoidea was available. The main aim of the present study was to investigate the phylogenetic position of Octostigmatidae, one of the three families of Projapygoidea, in Diplura and to test if Diplura are monophyletic. The complete 18S rRNA gene sequences of Octostigma sinensis (Projapygoidea: Octostigmatidae) from subtropical China, together with representative species of Campodeoidea and Japygoidea, and several species of Protura and Collembola were analyzed. The phylogenetic trees were obtained by different methods (neighbor-joining, maximum parsimony, and maximum likelihood) with a chelicerate species as outgroup. Our results suggested that Octostigma was closer to the genus Parajapyx (Japygoidea: Parajapygidae) than to the representative genus of Campodeidae (Campodeoidea). All phylogenetic trees supported the monophyly of Diplura.     

Identification of gadoid species in fish meat by polymerase chain reaction (PCR) on genomic DNA

Identification of fish species is significant due to the increasing interest of consumers in the meat of sea fish. Methods focusing on fish species identification help to reveal fraudulent substitution among economically important gadoid species in commercial seafood products. The objective of this work was to develop a conventional PCR method for the differentiation of the following gadoid fish species in fish products: Alaska pollack ( Theragra chalcogramma), blue whiting ( Micromesistius poutassou), hake spp. ( Merluccius spp.), Atlantic cod ( Gadus morhua), saithe ( Pollachius virens), and whiting ( Merlangius merlangus). The species-specific primer pairs for gadoid species determination were based on the partial pantophysin I ( PanI) genomic sequence. Sequence identification was confirmed by cloning and sequencing of the PCR products obtained from the species considered. For the simultaneous detection of Alaska pollack, blue whiting, and hake spp., a quadruplex PCR system was constructed. Other gadoid species were detected in separate PCR reactions. After optimization of the reactions, the developed PCR systems were used for the analysis of codfish samples obtained from the Czech market and the customs' laboratories. This method represents an alternative approach in the use of genomic DNA for the identification of fish species. This method is rapid, simple, and reliable without the need for further confirmative methods. Furthermore, the identification of a mixture of more than one species is possible. The PCR system has been optimized for routine diagnostic purposes.     

Comparison of the efficiency of A–PAGE and SDS–PAGE,ISSRs and RAPDs in resolving genetic relationships among <Emphasis Type="Italic">Triticum</Emphasis> and <Emphasis Type="Italic">Aegilops</Emphasis> species

Species that belong to the genus Triticum L. and the genetically related Aegilops L. genus are important genetic and economic resources because they have an evolutionary relationship with the two main agricultural crops T. aestivum (bread wheat) and T. durum (durum wheat). Therefore, it is important to understand the genetic relationships among the cultivated wheat species and their wild relatives. The latter have a great role in the improvement of cultivated wheat. Molecular markers are the best choice and most reliable means to study these relationships accurately. In this study, we compared the efficiency of the biochemical methods A–PAGE and SDS–PAGE on seed storage proteins and the molecular methods RAPDs and ISSRs to explore the genetic relationships among seven species of Triticum and 20 Aegilops species. Three phylogenetic trees obtained in this study were compared with available classifications and phylogenetic trees constructed earlier for these species. It was noted that the tree based on ISSRs data was the most congruent with those classification and trees. This may be attributed to the fact that ISSRs is more specific, and therefore more reliable. This study is the first to study genetic relationships among all species studied here using biochemical and molecular techniques.     

A phenetic analysis and lateral key of the genus Lolium (Gramineae)

88 herbarium specimens of the genus Lolium were scored for 22 characters, comprising 7 vegetative, 8 inflorescence and 7 seed characters. The aim was to review the species relationship and to produce a lateral key to the genus. The data were analysed using principal components analysis, discriminant function analysis and cluster analysis. All analyses separated L. perenne, L. multiflorum and L. temulentum from each other. The 2 varieties of L. rigidum showed varying degrees of separation from each other and from L. multiflorum. Their distinction as a species and as 2 varieties is discussed. L. perenne and L. rigidum were shown to contain the greatest similarity between species and L. temulentum was found to be the most distinct. A lateral key is proposed that separates the species on the basis of 11 morphological characters that are easily scored in the field.     

Identification of Hake species (Merluccius Genus) using sequencing and PCR-RFLP analysis of mitochondrial DNA control region sequences.

The use of DNA-based methodologies in identification of hake species belonging to the Merluccius genus was shown to be successful. A short fragment of the left hypervariable domain of the mitochondrial control region was amplified, sequenced, and digested from 11 hake species. The hake-specific PCR product, due to its limited size, was obtained in a variety of tissue samples with different levels of DNA concentration and degradation, including sterilized food products. On the basis of this phylogenetically informative 156-bp sequence were selected four restriction enzymes (ApoI, DdeI, DraIII, and MboII) that allow the hake species discrimination. Species identification by phylogenetic analysis of sequences or by PCR-RFLP methodologies is useful in a variety of scenarios including authentication of thermally processed food, detection of food components, and species determination of individuals whose morphological characters are removed.     

Phylogenetic position of the disjunct species <Emphasis Type="Italic">Musa ornata</Emphasis> (Musaceae): first approach to understand its distribution

Musa L. commonly known as the banana group is one of the most important and oldest food crops of humankind. Among the wild relatives with ornamental interest in the genus, Musa ornata Roxb. shows a disjunct distribution between Asia and North America (Mexico). The wild occurrence of this species in Mexico has led to speculation about the evolutionary relationships with its Asian relatives. This study examined the phylogenetic relationships between intercontinental specimens of this species and, based on registered evidence, explored the more likely hypothesis about the origins of its distribution. The phylogeny of intercontinental specimens, along with other representatives of the same genus, was carried out using three molecular markers (ITS, trnL-F, and atpB-rbcL) and applying three phylogenetic reconstruction methods: maximum parsimony, maximum likelihood, and Bayesian inference. The genetic analysis of the combined dataset grouped together all the Mexican and most Asian specimens, but the monophyly of the species was not supported. The relationships suggest that Mexican populations may have originated from an Asian invasion. However, several studies and historical documents suggest the presence of Musa in America long before the arrival of Europeans. Based on its current distribution, phylogenetic evidence, and fossil record, this species’ disjunct distribution could be explained in terms of an ancestral distribution range that encompassed America and Asia, followed by its subsequent restriction to the Old World and a secondary dispersal by humans. However, further studies are necessary to shed more light on the origins of this disjunct distribution.     

Determination of Ni (II) in beverages without any sample pretreatment by adsorptive stripping chronopotentiometry (AdSCP)

The purpose of this paper was to use adsorptive stripping chronopotentiometry for the determination of Ni (II) in worldwide consumed beverages without any sample pretreatment, using dimethilglyoxime (DMG) as complexing agent and a glassy carbon mercury film electrode as the working electrode. Ni (DMG)2 complex is adsorbed onto the mercury film at an electrolysis potential of -500 mV for 60 s and then reduced by a -5 microA constant cathodic current. The sensitivity of the method was studied for certified reference water and black tea in the pH range 6.5-11. At pH 9.5 in ammonia buffer, a detection limit of 0.2 microg L(-1) was achieved; the instrumental precision (expressed as rsd %) was 1.5%, and the accuracy, expressed as obtained recoveries both from certified and not certified matrixes, ranged from 93.0 to 95.5 %. The chronopotentiometric analysis executed on commercial beverages provided evidence that black tea samples were the richest source of Ni (II) (1500-3700 microg L(-1)), followed by coffee (100.0-300.5 microg L(-1)); bottled mineral water showed a Ni (II) concentration lower than 4.6 microg L(-1). Among alcoholic beverages, red wines presented the highest content of Ni (II) (55.5-105.0 microg L(-1)). Significant differences were noticed between Ni (II) levels of fermented and distillated alcoholic beverages; moreover, canned cola and beer did not show higher Ni (II) levels with respect to the glass-bottled products.     

Interlaboratory evaluation of a real-time multiplex polymerase chain reaction method for identification of salmon and trout species in commercial products

This interlaboratory study evaluated a real-time multiplex polymerase chain reaction (PCR) method for identification of salmon and trout species in a range of commercial products in North America. Eighty salmon and trout products were tested with this method by three independent laboratories. Samples were collected in the United States and Canada, and only the collecting institution was aware of the species declaration. Following analysis with real-time PCR, all three laboratories were able to identify species in 79 of the 80 products, with 100% agreement on species assignment. A low level of fraud was detected, with only four products (5%) found to be substituted or mixtures of two species. The results for two of the fraudulent products were confirmed with alternate methods, but the other two products were heavily processed and could not be verified with methods other than real-time PCR. Overall, the results of this study show the usefulness and versatility of this real-time PCR method for the identification of commercial salmon and trout species.     

Phylogenetic relationships among Triticum L. and Aegilops L. species as genome progenitors of bread wheat based on sequence diversity in trnT-F region of chloroplast DNA

Cultivated wheat, (Triticum aestivum L.), is one of the most important food crops in the world. The Aegilops L. genus is frequently utilized by plant breeders for improving the current wheat cultivars due to their close relationships. Therefore, understanding the phylogenetic relationships among the species of these genera is not only valuable for plant taxonomy, but also for plant breeding efforts. The presented phylogenetic analysis was based on the sequences of trnT-F chloroplast DNA containing three non-coding sub-regions. Twelve genotypes belonging to four species of Triticum L. genus and twenty-four genotypes belonging to eight species of Aegilops genus were used in the current study. The results postulated a close genetic relationship between diploid Aegilops species containing the BB genome and polyploid Triticum species. With the exception of Aegilops cylindrica Host (CCDD), all other Aegilops species having the CC genome were alienated from Aegilops speltoides Tausch (BB) and clustered together. These two clusters joined by a third cluster including the AA genome containing diploid Triticum species.     

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.     

山羊FSHR基因第10外显子的PCR-SSCP检测及其序列分析

利用PCR-SSCP技术检测山羊(Caprahircus)包括西农萨能奶山羊、关中奶山羊、陕南白山羊、安哥拉山羊和波尔山羊173个个体FSHR基因第10外显子的单核苷酸多态性(SNP)。结果未发现SNP位点。测序后获得山羊FSHR基因第10外显子的核苷酸序列,并在NCBI数据库中获得GenBank登录No.DQ069909和DQ069910。通过DNA序列分析发现,FSHR基因第10外显子第120位碱基不存在C→T的转换,也不存在颠换等其它遗传变化。山羊、绵羊(OvisariesL.)和普通牛(Bostaurus)FSHR基因第10外显子序列同源性比较和聚类分析结果表明,山羊、普通牛和绵羊该部分序列的相似性最高为99.3%;在物种间比较中,绵羊和普通牛纯合子该基因外显子序列的不相似性最高为3.4%;据FSHR基因外显子序列构建的分子系统树结果显示,山羊、绵羊和普通牛物种内的个体各自聚为一类;山羊和绵羊先聚为一类,然后再与普通牛聚为一类。提示FSHR基因第10外显子的核苷酸序列适合于物种间的动物分子树的构建。     

Differential detection of shrimp and crab for food labeling using polymerase chain reaction

Shrimp and crab are well-known as allergenic ingredients. According to Japanese food allergy labeling regulations, shrimp species (including prawns, crayfishes, and lobsters) and crab species must be differentially declared when ≥10 ppm (total protein) of an allergenic ingredient is present. However, the commercial ELISA tests for the detection of crustacean proteins cannot differentiate between shrimp and crab. Therefore, two methods were developed to discriminate shrimp and crab: a shrimp-PCR method with postamplification digestion and a crab-PCR method that specifically amplifies a fragment of the 16S rRNA gene. The sensitivity and specificity of both PCR methods were verified by experiments using DNA extracted from 15 shrimp species, 13 crab species, krill, mysid, mantis shrimp, other food samples (cephalopod, shellfish, and fish), incurred foods, and commercial food products. Both PCR methods could detect 5 pg of DNA extracted from target species and 50 ng of genomic DNA extracted from incurred foods containing 10 ppm (μg/g) total protein of shrimp or crab. The two PCR methods were considered to be specific enough to separately detect species belonging to shrimp and crab. Although false-positive and false-negative results were obtained from some nontarget crustacean species, the proposed PCR methods, when used in conjunction with ELISA tests, would be a useful tool for confirmation of the validity of food allergy labeling and management of processed food safety for allergic patients.     

Cytogenetic analysis of Kengyilia mutica (Keng) J. L. Yang, Yen and Baum (Poaceae: Triticeae)

Kengyilia mutica (Keng) Yang, Yen et Baum is a hexaploid perennial grass of the tribe Triticeae native to western central China. The analyzer species with known genomic constitution used to produce interspecific hybrids with the target taxon were Roegneria kamoji Ohwi (StStHHYY), K. hirsuta (Keng) J. L. Yang, Yen et Baum (PPStStYY) and K. rigidula (Keng) J. L. Yang, Yen et Baum (PPStStYY). Analysis of metaphase I pairing configurations in the F₁ hybrids indicates that K. mutica possesses the P, St and Y genomes, with only minor structural rearrangements. Chromosome pairing in hybrids supports the inclusion of K. mutica in the genus Kengyilia.     

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.