Application of the ITS2 region for barcoding plants of the genus Triticum L. and Aegilops L.

Molecular taxonomic studies have been performed in the past in order to identify different wheat species and construct a molecular phylogeny. These were based on universal but sufficiently divergent sequences from both the nuclear and chloroplastic genomes of wheat. They included two short plastid sequences from the plastid genes rbcL and matK which have been proposed as the core “barcode” sequences by the “barcoding” guidelines for general plant identification. Historically, in molecular plant taxonomy, plastidic sequences had been favored over nuclear sequences, due to their uniparental inheritance and consequently lower intra-molecular recombination. However recently, the short nuclear sequence from the internal transcribed spacer 2 (ITS2) has been used successfully for the accurate identification of many medicinal and other plant species. Herein, we have used the plastidic matK, rbcL trnL, and the nuclear ITS2 region for the identification of different wheat species of Triticum L. and goatgrass species of Aegilops L. We have successfully discriminated all species that were examined from both genera, thus, validating the ITS2 region as a ‘barcode tool’ for accurate distinction of plants in the genus Triticum L. and Aegilops L. The success rate of PCR amplification and sequencing of the ITS2 region was 100%. We report also that matK, rbcL and trnL regions could not discriminate all species in contrast to the ITS2 region which demonstrated full discriminatory capacity

Rapid and high throughput molecular identification of diverse mosquito species by igh resolution melting analysis

Mosquitoes are a diverse group of invertebrates, with members that are among the most important vectors of diseases. The correct identification of mosquitoes is paramount to the control of the diseases that they transmit. However, morphological techniques depend on the quality of the specimen and often unavailable taxonomic expertise, which may still not be able to distinguish mosquitoes among species complexes (sibling and cryptic species). High resolution melting (HRM) analyses, a closed-tube, post-polymerase chain reaction (PCR) method used to identify variations in nucleic acid sequences, has been used to differentiate species within the Anopheles gambiae and Culex pipiens complexes. We validated the use of PCR-HRM analyses to differentiate species within Anopheles and within each of six genera of culicine mosquitoes, comparing primers targeting cytochrome b (cyt b), NADH dehydrogenase subunit 1 (ND1), intergenic spacer region (IGS) and cytochrome c oxidase subunit 1 (COI) gene regions. HRM analyses of amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera (Aedes (Ae. vittatus from Ae. metallicus), Culex (Cx. tenagius from Cx. antennatus, Cx. neavei from Cx. duttoni, cryptic Cx. pipiens species), Anopheles (An. gambiae s.s. from An. arabiensis) and Mansonia (Ma. africana from Ma. uniformis)) based on their HRM profiles. However, PCR-HRM could not distinguish between species within Aedeomyia (Ad. africana and Ad. furfurea), Mimomyia (Mi. hispida and Mi. splendens) and Coquillettidia (Cq. aurites, Cq. chrysosoma, Cq. fuscopennata, Cq. metallica, Cq. microannulatus, Cq. pseudoconopas and Cq. versicolor) genera using any of the primers. The IGS and COI barcode region primers gave the best and most definitive separation of mosquito species among anopheline and culicine mosquito genera, respectively, while the other markers may serve to confirm identifications of closely related sub-species. This approach can be employed for rapid identification of mosquitoes

Between but not within species variation in the distribution of fitness effects

New mutations provide the raw material for evolution and adaptation. The distribution of fitness effects (DFE) describes the spectrum of effects of new mutations that can occur along a genome, and is therefore of vital interest in evolutionary biology. Recent work has uncovered striking similarities in the DFE between closely related species, prompting us to ask whether there is variation in the DFE among populations of the same species, or among species with different degrees of divergence, i.e., whether there is variation in the DFE at different levels of evolution. Using exome capture data from six tree species sampled across Europe we characterised the DFE for multiple species, and for each species, multiple populations, and investigated the factors potentially influencing the DFE, such as demography, population divergence and genetic background. We find statistical support for there being variation in the DFE at the species level, even among relatively closely related species. However, we find very little difference at the population level, suggesting that differences in the DFE are primarily driven by deep features of species biology, and that evolutionarily recent events, such as demographic changes and local adaptation, have little impact

A fast and accurate method for controlling the correct labeling of products containing buffalo meat using High Resolution Melting (HRM) analysis.

The substitution of high priced meat with low cost ones and the fraudulent labeling of meat products make the identification and traceability of meat species and their processed products in the food chain important. A polymerase chain reaction followed by a High Resolution Melting (HRM) analysis was developed for species specific detection of buffalo; it was applied in six commercial meat products. A pair of specific 12S and universal 18S rRNA primers were employed and yielded DNA fragments of 220bp and 77bp, respectively. All tested products were found to contain buffalo meat and presented melting curves with at least two visible inflection points derived from the amplicons of the 12S specific and 18S universal primers. The presence of buffalo meat in meat products and the adulteration of buffalo products with unknown species were established down to a level of 0.1%. HRM was proven to be a fast and accurate technique for authentication testing of meat products

A fast and accurate method for controlling the correct labeling of products containing buffalo meat using High Resolution Melting (HRM) analysis.

The substitution of high priced meat with low cost ones and the fraudulent labeling of meat products make the identification and traceability of meat species and their processed products in the food chain important. A polymerase chain reaction followed by a High Resolution Melting (HRM) analysis was developed for species specific detection of buffalo; it was applied in six commercial meat products. A pair of specific 12S and universal 18S rRNA primers were employed and yielded DNA fragments of 220bp and 77bp, respectively. All tested products were found to contain buffalo meat and presented melting curves with at least two visible inflection points derived from the amplicons of the 12S specific and 18S universal primers. The presence of buffalo meat in meat products and the adulteration of buffalo products with unknown species were established down to a level of 0.1%. HRM was proven to be a fast and accurate technique for authentication testing of meat products

High resolution melting analysis for quantitative detection of bovine milk in pure water buffalo mozzarella and other buffalo dairy products

Identification of buffalo dairy products has become an important issue to ascertain product quality, consumer rights and absence of food-borne allergic reactions. A polymerase chain reaction (PCR) followed by a high resolution melting (HRM) analysis was developed and applied for species specific detection of bovine milk in nine different commercial buffalo dairy products. A specific buffalo 12S rRNA and a bovine d-loop primer pair, targeting the mitochondrial genome, were employed in a duplex PCR assay. The analysis developed was found capable of identifying the presence of bovine milk down to 1% in commercial buffalo milk products and also of quantifying the ratio of bovine into buffalo milk. HRM was proven to be a fast and accurate technique for a routine authentication testing of mozzarella and other buffalo milk products

High resolution melting analysis for quantitative detection of bovine milk in pure water buffalo mozzarella and other buffalo dairy products

Identification of buffalo dairy products has become an important issue to ascertain product quality, consumer rights and absence of food-borne allergic reactions. A polymerase chain reaction (PCR) followed by a high resolution melting (HRM) analysis was developed and applied for species specific detection of bovine milk in nine different commercial buffalo dairy products. A specific buffalo 12S rRNA and a bovine d-loop primer pair, targeting the mitochondrial genome, were employed in a duplex PCR assay. The analysis developed was found capable of identifying the presence of bovine milk down to 1% in commercial buffalo milk products and also of quantifying the ratio of bovine into buffalo milk. HRM was proven to be a fast and accurate technique for a routine authentication testing of mozzarella and other buffalo milk products

Advances of DNA-based methods for tracing the botanical origin of food products

The need for accurate and reliable methods for plant species identification in nature and in food products has steadily increased during past decades, particularly with the recent food scares and the development of trade and technological progress in food production. Moreover, the development of high added value products based on plants has raised concerns about adulteration. Thus, reliable methods to protect the producer, the company and the customer are needed. Fresh food products without any processing are suitable for many types of analytical or molecular analyses. But as most of foodstuff samples are processed to some extent, DNA is usually altered and fragmented into small fragments. However, extensive research has been performed and DNA based methods for food authenticity are becoming the methods of choice. Herein DNA based methods for species identification and authenticity in foods as well as quantitation methods, are based on DNA. These methodologies progress extremely fast; thus a review on the current state of the art on DNA based methods is useful in order to assess the field. The problems, advantages and disadvantages of the methods are also discussed. The trend of high throughput DNA technologies is recognized

Genotyping of Listeria monocytogenes isolates from poultry carcasses using high resolution melting (HRM) analysis.

An outbreak situation of human listeriosis requires a fast and accurate protocol for typing Listeria monocytogenes. Existing techniques are either characterized by low discriminatory power or are laborious and require several days to give a final result. Polymerase chain reaction (PCR) coupled with high resolution melting (HRM) analysis was investigated in this study as an alternative tool for a rapid and precise genotyping of L. monocytogenes isolates. Fifty-five isolates of L. monocytogenes isolated from poultry carcasses and the environment of four slaughterhouses were typed by HRM analysis using two specific markers, internalin B and ssrA genes. The analysis of genotype confidence percentage of L. monocytogenes isolates produced by HRM analysis generated dendrograms with two major groups and several subgroups. Furthermore, the analysis of the HRM curves revealed that all L. monocytogenes isolates could easily be distinguished. In conclusion, HRM was proven to be a fast and powerful tool for genotyping isolates of L. monocytogenes

Advances of DNA-based methods for tracing the botanical origin of food products

The need for accurate and reliable methods for plant species identification in nature and in food products has steadily increased during past decades, particularly with the recent food scares and the development of trade and technological progress in food production. Moreover, the development of high added value products based on plants has raised concerns about adulteration. Thus, reliable methods to protect the producer, the company and the customer are needed. Fresh food products without any processing are suitable for many types of analytical or molecular analyses. But as most of foodstuff samples are processed to some extent, DNA is usually altered and fragmented into small fragments. However, extensive research has been performed and DNA based methods for food authenticity are becoming the methods of choice. Herein DNA based methods for species identification and authenticity in foods as well as quantitation methods, are based on DNA. These methodologies progress extremely fast; thus a review on the current state of the art on DNA based methods is useful in order to assess the field. The problems, advantages and disadvantages of the methods are also discussed. The trend of high throughput DNA technologies is recognized