De novo assembly and transcriptome analysis of five major tissues of Jatropha curcas L. using GS FLX titanium platform of 454 pyrosequencing

<p>Abstract</p> <p>Background</p> <p><it>Jatropha curcas </it>L. is an important non-edible oilseed crop with promising future in biodiesel production. However, factors like oil yield, oil composition, toxic compounds in oil cake, pests and diseases limit its commercial potential. Well established genetic engineering methods using cloned genes could be used to address these limitations. Earlier, 10,983 unigenes from Sanger sequencing of ESTs, and 3,484 unique assembled transcripts from 454 pyrosequencing of uncloned cDNAs were reported. In order to expedite the process of gene discovery, we have undertaken 454 pyrosequencing of normalized cDNAs prepared from roots, mature leaves, flowers, developing seeds, and embryos of <it>J. curcas</it>.</p> <p>Results</p> <p>From 383,918 raw reads, we obtained 381,957 quality-filtered and trimmed reads that are suitable for the assembly of transcript sequences. <it>De novo </it>contig assembly of these reads generated 17,457 assembled transcripts (contigs) and 54,002 singletons. Average length of the assembled transcripts was 916 bp. About 30% of the transcripts were longer than 1000 bases, and the size of the longest transcript was 7,173 bases. BLASTX analysis revealed that 2,589 of these transcripts are full-length. The assembled transcripts were validated by RT-PCR analysis of 28 transcripts. The results showed that the transcripts were correctly assembled and represent actively expressed genes. KEGG pathway mapping showed that 2,320 transcripts are related to major biochemical pathways including the oil biosynthesis pathway. Overall, the current study reports 14,327 new assembled transcripts which included 2589 full-length transcripts and 27 transcripts that are directly involved in oil biosynthesis.</p> <p>Conclusion</p> <p>The large number of transcripts reported in the current study together with existing ESTs and transcript sequences will serve as an invaluable genetic resource for crop improvement in jatropha. Sequence information of those genes that are involved in oil biosynthesis could be used for metabolic engineering of jatropha to increase oil content, and to modify oil composition.</p

DNA Barcoding of the Market Samples of Single-Drug Herbal Powders Reveals Adulteration with Taxonomically Unrelated Plant Species

Herbal drugs are increasingly becoming a viable alternative to allopathic medicine. Since powdered herbal drugs are more prone to adulteration than intact plant parts, their authentication becomes essential to ensure the safety and efficacy of herbal drugs. This study authenticated 107 single-drug herbal powders, representing 65 species from 60 genera and 35 families, collected from the markets in Tamil Nadu, India. DNA barcoding using the rbcL marker revealed that 58 samples (54%) were authentic, and 49 (46%) were adulterant. About 41% of the adulterant samples were a mixture of more than one species, possibly due to unintentional cross-contamination during processing. In 59% of the adulterant samples, the authentic species was entirely substituted with taxonomically and medicinally unrelated species, 72% of which belonged to different orders and families, while 28% were from other genera. Despite the taxonomic diversity, 20% of adulterant spe, cies had a morphological resemblance to the authentic species. It is not known whether their use as adulterants is intentional. In a detailed study on DNA barcoding of 17 powder samples from Ocimum tenuiflorum, 88% of the samples were authentic. These results indicate that the extent of adulteration is not high in all the species. Approximately, 95% of the samples collected for this study were produced by companies with limited resources and expertise in the unorganized sector. Hence, training them on species identification and providing simple and cost-effective authentication tools will likely reduce adulteration in the market samples

DNA Barcoding of the Market Samples of Single-Drug Herbal Powders Reveals Adulteration with Taxonomically Unrelated Plant Species

Herbal drugs are increasingly becoming a viable alternative to allopathic medicine. Since powdered herbal drugs are more prone to adulteration than intact plant parts, their authentication becomes essential to ensure the safety and efficacy of herbal drugs. This study authenticated 107 single-drug herbal powders, representing 65 species from 60 genera and 35 families, collected from the markets in Tamil Nadu, India. DNA barcoding using the rbcL marker revealed that 58 samples (54%) were authentic, and 49 (46%) were adulterant. About 41% of the adulterant samples were a mixture of more than one species, possibly due to unintentional cross-contamination during processing. In 59% of the adulterant samples, the authentic species was entirely substituted with taxonomically and medicinally unrelated species, 72% of which belonged to different orders and families, while 28% were from other genera. Despite the taxonomic diversity, 20% of adulterant spe, cies had a morphological resemblance to the authentic species. It is not known whether their use as adulterants is intentional. In a detailed study on DNA barcoding of 17 powder samples from Ocimum tenuiflorum, 88% of the samples were authentic. These results indicate that the extent of adulteration is not high in all the species. Approximately, 95% of the samples collected for this study were produced by companies with limited resources and expertise in the unorganized sector. Hence, training them on species identification and providing simple and cost-effective authentication tools will likely reduce adulteration in the market samples

DMSO and betaine significantly enhance the PCR amplification of ITS2 DNA barcodes from plants

ITS2 marker is highly efficient in species discrimination but its application in DNA barcoding is limited due to huge variations in the PCR success rate. We have hypothesized that higher GC content and the resultant secondary structures formed during annealing might hinder the PCR amplification of ITS2. To test this hypothesis, we selected 12 species from 12 different families in which ITS2 was not amplified under standard PCR reaction conditions. In these samples, DMSO, formamide, betaine, and 7-deaza-dGTP were evaluated for their ability to improve the PCR success rate. The highest PCR success rate (91.6%) was observed with 5% DMSO, followed by 1 M betaine (75%), 50 μM 7-deaza-dGTP (33.3%), and 3% formamide (16.6%). The one sample that did not amplify with DMSO was amplified by adding 1 M betaine. However, combining DMSO and betaine in the same reaction did not improve the PCR. Therefore, to achieve the highest PCR success rate for ITS2, it is recommended to include 5% DMSO by default and substitute it with 1 M betaine only in the case of failed reactions. When this strategy was tested in 50 species from 43 genera and 29 families, the PCR success rate of ITS2 increased from 42% to 100%.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Use of succinic & oxalic acid in reducing the dosage of colistin against New Delhi metallo-β-lactamase-1 bacteria

Background & objectives: New Delhi metallo-β-lactamase 1 (NDM-1) cleaves the beta-lactam ring, and confers bacterial resistance against most of the beta-lactam antibiotics, except tigecycline and colistin. Among these two antibiotics, colistin is considered toxic, and therefore, its clinical use and dosage need cautious approach. In the present study, six organic acids were screened individually and in combination of two acids for their effectiveness against NDM-1 Escherichia coli and a combination of colistin and oxalic or succinic acid was tested to find out the potential of combination therapy for reducing the dose of toxic colistin. Methods: Antibacterial activity of the organic acid and their combinations was tested by disc diffusion method against NDM-1 E. coli, and minimum inhibitory concentration (MIC) was determined by broth dilution method. Synergistic effect between organic acids and colistin was tested by checkerboard method. Results: Oxalic acid showed the highest zone of inhibition (15±1 mm) followed by succinic acid, tartaric acid, fumaric acid, citric acid and malic acid. The combination of two acids did not increase the zone of inhibition significantly. MIC was found to be the lowest with oxalic acid and succinic acid (320 μg/ml). In the presence of 160 μg/ml oxalic acid or succinic acid, MIC of colistin was reduced from 8 to 4 μg/ml, indicating synergistic effect. Interpretation & conclusions: Our findings showed that combination therapy using colistin and oxalic acid or succinic acid might find safe clinical application of this antibiotic in controlling infections due to NDM-1 bacteria

The complete chloroplast genome of Ocimum tenuiflorum L. subtype Rama Tulsi and its phylogenetic analysis

Ocimum tenuiflorum L. subtype Rama Tulsi is an important aromatic perennial herb. It belongs to the family of Lamiaceae. In this study, the complete chloroplast genome sequence of O. tenuiflorum subtype Rama Tulsi was assembled and annotated using Illumina paired-end sequencing data. The length of the complete circular chloroplast genome was 151,722 bp. It comprises an inverted repeat (IR) region with a repeat length of 25,677 bp, a large single-copy (LSC) region of 82,781 bp, and a small single-copy (SSC) region of 17,587 bp. The GC content of complete chloroplast genome, LSC, SSC, IR regions is 37.9%, 36.0%, 31.8%, and 43.1%, respectively. The chloroplast genome contains 134 genes, including 88 protein-coding genes, 38 transfer RNA genes, and eight ribosomal RNA genes. Phylogenetic analysis with the complete chloroplast genomes of other related species revealed that the O. tenuiflorum L. subtype Rama Tulsi is fully resolved in a clade with other Ocimum species classified under the Lamiaceae family

Genome-wide DNA polymorphisms in Kavuni, a traditional rice cultivar with nutritional and therapeutic properties

Although rice genome was sequenced in the year 2002, efforts in resequencing the large number of available accessions, landraces, traditional cultivars, and improved varieties of this important food crop are limited. We have initiated resequencing of the traditional cultivars from India. Kavuni is an important traditional rice cultivar from South India that attracts premium price for its nutritional and therapeutic properties. Whole genome sequencing of Kavuni using Illumina platform and SNPs analysis using Nipponbare reference genome identified 1,150,711 SNPs of which 377,381 SNPs were located in the genic regions. Non-synonymous SNPs (62,708) were distributed in 19,251 genes, and their number varied between 1 and 115 per gene. Large-effect DNA polymorphisms (7,769) were present in 3,475 genes. Pathway mapping of these polymorphisms revealed the involvement of genes related to carbohydrate metabolism, translation, protein-folding and cell death. Analysis of the starch biosynthesis related genes revealed that the granule-bound starch synthase I gene had T/G SNPs at the first intron/exon junction and a two-nucleotide combination, which were reported to favour high amylose content and low glycemic index. The present study provided a valuable genomics resource to study the rice varieties with nutritional and medicinal properties.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Desiccation and topping induced silencing of putrescine N-methyl transferase2 regulate nicotine biosynthesis in Nicotiana tabacum cv. Petite Havana

Abstract The current study was an attempt to understand the regulation of nicotine biosynthesis due to desiccation and meristem topping in Nicotiana tabacum cv. Petite Havana. Our results showed that desiccation induced silencing of Putrescine N-Methyl Transferase 2 (PMT2) (EC 2.1.1.53), a rate limiting enzyme in the nicotine biosynthetic pathway, regulates nicotine levels in tobacco. A hairpin siRNA (hpsiRNA) was designed and expressed against pmt using a stress inducible promoter rd29A from Arabidopsis thaliana. Stable transgenic RNAi lines, developed by Agrobacterium mediated transformation, showed reduced accumulation of nicotine due to desiccation and topping. The increase in the nicotine level in wild type was 115% while in RNAi lines, RNAi1, RNAi2 and RNAi3 the increase was 19.9%, 30% and 21.62%, respectively, due to desiccation. Furthermore, meristem topping of desiccated plants increased nicotine accumulation by 38% in wild type and only 7-9% in RNAi lines. No secondary surge in any related secondary metabolite was detected. No detectable change in the phenotype was observed in RNAi lines. This study revealed the critical role of pmt in nicotine biosynthesis under desiccation and topping and the inducible regulation of endogenous genes for crop improvement

Multi-locus DNA barcoding identifies matK as a suitable marker for species identification in Hibiscus L.

The genus Hibiscus L. includes several taxa of medicinal value and species used for the extraction of natural dyes. These applications require the use of authentic plant materials. DNA barcoding is a molecular method for species identification, which helps in reliable authentication by using one or more DNA barcode marker. In this study, we have collected 44 accessions, representing 16 species of Hibiscus, distributed in the southern peninsular India, to evaluate the discriminatory power of the two core barcodes, rbcLa and matK together with the suggested additional regions, trnH-psbA and ITS2. No intra-species divergence was observed among the accessions studied. Inter-species divergence was 0-9.6% with individual markers, which increased to 0-12.5% and 0.8-20.3% when using a two-, and three-marker combination, respectively. Differentiation of all the species of Hibiscus was possible with the matK DNA barcode marker. Also, in two-marker combinations, only those combinations with matK differentiated all the species. Though all the three-marker combinations showed 100% species differentiation, species resolution was consistently better when matK marker formed part of the combination. These results clearly showed that matK is more suitable when compared to rbcLa, trnH-psbA, and ITS2 for species identification in Hibiscus.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author