DNA Barcoding Markers Research Articles

Complete chloroplast genomes of 13 species of the Impatiens genus for genomic features and phylogenetic relationships studies

Impatiens spp. are well-known ornamental and medicinal plants that are widely distributed in the highlands and mountains of southwestern China. This area is one of the hotspots for the distribution of Impatiens species, with typical karst landforms and abundant wild resources. Many of these species are endemic to a narrow distribution area, but their classification and relationships are relatively unclear because of insufficient field investigations, diverse morphological characteristics and lack of molecular information. In this study, chloroplast genome analysis of 13 species (including 2 synonyms) in karst habitats was conducted to study their characteristics and phylogenetic relationships. The results revealed that these chloroplast genomes all had double-stranded tetrad structures ranging in length from 151,284 bp to 152,421 bp, including a total of 113 genes, including 80 protein-coding genes, 29 transfer RNAs, and 4 ribosomal RNAs. SSRs mainly consist of A/T repeats and AT/AT repeats, while INEs mainly consist of positive repeats and palindromic repeats. The frequency of codon usage was essentially the same, with a total of 31 high-frequency codons detected, the vast majority ending in A/U. Five mutation hotspots were detected: rps16-trnQ-UUG, ndhF, ccsA-ndhD, ycf1, and trnN-GUU, among which ycf1 had the highest Pi value and the greatest potential as a DNA barcode marker. Our phylogenetic tree shows that all 13 species belong to Section Impatiens. And supported the classification of I. reptans and I. rhombifolia should as synonyms (BS = 100/PP = 1.00). This study comprehensively analyzed the cp genomes of different taxa, sheds light on the taxonomic intricacies of Impatiens species, provide valuable information into its phylogenetic and taxonomy.

Development of a new primer cocktail for improved DNA barcoding of brachyuran crabs (Decapoda: Brachyura)

Abstract DNA barcoding has revolutionized species identification and molecular phylogeny, offering immense potential for elucidating the diversity of brachyuran crabs. We explored the utility of a new primer cocktail-based approach for amplifying DNA barcode regions of mitochondrial cytochrome c oxidase I (CO1) from 28 specimens representing brachyuran species across 21 major families collected along the Korean coast. The primer cocktail, comprising three forward and two reverse degenerate primers with M13 adapter tails, exhibited superior PCR amplification efficiency, being 100% successful. Sequencing of the PCR products yielded high-quality, 726 bp, DNA barcode sequences, facilitating accurate species identification with 23 out of 28 specimens matching precisely with their morphological taxonomy. Barcoding gap analysis revealed a clear gap between mean intra- and interspecific K2P distances, with a 148-fold difference, confirming the reliability of CO1 barcodes for achieving 100% success in species identification within the dataset. The study highlights the effectiveness and reliability of the new primer cocktail as a robust molecular marker for DNA barcoding of brachyuran crabs, essential for ecological investigations and conservation endeavors, and elucidating the taxonomic complexities among them.

The first initiative of dna barcoding of palaquium rostratum (miq.) burck from Bangka, Indonesia

Abstract Palaquium rostratum (Miq.) Burck with a local name Nyatoh, is the floral identity of Bangka Belitung Islands and has widely potential uses, making conservation efforts for this species important. However, there is still insufficient data about Nyatoh including information on its genetic diversity. In this study we used the chloroplast DNA (rbcL) as gene markers to identify P. rostratum in Bangka Island. The method included DNA extraction, PCR amplification, and DNA sequence analysis. Total 606 bp of rbcL sequences was obtained from Nyatoh leaves. When comparing to other DNA references using BLASTn, the barcode region of P. rostratum in this study showed up to 99% similarity with Palaquium sp. collected from Temburong, Brunei Darussalam. Further, phylogenetic analysis revealed the DNA barcode marker used were able to assign correctly to genus level while they showed a lower or no success in assigning taxa to species. For future, we recommend other DNA barcode or combination (e.g. ITS, rbcL + matK) to have a higher level of discriminatory power.

Lipid profiles and DNA barcoding markers reveal the genetic delimitation between species of the Carapa genus native from the Amazon

Authentic identification of Amazonian tree species is important for conservation and their sustainable exploitation. This work aimed to characterize the inter- and intraspecific divergence in two taxonomic units of the genus Carapa using the lipidomic profile of the seed oils, analyzed by gas chromatography-mass spectrometry (GC-MS) and DNA barcoding molecular markers to identify and discriminate differences between Carapa species. Seeds and leaves of 53 individuals were collected and morphologically grouped into Carapa guianensis and Carapa vasquezii. The oils extracted from seeds were esterified, and their lipidomic profiles were analyzed. Two chloroplast genome regions (psbA-trnH and rbcL) were amplified for genetic diversity analyses and the barcoding-gap test. The lipidomic profile allowed the identification of 40 substances present in the C. guianensis and C. vasquezii oils, with 20 shared among the species independent of the region of collection, two occurring only in the oil of C. guianensis from Acre, four in samples of C. guianensis and C. vasquezii from Amazonas, and 16 substances occurring only in samples of C. vasquezii oil. C. vasquezii presented a greater diversity of terpenoids. The genome regions made it possible to obtain 16 distinct haplotypes for the two species (C. guianensis and C. vasquezii), 999 conserved sites, and 32 polymorphic sites. The species presented significant molecular divergence (FST=0.87), and the rbcL region was considered promising for barcode studies at levels ranging from 0.1 to 0.2% of genetic distance. The results suggest that the two species morphologically identified C. guianensis and C. vasquezii constitute genetically divergent biological groups.

POTENTIAL FOR ACCELERATED REPLICATION OF FRAXINUS SOGDIANA USING IN VITRO CULTURE AND IDENTIFICATION OF A RELIC TREE USING DNA-BARCODING

Four species of Fraxinus including F. angustifolia subsp. syriaca (Boiss.) Yalt., F. sogdiana Bunge, F. pennsylvanica Marshall and F. americana L. grow in Kazakhstan. The study aimed to determine the conditions for awakening endogenous dormancy in vitro of F. sogdiana. In the control group without vernalization, the sterile culture yield at room temperature was 30%. However, with cold treatment, this yield increased to 60%, which is twice as high. It was observed that on MS medium without the addition of hormones, 22 microshoots were obtained from unwounded seeds, whereas 39 microshoots were obtained from wounded seeds during microclonal propagation.Phylogenetic relationships and species identification were analyzed using three DNA barcoding markers, comparing F. sogdiana with species from the NCBI database. rbcL and ITS markers did not show satisfactory interspecific genetic variability among F. species. Notably, matK gene sequences effectively differentiated F. sogdiana from other species. These results provide fundamental information that is valuable for future biotechnological and molecular studies.

Complete chloroplast genomes of Desmidorchis penicillata (Deflers) plowes and Desmidorchis retrospiciens Ehrenb.: comparative and phylogenetic analyses among subtribe Stapeliinae (Ceropegieae, Asclepiadoideae, Apocynaceae)

The succulent shrubs Desmidorchis penicillata and D. retrospiciens, part of the taxonomically challenging genus Desmidorchis, are well‐known for their ecological resilience and medicinal significance. This study sequences the first complete chloroplast genomes of these species, shedding light on their genomic characteristics and evolutionary relationships. The circular genomes of D. penicillata (161 776 bp) and D. retrospiciens (162 277 bp) display a quadripartite structure typical of Angiosperms. Gene content, order, and GC content are consistent, featuring 114 unique genes, including 80 protein‐coding, 30 transfer RNAs, and four ribosomal RNAs genes. Codon usage analysis underscores A/U‐rich preferences, while RNA editing sites, predominantly in ndhB and ndhD genes, suggest post‐transcriptional modifications. Analysis of long repeated sequences reveals a predominance of forward and palindromic repeats. Simple sequence repeats (SSRs), particularly A/T motifs, are abundant, with high presence of mononucleotide, offering potential molecular markers. Comparative analysis with their relatives in subtribe Stapeliinae identifies mutational hotspots such as ycf1, ndhF, trnG(GCC)‐trnfM(CGA) and ndhG‐ndhI that could be potential DNA barcoding markers. The inverted repeat (IR) boundaries analysis revealed an expansion of IR on the small single copy region, leading to the formation of a pseudogene. Overall, substitution rate analysis indicated purifying selection, with a few genes (rpl22, clpP and rps11) showing signatures of positive selection. Additionally, the phylogenetic analysis positioned Desmidorchis within the Stapeliinae clade and strongly supported the sister relationship between D. penicillata and D. retrospiciens. This study provides comprehensive molecular data for future research in Desmidorchis.

Aspergillus aureoterreus, A. pseudonomiae, A. siamensis, and A. flocculosus-ochraceopetaliformis as Four New Pathogens of Eye Infection in DNA Barcoding Markers

Background: The use of a morphological method to determine Aspergillus species in routine laboratory investigation has always been challenged with low sensitivity and low specificity. Time-consuming specimen preparation and insufficient expertise are the crucial reasons. DNA barcoding markers have been introduced for identification as another gold standard method. Objective: To investigate DNA barcoding markers in identifying Aspergillus strains isolated from patients with eye infection. Methods: This study extracted 12 DNA samples of 12 Aspergillus strains. DNA barcoding genes, i.e., ITS, betatubulin (BT2), calmodulin (CAL), and actin (ACT) genes, were amplified and sequenced by polymerase chain reaction and Sanger's sequencing methods. All gene sequences were compared to those of the type or reference species collected from public databases. The species were identified with phylogenetic analyses in free applications from public websites. Results: ITS, BT2 and CAL, and ACT identified 5, 6, and 3 species and 3, 2, and 1 species complex of Aspergillus, respectively. DNA barcoding with multi-locus sequence typing revealed four new pathogens of human eye infection, including A. aureoterreus, A. pseudonomiae, A. siamensis, and A. flocculosus-ochraceopetaliformis. Conclusion: The DNA barcoding markers identified Aspergillus species with better specificity than the conventional method. The markers and techniques need to be used as tools for routine Aspergillus identification.

In Silico Evaluation of rbcL, matK, and psbA-trnH Regions on the Genus Spatholobus (Fabaceae)

Spatholobus is a genus that belongs to the Fabaceae that is known to contain various bioactive compounds and distributed across Asia, including Indonesia. However, exploration of Spatholobus in Indonesia is still rare. Therefore, DNA barcoding is used to support the exploration and conservation of Spatholobus in Indonesia. However, there is no universal marker that can be used across all plant species. In addition, there are still few studies related to DNA barcoding within the genus Spatholobus. The purpose of this study was to evaluate the rbcL, matK, and intergenic spacer psbA-trnH regions in silico that can be used as DNA barcodes for the genus Spatholobus. This study began with a sequence search on the NCBI database including the rbcL, matK, and intergenic spacer psbA-trnH genes in the genus Spatholobus and Phaseolus coccineus as the outgroup. Each sequence was then aligned with ClustalW. Then, a phylogenetic tree was constructed using the Maximum-Likelihood (ML) with 1000× bootstrap. As a result, the rbcL, matK, and psbA-trnH regions can be used as markers for DNA barcoding in the genus Spatholobus with different specifications. The rbcL and matK can be used to distinguish Spatholobus at the genus level, while the psbA-trnH can be used to distinguish Spatholobus at the species level.

Genetic Diversity and Relationship of Native Phalaenopsis Orchids: A Case Study of Indonesian Archipelago

The native Phalaenopsis is valuable germplasm for future orchid breeding programs and for its conservation because it provides many beneficial traits or genes. This study aimed to determine and analyze the molecular diversity and phylogeny of Indonesian native Phalaenopsis by a DNA barcoding (matK) marker. A total of 19 samples of Phalaenopsis orchids were used in this study. All leaf samples of orchid were extracted and purified using a commercial DNA isolation kit from Geneaid Biotech Ltd., Taiwan (GP100). The DNAs were then amplified by specific matK primers: Forward (5’-CGTACAGTACTTTTGTGTTTACGAG-3’) and Reverse (5’-ACCCAGTCCATCTGGAAATCTTGGTTC-3’). The DNA targets or products (matK) were purified and sequenced by the Sanger-bidirectional method at 1st Base Ltd., Malaysia. Before further analysis, the matK sequences of Phalaenopsis were edited, reconstructed, and aligned with the assistance of Clustal W in the MEGA 11 software. Its genetic diversity was determined using the nucleotide diversity index (π%), and the phylogenetic analysis was performed using the maximum likelihood (ML) method with a statistical bootstrap. The phylogenetic relationship was also assessed using principal component analysis (PCA). Based on this marker, the native Phalaenopsis has a high genetic diversity (π% = 1.70). In addition, the phylogenetic analysis revealed that this germplasm was separated into seven clades, where P. pantherina has the closest relation to P. cornu-cervi and P. gigantea. Conversely, the highest genetic distance was to P. amabilis from South Kalimantan and to P. celebensis from Sulawesi, at a coefficient divergence of 0.084. Our findings provide an essential foundation for supporting future orchid breeding practices, including conservation, on local and global scales.

Use of a novel combination of multiplex PCR and DNA barcoding in assessing authenticity of ginseng products

Ginseng (Panax sp.) is a medicinal plant used for its purported health benefits, primarily in East Asian countries. The COVID-19 pandemic led to a significant increase in sales of dietary supplements, including ginseng supplements, to purportedly support immune health and provide other health benefits. However, this heightened demand has subsequently increased the risk of adulteration in these dietary supplements. This study aimed to determine the efficacy of a novel combination of DNA barcoding and multiplex PCR to identify species in ginseng supplements. A total of 50 commercial ginseng supplements containing Panax ginseng, Panax quinquefolius, or Panax notoginseng were obtained from an online vendor. Two composite samples per product were subjected to DNA extraction followed by multiplex PCR and DNA barcoding with three genetic targets (i.e., rbcL, matK, and ITS2). Of the three DNA barcoding markers, ITS2 had the highest amplification success at 74%, and matK had the highest sequencing success at 60%. DNA barcoding alone identified species in 68% of products, while multiplex PCR had an overall 60% identification rate. The combination of both methods resulted in a 72% species identification rate. In addition to improving the overall identification rate, the combination of both methods allowed for greater species resolution and the detection of undeclared plant species. Future research should explore combining DNA-based methods with chemical-based approaches to improve ginseng detection capabilities and enable the quantification of undeclared adulterants.

Molecular Characterization of Wild Tea Plants (Camellia spp.) in Thailand Using DNA Barcoding Markers

Molecular Characterization of Wild Tea Plants (Camellia spp.) in Thailand Using DNA Barcoding Markers

A First Approach for the In Vitro Cultivation, Storage, and DNA Barcoding of the Endangered Endemic Species Euonymus koopmannii.

Euonymus koopmannii is a rare and protected species in Kazakhstan, valued for its ecological role in soil stabilization and its ornamental properties. This study presents the first use of micropropagation and phylogenetic analysis for the endemic plant E. koopmannii. Seedlings of E. koopmannii proved to be more effective than internodes as primary explants for plant micropropagation of in vitro culture, with a multiplication coefficient of 28.5 from seedlings and 6.1 from internodes. On MSR I medium supplemented with 0.5 mg/L IBA and 0.05 mg/L IAA, a higher success rate of 67% was achieved for root formation of test tube-grown E. koopmannii plants. Using mannitol as an osmotic agent at a concentration of 8 mg/L prolonged the storage time of E. koopmannii under slow growth conditions when compared to CCC and abscisic acid. Phylogenetic relationships and species identification were analyzed using four DNA-barcoding markers, comparing E. koopmannii with species from NCBI. All candidate barcoding markers showed sufficient levels of interspecific genetic variation among Euonymus species. In addition, ITS region and rbcL gene sequences effectively distinguished E. koopmannii from other species. These results provide fundamental information that will be valuable for future biotechnological and molecular studies.

DNA barcodes are ineffective for species identification of Acropora corals from the aquarium trade.

Species identification of stony corals (Scleractinia), which are regulated under the Convention on International Trade in Endangered Species of Wild Fauna and Flora, is critical for effective control of harvest quotas, enforcement of trade regulations and species conservation in general. DNA barcoding has the potential to enhance species identification success, depending on the specific taxon concerned and genetic markers used. For Acropora, DNA barcoding, based on the mitochondrial putative control region (mtCR) and the nuclear PaxC intron (PaxC), has been commonly used for species identification and delimitation, but the reliability and robustness of these loci remain contentious. Therefore, we sought to verify the applicability of this approach. In this study, we obtained 127 Acropora colonies from the aquarium trade to test the effectiveness of barcoding mtCR and PaxC for species identification. We were able to recover sequences for both loci in over half of the samples (n = 68), while gene amplification and sequencing of mtCR (n = 125) outperformed PaxC (n = 70). Amongst the 68 samples with both loci recovered, just a single sample could be unambiguously identified to species. Preliminary identities, based on only one gene, were assigned for 40 and 65 samples with mtCR and PaxC, respectively. Further analyses of 110 complete mitochondrial genomes obtained from GenBank showed that, despite the full length of the sequences, only eight species were delimited, of which only three species were correspondingly monophyletic. Therefore, we conclude that the commonly used DNA barcoding markers for Acropora are ineffective for accurate species assignments due to limited variability in both markers and even across the entire mitochondrial genome. Therefore, we propose that barcoding markers should generally not be the only means for identifying corals.

Molecular identification and genetic diversity of biting midges (Diptera: Ceratopogonidae) in the tropical environment on Hainan Island, China.

Biting midges are hematophagous arthropods responsible for zoonotic infectious diseases and have a wide distribution in temperate and tropical latitudes of the world. The genomic DNA of midge samples was extracted using the Chelex method and the ITS1gene was amplified by PCR to identify the midge species via BLAST. The sequence characteristics and the genetic diversity were analyzed using ClustalOmega, DnaSP, Arlequin, PopART, and TCS software tool. The validity of the ITS1 gene as a DNA barcode marker was evaluated using DAMBE. The phylogenetic relationship was established in the MEGA software. The ABGD web determined the species boundary and the SDT software visualized the pairwise sequence comparisons. A total of 39 midge samples possessed the range from 364 to 429 bp of the ITS1 sequences. The midge samples were identified as Culicoides imicola, Culicoides oxystoma, Culicoides peregrinus, Culicoides jacobsoni, Forcipomyia peregrinator, and Culicoides fulvus, respectively. The ITS1 sequences had 288 conserved sites (60.25%), 167 variable sites (34.94%), 141 parsimony-informative sites (29.50%), and 26 singleton sites (5.44%), with a considerable sequence variation with a high haplotype diversity. Populations in Lingao, Haikou, Tunchang were relatively independent, with a low level of gene flow. A separate population of Forcipomyia genus in Danzhou was observed. The biting midges in Hainan, a tropical island, had abundant genetic diversity. Timely surveillance is a crucial control measure for the spread of midge-borne diseases.

Skmer approach improves species discrimination in taxonomically problematic genus Schima (Theaceae)

Genome skimming has dramatically extended DNA barcoding from short DNA fragments to next generation barcodes in plants. However, conserved DNA barcoding markers, including complete plastid genome and nuclear ribosomal DNA (nrDNA) sequences, are inadequate for accurate species identification. Skmer, a recently proposed approach that estimates genetic distances among species based on unassembled genome skims, has been proposed to effectively improve species discrimination rate. In this study, we used Skmer to identify species based on genomic skims of 47 individuals representing 10 out of 13 species of Schima (Theaceae) from China. The unassembled reads identified six species, with a species identification rate of 60%, twice as high as previous efforts that used plastid genomes (27.27%). In addition, Skmer was able to identify Schima species with only 0.5× sequencing depth, as six species were well-supported with unassembled data sizes as small as 0.5 Gb. These findings demonstrate the potential for the Skmer approach in species identification, where nuclear genomic data plays a crucial role. For taxonomically difficult taxa such as Schima, which have diverged recently and have low levels of genetic variation, Skmer is a promising alternative to next generation barcodes.

Versatile DNA extraction from diverse plant taxa using ionic liquids and magnetic ionic liquids: a methodological breakthrough for enhanced sample utility

BackgroundThere is a growing demand for fast and reliable plant biomolecular analyses. DNA extraction is the major bottleneck in plant nucleic acid-based applications especially due to the complexity of tissues in different plant species. Conventional methods for plant cell lysis and DNA extraction typically require extensive sample preparation processes and large quantities of sample and chemicals, elevated temperatures, and multiple sample transfer steps which pose challenges for high throughput applications.ResultsIn a prior investigation, an ionic liquid (IL)-based modified vortex-assisted matrix solid phase dispersion approach was developed using the model plant, Arabidopsis thaliana (L.) Heynh. Building upon this foundational study, the present study established a simple, rapid and efficient protocol for DNA extraction from milligram fragments of plant tissue representing a diverse range of taxa from the plant Tree of Life including 13 dicots and 4 monocots. Notably, the approach was successful in extracting DNA from a century old herbarium sample. The isolated DNA was of sufficient quality and quantity for sensitive molecular analyses such as qPCR. Two plant DNA barcoding markers, the plastid rbcL and nuclear ribosomal internal transcribed spacer (nrITS) regions were selected for DNA amplification and Sanger sequencing was conducted on PCR products of a representative dicot and monocot species. Successful qPCR amplification of the extracted DNA up to 3 weeks demonstrated that the DNA extracted using this approach remains stable at room temperature for an extended time period prior to downstream analysis.ConclusionsThe method presented here is a rapid and simple approach enabling cell lysis and DNA extraction from 1.5 mg of plant tissue across a broad range of plant taxa. Additional purification prior to DNA amplification is not required due to the compatibility of the extraction solvents with qPCR. The method has tremendous potential for applications in plant biology that require DNA, including barcoding methods for agriculture, conservation, ecology, evolution, and forensics.

Taxonomic accuracy and complementarity between bulk and eDNA metabarcoding provides an alternative to morphology for biological assessment of freshwater macroinvertebrates

Determining biological status of freshwater ecosystems is critical for ensuring ecosystem health and maintaining associated services to such ecosystems. Freshwater macroinvertebrates respond predictably to environmental disturbances and are widely used in biomonitoring programs. However, many freshwater species are difficult to capture and sort from debris or substrate and morphological identification is challenging, especially larval stages, damaged specimens, or hyperdiverse groups such as Diptera. The advent of high throughput sequencing technologies has enhanced DNA barcoding tools to automatise species identification for whole communities, as metabarcoding is increasingly used to monitor biodiversity. However, recent comparisons have revealed little congruence between morphological and molecular-based identifications. Using broad range universal primers for DNA barcode marker cox1, we compare community composition captured between morphological and molecular-based approaches from different sources — tissue-based (bulk benthic and bulk drift samples) and environmental DNA (eDNA, filtered water) metabarcoding — for samples collected along a gradient of anthropogenic disturbances. For comparability, metabarcoding taxonomic assignments were filtered by taxa included in the standardised national biological metric IBMWP. At the family level, bulk benthic metabarcoding showed the highest congruence with morphology, and the most abundant taxa were captured by all techniques. Richness captured by morphology and bulk benthic metabarcoding decreased along the gradient, whereas richness recorded by eDNA remained constant and increased downstream when sequencing bulk drift. Estimates of biological metrics were higher using molecular than morphological identification. At species level, diversity captured by bulk benthic samples were higher than the other techniques. Importantly, bulk benthic and eDNA metabarcoding captured different and complementary portions of the community — benthic versus water column, respectively — and their combined use is recommended. While bulk benthic metabarcoding can likely replace morphology using similar benthic biological indices, water eDNA will require new metrics because this technique sequences a different portion of the community.

Characterization of the Plastid Genomes of Four Caroxylon Thunb. Species from Kazakhstan.

The family Chenopodiaceae Vent. (Amaranthaceae s.l.) is known for its taxonomic complexity, comprising species of significant economic and ecological importance. Despite its significance, the availability of plastid genome data for this family remains limited. This study involved assembling and characterizing the complete plastid genomes of four Caroxylon Thunb. species within the tribe Salsoleae s.l., utilizing next-generation sequencing technology. We compared genome features, nucleotide diversity, and repeat sequences and conducted a phylogenetic analysis of ten Salsoleae s.l. species. The size of the plastid genome varied among four Caroxylon species, ranging from 150,777 bp (C. nitrarium) to 151,307 bp (C. orientale). Each studied plastid genome encoded 133 genes, including 114 unique genes. This set of genes includes 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Eight divergent regions (accD, atpF, matK, ndhF-ndhG, petB, rpl20-rpl22, rpoC2, and ycf3) were identified in ten Salsoleae s.l. plastid genomes, which could be potential DNA-barcoding markers. Additionally, 1106 repeat elements were detected, consisting of 814 simple sequence repeats, 92 tandem repeats, 88 forward repeats, 111 palindromic repeats, and one reverse repeat. The phylogenetic analysis provided robust support for the relationships within Caroxylon species. These data represent a valuable resource for future phylogenetic studies within the genus.

Assessment of Durian Diversity and Its Wild Relatives (Durio spp.) Based on Leaf Morphology and Molecular Marker

Durian (Durio spp.) is native to Southeast Asia and has potential for development. However, some species are threatened due to deforestation and extensive land conversion. This study aimed to determine the genetic diversity and relationships of durian and wild relatives (Durio spp.) on the Indonesia Island Borneo using a leaf morphology and DNA barcoding (matK) marker. In this study, 15 durian samples from this region were used, excluding ‘Monthong’ (Durio zibethinus) and ‘Bengang’ (Neesia strigosa) as the outgroups from the GenBank database. The leaf morphology was analyzed descriptively, whereas the genetic diversity was by the nucleotide diversity index (π%). The relationship of durians was revealed by the maximum likelihood (ML) method and examined with the bootstrap statistics for 1000 replicates, also confirmed by the PCA (principal component analysis). Based on the leaf morphology, the durians are divided into five forms, i.e., obovate-lanceolate, elliptic, ovate, oblong, and linear-oblong. ‘Pampaken’ and ‘Pampaken Burung Kecil’ indicated the earliest form (obovate-lanceolate), whereas the linear-oblong was by ‘Kamundai.’ Following the molecular marker, it was seen that the durians have low genetic diversity (π%) with only 0.015. However, phylogenetically, the durians were separated into four similar clades or groups for ML and PCA. In this instance, it has appeared that most of the durians evaluated in the current study have close relationships, except for the taxa with the farthest relationship. The results provide valuable information for the local and global durian conservation mission, including future breeding programs.

Cytochrome b gene as a potential DNA barcoding marker in ecoraces of tropical Tasar silkworm Antheraea mylitta Drury

BackgroundTropical Tasar silkworm Antheraea mylitta Drury plays a very crucial role in the economic development of the country employing around 9.76 million people in rural and semi-urban areas. Since the population of this species has spread over many states with varying environmental factors, the population has been isolated into many pockets, specifically adapted to that niche, and are called as “ecoraces”. Even though phenotypically these ecoraces may look similar to some level but the intra-generic genotypic variability between them is correlated to the economically important yield traits of their cocoons. The purpose of our study was to evaluate the effectiveness of Cytochrome b (Cyt b) gene of mitochondrial genome as a potential DNA barcoding marker for identification of these ecoraces as to are they really ecoraces, and attempts to establish the first country report of DNA barcode reference library of this silkworm species. Methods and resultsDNA was extracted from pupae of pure ecoraces collected from core ecozones. PCR amplicon was bead purified and further subjected to Sanger Sequencing. The parsimony tree was also created using the MEGA 11 software. The genetic distance, gene flow and relation among the ecoraces was evaluated. The conserved regions of Cyt b gene were identified along with the documentation of the mode and tempo of nucleotide variation and evolution. ConclusionThe present study confirms the efficiency of Cyt b gene as a potential DNA marker for identification of ecoraces of Tasar silkworm and is the first country report on barcoding of A. mylitta Drury. Apart from being used as an early-warning system for ecological degradation, this knowledge will help in assessing the evolutionary relationship of these economically important ecoraces and identify their position in the process of evolution aiding in the assessment of conservation priorities.