Overexpression of a Glutathione S-Transferase gene from P. vulgaris L. Improves salt stress Tolerance in Transgenic Tobacco Plants

Glutathione S-transferases (GSTs) are multifunctional proteins and forms major part, of plant cellular detoxification system and antioxidant enzyme network. Previously, a novel GST gene PvGSTU3-3 has been isolated from roots of Phaseolus vulgaris L. plants. The isoenzyme shows high antioxidant catalytic function and acts as hydroperoxidase, thioltransferase, and dehydroascorbate reductase. In the present study, with a view to investigate the biological function of PvGSTU3-3 a constitutive plant overexpression vector of PvGST3-3 was constructed and transferred into tobacco (Nicotiana tabacum. L. cv Xanthi) plants via A. tumefaciencs. The PvGSTU3-3 gene was successfully integrated into the genome of the transgenic tobacco lines as confirmed by Real time PCR and expressed in the transformants, validated through quantitative reverse transcription PCR. Three tobacco transgenic lines overexpressing PvGSTU3-3 tested for their salt tolerance (200mM NaCl) under in vitro conditions. All lines were more tolerant compared to wt plants, as demonstrated by the increased root length. These results suggest that PvGSTU3-3 isoenzyme can mediate physiological pathways that enhance salt stress tolerance

Molecular and catalytic characterization of the herbicide-inducible glutathione transferases from Phaseolus vulgaris

Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris

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

The identification of several dipterocarpaceae and fagaceae trees by barcode dna coupled with high-resolution melting analysis

The loss of forests is a major environmental, social, and economic problem. The disappear-ance has been occurring to an extreme degree in many parts of Southeast Asia, including Thailand. Logging and clearing of forests for agriculture, cash crops, and food production has destroyed much of the tropical forests in Thailand. Floristic inventory could provide essential information for forest conservation but species identification as a part of inventory creating could be challenging in some cases. Barcode DNA coupled with High Resolution Melting analysis (Bar-HRM) was used here in aiding species identification of plant in Dipterocarpaceae (Dipterocarpus alatus, D. costatu, D. intricatus, D. obtusifolius, Hopea ferrea, H. odorata, Shorea guiso, S. obtuse, S. roxburghii, and S. siamensis) and Fagaceae (Castanopsis echinocarpa, C. inermis, Lithocarpus wallichianus, Quercus aliena and Q. oidocarpa) families. Two main experiments were conducted including: (1) a comparing method for primer design and (2) testing the robustness of the Bar-HRM by trying to identify tree samples that did not have sequences in the GenBank. In experiment 1, the manual design primer pair was found to be the best fit for the work. Of key importance is finding the primers which give the most nucleotide variations within the generated amplicon; this is a parameter that cannot be set in any web-based tools. Next, in experiment 2, Bar-HRM using primers of ITS1 and ITS2 regions were able to discriminate all 10 tested tree species without any problem, even when there were no sequences of the samples to be analysed before performing the HRM. Here, Bar-HRM poses potential to be a game-changer in tropical forest conservation, as it will be useful for species identification. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Environmental DNA detection of giant snakehead in Thailand’s major rivers for wild stock assessment

Capture-based aquaculture is now gaining much attention in Southeast Asia. This system was used to produce several fish species with social and economic implications, including the giant snakehead (Channa micropeltes). As wild harvesting of organisms for seed stock is one of main practices in capture-based aquaculture, abundance and distribution of the wild stock are essential for both environmental impact evaluation and stock management. Mark and recapture, visual observation and physical capture of target species are costly, ineffective, and labour intensive for fish surveys in several cases. Detection of target organisms using eDNA (environmental DNA) could be a good alternative as it has proved to be a non-invasive, rapid, and sensitive method for aquatic species monitoring and surveying. Here, we developed a TaqMan assay that targets the 16S region of giant snakehead DNA to amplify eDNA captured in water samples. 300 µl of water samples were collected from 15 sites located in the Chao Phraya River Basin (Ping, Wang, Yom, Nan, and Chao Phraya River) and filtered with 0.7 µm glass fibre membrane filter. Giant snakehead eDNA was detected in most tributaries (60%) with concentrations ranging from 74.0 copies/ml in Wang River sites to 7.4 copies/ml in Nan River sites. As intensification of capture-based aquaculture could lead to depleting of wild fish stocks, urgent management is needed. However, the existing conventional approaches for assessment of fish overexploitation, survey and monitoring have several limitations. Copyright: © 2022 Osathanunkul, Madesis. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Rapid analysis for the identification of the seagrass Halophila ovalis (Hydrocharitaceae)

Seagrasses are considered as one of the most important species as they play key ecological roles in various types of ecosystems and also provide a food source for endangered animal species. There are two main  characteristics of seagrasses that hinder efforts to correctly identify species based on conventional identification keys alone: i) the variability of  morphological characteristics and ii) lack of needed morphological  characters especially flowers. A taxonomically unresolved complex such as Halophila spp. is reported. Plant DNA barcoding regions (rbcL and trnH-psbA) were used to confirm species of collected seagrasses from the southern coast of Thailand. Small and big-leaved samples of Halophila spp. were analysed in this study. The big-leaved samples were identified on the field as Halophila ovalis whilst it was uncertain whether the small-leaved samples belonged to H. ovalis. DNA analysis revealed that the small-leaved samples could be H. ovalis. We also coupled PCR with high resolution melt (HRM) to more cost-effectively identify individuals of H. ovilis than using barcoding alone. Using HRM to resolve differences in the sequence of two genes showed that the two unknown seagrasses belonged to the same species as H. ovalis.  In conclusion, using HRM proved to pose great potential in seagrass identification. Key words: DNA barcoding, Halophila ovalis, rbcL, trnH-psbA, species identification

SNPs identification and association study of fruit quality genes from selected whole-genome sequenced peach cultivars grown in Greece

In Greece, peach is one of the largest cultivations both in terms of hectares and in economic profit, with table peaches representing a 40% and industrial peaches the 60%. Next generation sequence technologies can prove valuable in evaluating present peach cultivars and getting the necessary knowledge to develop new cultivars with appreciable quality traits. Whole genome sequencing was performed on selected peach cultivars (‘Romea’, ‘Virgil’, ‘Catherina’, ‘Fergold’, ‘Crimson Lady’, ‘Rubirich’, ‘Rebus 028’, ‘Early Top’) and a local cultivar called ‘Lemonato’. These cultivars were selected according to their differences in specific fruit quality traits, such as slow melting flesh, stone adhesion and hardness, fruit color (skin and flesh), fruit size, along with maturity date and fruit productivity. Sequencing was performed using Illumina Nextseq 550 platform, with Nextera DNA Flex indexes, resulting in over 150 million reads with an average GC of 38.5%. The aim of this study is the identification of SNPs associated with genes that control important fruit quality characteristics and the development of molecular markers linked to these traits. The number of SNPs associated with each of these genes and the number of useful SNPs for marker-assisted selection varied accordingly. A total of 1,535 SNPs with high impact effect on known and putative genes were identified across all cultivars. SNPs identified in genes that are involved in biological processes (cellular and metabolic process) were the most abundant. © 2022 International Society for Horticultural Science. All rights reserved

Bar-HRM for Species Confirmation of Native Plants Used in Forest Restoration in Northern Thailand

Plant species confirmation is a crucial step in using native plant species for forest restoration. To enhance this, a hybrid method of DNA barcoding and high-resolution melting analysis (Bar-HRM) was investigated in this study. In total, 12 native plant species samples were collected from forest restoration sites in Nan, a province in Northern Thailand. Simulation HRM analysis was performed to find the most appropriate region for in vitro Bar-HRM analysis. After that, in vitro Bar-HRM was carried out to validate the performance of native plant species. Results from both simulation and in vitro analyses revealed that the nuclear ribosomal internal transcribed spacer (ITS) region can be used as a primer set that can clearly discriminate native plant species in this study. With our study, Bar-HRM was proved of use in native plant species confirmation, even if that species had no molecular data available. In this con-text, Bar-HRM would be useful for the identification of native plant species used in tropical forest restoration not only in Thailand but also in any areas with similar plant groups. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

Fruit quality traits and genotypic characterization in a pomegranate ex situ (Punica granatum l.) collection in greece

Pomegranate is one of the oldest known fruit crops, well adapted to hot and dry areas and successfully grown in Mediterranean countries. During the last two decades, numerous pub-lications have revealed the traditionally known associations between pomegranate consumption and health benefits, which led to increased demand by consumers and expansion in cultivation areas. Pomegranate is well adapted to areas with diverse pedoclimatic conditions and local cultivars could provide an essential source of genes for breeding. In this study, fruit phenotypic and genetic variability, and relationships, were studied in 26 Greek and foreign pomegranate cultivars/accessions grown in an ex situ collection located in Naoussa Greece, using Inter Simple Sequence Repeat (ISSR) and Start Codon Targeted (SCoT) molecular markers. Results from the principal component analysis made on fruit phenotypic characters revealed five components that accounted for 74.8% of the total variance, the first being related to skin color parameters and the second to juice antioxidant contents and aril color. Clustering from phenotypic data allocated individuals into four clusters. A total of 184 bands were generated for all markers applied across the 26 pomegranate cultivars/accessions, with an average of 77 bands per ISSR markers and 82 bands for SCoT markers. Low variability in the phenotypic and genotypic level was indicated; nevertheless, results from the association study between phenotypic traits and molecular markers that were obtained using Multiple Regression Analysis (MRA) could prove valuable for marker-assisted breeding programs, especially from SCoT markers that were found to be strongly or averagely associated with the morphological traits and chemical components. © 2021 by the authors. Licensee MDPI, Basel, Switzerland