Phylogenetic Analysis of Black Peper (Piper Spp.) Population Collected in Different Locations of Vietnam Based on the ITSU1-4 Gene Region

Background: The internal transcribed spacer (ITS) of nuclear ribosomal DNA is one of the most commonly used DNA markers in plant phylogenetic and DNA barcoding analyses, and it has been recommended as a core plant DNA barcode. To compare and find out the analysis genetic diversity difference some pepper individuals collected in different localities in Vietnam when using the ITS of nuclear ribosomal DNA. The ITS gene region from the nuclear genomes were tested for their suitability as DNA barcoding regions of thirty-nine pepper individuals. Universal primers were used, and sequenced products were analyzed using the Maximum Likelihood method and Tamura-Nei model in the MEGA X program.Results: We did not observe high variability in intraspecific distance within the ITSu1-4 gene region between individuals, ranged from 0.000 to 0.155 (mean = 0.033). The size of the gene region has fluctuated from 667 to 685 bp between different individuals with the percentage (G + C) contained in the ITSu1-4 gene region was ranged from 54.776% to 60.805%, mean = 60.174%. The values of Fu’s Fs, D, Fu and Li’s D* and F* were negative as well (Fs = -0.209, D = -1.824; P < 0.05, D* = -1.205; not significant, P > 0.10 and F* = -1.699; not significant, 0.10 > P > 0.05), indicating an excess of recently derived haplotypes and suggesting that either population expansion or background selection has occurred. The value Strobeck’s S the obtained between individuals in a population is high (S = 0.684). The results of evolutionary relationships of taxa obtained 3 groups with the highest value of Fst is shown in the pairs of groups II and III (Fst = 0.151), and the lowest is in groups II and I (Fst = 0.015). All of the new sequences have been deposited in GeneBank under the following accession numbers MZ636718 to MZ636756.Conclusions: This database is an important resource for researchers working on Species of pepper in Vietnam and also provides a tool to create ITSu1-4 databases for any given taxonomy.

A DNA barcode library for the butterflies of North America

Although the butterflies of North America have received considerable taxonomic attention, overlooked species and instances of hybridization continue to be revealed. The present study assembles a DNA barcode reference library for this fauna to identify groups whose patterns of sequence variation suggest the need for further taxonomic study. Based on 14,626 records from 814 species, DNA barcodes were obtained for 96% of the fauna. The maximum intraspecific distance averaged 1/4 the minimum distance to the nearest neighbor, producing a barcode gap in 76% of the species. Most species (80%) were monophyletic, the others were para- or polyphyletic. Although 15% of currently recognized species shared barcodes, the incidence of such taxa was far higher in regions exposed to Pleistocene glaciations than in those that were ice-free. Nearly 10% of species displayed high intraspecific variation (>2.5%), suggesting the need for further investigation to assess potential cryptic diversity. Aside from aiding the identification of all life stages of North American butterflies, the reference library has provided new perspectives on the incidence of both cryptic and potentially over-split species, setting the stage for future studies that can further explore the evolutionary dynamics of this group.

DNA Mini-Barcodes, a Potential Weapon for Conservation and Combating Illegal Trade of Pangolin

Background Smuggling and illegal trade of pangolins and their scales has drastically reduced the wild population of pangolins. Accurate species identification is currently in urgent need as a powerful weapon for combating pangolin smuggling and trade and conserving the already endangered pangolin species. Aim of the study To develop an efficient method based on DNA mini-barcodes for accurate pangolin species identification and authentication of processed pangolin scales against the non-target species. Materials and methods The primers for amplifying the DNA mini-barcodes were designed based on cytochrome C oxidase subunit I (COI) gene fragments. The mini-barcodes were compared with the two universal barcodes (COI and Cytb) for performance in pangolin species identification by calculating the Kimura-2-parameter (K2P) distance, assessing the clustering dendrogram, and analyzing the BLAST similarity and barcoding gap. The accuracy of the three barcodes was also compared for authentication of pangolin scales against non-target species. Results Comparison of the three barcodes showed that the mini-barcode form COI had the highest amplification success rate (100%) and high variable sites (40.0%), with the ratio of mean inter- to intraspecific distance ratio was 25 and a distinct DNA barcoding gap. In the neighbor-joining (NJ) tree constructed based on the mini-barcode regions, each species of the pangolin family formed an obvious clade respectively, and the clades were all separated from those of the non-target species, indicating that the genetic information in the mini-barcode was sufficient for species identification. Conclusion The DNA mini-barcodes based on COI gene fragments provide an effective and accurate method for identification of pangolin species and authentication of pangolin scale products.

Genetic identification of Javan hawk-eagle (Nisaetus bartelsi) from Indonesia using mitochondrial COI gene

Nisaetus bartelsi is a native hawk from Java which its genetic information is not commonly understood yet. As a part of the conservation effort to maintain genetic diversity of this endangered species, this research aimed to obtain COI gene sequences from five individuals of N. bartelsi to confirm its position in the phylogenetic tree. DNA isolation from 5 N. bartelsi blood sample was performed and its COI gene sequence was amplified, sequenced, and used to reconstruct phylogenetic tree using MEGA6 with several other members of Nisaetus, Aquila, and Saggitaridae family. Furthermore, the intraspecific distance between 5 N. bartelsi samples and interspecific distance with other species were calculated using MEGA6. The result suggested that all five individuals belonged to the species Javan hawk-eagle (N. bartelsi) and were closely related to the Blyth’s hawk-eagle (Nisaetus alboniger). The DNA barcoding of the Javan hawk-eagle conducted in this study is a stepping stone to conservation efforts for the Javan hawk-eagle.

DNA barcoding and phylogenetic relationships in Omphalogramma (Primulaceae) from the Hengduan Mountain region of China

One nuclear and three chloroplast DNA markers (internal transcribed spacer [ITS], rbcL, matK, and trnH-psbA) were evaluated from 39 accessions of Omphalogramma (Primulaceae) from the Hengduan Mountains, representing seven species. The discrimination power of the markers at the species level was 14.3% (rbcL), 28.4% (trnH-psbA), 42.9% (matK), and 100% (ITS). The mean value of genetic distance between species for ITS was more than 3-fold that of intraspecific distance; all species were distinguished. Species were differentiated by unique characters, and phylogenetic analysis showed that the Omphalogramma species formed two clear monophyletic clades comprising O. vincaeflora (bootstrapping: 96.8%) and remaining Omphalogramma species (bootstrapping: 97.5%). This study indicated that DNA barcoding is a useful technique in the phylogenetic and taxonomic description of Omphalogramma species.

Molecular Identification of some Indian Muscid flies (Diptera: Muscidae) based on mitochondrial gene COII

ABSTRACTTaxonomic identification of flies having medical and veterinary importance is often complicated due to the lookalike morphological characters. Molecular identification of five Indian muscid flies (Musca domestica, Musca sorbens, Musca crassirostris, Stomoxys calcitrans and Haematobia irritans) has been attempted on the basis of mitochondrial COII gene. Sequences of 500-520 bp were analysed and found to be A+T rich. Rate of transitions was higher than transversions. The average haplotype diversity was 0.833 and nucleotide diversity was 0.02547 within the different species, which were calculated with the DnaSP Version 5.0. The genetic distances calculated by K2P model, showed the interspecific distances range more than 8.2%, while the intraspecific distance range had not exceed 0.8%. The 1000 bootstrapped Neighbour-joining tree and Maximum likelihood tree were constructed to establish the phylogenetic relationship between the different muscid species. The results show the robustness of COII gene as a diagnostic marker. The data obtained from this study would be worthy for medical and veterinary entomologists for precise identification of imperative muscid species.

Using DNA Barcoding to Identify the Genus Lolium

Seeds of the genus Lolium are difficult to identify based on morphology for morphological likeness and some physical deformation such as friction and flattening during storage and transport. DNA barcoding, a newly-established method, has been used to discriminate a variety of agricultural crops with its own advantages. In present study, DNA barcodes for the genus Lolium were investigated for the first time. DNA sequences of psbA-trnH, rbcL, atpF-atpH, and the ITS2 region were evaluated for their ability to differentiate Lolium from the related genus Festuca. As confirmed by inter-intraspecific divergence and Kimura 2 parameter analysis, the greatest divergence existed in ITS2, followed by psbA-trnH. On the contrary, rbcL and atpF-atpH possessed poor genetic variation of 0-0.0115, and was relatively difficult in discrimination of genus Lolium. For ITS2 sequence, no inter-intraspecific distance overlaps were observed and each species has a distinct barcoding gap. ITS2 could effectively discriminate all species based on a neighbor-joining tree. Thus, the ITS2 region is a candidate for DNA barcoding of Lolium.