Identification of full-length genes involved in the biosynthesis of ß-caryophyllene and Lupeol from the leaf transcriptome of Ayapana triplinervis.

ß-Caryophyllene possesses potential anticancer properties against various cancers, including breast, colon, and lung cancer. Therefore, the essential oil of Ayapana triplinervis, which is rich in ß-caryophyllene, can be a potential herbal remedy for treating cancer. However, molecular and genomic studies on A. triplinervis are still sparse. In this study, we obtained 14.7 Gb of RNA-Seq data from A. triplinervis leaf RNA and assembled 1,37,554 transcripts with an N50 value of 1,437 bp. We annotated 72,436 (52.7%) transcripts and mapped 10,640 transcripts to 156 biochemical pathways. Among them, 218 were related to terpenoid backbone biosynthesis, while 27 were linked to sesquiterpenoid and triterpenoid pathways. Ninety-four transcripts were annotated in the ß-caryophyllene and lupeol pathways. From these transcripts, for the first time, we identified 25 full-length genes encoding all the 17 enzymes involved in ß-caryophyllene biosynthesis and an additional five genes involved in lupeol biosynthesis. These genes will be useful for the metabolic engineering of ß-caryophyllene and lupeol biosynthesis, not just in A. triplinervis but also in other species. Keywords: ß-caryophyllene, Eupatorium ayapana, Eupatorium triplinervis, lupeol, transcriptome.

From swamp to field: how genes from mangroves and its associates can enhance crop salinity tolerance.

Salinity stress is a critical challenge in crop production and requires innovative strategies to enhance the salt tolerance of plants. Insights from mangrove species, which are renowned for their adaptability to high-salinity environments, provides valuable genetic targets and resources for improving crops. A significant hurdle in salinity stress is the excessive uptake of sodium ions (Na+) by plant roots, causing disruptions in cellular balance, nutrient deficiencies, and hampered growth. Specific ion transporters and channels play crucial roles in maintaining a low Na+/K+ ratio in root cells which is pivotal for salt tolerance. The family of high-affinity potassium transporters, recently characterized in Avicennia officinalis, contributes to K+ homeostasis in transgenic Arabidopsis plants even under high-salt conditions. The salt overly sensitive pathway and genes related to vacuolar-type H+-ATPases hold promise for expelling cytosolic Na+ and sequestering Na+ in transgenic plants, respectively. Aquaporins contribute to mangroves' adaptation to saline environments by regulating water uptake, transpiration, and osmotic balance. Antioxidant enzymes mitigate oxidative damage, whereas genes regulating osmolytes, such as glycine betaine and proline, provide osmoprotection. Mangroves exhibit increased expression of stress-responsive transcription factors such as MYB, NAC, and CBFs under high salinity. Moreover, genes involved in various metabolic pathways, including jasmonate synthesis, triterpenoid production, and protein stability under salt stress, have been identified. This review highlights the potential of mangrove genes to enhance salt tolerance of crops. Further research is imperative to fully comprehend and apply these genes to crop breeding to improve salinity resilience.

Development of an allele-specific PCR (AS-PCR) method for identifying high-methyl eugenol-containing purple Tulsi (Ocimum tenuiflorum L.) in market samples.

BACKGROUND: Ocimum tenuiflorum L. is a highly traded medicinal with several therapeutic values. Green Tulsi and purple Tulsi are two subtypes in O. tenuiflorum and both have the same medicinal properties. Recent reports have revealed that purple Tulsi contains higher quantities of methyl eugenol (ME), which is moderately toxic and potentially carcinogenic. Therefore, we developed an allele-specific PCR (AS-PCR) method to distinguish the green and purple Tulsi. METHODS AND RESULT: Using the green Tulsi as a reference, 12 single nucleotide polymorphisms (SNPs) and 10 insertions/deletions (InDels) were identified in the chloroplast genome of the purple Tulsi. The C > T SNP at the 1,26,029 position in the ycf1 gene was selected for the development of the AS-PCR method. The primers were designed to amplify 521 bp and 291 bp fragments specific to green and purple Tulsi, respectively. This AS-PCR method was validated in 10 accessions from each subtype and subsequently verified using Sanger sequencing. Subsequently, 30 Tulsi powder samples collected from the market were subjected to molecular identification by AS-PCR. The results showed that 80% of the samples were purple Tulsi, and only 3.5% were green Tulsi. About 10% of the samples were a mixture of both green and purple Tulsi. Two samples (6.5%) did not contain O. tenuiflorum and were identified as O. gratissimum. CONCLUSION: The market samples of Tulsi were predominantly derived from purple Tulsi. The AS-PCR method will be helpful for quality control and market surveillance of Tulsi herbal powders.

Chloroplast genome of Ocimum basilicum var. purpurascens Bentham 1830 (Lamiaceae).

Ocimum basilicum var. purpurascens Bentham 1830 (Red Rubin Basil) is an aromatic herb belonging to the family Lamiaceae and is known for its medicinal uses. It is commonly used in traditional medicine to treat cardiovascular diseases and obesity. It possesses anti-inflammatory, antioxidant, antifungal, and anti-spasmodic properties. In our recent study, we assembled the chloroplast genome sequence of O. basilicum var. purpurascens using Illumina paired-end sequencing technology. The assembled chloroplast genome was 152,407 base pairs (bp), inclusive of a large single-copy (LSC) region accounting for 83,409 bp and a small single-copy (SSC) region spanning 17,604 bp. Two inverted repeats (IRs) interspersed these regions, each 25,697 bp long. The chloroplast genome harbored 132 genes, comprising 88 protein-coding genes, 36 transfer RNA (tRNA), and eight rRNA genes. Among these, nine genes encompassed a single intron, two presented with two introns, with the remaining devoid of any introns. The overall GC content of the chloroplast genome was determined to be 38%. The GC content in the LSC, SSC, and IR regions was 35.9%, 31.6%, and 43.1%, respectively. Our phylogenetic exploration of the chloroplast genomes elucidated that O. basilicum var. purpurascens exhibits close genetic affinity with O. basilicum var. basilicum and other constituents of the Ocimum genus within the Lamiaceae family.

Identification of adulteration in the market samples of saffron using morphology, HPLC, HPTLC, and DNA barcoding methods.

Saffron, the stigma of Crocus sativus 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 complete chloroplast genome of Phyla nodiflora (Linnaeus) Greene (1899) from the Verbenaceae family and its phylogenetic analysis.

Phyla nodiflora (Linnaeus) Greene (1899) is a perennial creeping herb belonging to the family Verbenaceae. It has numerous pharmacological properties, including anti-dandruff, anti-inflammatory, anti-oxidant, anti-melanogenesis, anti-hypertensive, and anti-hyperuricemic properties. We generated the complete chloroplast genome sequence of P. nodiflora using Illumina paired-end sequencing data. The P. nodiflora chloroplast genome is 154,341 bp in length, containing a large single copy (LSC) region of 85,185 bp and a small single copy (SSC) region of 17,222 bp, separated by a pair of inverted repeats (IRs) of 25,967 bp. The genome contained 128 genes, including 86 protein-coding, 34 tRNA, and eight rRNA genes. Six genes had one intron, one gene had two introns, and the others did not have an intron. Overall GC content of the chloroplast genome was 39%, while those of LSC, SSC, and IR regions were 38.2%, 33.7%, and 44%, respectively. Phylogenetic analysis of the chloroplast genome revealed that P. nodiflora is closely related to the other species from Verbenaceae.

The complete chloroplast genome of Ocimum americanum Linnaeus 1755 and phylogenetic analysis among the Lamiaceae family.

Ocimum americanum Linnaeus 1755 (Lemon basil) is an essential medicinal species in the Ocimum genus. Its leaf decoction is traditionally used to treat diabetes, constipation, diarrhea, piles, and dysentery. The essential oils from this species have intense fungicidal activity. The complete chloroplast genome sequence of O. americanum was assembled from Illumina paired-end sequencing data. The O. americanum chloroplast genome was 152,460 bp in length, containing a large single copy (LSC) region of 83,459 bp and a small single copy (SSC) region of 17,607 bp, separated by a pair of inverted repeats (IRs) of 25,697 bp. The genome contained 134 unique genes, including 89 protein-coding, 37 tRNA, and eight rRNA genes. Among them, nine genes had a single intron, and two genes contained two introns. The overall GC content of the chloroplast genome was 38%, while the corresponding values of LSC, SSC, and IR regions were 35.8%, 31.7%, and 43.1%, respectively. In the phylogenetic analysis, all the Ocimum species formed a group closely related to Plectranthus barbatus. O. americanum was more closely related to O. gratissimum and O. basilicum than the other species of Ocimum included in this study.

Whole transcriptome analysis identifies full-length genes for neoandrographolide biosynthesis from Andrographis alata, an alternate source for antiviral compounds.

Andrographolide and related compounds are effective against several viral diseases, including dengue, COVID-19, influenza, and chikungunya. Andrographis paniculata is the primary source for these compounds, but its availability is limited. A. alata is a potential alternative source, and neoandrographolide (NAG) is the major antiviral compound in this species. Since molecular studies in A. alata are scarce, we sequenced its leaf transcriptome to identify the full-length genes involved in neoandrographolide biosynthesis. We assembled 13.6 Gb RNA-Seq data and generated 81,361 transcripts with 1007 bp average length and 1,810 bp N50. The transcripts were categorized under biological processes (2,707), cellular components (678), and molecular functions (2,036). KEGG analysis mapped 975 transcripts to the secondary metabolite pathways. Among the 420 transcripts mapped to terpenoids and polyketides pathways, 142 transcripts were related to the biosynthesis of andrographolide and its derivatives. After a detailed analysis of these transcripts, we identified 32 full-length genes coding for all the 22 enzymes needed for andrographolide biosynthesis. Among them, 15 full-length genes were identified for the first time from Andrographis species. These full-length genes and the transcripts shall serve as an invaluable resource for the metabolic engineering of andrographolides and neoandrographolide in Andrographis and other species.

The complete chloroplast genome of Ocimum basilicum L. var. basilicum (Lamiaceae) and its phylogenetic analysis.

Ocimum basilicum L. var. basilicum (Sweet Basil) is an aromatic herb belonging to the family Lamiaceae and is known for its medicinal uses. It is commonly used in traditional medicine for its therapeutic value, including anti-allergic, anti-inflammatory, antioxidant, antitumor, and antimicrobial properties. In this study, we generated the complete chloroplast genome sequence of O. basilicum var. basilicum using Illumina paired-end sequencing data. The chloroplast genome was 152,407 bp in length, containing a large single-copy (LSC) region of 83,409 bp and a small single-copy region (SSC) of 17,604 bp, separated by a pair of inverted repeats (IRs) of 25,697 bp. The genome contained 134 genes, including 89 protein-coding, 37 tRNA, and eight rRNA genes. Nine genes had one intron, two genes had two introns, and others did not have any intron. Overall GC content of the chloroplast genome was 38%, while that of LSC, SSC, and IR regions was 35.9%, 31.6%, and 43.1%, respectively. Phylogenetic analysis of the chloroplast genomes revealed that O. basilicum var. basilicum was closely related to Ocimum basilicum from the Ocimum species.

Transcriptome analysis of five different tissues of bitter gourd (Momordica charantia L.) fruit identifies full-length genes involved in seed oil biosynthesis.

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.

Case-Control Genotyping of the c.788C>T Variant of Transforming Growth Factor-Beta 1 Gene in Otosclerosis in the South Indian Population.

BACKGROUND: Otosclerosis is a common conductive hearing loss resulting from abnormal bone metabolism. The c.788C>T variant in the transforming growth factor-beta 1 gene is associated with otosclerosis in all studied populations, except the Indian population. In this study, we predicted the functional effects of reported variants in transforming growth factor-beta 1 and analyzed the c.788C>T variant in a case-control cohort from India and in the genomes present in public databases. METHODS: Clinically confirmed otosclerosis cases (n=120) and controls (n=120) were recruited and genotyped by polymerase chain reactionrestriction fragment length polymorphism and DNA sequencing. In addition, Ensembl 1000 Genome, Ensembl NHLBI Exome, GnomAD, and Genome Asia 100K human genome databases were analyzed for allele frequency. RESULTS: Among the 3 variants studied, a significant functional effect was observed only for the c.788C>T variant. This variant was found in 1 case but absent in all others and controls. Odds ratio, 95% CI, and P-value under the dominant model were 1.00, 0.0197-50.8116, and 1.00, respectively. Analysis of genomic databases showed a frequency of 0-11.21% and 0-1.25% for the c.788C>T variant and the individuals homozygous for this variant, respectively. CONCLUSION: We did not find any genetic association between the c.788C>T variant and otosclerosis in the South Indian population; however, it was not monomorphic as had previously been reported from the Odisha population of Eastern India. Moreover, contrary to an earlier report that the c.788C>T variant was never found in a homozygous condition, homozygous individuals were found in the European, Asian, Latin American, and Ashkenazi Jews populations.

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

The complete chloroplast genome (cp) of Ocimum tenuiflorum L. subtype Krishna Tulsi was sequenced and assembled using Illumina paired-end sequencing data. The cp genome is 151,758 bp in length, including a large single copy (LSC) region of 82,794 bp, a small single-copy region (SSC) of 17,592 bp, and a pair of inverted repeated (IR) region of 25,686 bp. The cp genome of Krishna Tulsi encodes 129 genes, including 90 protein-coding, 31 transfer RNA (tRNA), and eight ribosomal RNA (rRNA) genes. While the overall GC content was 37.9%, it is 36.0%, 31.8%, and 43.1% in the LSC, SSC, and IR regions, respectively. Phylogenetic analysis based on chloroplast genome sequences of 17 species from Lamiaceae revealed that O. tenuiflorum subtype Krishna Tulsi is clustered with other Ocimum species, and forms a clade with genera from family Lamiaceae.

A reference-grade genome identifies salt-tolerance genes from the salt-secreting mangrove species Avicennia marina.

Water scarcity and salinity are major challenges facing agriculture today, which can be addressed by engineering plants to grow in the boundless seawater. Understanding the mangrove plants at the molecular level will be necessary for developing such highly salt-tolerant agricultural crops. With this objective, we sequenced the genome of a salt-secreting and extraordinarily salt-tolerant mangrove species, Avicennia marina, that grows optimally in 75% seawater and tolerates >250% seawater. Our reference-grade ~457 Mb genome contains 31 scaffolds corresponding to its chromosomes. We identified 31,477 protein-coding genes and a salinome consisting of 3246 salinity-responsive genes and homologs of 614 experimentally validated salinity tolerance genes. The salinome provides a strong foundation to understand the molecular mechanisms of salinity tolerance in plants and breeding crops suitable for seawater farming.

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.

The complete chloroplast genome and phylogenetic analysis of Ocimum kilimandscharicum Gurke (Camphor Basil) from India.

Ocimum kilimandscharicum Gurke commonly known as Camphor Basil, is a medicinal plant species that belongs to the Lamiaceae family. Here, the sequencing and characterization of complete chloroplast genome sequence of O. kilimandscharicum is reported for the first time using Illumina paired-end sequencing data. The size of the chloroplast (cp) genome is 151,741 bp in length, with a large single-copy (LSC) region of 82,882 bp and a small single-copy (SSC) region of 17,587 bp, separated by a pair of 25,636 bp inverted repeat (IR) regions. There are 135 predicted genes, including 90 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes in the genome, and the overall GC content of the genome is 37.9%. The phylogenetic analysis based on the chloroplast genome data indicated that O. kilimandscharicum is closer to O. tenuiflorum and clustered to other Ocimum species in Lamiaceae.

The complete chloroplast genome of Ocimum gratissimum from India - a medicinal plant in the Lamiaceae.

Ocimum gratissimum L. is an important medicinal species with several therapeutic applications. It is used in traditional medicine as a single drug and in formulations. We generated the complete chloroplast genome sequence of O. gratissimum by using Illumina paired-end sequencing data. The O. gratissimum chloroplast genome is 152,469 bp in length, containing a large single copy (LSC) region of 83,614 bp and a small single copy region (SSC) of 17,607 bp, separated by a pair of inverted repeats (IRs) of 25,624 bp. The genome contains 138 unique genes, including 85 protein-coding, 45 tRNA, and eight rRNA genes. Among them, six genes have one intron each, and two genes contain two introns. The overall GC content of the chloroplast genome is 37.8%, while the corresponding values of LSC, SSC, and IR regions are 35.6%, 31.7%, and 43.2%, respectively. Phylogenetic analysis with the complete chloroplast genomes of other related species revealed that O. gratissimum is fully resolved in a clade with other Ocimum species classified to the family Lamiaceae.

Forensic application of DNA barcoding in the identification of commonly occurring poisonous plants.

Exposure to poisonous plants is hazardous to health; thus, reliable species identification is required to decide the most appropriate treatment. Since ingested plants are too much degraded for visual observation, DNA barcoding can be used as a molecular tool for species identification. Considering the universal primers, PCR and sequencing success rate, and diversity of the poisonous plants, the rbcL DNA marker was selected for molecular identification. A reference DNA barcode library for 100 poisonous plant species was created using rbcL DNA barcodes. For the poisonous plants represented in the library, 100% and 89% species differentiation was observed at the genus and species level, respectively. All the undifferentiated species were congeneric species. Mapping the metabolites of the poisonous plants to the DNA based phylogenetic tree indicated that the phylogenetically related species also had related toxic compounds. Therefore, genus-level identification may be sufficient in the practical application of DNA barcoding in poisoning cases. We conclude that rbcL can be used as a primary marker, and if required, ITS2 or trnH-psbA may be used as a secondary marker to identify the poisonous plants. The present study provides the foundation to develop a reliable molecular method to identify the poisonous species from the vomit samples of poisoning cases.

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%.

Severe acute respiratory syndrome-coronavirus-2: Current advances in therapeutic targets and drug development.

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.

Protein extracts from Momordica charantia var. charantia and M. charantia var. muricata show anti-lipidemic and antioxidant properties in experimental type 2 diabetic rats.

Plant-derived compounds are used to manage dyslipidemia and oxidative stress in type 2 diabetic condition. In this study, anti-lipidemic and antioxidant properties of the protein extracts from "Charantia" (PEC) and "Muricata" (PEM) varieties of Momordica charantia were analyzed by quantifying lipids, hepatic, renal, and oxidative stress markers, and histopathological examination of liver and kidney tissues. Protein extracts were orally administered at 10 (PEC10, PEM10) or 20 mg/kg body weight (PEC20, PEM20). Levels of cholesterol, low-density lipoprotein, and triglycerides decreased but high-density lipoprotein increased significantly in treated rats as compared to untreated diabetic rats (p < .01), and attained normal physiological range in both doses. Levels of superoxide dismutase, catalase, glutathione peroxidase, and reduced glutathione increased but thiobarbituric acid reactive substances decreased significantly in treated rats as compared to untreated diabetic rats (p < .01), and attained normal physiological range in PEM20 only. Histopathological examinations supported a protective role for the protein extracts against oxidative stress. PRACTICAL APPLICATIONS: Momordica charantia, a well-known medicinal plant is traditionally used for treating diabetes in India as well as other countries. The whole plant was shown to have medicinal importance. Anti-diabetic potential of this plant was scientifically established largely using organic extracts and water extract mainly from the fruits of this plant. However, protein extracts from the seeds and fruit pulp of this plant were also proven to have anti-diabetic activity. The present study illustrates the anti-lipidemic and antioxidant effect of protein extracts from the fruit pulp of two varieties of M. charantia (Charantia and Muricata) in Streptozotocin-induced type 2 diabetic rats. This study provides experimental evidence in support of its use in the management of type 2 diabetes-related complications.