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%.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Abstract
The bitter gourd seed oil, rich in conjugated fatty acids, has therapeutic value to treat cancer, obesity, and aging. It also has an industrial application as a drying agent. Despite its ...significance, genomics studies are limited, and the genes for seed oil biosynthesis are not fully understood. In this study, we assembled the fruit transcriptome of bitter gourd using 254.5 million reads (Phred score > 30) from the green rind, white rind, pulp, immature seeds, and mature seeds. It consisted of 125,566 transcripts with N50 value 2,751 bp, mean length 960 bp, and 84% completeness. Transcript assembly was validated by RT-PCR and qRT-PCR analysis of a few selected transcripts. The transcripts were annotated against the NCBI non-redundant database using the BLASTX tool (E-value < 1E−05). In gene ontology terms, 99,443, 86,681, and 82,954 transcripts were classified under biological process, molecular function, and cellular component. From the fruit transcriptome, we identified 26, 3, and 10 full-length genes coding for all the enzymes required for synthesizing fatty acids, conjugated fatty acids, and triacylglycerol. The transcriptome, transcripts with tissue-specific expression patterns, and the full-length identified from this study will serve as an important genomics resource for this important medicinal plant.
Jatropha curcas 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 J. curcas.
From 383,918 raw reads, we obtained 381,957 quality-filtered and trimmed reads that are suitable for the assembly of transcript sequences. De novo 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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Saffron, the stigma of
L., is the most expensive spice used for culinary, medicinal, dye, and cosmetics purposes. It is highly adulterated because of its limited production and high commercial value. ...In this study, 104 saffron market samples collected from 16 countries were tested using morphology, high-performance liquid chromatography (HPLC), high-performance thin-layer chromatography (HPTLC), and deoxyribonucleic acid (DNA) barcoding. Overall, 45 samples (43%) were adulterated. DNA barcoding identified the highest number of adulterated saffron (44 samples), followed by HPTLC (39 samples), HPLC (38 samples), and morphology (32 samples). Only DNA barcoding identified the adulterated samples containing saffron and other plants' parts as bulking agents. In addition, DNA barcoding identified 20 adulterant plant species, which will help develop quality control methods and market surveillance. Some of the adulterant plants are unsafe for human consumption. The HPLC method helped identify the saffron samples adulterated with synthetic safranal. HPLC and HPTLC methods will help identify the samples adulterated with other parts of the saffron plant (auto-adulteration).
The underrepresentation of non-Europeans in human genetic studies so far has limited the diversity of individuals in genomic datasets and led to reduced medical relevance for a large proportion of ...the world's population. Population-specific reference genome datasets as well as genome-wide association studies in diverse populations are needed to address this issue. Here we describe the pilot phase of the GenomeAsia 100K Project. This includes a whole-genome sequencing reference dataset from 1,739 individuals of 219 population groups and 64 countries across Asia. We catalogue genetic variation, population structure, disease associations and founder effects. We also explore the use of this dataset in imputation, to facilitate genetic studies in populations across Asia and worldwide.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Being an expensive spice, saffron is highly prone to adulteration. Crocin, picrocrocin and safranal, which are commonly used as chemical markers for authentication, suffer from a lack of stability ...and uniqueness to saffron. Inexpensive synthetic safranal poses an additional concern in adulteration. Therefore, identification of other metabolites becomes essential for saffron authentication. Available metabolite extraction methods are laborious, time-consuming and require a large quantity of samples. We report a simple protocol for extracting the metabolites from as little as 5 mg of saffron using 100 μl of solvent in 2 h. High-performance liquid chromatography (HPLC) of the metabolites at 1 ml·min-1 flow rate with 70% acetonitrile for 10 min and detection at 308 nm yielded an HPLC fingerprint specific to saffron's stigma. The fingerprint was stable after at least 6 years of storage at room temperature and successfully differentiated the authentic from inauthentic market samples.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Summary
The current pandemic of severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) has quickly emerged as a global health concern with government bodies worldwide taking drastic control ...measures. Understanding the virology of SARS‐CoV‐2, its molecular mechanisms, and its pathogenesis are required for a targeted therapeutic approach. In this review, we highlight the current molecular and drug advances that target SARS‐CoV‐2 at the genome level. We also summarize studies that therapeutically target the host angiotensin‐converting enzyme 2 and proteases. Finally, we summarize antibody‐mediated therapeutic approaches, as well as recent trends in vaccine development. Hence, the purpose of this study is to investigate different molecular targets in SARS‐CoV‐2 pathogenesis and their usefulness in developing strategies for drug development.
Fragrance of rice is an important trait that confers a large economic benefit to the farmers who cultivate aromatic rice varieties. Several aromatic rice varieties have limited geographic ...distribution, and are endowed with variety-specific unique fragrances. BADH2 was identified as a fragrance gene in 2005, and it is essential to identify the fragrance alleles from diverse geographical locations and genetic backgrounds. Seeragasamba is a short-grain aromatic rice variety of the indica type, which is cultivated in a limited area in India. Whole genome sequencing of this variety identified a new badh2 allele (badh2-p) with an 8 bp insertion in the promoter region of the BADH2 gene. When the whole genome sequences of 76 aromatic varieties in the 3000 rice genome project were analyzed, the badh2-p allele was present in 13 varieties (approximately 17%) of both indica and japonica types. In addition, the badh2-p allele was present in 17 varieties that already had the loss-of-function allele, badh2-E7. Taken together, the frequency of badh2-p allele (approximately 40%) was found to be greater than that of the badh2-E7 allele (approximately 34%) among the aromatic rice varieties. Therefore, it is suggested to include badh2-p as a predominant allele when screening for fragrance alleles in aromatic rice varieties.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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