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The heterogeneity and separability for individual taxa of ITS2 based on 45 Physalis species by TaxonGap. The left side shows the complete list of Physalis species used in this study. The right side depicts the within species heterogeneity (presented as light gray horizontal bar) and between-species separability (presented as dark gray horizontal bar) values with different OTUs as matrix rows for ITS2. The names of the closest relatives (the taxon with the smallest separability) are listed at the right side of the dark gray bar.
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Recently, commercial interest in Physalis species has grown worldwide due to their high nutritional value, edible fruit, and potential medicinal properties. However, many Physalis species have similar shapes and are easily confused, and consequently the phylogenetic relationships between Physalis species are poorly understood. This hinders their sa...
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... The dataset was thoroughly reviewed and any positions with gaps or missing data were excluded. The K2P model was utilized to quantify interspecific divergences, with the average and minimum interspecific distances as well as Theta prime serving as representative measures [21,38]. Intraspecific variation was assessed through the calculation of average intraspecific distance, coalescent depth, and theta [21,38]. ...
... The K2P model was utilized to quantify interspecific divergences, with the average and minimum interspecific distances as well as Theta prime serving as representative measures [21,38]. Intraspecific variation was assessed through the calculation of average intraspecific distance, coalescent depth, and theta [21,38]. We compared the differences in variability within and between species by analyzing DNA barcoding gaps [21,38]. ...
... Intraspecific variation was assessed through the calculation of average intraspecific distance, coalescent depth, and theta [21,38]. We compared the differences in variability within and between species by analyzing DNA barcoding gaps [21,38]. Paired Wilcoxon signed-rank tests were conducted as previously described [21]. ...
Numerous Cymbidium species have significant commercial value globally due to their exotic ornamental flowers. Identifying Cymbidium species is challenging due to their similar shapes, which hinders their rational use and the conservation of germplasm resources. In the present study, firstly, four plastid loci (matK, rbcL, psbA-trnH, and atpF-atpH) and a nuclear locus (internal transcribed spacer, ITS) were initially examined to identify Cymbidium species. Secondly, we inferred the interspecific phylogeny of Cymbidium species using ITS sequences. All of these DNA regions, with the exception of atpF-atpH, could be readily amplified from Cymbidium, and the corresponding DNA sequences can be successfully obtained by sequencing. Our research demonstrated that ITS exhibited the highest intra- and interspecific divergences, the greatest barcoding gap, and the highest proportion of species identification. The phylogenetic analysis of Cymbidium species based on the ITS regions primarily corroborated the results obtained using traditional morphological methods. A comparative analysis of candidate DNA barcodes has shown that the ITS can be used not only for barcoding Cymbidium species but also for the phylogenetic analysis of Cymbidium.
... According to these standards mentioned above, the Consortium for the Barcode of Life (CBOL) recommended two chloroplast loci as the central DNA barcodes in plants: the region of the maturase K gene (matK) and the ribulosebisphosphate carboxylase gene (rbcL) (CBOL Plant Working Group et al., 2009). Additionally, The ITS2 region was selected as a barcode candidate because the sequences generated with this region have been widely used for phylogenetic reconstructions at the genus and species levels (Schultz & Wolf, 2009;Keller et al., 2010;Marghali et al., 2015;Feng et al., 2016). Chen et al., (2010) validated and proposed the inclusion of the ITS2 region as a complementary region in DNA barcode studies as it seems to be essential in the identification of species. ...
... Chen et al. (2010) found that using ITS2 as a barcoding region could identify medicinal plants accurately with >90% success for species-level identification (6600 specimens belonging to 4800 plant species). Similarly, several studies testing the efficacy of ITS2 confirmed its ability to identify plant species in a variety of families including dicotyledons, monocotyledons, gymnosperms, ferns, and mosses (Gao et al. 2010;Pang et al. 2011;Gu et al. 2013;Feng et al. 2016). Although trnL region is frequently used for metabarcoding diet analysis, some studies have found that it was limited in differentiating congeners, which limited prey/food item identification to the genus or family level (Scasta et al. 2019;Tosa et al. 2023). ...
Metabarcoding-based diet analysis is a valuable tool for understanding the feeding behavior of a wide range of species. However, many studies using these methods for wild animals assume accuracy and precision without experimental evaluation with known positive control food items. Here, we conducted a feeding trial experiment with a positive control community in pasture-raised chickens and assessed the efficacy of several commonly used DNA extraction kits and primer sets. We hand-fed 22 known food items, including insects and plants, to six backyard laying hens and collected their excreta for eight h. We evaluated the efficacy of three DNA extraction kits, three primer sets for plant identification (targeting rbcL, trnL, and internal transcribed spacer 2 [ITS2]), and three primer sets for arthropod identification (targeting cytochrome oxidase subunit I [COI]). The detection success rate of our positive control food items was highly variable, ranging from 2.04% to 93.88% for all kit/primer combinations and averaging 37.35% and 43.57% for the most effective kit/primer combination for plants and insects, respectively. Extraction kits using bead-based homogenization positively affected the recovery proportion of plant and insect DNA in excreta samples. The minimum time to detect known food items was 44 min post-feeding. Two COI primer sets significantly outperformed the third, and both recovery proportion and taxonomic resolution from ITS2 were significantly higher than those from rbcL and trnL. Taken together, these results display the potential variability that can be inherently present in DNA-based diet analyses and highlight the utility of experimental feeding trials in validating such approaches, particularly for omnivores with diverse diets.
... DNA barcoding, which utilizes the ribosomal DNA ITS2 region as a tag to identify species, has gained a lot of attention recently [34]. ITS2 has several benefits including strong universality, low intraspecific variance but significant interspecific divergence, and a short fragment length (200 bp) over other suggested DNA barcodes such as psbA-trnH, matK, rbcL, and ITS [35][36][37]. DNA barcoding technology is used to identify the known as well as unknown species of berry fruit products. ...
Background. DNA barcoding is a useful technique for the identification, conservation, and diversity estimation at the species level in plants. e current research work was carried out to characterize selected Fragaria species from northern Pakistan using DNA barcode markers. Methodology. Initially, the efficacy of eight DNA barcode markers was analyzed based on the amplification and sequencing of the genome of selected Fragaria species. e resultant sequences were analyzed using BLAST, MEGA 7.0, and Bio Edit software. e phylogenetic tree was constructed by using Fragaria current species sequences and reference sequences through the neighbor-joining method or maximum likelihood method. Results. Among eight DNA barcode markers, only two (ITS2 and rbclC) were amplified, and sequences were obtained. ITS2 sequence was BLAST in NCBI for related reference species which ranged from 89.79% to 90.05% along with Fragaria vesca (AF163517.1) which have 99.05% identity. Similarly, the rbclC sequence of Fragaria species was ranged from 96% to 99.58% along with Fragaria × ananassa (KY358226.1) which had 99.58% identity. Conclusion. It is recommended that DNA barcode markers are a useful tool to identify the genetic diversity of a species. Moreover, this study could be helpful for the identification of the Fragaria species cultivated in other regions of the world.
... In addition, Physalis ixocarpa, commonly referred to as tomatillo, is a source of nutrients used in the preparation of sauces and salads [18]. Due to the wide diversity of Physalis species and species-specific applications, there is a need to authenticate and discriminate the different Physalis species in particular regions for efficient utilization, genetic resource conservation, and effective utilization in breeding programs [19]. ...
... The identification of Physalis species using morphological properties has resulted in misidentifications due to similarities in the phenotypic characteristics of the different species [19]. For example, Physalis minima and Physalis pubescens are morphologically similar, which presents a challenge in their differentiation using their phenotypic characteristics [19]. ...
... The identification of Physalis species using morphological properties has resulted in misidentifications due to similarities in the phenotypic characteristics of the different species [19]. For example, Physalis minima and Physalis pubescens are morphologically similar, which presents a challenge in their differentiation using their phenotypic characteristics [19]. Morphological identification is also affected by the environmental/physiological factors, stage of growth and development of plants [20,21]. ...
Plants of the genus Physalis are of economic interest because of their fleshy edible fruits with high nutritional value. Some species have high medicinal value with a long history of ethno-medicinal use to treat diverse diseases. There is therefore a need to correctly discriminate the different species of Physalis for proper utilization. Although most Physalis species have unique morphologies, their vegetative stages are identical, making it difficult to accurately identify them based on morphological characteristics. DNA barcoding has the potential to discriminate species accurately. In this study, ribulose bisphosphate carboxylase large (rbcL) and internal transcribed spacer 2 (ITS2) regions were used to discriminate Physalis species and to reveal their phylogenetic relationships and genetic diversity. Physalis plant samples were collected from seven counties in Kenya based on the availability of the germplasm. The voucher specimens were identified using the botanical taxonomy method and were deposited in the University of Nairobi herbarium. Genomic DNA was isolated from leaf samples of 64 Physalis accessions and used for PCR amplification and the sequencing of rbcL and ITS2 barcode regions. The discriminatory ability of the barcodes was based on BLASTn comparison, phylogenetic reconstruction and cluster analysis, and the determination of inter- and intra-specific distances. The nucleotide polymorphism, genetic diversity and distance of the identified Physalis species were determined using DnaSP and MEGA 11.0 software. Species discrimination was more robust using ITS2 sequences. The species identified and discriminated by ITS2 sequences were Physalis purpurea, Physalis peruviana and Physalis cordata. The rbcL sequences were only able to identify Physalis to the genus level. There was high interspecific and low intraspecific divergence within the identified Physalis species based on ITS2 sequences. The ITS2 barcode is an ideal DNA barcode for use in the discrimination of species, as well as in genetic diversity studies of Physalis accessions in Kenya.
... One such underutilized wild plant species is of the genus Physalis and belongs to the Nightshade (Solanaceae) family [1]. Physalis species are native to the Peruvian and Ecuadorian Andes region of South America; hence, it is referred to as the Peruvian gooseberry [2], although some are also native to Southeast Asia and Eurasia [3]. Studies of Physalis in China have identified five species including Physalis alkekengi, P. angulata, P. pubescens, P. peruviana, and P. minima and two variants of P. alkekengi [4]. ...
... The high conservation of the rbcL barcode gene in Physalis accessions makes it a less ideal candidate for DNA barcoding when compared to other barcode genes such as ITS2 [43]. The ability of ITS2 to emerge as a better barcode than rbcL is clearly supported in other studies on Physalis and other plants [2,13,41]. ...
... Commercial interest in plants of the genus Physalis has been rising worldwide due to its nutritional value, edible fruits, and the current and potential medicinal uses [2]. The Physalis accessions were investigated for mineral content and the analysis revealed that P. purpurea fruits are rich in potassium, sodium, calcium, and magnesium, which concurs with reports from other studies on the wild edible fruits of Physalis [49]. ...
Physalis species are used as an indigenous food and medicine in Kenya. However, species identification and an analysis of the health-promoting bioactive compounds and antioxidant properties are lacking. In this study, we report the molecular identification and mineral and phytochemical profiling of wild Physalis accessions. Leaf samples of 10 Physalis accessions were collected and used for species identification using nuclear ITS2 and plastid rbcL barcodes. Ripe fruits were collected from the same accessions and analyzed for mineral, total phenolic, tannin, and flavonoid contents, and antioxidant activities. The Physalis species were discriminated based on the ITS2 barcode and identified as Physalis purpurea. The genetic diversity, distance, and polymorphism of the ITS2 region of Physalis accessions were high due to the high rate of singleton and parsimony mutations. No genetic diversity, distance, or polymorphism was observed based on the rbcL barcode. The mineral content was significantly different (p < 0.05) for calcium, zinc, nickel, copper, and lithium among the Physalis accessions. No significant variation (p > 0.05) was found for phenolic acids or flavonoids, but the tannic acid content varied significantly (p < 0.05). DPPH free radical scavenging varied significantly (p < 0.05) among Physalis accessions. In conclusion, nuclear ITS2 was used to successfully identify the Physalis species of all the accessions as Physalis purpurea. The present study confirmed that Physalis purpurea has a significantly high mineral and phytochemical content and antioxidant activity. The findings from this study can be used to facilitate exploitation of Physalis purpurea in genetic breeding, their application in pharmaceutical, cosmetic, and nutritional value as well as conservation and sustainable use.
... Due to the high percentage of substitution of nucleotide, reasonably easier amplification, and availability of a greater amount of sequence, the interior transcribed spacer (ITS) regions of the nuclear ribosomal cistron (18S-5.8S-26S) regions of flowering plants are useful for species identification across flowering plants [106]. Nuclear barcodes are useful in identifying distinct allelic variations in a sample, and as a result, they are frequently utilized in cases of new hybridization or continuing introgression. ...
Nature contains a huge array of unique phytomolecules, many of which have previously evolved into lead compounds and have been transformed into herbal formulations or are currently undergoing clinical studies. Plant-based pharmaceuticals account for 25% of all drugs on the market, either directly or indirectly. The herbal drug sector has gotten a lot of attention in recent years, which has resulted in its exponential rise. People increasingly prefer herbal treatments to synthetic drugs, highlighting their safety and efficacy. Standardization of these herbal medications has become a significant component of the process involved in herbal drug development, not just for Asians but also for westerners, due to increased interest. The role of various analytical methods, such as chromatographic methods, spectroscopic methods, metabolomics, DNA barcoding, and so on, has been explored in this chapter. These methods aid in not only authentication but also quality assurance during the herbal medicine research process.
... Each helix had stem-ring structures of different sizes and numbers, like M. oleifera ( Figure 12). Previous reports highlighted that the ITS2 region and its secondary structure were conserved, which provided molecular and morphological characteristics that could be used for further improvements in species resolution (28), (9). The present study revealed that the ITS2 secondary structures from all the tea products had helices and motifs similar to those of the M. oleifera plant. ...
Adulteration is a severe issue affecting the herbal industry. Therefore, a robust tool is needed to address this problem. In the current study, Moringabased products (tea) authentication was conducted using DNA barcoding. Two different barcode regions, rbcL and ITS2, were investigated in terms of their effectiveness in authenticating the herbal products. To proceed with the DNA barcoding, a good quality of gDNA is a prerequisite for PCR amplification. Hence, two lysis buffers, PL1 and PL2, were compared to obtain good quality gDNA. Results revealed that a higher gDNA yield was obtained from the fresh plants using PL2, but a lower gDNA yield was obtained for the tea products except for sample P3. The PCR reaction was successfully conducted by amplifying two different barcodes, rbcL and ITS2. The amplicon size for the fresh plant was 643 bp for rbcL and 404 bp for ITS2. In contrast, the generated rbcL amplicon sizes for herbal tea products were 672 bp for P1, 679 bp for P2, 679 bp for P3, and 674 bp for P4. For rbcL and ITS2 amplicon sizes were 395 bp for P1, 406 bp for P2, 398 bp for P3, and 413 bp for P4. The amplified PCR products were analyzed using bioinformatic tools. The neighbour-joining (NJ) analysis for the rbcL barcode indicated that the P2, P3, and P4 tea products were categorized in the same clade with the M. oleifera sequence obtained from GenBank. Simultaneously, P1 was clustered individually with other closely related species. The analysis for the ITS2 barcode showed that all samples were grouped in the same clade. Incorporating secondary structure prediction after NJ analysis improved the discrimination between species.
... Cutleaf groundcherry (Physalis angulata L.) is an important annual herbaceous plant from the Solanaceae family, mainly distributed in China, Japan, India, Australia, and the Americas [1,2]. P. angulata has high potential medicinal value and a very long history of being used in traditional medicines around the world. ...
... In total, 203 samples of wild P. angulata representing 16 populations were randomly collected from their main areas of distribution in China (Table 8 Fresh, young leaf tissues from three individuals of each sample were randomly collected for genomic DNA isolation. Genomic DNA was isolated from these samples as described in our previous studies [1,48]. The DNA quality was evaluated using 1.0% agarose gel electrophoresis, and the DNA quantity was determined using a UV spectrophotometer. ...
Cutleaf groundcherry (Physalis angulata L.), an annual plant containing a variety of active ingredients, has great medicinal value. However, studies on the genetic diversity and population structure of P. angulata are limited. In this study, we developed chloroplast microsatellite (cpSSR) markers and applied them to evaluate the genetic diversity and population structure of P. angulata. A total of 57 cpSSRs were identified from the chloroplast genome of P. angulata. Among all cpSSR loci, mononucleotide markers were the most abundant (68.24%), followed by tetranucleotide (12.28%), dinucleotide (10.53%), and trinucleotide (8.77%) markers. In total, 30 newly developed cpSSR markers with rich polymorphism and good stability were selected for further genetic diversity and population structure analyses. These cpSSRs amplified a total of 156 alleles, 132 (84.62%) of which were polymorphic. The percentage of polymorphic alleles and the average polymorphic information content (PIC) value of the cpSSRs were 81.29% and 0.830, respectively. Population genetic diversity analysis indicated that the average observed number of alleles (Na), number of effective alleles (He), Nei’s gene diversity (h), and Shannon information indices (I) of 16 P. angulata populations were 1.3161, 1.1754, 0.1023, and 0.1538, respectively. Moreover, unweighted group arithmetic mean, neighbor-joining, principal coordinate, and STRUCTURE analyses indicated that 203 P. angulata individuals from 16 populations were grouped into four clusters. A molecular variance analysis (AMOVA) illustrated the considerable genetic variation among populations, while the gene flow (Nm) value (0.2324) indicated a low level of gene flow among populations. Our study not only provided a batch of efficient genetic markers for research on P. angulata but also laid an important foundation for the protection and genetic breeding of P. angulata resources.
... In previous studies, internal transcribed spacer (ITS) region has been used not only for the phylogenetic and species relationship studies, but also for recognition of intraspecific variation among the different populations (Goel et al. 2002;Álvarez and Wendel 2003;Saini and Jawali 2009;Gao et al. 2010;Yu et al. 2017;Qin et al. 2017;Mania et al. 2019). Furthermore, the secondary structure of the ITS2 region has also been more effectively utilized in species authentication (Grajales et al. 2007;Chen et al. 2010;Koetschan et al. 2012;Feng et al. 2016;Umdale et al. 2019). ...
For the study of genetic diversity at the molecular level and species relationships among plant genera, internal transcribed spacer (ITS) sections, particularly ITS2 of nuclear DNA, are recommended. Based on ITS sequences and their secondary structures, we looked at the species relationships and intraspecific variations among 221 different germplasm representing crop wild relatives (CWR) of Asian Vigna species (subgenus Ceratotropis; genus Vigna) in India. The distinctness of various Asian Vigna species was supported by the significant nucleotide and haplotype diversity among populations and the maximum parsimony tree based on ITS2 barcode. The nucleotide sequences obtained from ITS2 data of Asian Vigna species showed remarkable variations in nucleotide composition and sequence length. A total of forty haplotypes were found within the 221 diverse accessions of Vigna species. Our findings suggest that ITS2 secondary structure analysis and haplotype diversity is an effective and trustworthy method for examining the genetic diversity of Asian Vigna species. Additionally, the secondary structures of ITS2 offered a different molecular standard for defining species in Asian Vigna. The interspecific variation and species relationships of the Asian Vigna species are better understood as a result of the current study, which opens up new possibilities for the management and conservation of CWR.
