Larches of Kuzhanovo Have a Unique Mutation in the atpF-atpH Intergenic Spacer.

The larches of Kuzhanovo (Larix sibirica Ledeb.) are protected trees with a round crown growing in the Southern Urals. In 2020 vandals sawed the sapwood of these trees, which exposed the problem of insufficient conservation measures. Their origin and genetic characteristics have been of particular interest to breeders and scientists. The larches of Kuzhanovo were screened for polymorphisms using SSR and ISSR analyses and the sequencing of genetic markers and genes GIGANTEA and mTERF, associated with wider crown shape. A unique mutation was discovered in the atpF-atpH intergenic spacer of all protected trees, but it was absent in some of their descendants and larches with similar crown shape. Mutations were discovered in the rpoC1 and mTERF genes of all samples. Flow cytometry did not reveal any changes in genome size. Our results suggest that the unique phenotype arose from point mutations in L. sibirica, but they are yet to be found in the nuclear genome. The concurrent mutations in the rpoC1 and mTERF genes may indicate that the round crown shape originates from the Southern Urals. The atpF-atpH and rpoC1 genetic markers are not common in studies of Larix sp., but their wider use could help to establish the origin of these endangered plants. The discovery of the unique atpF-atpH mutation also allows for stronger conservation and crime detection efforts.

Differentiation of Lingxiaohua and Yangjinhua by chloroplast genome sequencing and DNA barcoding markers.

Lingxiaohua (Campsis Flos, Campsis grandiflora (Thunb.) K. Schum) is a medicinal herb used for promoting diuresis and treating blood-related disorders by the promotion of blood circulation. It also possesses anti-inflammatory and antioxidative properties. This non-poisonous plant is frequently confused with poisonous Yangjinhua (Daturae Metelis Flos, Datura metel Linnaeus) in the market, resulting in serious anticholinergic poisoning. The confusion of these two herbs is due to the similarity in their appearances. In our study, we compared the complete chloroplast genomes of the two plants and found that they are very different in terms of their gene content and gene arrangement. There were also significant differences in the number and repeating motifs of microsatellites and complex repeats. We used universal primers for the amplification of rbcL, matK, psbA-trnH, and ITS2 regions and successfully differentiated the two plants. Furthermore, we designed two pairs of primers based on the nucleotide differences in chloroplast genomes at the rps14 and rpoC1 regions to provide additional authentication markers. The universal primers and specific primers when used together can accurately discriminate Lingxiaohua and Yangjinhua.

Seasonal and spatial variations of Synechococcus in abundance, pigment types, and genetic diversity in a temperate semi-enclosed bay.

Synechococcus is abundant and globally widespread in various marine environments. Seasonal and spatial variations in Synechococcus abundance, pigment types, and genetic diversity were investigated based on flow cytometric analysis and high-throughput sequencing of cpcBA operon (encoding phycocyanin) and rpoC1 gene (encoding RNA polymerase) in a temperate semi-enclosed bay. Synechococcus abundance exhibited seasonal variations with the highest value in summer and the lowest value in winter, which was consistent with temperature variation. Three pigment types of Synechococcus type 1, type 2, and type 3 were distinguished based on cpcBA operon, which displayed obvious variations spatially between the inner and the outer bay. Freshwater discharge and water turbidity played important roles in regulating Synechococcus pigment types. Synechococcus assemblages were phylogenetically diverse (12 different lineages) based on rpoC1 gene and dominated by three core lineages S5.1-I, S5.1-IX, and S5.2-CB5 in different seasons. Our study demonstrated that Synechococcus abundance, pigment types, and genetic diversity displayed variations seasonally and spatially by different techniques, which were mainly driven by temperature, salinity, nutrients, and turbidity. The combination of more technical means provides more information for studying Synechococcus distribution. In this study, three pigment types of Synechococcus were discriminated simultaneously by dual lasers flow cytometer for the first time.

DNA-Based Identification of Eurasian Vicia Species Using Chloroplast and Nuclear DNA Barcodes.

Many legume species of the Vicia L. genus (Fabaceae Lindl.) are key components of the Mediterranean diet and have an integral role in sustainable agriculture. Given the importance of the Vicia species for Eurasian culture, it is necessary to implement methodologies, such as DNA barcoding, that can enable the effective authentication and identification of species in the genus. In this study, we analysed the chloroplast trnL and rpoC1, as well as the nuclear ITS2 DNA barcoding regions, to identify 71 Vicia specimens of Eurasian descent. Both the trnL and ITS2 regions were highly effective in discriminating the analysed taxa, while the more conserved rpoC1 region could not identify all of the selected species due to high sequence conservation or non-annotated or absent rpoC1 species sequences in GenBank. A dendrographic representation of the generated trnL data showed sufficient clustering for most of the analysed taxa, although some topological discrepancies were observed. ITS2 and rpoC1 reconstructions were also used for resolving the topological discrepancies observed in the trnL tree. Our analysis suggests that a combination of DNA barcoding regions is essential for accurate species discrimination within the Vicia genus, while single-locus analyses do not provide the necessary resolution.

DNA Barcoding of Two Thymelaeaceae Species: Daphne mucronata Royle and Thymelaea hirsuta (L.) Endl.

Daphne mucronata Royle and Thymelaea hirsuta (L.) Endl both belong to the Thymelaeaceae family. Both species are used traditionally to treat several diseases along with various daily applications by Jordanian Bedouins. Traditionally, those species are identified through personal proficiency, which could be misleading due to human errors or lack of expertise. This study aims to investigate an effective DNA barcoding method to identify and characterize Daphne mucronata Royle and Thymelaea hirsuta plant species at the molecular level. Daphne mucronata Royle and Thymelaea hirsuta were collected from the ancient city of Petra in the Southern part of Jordan. Sequences of candidate DNA barcodes were amplified (rbcL, matK, and rpoC1), sequenced, and aligned to the blastn database. Moreover, the obtained sequences were compared with available sequences of related species at the GenBank database. Our results showed that DNA barcoding successfully identifies the two plant species using any of chloroplast genes (rbcL, matK, or rpoC1). The results emphasize the ability of DNA barcoding for identifying and characterizing different plant species through the recruitment of different barcode loci in molecular identification.

A rice mTERF protein V14 sustains photosynthesis establishment and temperature acclimation in early seedling leaves.

BACKGROUND: Plant mitochondrial transcription termination factor (mTERF) family members play important roles in development and stress tolerance through regulation of organellar gene expression. However, their molecular functions have yet to be clearly defined. RESULTS: Here an mTERF gene V14 was identified by fine mapping using a conditional albino mutant v14 that displayed albinism only in the first two true leaves, which was confirmed by transgenic complementation tests. Subcellular localization and real-time PCR analyses indicated that V14 encodes a chloroplastic protein ubiquitously expressed in leaves while spiking in the second true leaf. Chloroplastic gene expression profiling in the pale leaves of v14 through real-time PCR and Northern blotting analyses showed abnormal accumulation of the unprocessed transcripts covering the rpoB-rpoC1 and/or rpoC1-rpoC2 intercistronic regions accompanied by reduced abundance of the mature rpoC1 and rpoC2 transcripts, which encode two core subunits of the plastid-encoded plastid RNA polymerase (PEP). Subsequent immunoblotting analyses confirmed the reduced accumulation of RpoC1 and RpoC2. A light-inducible photosynthetic gene psbD was also found down-regulated at both the mRNA and protein levels. Interestingly, such stage-specific aberrant posttranscriptional regulation and psbD expression can be reversed by high temperatures (30 ~ 35 °C), although V14 expression lacks thermo-sensitivity. Meanwhile, three V14 homologous genes were found heat-inducible with similar temporal expression patterns, implicating their possible functional redundancy to V14. CONCLUSIONS: These data revealed a critical role of V14 in chloroplast development, which impacts, in a stage-specific and thermo-sensitive way, the appropriate processing of rpoB-rpoC1-rpoC2 precursors and the expression of certain photosynthetic proteins. Our findings thus expand the knowledge of the molecular functions of rice mTERFs and suggest the contributions of plant mTERFs to photosynthesis establishment and temperature acclimation.

A new cyanobacterial genus Altericista and three species, A. lacusladogae sp. nov., A. violacea sp. nov., and A. variichlora sp. nov., described using a polyphasic approach.

Several strains of unicellular cyanobacteria from the culture collection of St. Petersburg State University, St. Petersburg, Russia (CALU), which were preliminary identified as Synechocystis sp., are reclassified in the new genus Altericista. Three new species are proposed, A. lacusladogae, A. violacea, and A. variichlora. The last species produces accessory chlorophylls d and f in cultures illuminated by far-red light, an attribute rarely observed in cyanobacteria, especially in unicellular strains. This genus is morphologically similar to Synechocysis having coccoid cells that divide in two successive planes at right angles, containing no sheath or capsule, and having the lamellar system represented by peripheral concentric thylakoids. Altericista shows ecological, biochemical, and physiological characters unlike those in Synechocystis and has the distinguishing phenotypic characters as follows: freshwater, non-halotolerant ecotype; palmitate and α-linoleate as major fatty acids; and the ability to photoacclimate, including several types of complementary chromatic adaptation. Genetic differences from Synechocystis sp. include 16S rRNA, rpoC1, and rbcL gene sequences, as well as sequence and folding of 16S-23S ITS.

Molecular barcode and morphological analysis of Smilax purhampuy Ruiz, Ecuador.

Smilax plants are distributed in tropical, subtropical, and temperate regions in both hemispheres of the world. They are used extensively in traditional medicines in a number of countries. However, morphological and molecular barcodes analysis, which may assist in the taxonomic identification of species, are lacking in Ecuador. In order to evaluate the micromorphological characteristics of these plants, cross sections of Smilax purhampuy leaves were obtained manually. The rhizome powder, which is typically used in traditional medicines, was analyzed for micromorphological characteristics. All samples were clarified with 1% sodium hypochlorite. Tissues were colored with 1% safranin in water and were fixed with glycerinated gelatin. DNA was extracted from the leaves using a modified CTAB method for molecular barcode characterization and PCR was performed using primers to amplify the different loci including the plastid genome regions atpF-atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK-psbI spacer, and trnH-psbA spacer; and the nuclear DNA sequence ITS2. A DNA sequence similarity search was performed using BLAST in the GenBank nr database and phylogenetic analysis was performed using the maximum likelihood method according to the best model identified by MEGAX using a bootstrap test with 1,000 replicates. Results showed that the micromorphological evaluation of a leaf cross section depicted a concave arrangement of the central vein, which was more pronounced in the lower section and had a slight protuberance. The micromorphological analysis of the rhizome powder allowed the visualization of a group of cells with variable sizes in the parenchyma and revealed thickened xylematic vessels associated with other elements of the vascular system. Specific amplicons were detected in DNA barcoding for all the barcodes tested except for the trnH-psbA spacer. BLAST analysis revealed that the Smilax species was predominant in all the samples for each barcode; therefore, the genus Smilax was confirmed through DNA barcode analysis. The barcode sequences psbK-psbI, atpF-atpH, and ITS2 had a better resolution at the species level in phylogenetic analysis than the other barcodes we tested.

Untargeted metabolomics and DNA barcoding for discrimination of Phyllanthus species.

ETHNOPHARMACOLOGICAL RELEVANCE: Phyllanthus species is extensively cultivated and used as edible fruits and herbal drugs. The Phyllanthus species are used extensively as ethnopharmacologically important materials in several countries, especially in Asia. Various Phyllanthus species are broadly used in the Ayurvedic system of medicine and deliberated as bitter, astringent, stomachic, diuretic, febrifuge, deobstruent, and antiseptic, and used for the treatment of digestive, genitourinary, respiratory, skin diseases, hepatopathy, jaundice, and renal calculus in India. Precise authentification of Phyllanthus species is a challenge due to morphological similarities and is important to avoid adulteration found in herbal drugs. Hence, there is a need to establish comprehensive methods for the identification of Phyllanthus species. AIM OF THE STUDY: In this study, we attempted to integrate untargeted metabolomics to identify species-specific metabolites with traditional phylogenetic analysis for identification and discrimination of nine Phyllanthus species. MATERIALS AND METHODS: Phyllanthus species such as P. acidus, P. amarus, P. debilis, P. emblica, P. virgatus, P. urinaria, P. lawii, P. myrtifolius, and P. reticulatus were collected. The liquid chromatography coupled mass spectrometry (LC-MS) was performed for untargeted metabolite profiling and MS/MS fragmentation analysis was performed for selected compounds. Further, the barcoding analysis was executed using plastid loci, rpoC1 to integrate with metabolite profiling data. RESULTS: The Principal Component Analysis (PCA) of leaf metabolites showed distinct clusters in different species. Through further analysis, we have also identified the qualitative and quantitative status of unique metabolites across the species, and the majority of the selected compounds were annotated. The metabolic fingerprinting and the hierarchical clustering indicated that though the P. deblis and P. virgatus are distantly related to each other, they are closely associated with their metabolic profiling. Similarly, P. myrtifolius and P. urinaria are closely related to each other with their metabolic fingerprints than the genetic alignment. Further, we performed barcoding with rpoC1 across nine Phyllanthus species (P. acidus, P. amarus, P. debilis, P. emblica, P. virgatus, P. urinaria, P. lawii, P. myrtifolius, and P. reticulatus). Sequence similarity search in the GenBank database showed rpoC1 barcode loci from nine Phyllanthus species showed significant identity (>97%) with the sequences of various Phyllanthus species. CONCLUSIONS: The bioactive metabolites and their abundance can be assigned to specific species thereby serving as a biological signature and indicators for potential therapeutic use. This study identified differential expression of 14 secondary metabolites from nine Phyllanthus species. Alkaloid compound zeatin was found specific to P. virgatus and delphinidin-3-O- ß -D-glucoside was not found in P. myrtifolius. Barcoding and phylogenetic analysis showed P. acidus is the most genetically distinct among the groups and the sequence pair between P.emblica-P.reticulatus and P.emblica-P.urinaria showed the least difference.

Molecular Assessment of Genetic Stability Using CDDP and DNA-barcoding Assays in Long-term Micropropagated Rose Plant.

BACKGROUND AND OBJECTIVE: Roses are the world's best-known garden plants, established as ornamental plants cultivated for their blooms. Taif rose (Rosa damascena trigintipetala) refers to the Damascus Rose species and is regarded one of Taif Governorate's most significant financial goods, which produces an extremely fragrant commercially precious essential oil. The objective of current study was to assess the genetic stability of micropropagated Taif rose and to assess the usefulness of Conserved DNA Derived Polymorphism (CDDP) and DNA-barcoding genes such as; rpoC1 (chloroplast gene RNA polymerase1) in the detection of somaclonal variation. MATERIALS AND METHODS: Ten combinations of CDDP PCR primers were employed and the rpoC1 gene region was sequenced for mother plant (control) and micropropagated plantlets of Taif rose plant. RESULTS: Based on CDDP data, phylogenetic divergence indicated that the distinct specimens of Taif rose micro-propagated plantlets and control were genetically differentiated by a difference of 1% of genetic dissimilarity. Phylogenetic tree which developed using rpoC1 DNA showed that rpoC1 DNA sequencing discovered a genetic difference between the control and micro-propagated plantlets of Taif rose. CONCLUSION: Furthermore, CDDP and DNA barcoding using rpoC1 gene have demonstrated their usefulness in investigating the genetic history of Rosa species and their ability to explore genetic mutation.

Genomic Comparison and Spatial Distribution of Different Synechococcus Phylotypes in the Black Sea.

Picocyanobacteria of the genus Synechococcus are major contributors to global primary production and nutrient cycles due to their oxygenic photoautotrophy, their abundance, and the extensive distribution made possible by their wide-ranging biochemical capabilities. The recent recovery and isolation of strains from the deep euxinic waters of the Black Sea encouraged us to expand our analysis of their adaptability also beyond the photic zone of aquatic environments. To this end, we quantified the total abundance and distribution of Synechococcus along the whole vertical profile of the Black Sea by flow cytometry, and analyzed the data obtained in light of key environmental factors. Furthermore, we designed phylotype-specific primers using the genomes of two new epipelagic coastal strains - first described here - and of two previously described mesopelagic strains, analyzed their presence/abundance by qPCR, and tested this parameter also in metagenomes from two stations at different depths. Together, whole genome sequencing, metagenomics and qPCR techniques provide us with a higher resolution of Synechococcus dynamics in the Black Sea. Both phylotypes analyzed are abundant and successful in epipelagic coastal waters; but while the newly described epipelagic strains are specifically adapted to this environment, the strains previously isolated in mesopelagic waters are able, in low numbers, to withstand the aphotic and oxygen depleted conditions of deep layers. This heterogeneity allows different Synechococcus phylotypes to occupy different niches and underscores the importance of a more detailed characterization of the abundance, distribution, and dynamics of individual populations of these picocyanobacteria.

Reappraising plastid markers of the red algae for phylogenetic community ecology in the genomic era.

Selection of appropriate genetic markers to quantify phylogenetic diversity is crucial for community ecology studies. Yet, systematic evaluation of marker genes for this purpose is scarcely done. Recently, the combined effort of phycologists has produced a rich plastid genome resource with taxonomic representation spanning all of the major lineages of the red algae (Rhodophyta). In this proof-of-concept study, we leveraged this resource by developing and applying a phylogenomic strategy to seek candidate plastid markers suitable for phylogenetic community analysis. We ranked the core genes of 107 published plastid genomes based on various sequence-derived properties and their tree distance to plastid genome phylogenies. The resulting ranking revealed that the most widely used marker, rbcL, is not necessarily the optimal marker, while other promising markers might have been overlooked. We designed and tested PCR primers for several candidate marker genes, and successfully amplified one of them, rpoC1, in a taxonomically broad set of red algal specimens. We suggest that our general marker identification methodology and the rpoC1 primers will be useful to the phycological community for investigating the biodiversity and community ecology of the red algae.

Polyphasic approach using multilocus analyses supports the establishment of the new aerophytic cyanobacterial genus Pycnacronema (Coleofasciculaceae, Oscillatoriales).

A new Phormidium-like genus was found during an investigation of Oscillatoriales diversity in Brazil. Eight aerophytic populations from south and southeastern regions were isolated in monospecific cultures and submitted to polyphasic evaluation. The populations presented homogeneous morphology with straight trichomes, not attenuated, and apical cell with thickened cell wall. Phylogenetic analyses based on 16S rRNA gene sequences showed that these populations, plus the Brazilian strain Phomidium sp. B-Tom from GenBank, formed a highly supported and distinctive clade, which corresponds to the new genus Pycnacronema, comprising six new species: P. brasiliensis (type species), P. arboriculum, P. conicum, P. marmoreum, P. rubrum, and P. savannensis. These results were confirmed and supported by rpoC1 and rbcL genes evaluated independently and by the concatenated analysis of 16S rRNA, rpoC1 and rbcL genes (for all species but P. savannensis). Secondary structures of the D1-D1', box-B, and V3 regions of the internal transcribed spacer were informative at specific level, being conserved in P. brasiliensis and variable among the other strains, also confirming the phylogenetic analyses. The generic name and specific epithets of the new taxa are proposed under the provisions of the International Code of Nomenclature of algae, fungi, and plants.

The complete plastid genome sequence of Chrysanthemum lucidum (Asteraceae): an endemic species of Ulleung Island of Korea.

The characteristic of complete chloroplast (cp) genome sequence of Chrysanthemum lucidum, one of famous insular plant and an endemic to Ulleung Island of Korea, was firstly introduced in the present study. It was 150,985 bp and contained a large single copy region (82,786 bp) and a small single copy region (18,281 bp) which were separated by two inverted repeat regions (24,959 bp). In total, 131 genes were identified and they were consisted of 76 coding genes, eight rRNA genes, and 36 tRNA genes. Comparing to the previously reported Chrysanthemum indicum and C. x morifolium cp genomes, we found complete inversion of SSC region in this taxa. rpoC1 gene was pseudogenes due to 1 bp insertion of poly-A sequence in the 3' of exon 2.

High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates.

Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Genome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genome and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar transcripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3'-end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C. reinhardtii identified 2658 variants in the organellar genomes, which is 1/10th as much genetic diversity as is found in the nucleus.

Synechococcus Assemblages across the Salinity Gradient in a Salt Wedge Estuary.

Synechococcus are the most abundant and widely distributed picocyanobacteria in the ocean. The salt-wedge type of estuary possesses the complete horizontal and vertical gradient of salinity together with other physical and chemical parameters. In order to reveal whether such a complex environmental gradient harbors a high diversity of Synechococcus, we investigated the abundance, taxonomic composition and pigment genetic diversity of Synechococcus in surface and bottom waters across the salinity gradient in a salt-wedge estuary by flow cytometric analysis and pyrosequencing of the rpoC1 gene and cpcBA operon (encoding phycocyanin). Synechococcus were ubiquitously distributed in the studied region, with clear spatial variations both horizontally and vertically. The abundance and diversity of Synechococcus were low in the freshwater-dominated low salinity waters. By pyrosequencing of the rpoC1 gene, we have shown that with the increase of salinity, the dominant Synechococcus shifted from the freshwater Synechococcus to the combination of phylogenetic subcluster 5.2 and freshwater Synechococcus, and then the strictly marine subcluster 5.1 clade III. Besides, the composition of Synechococcus assemblage in the deep layer was markedly different from the surface in the stratified waters (dissimilarities: 40.32%-95.97%, SIMPER analysis). High abundance of clade III Synechococcus found in the brackish waters may revise our previous understanding that strains of this clade prefers oligotrophic environment. Our data also suggested that both the phylogenetic subcluster 5.3 Synechococcus, a lineage that was not well understood, and subcluster 5.1 clade I, a typical cold water lineage, were widely distributed in the bottom layer of the estuary. Clade I detected in the studied region was mainly contributed by subclade IG. Analysis of the cpcBA operon sequences revealed niche partitioning between type 1 and type 3 Synechococcus, with type 2 distributed broadly across the whole environmental gradients. Our results suggest that the salt wedge estuary provides various niches for different lineages of Synechococcus, making it an environment with high Synechococcus diversity compared with adjacent freshwater and shelf sea environments.

DNA barcoding of selected UAE medicinal plant species: a comparative assessment of herbarium and fresh samples.

It is commonly difficult to extract and amplify DNA from herbarium samples as they are old and preserved using different compounds. In addition, such samples are subjected to the accumulation of intrinsically produced plant substances over long periods (up to hundreds of years). DNA extraction from desert flora may pause added difficulties as many contain high levels of secondary metabolites. Herbarium samples from the Biology Department (UAE University) plant collection and fresh plant samples, collected from around Al-Ain (UAE), were used in this study. The three barcode loci for the coding genes matK, rbcL and rpoC1-were amplified. Our results showed that T. terresteris, H. robustum,T. pentandrus and Z. qatarense were amplified using all three primers for both fresh and herbaium samples. Both fresh and herbarium samples of C. comosum, however, were not amplified at all, using the three primers. Herbarium samples from A. javanica, C. imbricatum, T. aucherana and Z. simplex were not amplified with any of the three primers. For fresh samples 90, 90 and 80% of the samples were amplified using matK, rbcL and rpoC1, respectively. In short, fresh samples were significantly better amplified than those from herbarium sources, using the three primers. Both fresh and herbarium samples from one species (C. comosum), however, were not successfully amplified. It is also concluded that the rbcL regions showed real potentials to distinguish the UAE species under investigation into the appropriate family and genus.

Identification and genetic diversity analysis of Memecylon species using ISSR, RAPD and Gene-Based DNA barcoding tools

Background: Memecylon species are commonly used in Indian ethnomedical practices. The accurate identification is vital to enhance the drug's efficacy and biosafety. In the present study, PCR based techniques like RAPD, ISSR and DNA barcoding regions, such as 5s, psbA-trnH, rpoC1, ndh and atpF-atpH, were used to authenticate and analyze the diversity of five Memecylon species collected from Western Ghats of India. Results: Phylogenetic analysis clearly distinguished Memecylon malabaricum from Memecylon wightii and Memecylon umbellatum from Memecylon edule and clades formed are in accordance with morphological keys. In the RAPD and ISSR analyses, 27 accessions representing five Memecylon species were distinctly separated into three different clades. M. malabaricum and M. wightii grouped together and M. umbellatum, M. edule and Memecylon talbotianum grouped in the same clade with high Jaccard dissimilarity coefficient and bootstrap support between each node, indicating that these grouped species are phylogenetically similar. Conclusion: Data from the present study reveals that chloroplast psbA-trnH region could be used as a potential candidate region for identifying Memecylon species, and ISSR marker system could be used for estimating genetic diversity since it has high percent polymorphism compared to RAPD marker.

A polyphasic approach leading to the revision of the genus Planktothrix (Cyanobacteria) and its type species, P. agardhii, and proposal for integrating the emended valid botanical taxa, as well as three new species, Planktothrix paucivesiculata sp. nov.ICNP, Planktothrix tepida sp. nov.ICNP, and Planktothrix serta sp. nov.ICNP, as genus and species names with nomenclatural standing under the ICNP.

Twenty strains of Planktothrix and five of 'Oscillatoria' were characterized by a polyphasic approach, for clarification of their taxonomic relationships. Emphasis was given to the strains (17) of the Pasteur Culture Collection of Cyanobacteria (PCC). Phenotypic characters analyzed comprised morphology, phycobiliprotein composition, temperature and salinity tolerance. The gvpA gas vesicle gene was detected by PCR in all strains, and transmission electron microscopy confirmed gas vesicle formation in the strains of 'Oscillatoria'. MALDI-TOF mass spectrometry revealed 13 chemotypes, nine of which produce microcystins. A multi-locus sequence typing (MLST) analysis was conducted using individual and concatenated nucleotide sequences of the 16S rDNA, internal transcribed spacer (ITS), gyrB, rpoC1 and rpoB. The results highlighted an unexpected diversity within the genus Planktothrix, showing that the five strains of 'Oscillatoria' need to be included in this taxon. Consequently, the genus consists of seven phylogenetic clusters, three of which represent new species, named Planktothrix paucivesiculata sp. nov.ICNP (type strain: PCC 8926T), Planktothrix tepida sp. nov.ICNP (type strain: PCC 9214T) and Planktothrix serta sp. nov.ICNP (type strain: PCC 8927T). These, together with the emended genus Planktothrix and its type species P. agardhii, valid taxa under the ICN, are described/re-described for gaining nomenclatural standing under the ICNP.

Polyphasic identification of cyanobacterial isolates from Australia.

Reliable identification of cyanobacterial isolates has significant socio-economic implications as many bloom-forming species affect the aesthetics and safety of drinking water, through the production of taste and odour compounds or toxic metabolites. The limitations of morphological identification have promoted the application of molecular tools, and encouraged the adoption of combined (polyphasic) approaches that include both microscopy- and DNA-based analyses. In this context, the rapid expansion of available sequence data is expected to allow increasingly reliable identification of cyanobacteria, and ultimately resolve current discrepancies between the two approaches. In the present study morphological and molecular characterisations of cyanobacterial isolates (n = 39), collected from various freshwater sites in Australia, were compared. Sequences were obtained for the small ribosomal subunit RNA gene (16S rDNA) (n = 36), the DNA-dependent RNA polymerase gene (rpoC1) (n = 22), and the phycocyanin operon, with its intergenic spacer region (cpcBA-IGS) (n = 19). Phylogenetic analyses identified three cyanobacterial orders: the Chroococcales (n = 8), Oscillatoriales (n = 6), and Nostocales (n = 25). Interestingly, multiple novel genotypes were identified, with 22% of the strains (17/77) having <95% similarity to available sequences in GenBank. Morphological and molecular data were in agreement at the species level for only 26% of the isolates obtained (10/39), while agreement at the genus level was obtained for 31% (12/39). Confident identification of the remaining 44% of the strains (17/39) beyond the order level was not possible. The present study demonstrates that, despite the taxonomic revisions, and advances in molecular-, and bioinformatics-tools, the lack of reliable morphological features, culture-induced pleomorphism, and proportion of misidentified or poorly described sequences in GenBank, still represent significant factors, impeding the confident identification of cyanobacteria species.