rpoC1 Sequences Research Articles

THE ANALYSIS OF DNA BARCODING AND THE EVOLUTIONARY DIVERGENCE OF ANNONA BY ISSR MARKERS

The genus Annona includes important commercially grown tropical crops but also has been grown popularly in Vietnam due to its wide range of clinical applications. In this study, two DNA barcodes (rbcL and rpoC1) and two ISSR markers were used to assess the interspecies of eleven Annona samples collected from different provinces in the Mekong Delta. As a result, the rbcL marker was successfully amplified and sequenced for identifying Annona species, while rpoC1 sequences required extensive editing and could not distinguish between A. muricata, A. glabra, and A. squamosa samples in BLASTN analysis. The rbcL and rpoC1 sequences harbor 9 and 7 SNPs, respectively, and showed cross evolution properties of A. squamosal NDL-NB among three species. The phylogenetic analysis revealed that sample NDL-NB, previously recognized as A. squamosa morphologically, was grouped in the cluster of A. muricata based on rbcL sequences, whereas it was distinguished in the clusters of A. squamosal and A. glabra based on rpoC1 sequences. Among 33 bands generated from ISSR primers, 20 bands were polymorphic, accounting for 60.60%. Dendrogram analysis of ISSR data could classify seven amplified DNA samples into three genotypes that were not geographically dependent. In conclusion, the performance of the approved DNA barcodes and ISSR analysis appeared to be species-specific or genus-specific, especially indicating the evolutionary divergence of Annona species derived from a neighboring country and its adaptation to certain geographical locations.

DNA barcoding of Indian Alysicarpus (Fabaceae): ITS alone distinguishes species

In India, the genus Alysicarpus (Fabaceae) is represented by approximately 18 species and seven varieties. It includes some species of considerable economic importance, including two of therapeutic value. The species of this genus are difficult to identify because of overlapping morphological characters. Moreover, it becomes difficult to establish the botanical identity of a herbal medicine available in powdered/fragmented form. In the present study, DNA barcoding was used to identify 42 accessions belonging to 15 species of Alysicarpus and two varieties. The DNA regions that were tested as potential barcodes were matK (Maturase K), rbcL (Rubisco Large Sub-unit), rpoC1 (RNA Polymerase-β subunit, the main catalytic subunit) and nrITS (Nuclear Ribosomal Internal Transcribed Spacer). In BLAST search on National Centre for Biotechnology Information (NCBI), ITS and rpoC1 sequences best matched with the respective sequences of species of other genera belonging to the family Fabaceae with less than 100% similarity, as no corresponding sequences of any Alysicarpus species were available. The rbcL and matK sequences had first match, with the respective sequences of Alysicarpus species, however, with less than 100% similarity. Among the loci tested, ITS alone discriminated all species on the basis of genetic distance as well as phylogenetic tree methods.

Employing barcoding markers to authenticate selected endangered medicinal plants traded in Indian markets.

The high demand of medicinal plants and their unrestricted collection have rendered many of these as rare or endangered. The restrictions imposed on their collection and trade are difficult to implement because of the inability to identify them in fragmented form. The rarity of these plants in nature and lack of their cultivation raise doubt about the authenticity of the herbals sold in markets. Therefore, in the present investigation, ITS/ITS2, matK, rbcL and rpoC1 sequences of fourteen species of important medicinal plants, some of which are endangered, were generated and checked for their species-specificity (sequences having maximum similarity only with their own) by BLAST1 and/or BOLD identifications. ITS sequences of 12 species were species-specific. However, ITS2 of only 10 of these 12 species were species-specific. As for the chloroplast loci, rbcL and rpoC1 sequences of all 14 species could be obtained, while matK sequences of only 10 of these could be generated. Of the retrieved sequences, rbcL, rpoC1 and matK sequences of 7, 11 and 7 species, respectively, were species-specific. The sequences of the targeted loci from the herbal samples of these species were difficult to retrieve because of failure in the amplification or sequencing. Nevertheless, based on ITS2 and/or one or more of the chloroplast loci targeted, the botanical identities of 22 herbal market samples were checked by phylogenetic tree, BLAST1 and BOLD identification methods. Of these 22 samples, only one of each of Rauvolfia serpentina and Picrorhiza kurroa were found to be authentic.

Utility of DNA barcoding to identify rare endemic vascular plant species in Trinidad.

The islands of the Caribbean are considered to be a “biodiversity hotspot.” Collectively, a high level of endemism for several plant groups has been reported for this region. Biodiversity conservation should, in part, be informed by taxonomy, population status, and distribution of flora. One taxonomic impediment to species inventory and management is correct identification as conventional morphology‐based assessment is subject to several caveats. DNA barcoding can be a useful tool to quickly and accurately identify species and has the potential to prompt the discovery of new species. In this study, the ability of DNA barcoding to confirm the identities of 14 endangered endemic vascular plant species in Trinidad was assessed using three DNA barcodes (matK, rbcL, and rpoC1). Herbarium identifications were previously made for all species under study. matK, rbcL, and rpoC1 markers were successful in amplifying target regions for seven of the 14 species. rpoC1 sequences required extensive editing and were unusable. rbcL primers resulted in cleanest reads, however, matK appeared to be superior to rbcL based on a number of parameters assessed including level of DNA polymorphism in the sequences, genetic distance, reference library coverage based on BLASTN statistics, direct sequence comparisons within “best match” and “best close match” criteria, and finally, degree of clustering with moderate to strong bootstrap support (>60%) in neighbor‐joining tree‐based comparisons. The performance of both markers seemed to be species‐specific based on the parameters examined. Overall, the Trinidad sequences were accurately identified to the genus level for all endemic plant species successfully amplified and sequenced using both matK and rbcL markers. DNA barcoding can contribute to taxonomic and biodiversity research and will complement efforts to select taxa for various molecular ecology and population genetics studies.

Distribution of Prochlorococcus Ecotypes in the Red Sea Basin Based on Analyses of rpoC1 Sequences

The marine picocyanobacteria Prochlorococcus represent a significant fraction of the global pelagic bacterioplankton community. Specifically, in the surface waters of the Red Sea, they account for around 91% of the phylum Cyanobacteria. Previous work suggested a widespread presence of high-light (HL)-adapted ecotypes in the Red Sea with the occurrence of low-light (LL)-adapted ecotypes at intermediate depths in the water column. To obtain a more comprehensive dataset over a wider biogeographical scope, we used a 454-pyrosequencing approach to analyze the diversity of the Prochlorococcus rpoC1 gene from a total of 113 samples at various depths (up to 500 m) from 45 stations spanning the Red Sea basin from north to south. In addition, we analyzed 45 metagenomes from eight stations using hidden Markov models based on a set of reference Prochlorococcus genomes to 1) estimate the relative abundance of Prochlorococcus based on 16S rRNA gene sequences, and 2) identify and classify rpoC1 sequences as an assessment of the community structure of Prochlorococcus in the northern, central and southern regions of the basin without amplification bias. Analyses of metagenomic data indicated that Prochlorococcus occurs at a relative abundance of around 9% in samples from surface waters (25, 50, 75 m), 3% in intermediate waters (100 m) and around 0.5% in deep-water samples (200 – 500 m). Results based on rpoC1 sequences using both methods showed that HL II cells dominate surface waters and were also present in deep-water samples. Prochlorococcus communities in intermediate waters (100 m) showed a higher diversity and co-occurrence of low-light and high-light ecotypes. Prochlorococcus communities at each depth range (surface, intermediate, deep sea) did not change significantly over the sampled transects spanning most of the Saudi waters in the Red Sea. Statistical analyses of rpoC1 sequences from metagenomes indicated that the vertical distribution of Prochlorococcus in the water column is correlated with physicochemical gradients: temperature and oxygen are positively correlated with HL II (R2= 0.71, p-value ≤ 0.05) while Chl a concentration, nutrient concentrations and salinity correlate with the prevalence of LL clades.

Fine spatial structure of genetically distinct picocyanobacterial populations across environmental gradients in the Costa Rica Dome

We investigated the spatial variability of picocyanobacterial community structure across the Costa Rica Dome (CRD), an offshore upwelling system characterized by high seasonal abundance of Synechococcus spp. We constructed clone libraries of the rpoC1 gene to survey picocyanobacterial diversity and developed specific real‐time quantitative polymerase chain reaction assays to assess the distribution of genetically distinct Synechococcus (SYN) and Prochlorococcus (PRO) populations across vertical and horizontal physicochemical gradients. Flow cytometry data showed that cell abundances for both SYN and PRO were highest near the dome center. Phylogenetic analysis of rpoC1 sequences revealed a remarkably high and distinctive picocyanobacterial diversity (FLU1‐3, CRD1, Clade II, XV, XVI) that included “novel” SYN and PRO genotypes. Furthermore, genetically different populations exhibited vertical and horizontal spatial partitioning. Abundances of distinct SYN genotypes peaked at subsequent depth horizons, leading to a fine vertical structure with at least three populations stacked within the upper 30–40 m at the dome. Clade II and FLU1A peaked in surface waters, while maximum concentrations of CRD1, FLU1B, and Clade XVI occurred in the upper and lower thermocline, respectively. Horizontally, Clade II abundance in surface waters remained high across the entire region, while SYN genotypes CRD1 and FLU1A increased with shoaling of the thermo‐ and nutricline toward the center of the dome to become the dominant genotypes of the SYN assemblage in the dome. Below the mixed layer, Clade XVI and PRO genotype FLU2, virtually absent outside the dome, became abundant components of the picocyanobacterial assemblage. Despite their phylogenetic relatedness, FLU1A and FLU1B subclades followed different distributional patterns, suggesting ecological significance of the microdiversity within the clade. The unprecedented fine vertical structure demonstrated for SYN genotypes is driven by sharp physicochemical gradients (e.g., density, nutrient, oxygen, and trace metals) created by the dome and the presence of a shallow oxycline that enhances habitat diversification.

DNA barcoding based on plastid matK and RNA polymerase for assessing the genetic identity of date (Phoenix dactylifera L.) cultivars

The cultivated date palm is the most agriculturally important species of the Arecaceae family. The standard chloroplast DNA barcode for land plants recommended by the Consortium for the Barcode of Life plant working group needs to be evaluated for a wide range of plant species. Therefore, we assessed the potential of the matK and rpoC1 markers for the authentication of date cultivars. There is not one universal method to authenticate date cultivars. In this study, 11 different date cultivars were sequenced and analyzed for matK and rpoC1 genes by using bioinformatic tools to establish a cultivar-specific molecular monogram. The chloroplast matK marker was more informative than the rpoC1 chloroplast DNA markers. Phylogenetic trees were constructed on the basis of the matK and rpoC1 sequences, and the results suggested that matK alone or in combination with rpoC1 can be used for determining the levels of genetic variation and for barcoding.

Recombination Signals In The rpoC1 Gene Indicate Gene-Flow Between Planktothrix (Cyanoprokaryota) Species.

The delineation of species boundaries in the potentially harmful cyanobacterium Planktothrix Anagnostidis et Komárek 1988 is particularly tangled. Genetic recombination has been invoked to explain the occurrence of overlapping biological traits among recognized species. Although horizontal gene transfer is shown as a driver of diversification in this genus, clear evidence for homologous recombination at the single gene level is still lacking. Several Planktothrix strains (n=244) sampled in eight fresh water lakes in north Italy were characterized by sequencing the rpoC1 gene, a molecular marker previously proposed to discriminate between species. Six haplotypes were detected, four of which are newly described. A relevant number of rpoC1 sequences (n=54) showed evidence of homologous recombination. By comparing the sequences produced in the work presented here to those available on GenBank for the genus, multiple recombination events were tracked between haplotypes associated to P. rubescens, P. suspensa and P. agardhii, the latter being a species not found in our survey. Recombination signals were in form of (i) a vast mosaic structure present in the alignment of rpoC1 haplotypes, (ii) multiple and statistically significant paths in the split decomposition network connecting these haplotypes and (iii) many individual crossing-over events detected by means of recombination detection tests. Data suggest that the molecular evolution of the rpoC1 gene in the genus Planktothrix appears as strongly influenced by homologous recombination. In addition, rpoC1 diversity effectively tracks recombinational processes among species in the complex made up by P. rubescens, P. agardhii and P. suspensa, which are not isolated in terms of gene-flow.

Isoflavone determination in spontaneous legumes identified by DNA barcodes

Isoflavones have been associated with several health protective effects. In this work spontaneous legume plants were screened as putative sources of dietary isoflavones. A molecular identification of the collected species was performed throughout DNA barcoding using ITS, rbcL, rpoC1 and matK sequences. The use of a multi-locus barcoding system complemented with basic morphological information allowed the unequivocal identification at the species level of 90% of the samples. The determination of isoflavone content was performed by high-performance liquid chromatography with diode-array detection. Total average contents in the studied species were significantly different, Ononis natrix and Cytisus scoparius possessing the highest total isoflavones content (396 and 273 mg kg(-1), respectively) and Lotus creticus, the lowest (20 mg kg(-1)). The correlation of total isoflavone content with the phylogeny of this set of plants as determined by the rpoC1 sequences was evaluated for the first time.

Molecular and phylogenetic characterization of potentially toxic cyanobacteria in Tunisian freshwaters

This study presents a genetic characterization of 27 potentially toxic cyanobacterial strains isolated from seven reservoirs located in the north and centre of Tunisia. These strains belonged mainly to Microcystis aeruginosa, Cylindrospermopsis raciborskii and Planktothrix agardhii species. Their toxicological potential was evaluated by molecular biology tools, which showed that none of the isolated strains carried segments of the gene cluster responsible for the production of cylindrospermopsin and saxitoxin. The majority of Microcystis isolates were able to synthesize microcystin, since they presented the six characteristic segments of the microcystin synthetase mcy cluster ( mcyA, - B, - C, - D, - E and - G). This was further confirmed by MALDI-TOF analysis that showed the presence of eight microcystin variants, including microcystin-LR. The taxonomic identification of the strains was assessed based on the variability of the 16S rRNA gene sequences. Furthermore, the 16S–23S rRNA ITS sequences of Microcystis isolates and rpoC1 sequences of Cylindrospermopsis strains were also used in the phylogenetic analysis.

High resolution genetic diversity studies of marine Synechococcus isolates using rpoC1-based restriction fragment length polymorphism

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 45:263-275 (2006) - doi:10.3354/ame045263 High resolution genetic diversity studies of marine Synechococcus isolates using rpoC1-based restriction fragment length polymorphism Martin Mühling1,2,*, Nick J. Fuller2, Paul J. Somerfield1, Anton F. Post3, William H. Wilson1, David J. Scanlan2, Ian Joint1, Nicholas H. Mann2 1Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1 3DH, Devon, UK 2Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, Midlands, UK 3InterUniversity Institute for Marine Science, Coral Beach, 88103 Eilat, Israel *Email: mmuh@pml.ac.uk ABSTRACT: Synechococcus-specific polymerase chain reaction (PCR) primers were developed and used to amplify fragments of the RNA polymerase core subunit gene rpoC1 of a set of 23 marine isolates from different oceanographic locations. Restriction fragment length polymorphism (RFLP) analysis of the PCR products differentiated between most strains and all phylogenetic clades, thus allowing the screening of large sample sets at high genetic resolution. The method was used to analyse changes in the genetic diversity of marine Synechococcus strains through an annual cycle at 100 m depth in the Gulf of Aqaba, Red Sea, and along the depth profile of a sample from June 1999. RFLP analysis of 36 clones of each of the 8 rpoC1 clone libraries screened showed that the Synechococcus population during summer and autumn was dominated by 1 to 3 genetically different clones, while maximum genetic richness was found during spring and winter. Analysis of the depth profile during the period of stratification revealed differences in relative abundance of particular RFLP types among depths (10, 30, 100 m), although the same genotypes dominated the assemblage throughout. Multivariate analysis of environmental variables and Synechococcus assemblage composition showed that Synechococcus genetic diversity was most closely, and significantly, correlated to a combination of temperature and NO3– (ρ = 0.645, p = 0.038). Phylogenetic analysis of the marine rpoC1 nucleotide sequences of the 40 different RFLP types identified in this study, and all Synechococcus rpoC1 sequences in the databases, demonstrates that the new PCR primers amplify rpoC1 gene fragments from all MC-A and some Cyanobium lineages of marine Synechococcus strains and are also useful for the identification of potentially novel lineages. KEY WORDS: Synechococcus spp. · Marine · rpoC1 · Genetic diversity · Population dynamics Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 45, No. 3. Online publication date: December 21, 2006 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2006 Inter-Research.