trnL Gene Research Articles

Modulating the gut microbiota in Crohn’s disease: a pilot study on the impact of a plant-based diet with DNA-based monitoring

IntroductionCrohn’s Disease (CD) is characterized by chronic intestinal inflammation and dysbiosis. This study aimed to investigate the effects of a plant-based diet (PBD) on gut microbiota composition and inflammation in CD patients and assess the utility of trnL gene sequencing for monitoring dietary adherence.MethodsFourteen CD patients participated in a 12-week PBD intervention. Dietary adherence was monitored through self-reported food diaries and trnL sequencing, which detects plant residues in fecal samples. Gut microbiota was analyzed using 16S rRNA sequencing, and fecal calprotectin levels were measured as an indicator of intestinal inflammation.ResultsTrnL sequencing identified 55 plant genera in fecal samples, compared to 41 reported in food diaries, highlighting its accuracy in assessing plant residue diversity. By week 4, participants demonstrated a 1.4-fold increase in plant intake, correlating with a significant increase in microbial diversity. Key genera associated with gut health, such as Faecalibacterium and Bacteroides, increased in abundance. Additionally, fecal calprotectin levels decreased from 472 mg/kg at baseline to 207 mg/kg at week 12, indicating reduced intestinal inflammation.DiscussionA PBD positively influenced gut microbiota composition and decreased intestinal inflammation in CD patients. The study also demonstrated that trnL sequencing is an effective tool for assessing dietary adherence in clinical settings, offering a more objective measure than self-reported food diaries.

DNA Metabarcoding Approach as a Potential Tool for Supporting Official Food Control Programs: A Case Study.

Food authentication significantly impacts consumer health and the credibility of Food Business Operators (FBOs). As European regulations mandate the verification of food authenticity and supply chain integrity, competent authorities require access to innovative analytical methods to identify and prevent food fraud. This study utilizes the DNA metabarcoding approach on meat preparations, sampled during an official control activity. It assesses animal and plant composition by amplifying DNA fragments of the 12S rRNA and trnL (UAA) genes, respectively. The results not only confirmed the declared species but also revealed undeclared and unexpected taxa in products labelled as containing a single animal species and various unspecified plant species. Notable findings such as the presence of Murinae, Sus scrofa, Ovis aries, and Pisum sativum could raise public health concerns, compromise consumer choices made for ethical or religious reasons, and reflect the hygienic conditions of the processing plant. This study demonstrates that the DNA metabarcoding approach looks to be a promising support tool for official control authorities to ensure food authenticity and safety, and to develop risk profiles along the supply chain.

Comparison of Metabarcoding Techniques for Dietary Assessment in Herbivores and Omnivores

Dietary assessment plays a crucial role in comprehending the ecological dynamics and nutritional needs of herbivores and omnivores. The metabarcoding technique has emerged as a potent tool for exploring the dietary composition of these animals. However, various metabarcoding techniques have been developed, each with its own advantages and limitations. This study aims to compare the performance of different metabarcoding techniques in herbivores and omnivores diet. We systematically reviewed 159 published manuscripts in Scopus and Google Scholar, and thematic analysis was conducted across several categories, including the marker, platform, and database utilized. Preliminary findings reveal significant variations among metabarcoding techniques across these two animal groups. The trnL gene exhibited higher taxonomic resolution for herbivorous species, whereas the combination of the 'trnL + 16s rRNA' gene exhibited superior performance for omnivorous species. The Illumina platform emerged as the most commonly used method for analyzing the diets of both herbivores and omnivores, with the primary reference database being the National Centre for Biotechnology Information (NCBI). This study offers valuable insights into the strengths and limitations of different metabarcoding techniques for dietary assessment in herbivores and omnivores and optimizing metabarcoding protocols, facilitating more precise and reliable diet analyses within these ecological groups.

USE OF THE rpl32-trnL REGION OF THE CHLOROPLAST GENOME IN THE MOLECULAR TAXONOMY OF HERACLEUM SPECIES

In recent decades, the invasion of alien plant species has acquired the status of one of the greatest threats to biodiversity. Invasive plants can not only displace native species but also transform ecosystems, which leads to more global negative consequences. An additional danger is the hybridization of invasive species with closely related native plants, which can contribute to the emergence of more aggressive invasive forms. The territory of Ukraine is also actively colonized by alien plants, among which the group of giant borschts (Heracleum, Apiaceae) attracts special attention. In addition to the impact on ecosystems, these plants are dangerous for people, because they can cause photochemical burns when in contact with the skin. For one of the species of this group, namely H. mantegazzianum Sommier & Levier, the possibility of hybridization with the aboriginal European species H. sphondylium L. However, both parental species and potential hybrid forms from the territory of Ukraine remain unexplored using molecular genetic methods. In this work, for the first time, we amplified and sequenced the spacer region of the chloroplast genome between the rpl32 and trnL genes for samples of H. mantegazzianum and H. sphondylium. The obtained sequences were compared with each other and with rpl32-trnL sequences available in the GenBank database for other species of the genus Heracleum. Phylogenetic analysis showed that the use of the rpl32-trnL region allows dividing all species of the genus into two main groups, one of which includes H. mantegazzianum and H. sphondylium Thus, the rpl32-trnL site can be successfully used for molecular identification of Ukrainian representatives of the genus Heracleum, as well as for determining the direction of hybridization for potential hybrid forms between the invasive species H. mantegazzianum and the native representative of the Ukrainian flora H. sphondylium. Key words: bioinformatic analysis, genetic polymorphism, molecular markers, molecular genomics, rpl32-trnL intergenic spacer, interspecific hybridization, Heracleum, Apiaceae.

Characterization of the Complete Mitochondrial Genome of Agelas nakamurai from the South China Sea.

The Agelas genus sponges are widely distributed and provide shelter for organisms that inhabit reefs. However, there is a lack of research on the genetic diversity of the Agelas sponges. Additionally, only one Agelas mitochondrial genome has been documented, leaving the characteristics of the Agelas genus's mitogenome in need of further clarification. To address this research gap, we utilized Illumina HiSeq4000 sequencing and de novo assembly to ascertain the complete mitochondrial genome of Agelas sp. specimens, sourced from the South China Sea. Our analysis of the cox1 barcoding similarity and phylogenetic relationship reveals that taxonomically, the Agelas sp. corresponds to Agelas nakamurai. The mitogenome of Agelas nakamurai is 20,885 bp in length, encoding 14 protein-coding genes, 24 transfer RNA genes, and 2 ribosomal RNA genes. Through a comparison of the mitochondrial genes, we discovered that both Agelas nakamurai and Agelas schmidti have an identical gene arrangement. Furthermore, we observed a deletion in the trnD gene and duplication and remodeling of the trnL gene in the Agelas nakamurai's mitogenome. Our evolutionary analysis also identified lineage-specific positive selection sites in the nad3 and nad5 genes of the Agelas sponges' mitogenome. These findings shed light on the gene rearrangement events and positive selection sites in the mitogenome of Agelas nakamurai, providing valuable molecular insights into the evolutionary processes of this genus.

Mitogenomes of Eight Nymphalidae Butterfly Species and Reconstructed Phylogeny of Nymphalidae (Nymphalidae: Lepidoptera).

The Nymphalidae family of cosmopolitan butterflies (Lepidoptera) comprises approximately 7200 species found on all continents and in all habitats. However, debate persists regarding the phylogenetic relationships within this family. In this study, we assembled and annotated eight mitogenomes of Nymphalidae, constituting the first report of complete mitogenomes for this family. Comparative analysis of 105 mitochondrial genomes revealed that the gene compositions and orders were identical to the ancestral insect mitogenome, except for Callerebia polyphemus trnV being before trnL and Limenitis homeyeri having two trnL genes. The results regarding length variation, AT bias, and codon usage were consistent with previous reports on butterfly mitogenomes. Our analysis indicated that the subfamilies Limenitinae, Nymphalinae, Apaturinae, Satyrinae, Charaxinae, Heliconiinae, and Danainae are monophyletic, while the subfamily the subfamily Cyrestinae is polyphyletic. Danainae is the base of the phylogenetic tree. At the tribe level, Euthaliini in Limenitinae; Melitaeini and Kallimini in Nymphalinae; Pseudergolini in Cyrestinae; Mycalesini, Coenonymphini, Ypthimini, Satyrini, and Melanitini in Satyrinae; and Charaxini in Charaxinae are regarded as monophyletic groups. However, the tribe Lethini in Satyrinae is paraphyletic, while the tribes Limenitini and Neptini in Limenitinae, Nymphalini and Hypolimni in Nymphalinae, and Danaini and Euploeini in Danainae are polyphyletic. This study is the first to report the gene features and phylogenetic relationships of the Nymphalidae family based on mitogenome analysis, providing a foundation for future studies of population genetics and phylogenetic relationships within this family.

Metabarcoding of soil environmental DNA to estimate plant diversity globally.

Traditional approaches to collecting large-scale biodiversity data pose huge logistical and technical challenges. We aimed to assess how a comparatively simple method based on sequencing environmental DNA (eDNA) characterises global variation in plant diversity and community composition compared with data derived from traditional plant inventory methods. We sequenced a short fragment (P6 loop) of the chloroplast trnL intron from from 325 globally distributed soil samples and compared estimates of diversity and composition with those derived from traditional sources based on empirical (GBIF) or extrapolated plant distribution and diversity data. Large-scale plant diversity and community composition patterns revealed by sequencing eDNA were broadly in accordance with those derived from traditional sources. The success of the eDNA taxonomy assignment, and the overlap of taxon lists between eDNA and GBIF, was greatest at moderate to high latitudes of the northern hemisphere. On average, around half (mean: 51.5% SD 17.6) of local GBIF records were represented in eDNA databases at the species level, depending on the geographic region. eDNA trnL gene sequencing data accurately represent global patterns in plant diversity and composition and thus can provide a basis for large-scale vegetation studies. Important experimental considerations for plant eDNA studies include using a sampling volume and design to maximise the number of taxa detected and optimising the sequencing depth. However, increasing the coverage of reference sequence databases would yield the most significant improvements in the accuracy of taxonomic assignments made using the P6 loop of the trnL region.

The complete mitochondrial genome of Hyotissasinensis (Bivalvia, Ostreoidea) indicates the genetic diversity within Gryphaeidae.

Different from the true oyster (family Ostreidae), the molecular diversity of the gryphaeid oyster (family Gryphaeidae) has never been sufficiently investigated. In the present study, the complete mitochondrial (mt) genome of Hyotissasinensis was sequenced and compared with those of other ostreoids. The total length of H.sinensis mtDNA is 30,385 bp, encoding 12 protein-coding-genes (PCGs), 26 transfer RNA (tRNA) genes and two ribosomal RNA (rRNA) genes. The nucleotide composition and codon usage preference of H.sinensis mtDNA is similar to that of H.hyotis within the same genus. On the other hand, the presence of three trnM and three trnL genes of H.sinensis was not detected neither in H.hyotis nor other ostroid species. Another unique character of H.sinensis mtDNA is that both rrnS and rrnL have a nearly identical duplication. The PCG order of H.sinensis is identical to H.hyotis and the two congener species also share an identical block of 12 tRNA genes. The tRNA rearrangements mostly happen in the region from Cox1 to Nad3, the same area where the duplicated genes are located. The rearrangements within Gryphaeidae could be explained by a "repeat-random loss model". Phylogenetic analyses revealed Gryphaeidae formed by H.sinensis + H.hyotis as sister to Ostreidae, whereas the phylogenetic relationship within the latter group remains unresolved. The present study indicated the mitogenomic diversity within Gryphaeidae and could also provide important data for future better understanding the gene order rearrangements within superfamily Ostreoidea.

Flavonoid-producing tomato plants have a direct negative effect on the zoophytophagous biological control agent Orius sauteri.

Orius sauteri (Poppius) (Hemiptera: Anthocoridae) is often used for biological control of small arthropod pests in greenhouse vegetable production systems in Asia. In addition to feeding on arthropod prey, O. sauteri consumes small quantities of plant material. Previous studies demonstrated that tomato plant chemistry confers antixenosis resistance to phloem-feeding whiteflies, but the potential nontarget effects of phytochemicals on the beneficial predator O. sauteri are unknown. Comparison of O. sauteri confined to near-isogenic lines (NILs) of tomatoes producing high levels of flavonoids (NIL-purple hypocotyl; resistant to whiteflies) and low levels of flavonoids (NIL-green hypocotyl; susceptible to whiteflies) revealed that O. sauteri had reduced oviposition, nymphal survival, and development on resistant plants, even if they were also provided with prey that did not feed on the host plant. Moreover, O. sauteri showed a significant ovipositional preference in choice assays, laying significantly more eggs on susceptible than on resistant plants. Molecular gut content analysis using the specific chloroplast trnL gene from tomato confirmed that adult and immature O. sauteri feed on both resistant and susceptible genotypes, and feeding behavior assays revealed that resistance did not affect plant feeding or prey acceptance by O. sauteri adults. These results demonstrate a direct negative effect of phytochemicals on a nontarget beneficial species and indicate that resistance mediated by phytochemicals can affect organisms that do not solely feed on phloem sap. The results also indicate that the mode of action and the potential ecological effects of phytochemical-mediated resistance are broader than previously recognized.

DNA metabarcoding uncovers the diet of subterranean rodents in China.

A type of rodent called a zokor causes great harm to agriculture and forestry production due to its large and sophisticated diet. As this rodent lives subterrane for most of its life, researchers know little about its dietary habits. Further understanding of its diet is important for developing green and sustainable control strategies for the zokor. Chloroplast trnL gene and internal transcription spacer 1 primers were selected for high-throughput sequencing of stomach contents of captured zokor by DNA metabarcoding. A total of 25 zokors were selected, the food list of 32 families, 80 genera, and 154 species was obtained. At the family level, it was found that zokors mainly fed on Asteraceae, Poaceae, Rosaceae, Pinaceae, Brassicaceae, and Apiaceae. At the genus level, zokors mainly fed on Echinops, Littledalea, Artemisia, Picea, Cirsium, and Elymus. The diet alpha diversity of Eospalax cansus was slightly higher than that of Eospalax cansus (P>0.05). The zokor's diet is highly phconsistent with the resources of its habitat. Most food choices tend to be the same between the two zokors. They fed primarily on Calamagrostis, Cirsium, Echinops, Medicago, Sanguisorba, and Taraxacum. Zokors mainly fed on the roots of perennial herbs, which are important source of energy. High-throughput sequencing-based DNA metabarcoding technology has effectively revealed the diet of zokors and indicated that zokors are food generalists.

The Phylogeography of Rhizophora in Peninsular Malaysia: High Genetic Variation between West and East

Geographic history of Rhizophora species and hybrid is effect on their genetic structure. In addition, determining population genetic variation is essential for phylogeography research. For this phylogeography investigation, Four natural population of Rhizophora were collected from five different regions in peninsular Malaysia for this phylogeography study. This study aims to evaluate the genetic diversity of Rhizophora species namely Rhizophora apiculata , Rhizophora mucronata , Rhizophora stylosa and hybrid R. × lamarcki and clarify their genetic structure of its populations using Nucleotide polymorphism in chloroplast intergenic spacer between trnL and trnF genes was studied.The populations of Rhizophora species and hybrids were divided into two groups: the east coast populations, which were represented by samples collected from Mersing, Tanjang Piai, and Sedili; and the west coast populations, which were represented by samples collected from Mersing, Tanjang Piai, and Sedili. The population of the west coast was the second cluster. This result was supported by UPGMA analysis using MEGA5 software.

Fast DNA biosensing based on isothermal amplification, unmodified gold nanoparticles, and smartphone detection

The protection of the consumer against foodborne illnesses and fraudulent practices requires methods capable of detecting critical components. Classical instrumental and DNA-based technologies have assay time, portability, and cost limitations. Thus, food control needs alternative methodologies for massive and cost-effective screening. Herein, we report an instrument-free method based on detecting genes that encode proteins related to food allergies and ingredients associated with illegal practices. Tailed recombinase polymerase amplification (tailed-RPA) provided the selective isothermal amplification of target regions. A reproducible and fast optical detection was developed based on the salt-induced aggregation of 15 nm gold nanoparticles (AuNPs). The target presence was directly observed (naked-eye detection) and quantified using a smartphone and RGB image decomposition. The advantages arise from the effective formation of a hybrid complex between non-functionalized nanoparticles and amplification products with a single-strand tail, featuring short incubation time, simplicity, and low sample and reagent volumes. As proof of concept, two targets were determined: trnL gene and ITS region. The first sequence is located in the chloroplast genome of cereals and is valuable to control the adulteration of meat products. The second is a fragment common to the three main gluten-containing cereals, applicable to the indirect detection of this allergen. The novelty also is the integration of a low-cost assay platform and a straightforward interpretation system to foster its implementation in the industry. The RPA-AuNP assay offered a good sensitivity (genomic DNA 0.8 ng), selectivity (absence of unspecific response), reproducibility (standard deviation 2–11%), and accuracy in marketed food products (100%). Therefore, a competitive biosensing system enables better control according to food industry/consumers demands from Farm to Fork strategy.

Plant Sedimentary Ancient DNA From Far East Russia Covering the Last 28,000 Years Reveals Different Assembly Rules in Cold and Warm Climates

Woody plants are expanding into the Arctic in response to the warming climate. The impact on arctic plant communities is not well understood due to the limited knowledge about plant assembly rules. Records of past plant diversity over long time series are rare. Here, we applied sedimentary ancient DNA metabarcoding targeting the P6 loop of the chloroplast trnL gene to a sediment record from Lake Ilirney (central Chukotka, Far Eastern Russia) covering the last 28 thousand years. Our results show that forb-rich steppe-tundra and dwarf-shrub tundra dominated during the cold climate before 14 ka, while deciduous erect-shrub tundra was abundant during the warm period since 14 ka. Larix invasion during the late Holocene substantially lagged behind the likely warmest period between 10 and 6 ka, where the vegetation biomass could be highest. We reveal highest richness during 28–23 ka and a second richness peak during 13–9 ka, with both periods being accompanied by low relative abundance of shrubs. During the cold period before 14 ka, rich plant assemblages were phylogenetically clustered, suggesting low genetic divergence in the assemblages despite the great number of species. This probably originates from environmental filtering along with niche differentiation due to limited resources under harsh environmental conditions. In contrast, during the warmer period after 14 ka, rich plant assemblages were phylogenetically overdispersed. This results from a high number of species which were found to harbor high genetic divergence, likely originating from an erratic recruitment process in the course of warming. Some of our evidence may be of relevance for inferring future arctic plant assembly rules and diversity changes. By analogy to the past, we expect a lagged response of tree invasion. Plant richness might overshoot in the short term; in the long-term, however, the ongoing expansion of deciduous shrubs will eventually result in a phylogenetically more diverse community.

Complete chloroplast genome of novel Adinandra megaphylla Hu species: molecular structure, comparative and phylogenetic analysis

Adinandra megaphylla Hu is a medicinal plant belonging to the Adinandra genus, which is well-known for its potential health benefits due to its bioactive compounds. This study aimed to assemble and annotate the chloroplast genome of A. megaphylla as well as compare it with previously published cp genomes within the Adinandra genus. The chloroplast genome was reconstructed using de novo and reference-based assembly of paired-end reads generated by long-read sequencing of total genomic DNA. The size of the chloroplast genome was 156,298 bp, comprised a large single-copy (LSC) region of 85,688 bp, a small single-copy (SSC) region of 18,424 bp, and a pair of inverted repeats (IRa and IRb) of 26,093 bp each; and a total of 51 SSRs and 48 repeat structures were detected. The chloroplast genome includes a total of 131 functional genes, containing 86 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes. The A. megaphylla chloroplast genome indicated that gene content and structure are highly conserved. The phylogenetic reconstruction using complete cp sequences, matK and trnL genes from Pentaphylacaceae species exhibited a genetic relationship. Among them, matK sequence is a better candidate for phylogenetic resolution. This study is the first report for the chloroplast genome of the A. megaphylla.

Hidden below‐ground plant diversity buffers against species loss during land‐use change in species‐rich grasslands

AbstractQuestionsTheory suggests that while plant diversity of grasslands decreases in response to increased competition for light, many plant species persist below‐ground even in the temporary absence of shoots. Thus, we hypothesized that below‐ground, diversity is less affected by increased competition during land‐use change compared to above‐ground.LocationSpecies‐rich, oligotrophic grasslands in the Železné hory Mts, eastern Bohemia, Czech Republic.MethodsWe quantified above‐ and below‐ground plant richness (roots and rhizomes, seed and bud bank) in grasslands subjected to seven years of experimental abandonment and fertilization. We used conventional species identification to measure richness above‐ground and 454 sequencing of the chloroplast trnL (UAA) gene to measure richness of roots and rhizomes; additionally, we used the shoot emergence method to measure soil seed and bud bank richness.ResultsWhile above‐ground richness decreased with abandonment and fertilization, below‐ground richness remained less affected or even increased compared to the control (traditional mowing). Overall, compared to above‐ground the proportion of plant species found only below‐ground as roots and rhizomes increased, particularly in the abandonment with fertilization treatment.ConclusionsBelow‐ground plant diversity could potentially buffer against biodiversity losses during short‐term land‐use change. Our findings highlight that measuring the complete plant species diversity improves our understanding of processes that underlie plant diversity and coexistence and refine predictions of vegetation responses to biodiversity threats.

Detecting Ingested Host Plant DNA in Potato Leafhopper (Hemiptera: Cicadellidae): Potential Use of Molecular Markers for Gut Content Analysis.

Detection of host plant DNA from sap-feeding insects can be challenging due to potential low concentration of ingested plant DNA. Although a few previous studies have demonstrated the possibility of detecting various fragments of plant DNA from some sap-feeders, there are no protocols available for potato leafhopper, Empoasca fabae (Harris) (Hemiptera: Cicadellidae), a significant agricultural pest. In this study we focused on optimizing a DNA-based method for host plant identification of E. fabae and investigating the longevity of the ingested plant DNA as one of the potential applications of the protocol. We largely utilized and modified our previously developed PCR-based method for detecting host plant DNA from grasshopper and the spotted lanternfly gut contents. We have demonstrated that the trnL (UAA) gene can be successfully utilized for detecting ingested host plant DNA from E. fabae and determining plant DNA longevity. The developed protocol is a relatively quick and low-cost method for detecting plant DNA from E. fabae. It has a number of important applications-from determining host plants and dispersal of E. fabae to developing effective pest management strategies.

Contradictory between morphology and phylogenetic trees of Orthosiphon spp. (Lamiaceae) from Indonesia

The nuclear ribosomal Internal Transcribed Spacer region (ITS), and three chloroplast loci (trnL-trnF, rps16 and trnL) were also carried out in this study. Morphological analysis of leaves, stems, and flowers is used to identify visual differences. This study was the purpose of analyzed morphological and phylogenetic relationships among ten taxa of Orthosiphon from the analysis of DNA sequences. The results that the difference in leaves, flowers and stems is very striking in seven taxa of Orthosiphon aristatus (OGP and OGW) and O. endanghidayatae (OWW, OG + W and OW + W). The phylogenetic trees constructed from cpDNA (trnL gene, trnL-trnF intergenic spacer region, and rps16 region) of using NJ methods among taxa of Orthosiphon aristatus and O. endanghidayatae collected from Indonesia showed constant topologies with high bootstrap values (BS 86% and 100%, respectively), but were different from those ITS of nrDNA that the phylogenetic trees supported low bootstrap values (65% by NJ). Although differing from morphology to species of Orthosiphon spp. but still similar in DNA analysis. The results of this study indicate that the speciation process of O. endanghidayatae (OWW, OG + W and OW + W) is as a process of morphological changes faster than the process of genetic change.

Full plastome sequence of the fern Vandenboschia speciosa (Hymenophyllales): structural singularities and evolutionary insights.

We provide here the first full chloroplast genome sequence, i.e., the plastome, for a species belonging to the fern order Hymenophyllales. The phylogenetic position of this order within leptosporangiate ferns, together with the general scarcity of information about fern plastomes, places this research as a valuable study on the analysis of the diversity of plastomes throughout fern evolution. Gene content of V. speciosa plastome was similar to that in most ferns, although there were some characteristic gene losses and lineage-specific differences. In addition, an important number of genes required U to C RNA editing for proper protein translation and two genes showed start codons alternative to the canonical AUG (AUA). Concerning gene order, V. speciosa shared the specific 30-kb inversion of euphyllophytes plastomes and the 3.3-kb inversion of fern plastomes, keeping the ancestral gene order shared by eusporangiate and early leptosporangiate ferns. Conversely, V. speciosa has expanded IR regions comprising the rps7, rps12, ndhB and trnL genes in addition to rRNA and other tRNA genes, a condition shared with several eusporangiate ferns, lycophytes and hornworts, as well as most seed plants.

CpDNA Barcoding by Combined End-Point and Real-Time PCR Analyses to Identify and Quantify the Main Contaminants of Oregano (Origanum vulgare L.) in Commercial Batches

Oregano (Origanum vulgare L.) is a flowering plant that belongs to the mint family (Lamiaceae). It is used as a culinary herb and is often commercialized as a fine powder or a mixture of small fragments of dried leaves, which makes morphological recognition difficult. Like other commercial preparations of drugs and spices, the contamination of oregano mixtures with vegetable matter of lower quality, or the use of generic misleading names, are frequent and stress the need to develop a molecular traceability system to easily, quickly, and cheaply unveil these scams. The DNA-based analytical approach known as cpDNA barcoding is particularly suited for fraud identification in crop plant species (fresh products and food derivatives), and it represents a promising traceability tool as an alternative or complement to traditional detection methods. In the present study, we used a combined approach based on both qualitative and quantitative cpDNA barcoding with end-point and real-time polymerase chain reaction (PCR) analyses to assess the type and degree of contamination in commercial batches of common oregano. In a preliminary qualitative screening, we amplified, cloned, and sequenced a number of universal trnH-psbA- and trnL-barcoded regions, to identify the main contaminants in the samples under investigation. On the basis of these findings, we then developed and validated a species-specific and sequence-targeted method of testing for the quantitative assessment of contaminants, using trnL gene intron assays. Surprisingly, the results obtained in our case study indicated an almost total absence of O. vulgare in the commercial batches analyzed, but a high presence of group I contaminants (Satureja pilosa Velen.), and a moderate presence of group II contaminants (Cistus lanidifer L./Cistus albidus).

Efficient chloroplast transformation in Scoparia dulcis L. using pFaadAII vector

The plastid transformation is used for high level expression of certain metabolically and industrially important recombinant proteins in plants. The vector, pFaadAII, a tobacco based vector system, harbouring a chimeric gene consisting of aadA coding region from Escherichia coli with 5′ 16S rDNA promoter and 3′ untranslated transcript region (UTR) of chlamydomonas rbcL gene, located in between the intergenic regions of rp132 and trnL genes. This vector used for transformation of plastids targets the foreign sequences to the small single-copy region of the plastome. Biolistic mode of approach for chloroplast transformation in Scoparia dulcis L., was achieved by bombarding the leaf explants and spectinomycin based selection system was used for regeneration of transformed plants. Transplastomic lines have been successfully established with overall efficiency of two transgenic lines for twenty-five bombarded explants. Integration of aadA in selection based regenerants was characterized by PCR and protein accumulation analysis along with seedlings experiment obtained from selfing. The chloroplast transformation developed in this plant system will provide scope for research in plastid based metabolic engineering pathways.