Department Of Botany
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Item Selection constraints determine preference for A/U-ending codons in Taxus contorta(Canadian Science Publishing, 2020) Majeed, Aasim; Kaur, Harpreet; Bhardwaj, PankajUnequal utilization of synonymous codons is a well-known phenomenon among living organisms. This phenomenon plays a major role in the enhancement of the accuracy and efficiency of translation. Gymnosperms are rarely paid attention in this aspect. Understanding the degree of and determining the forces influencing codon usage bias (CUB) in Taxus contorta, an endangered Himalayan gymnosperm, will prove useful in interpreting the evolutionary characteristics of this species. Using RNAseq data, 93?790 assembled transcripts were clustered into 32?701 unigenes. Around 13?061 full-length sequences were utilized for the analysis of CUB. Compositional properties showed that GC-content ranged from 28.76% to 65.22%, with an average value of 44.28%, suggesting an AT-rich genome. The mean effective number of codons (ENC) value revealed that CUB is not strong in T. contorta. The preferred codons tended to be A/U ending, whereas the avoided codons tended to be G/C ending. A P2 index of 0.54 and a Mutation Responsive Index (MRI) value of-0.02 in addition to the results revealed by the neutrality, ENC, and parity plots showed that natural selection is a predominating factor governing CUB. Mutational pressure, gene length, hydropathiciy, aromaticity, and nucleotide composition influence CUB weaklyItem Comparative transcriptome profiling reveals the reprogramming of gene networks under arsenic stress in Indian mustard.(Canadian Science Publishing, 2019) Thakur, Sapna; Choudhary, Shruti; Dubey, Preeti; Bhardwaj, PankajArsenic is a widespread toxic metalloid that is classified as a class I carcinogen known to cause adverse health effects in humans. In the present study, we investigated arsenic accumulation potential and comparative gene expression in Indian mustard. The amount of arsenic accumulated in shoots varied in the range of 15.99-1138.70 mg/kg on a dry weight basis among five cultivars. Comparative expression analysis revealed 10?870 significantly differentially expressed genes mostly belonging to response to stress, metabolic processes, signal transduction, transporter activity, and transcription regulator activity to be up-regulated, while most of the genes involved in photosynthesis, developmental processes, and cell growth were found to be down-regulated in arsenic-treated tissues. Further, pathway analysis using the KEGG Automated Annotation server (KAAS) revealed a large-scale reprogramming of genes involved in genetic and environmental information processing pathways. Top pathways with maximum KEGG orthology hits included carbon metabolism (2.5%), biosynthesis of amino acids (2.1%), plant hormone signal transduction (1.4%), and glutathione metabolism (0.6%). A transcriptomic investigation to understand the arsenic accumulation and detoxification in Indian mustard will not only help to improve its phytoremediation efficiency but also add to the control measures required to check bioaccumulation of arsenic in the food chain.Item Molecular basis of transitivity in plant RNA silencing(Springer, 2019) Choudhary, S; Thakur, S; Bhardwaj, PankajThe discovery of small RNAs has offered exciting opportunities in manipulating gene expression. The non-coding RNAs cause target gene inactivation at the transcriptional, post-transcriptional or translational level. In addition to the default silencing approach, they provide another mode of gene regulation by transitivity. Here, gradual amplification in effector RNAs number allows regulation of genes other than the original target and causes the outspread of silencing from its origin to aid a robust response. Unlike the short-range cell-to-cell movement of silencing signal (through plasmodesmata), little is known of the mediators of systemic silencing (usually through phloem). Through the present review, we combine the reports available so far to better understand the characteristics of secondary silencing, factors involved, and summarize the instances where it has been employed in plants. Understanding the molecular mechanism behind transitivity has led to the designing of efficient transgenes for targeted gene inactivation, utilized in silencing of a multigene family, and in the field of functional genomics. Studies uncovering the origin of distinct secondary silencing pathways in plants have been exploited for developing artificial RNA silencing methods such as hairpin RNA, artificial microRNA, intrinsic direct repeat, inverted repeat, artificial trans-acting siRNA, phased siRNA, and virus-induced gene silencing. The techniques have facilitated the spread of traits such as pathogenic resistance or alter plant growth and development features. The mechanism of reprogramming in the silencing machinery and the consequent genetic manipulation through transitive RNA is still not completely understood and its exploitation in crop improvement programmes is still in a developing phase. © 2019, Springer Nature B.V.Item Transcriptome characterization and development of functional polymorphic SSR marker resource for Himalayan endangered species, Taxus contorta (Griff)(Elsevier, 2019) Majeed, A; Singh, A; Choudhary, S; Bhardwaj, PankajTaxus contorta is an important medicinal plant currently listed as endangered in IUCN Red Data List. It produces an anticancer drug, paclitaxel which is well known in the industrial sector. Due to habitat destruction and overexploitation, it is at the verge of extinction. Genomic and transcriptomic data for this species is scarce which has hampered its genomic studies. Moreover, large scale polymorphic informative codominant marker resource is also scarce which hinders its population and landscape genetic analysis. Here, we generated a reference transcriptome for this species which would facilitate the understanding of the functional elements and promote genomic research in this species. Also, a robust polymorphic SSR marker resource was characterized which can be used in conservation of this species. More than 100 million paired end raw reads were obtained through Illumina sequencing. A total of 129,869 unigenes with mean sequence length of 1244 nt were obtained from 209,860 de novo assembled transcripts. Of these, 35,752 transcripts were assigned 5971 unique GO terms. Around 40,386 transcripts were found to have 2163 unique Pfam Ids. Pathway analysis against KEGG database yielded 3721 unique enzyme numbers. Screening of the transcripts for microsatellite regions yielded 7041 SSRs. Among the 100 SSRs selected for characterization on 30 genotypes, 37 polymorphic markers showed a total of 214 alleles with mean of 5.78 alleles per locus. Mean effective number of alleles (Ne) was found to be 3.64 and average PIC value of 0.64 was observed. Observed heterozygosity (0.57) was found to be lower than expected (0.69). This effective polymorphic SSR marker resource will act as valuable tool for deciphering its genetic diversity. © 2019 Elsevier B.V.Item SSR marker based DNA fingerprinting and diversity assessment in superior tea germplasm cultivated in western Himalaya(Indian National Academic Science, 2014) Bhardwaj, Pankaj; Sharma, Rajesh Kumar; Kumar, Rajendra; Sharma, Hemlata; Ahuja, Paramvir SinghTwenty one microsatellites (genomic & genic) markers were used to evaluate genetic diversity and DNA fingerprinting of 15 popular tea accessions. Each accession had a unique marker profile, indicating that microsatellite markers were useful in differentiation studies among the tea collections. A total of 127 polymorphic alleles were scored with an allele frequency of 6.05 per primer. The polymorphism information content (PIC) ranged from 0.2 (CamsinM1) to 0.60 (TUGMS12), with an average of 0.359. SSR markers analysis detected a high level of heterozygosity (av Ho 0.775; He 0.847) in tea. The Jaccard's similarity coefficients ranged from 0.15 to 0.56 with an average similarity index (ASI) of 0.234. The first two coordinate explained 54.33% of the total variance. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram and the PCoA (Principle coordinate analysis) indicated that the populations formed two major groups with exclusive China and China hybrids (I) and Assam types (II). The collections from western Himalayan possessed a moderate to high level of genetic diversity which could provide valid guidelines for genetic improvement of teaItem RNAseq-based phylogenetic reconstruction of Taxaceae and Cephalotaxaceae(Wiley, 2018) Majeed, Aasim; Singh, Amandeep; Choudhary, Shurti; Bhardwaj, PankajTaxaceae and Cephalotaxaceae are the two economically important conifer families. Over the years there has been much controversy over the issue of merging these families. The position of Amentotaxus and Torreya is also ambiguous. Some authors consider them closer to Taxaceae while others deemed them to fit within Cephalotaxaceae. Still, others prefer to raise them to their own tribe. Different morphological, anatomical, embryological and phylogenetic evidence supports one or the other view, making the precise delineation between them unresolved. Here we used an RNAseq?based approach to obtain orthologous genes across the selected species to reconstruct a more robust phylogeny of these families. A total of 233.123 million raw reads were de novo assembled to generate nine different transcript assemblies for the corresponding species. Of the 940 191 assembled transcripts across nine species, we generated 409 734 unigenes, which were clustered into orthologous groups. A total of 331 single?copy complete orthologous groups were selected for phylogenetic analysis. Maximum?likelihood, maximum?parsimony and Bayesian phylogenetic trees showed a sister relationship between Taxaceae and Cephalotaxaceae. Our analysis supports their distinctiveness at the family level and also shows that Amentotaxus and Torreya fit within Cephalotaxaceae.Item Gene and metabolite profiling reveals flowering and survival strategies in Himalayan Rhododendron arboreum(Elsevier, 2019) Choudhary, Shurti; Thakur, Sapna; Jaitak, Vikas; Bhardwaj, PankajRhododendron arboreum inhabits the Himalayan climate otherwise detrimental to many species, though the underlying survival mechanism remains unclear. Such temperate species have an inherent endurance towards freezing temperature which is prerequisite for an initiation and transition to flowering phase. Orchestrating the molecular architecture is vital towards managing distinct abiotic signals. To determine the molecular factors directing growth, development, and tolerance under environmental extremes in the species, the high-throughput transcriptome and metabolome data from vegetative as well as cold-acclimatized flowering season tissues were generated. Firstly, the de novo assembly pertaining to the foliar and floral tissues comprising of 157,427 unigenes was examined for a comparative analysis. 4149 of 12,577 transcripts observed with a significant fluctuating expression corresponded to seasonal retorts. Following the interactive network, 525 genes were distinguished as the epicenters of sense, response, and tolerance. Secondly, liquid chromatography coupled to mass spectrometry was adopted to profile the extent of metabolite richness across the tissues of two seasons. Taking into account the formula-based mappings offered by MetaboSearch tool, 421 unique ions obtained were annotated to 173 KEGG compounds, especially secondary metabolites. Moreover, by integrating the transcript and metabolite annotations, it was found that right from active metabolism, signaling, development, and their regulations, supplementary response to abiotic/biotic stimuli was induced. A multifaceted response displayed during flowering not only sponsored the climatic encounters but brought the shift from vegetative to reproductive growth. Overall, this comprehensive approach following transcriptome and non-targeted metabolome elucidated the contribution of genetic and metabolic factors in environmental responsesItem Comparative Transcriptome Profiling Under Cadmium Stress Reveals the Uptake and Tolerance Mechanism in Brassica juncea(Springer, 2019) Thakur, Sapna; Choudhary, Shurti; Bhardwaj, PankajCadmium (Cd) is a biologically non-essential and phytotoxic heavy metal pollutant. In this study, we estimated the Cd accumulation potential of Indian mustard and identified factors responsible for Cd uptake, tolerance, and detoxification. Eight transcriptomic libraries were sequenced and ??230 million good quality reads were generated. The alignment rate against B. juncea reference genome V1.5 varied in the range of 85.03-90.06%. Comparative expression analysis using DESeq2 revealed 11,294 genes to be significantly differentially expressed under Cd treatment. The agriGO singular enrichment analysis revealed genes related to response to chemical, oxidative stress, transport, and secondary metabolic process were upregulated, whereas multicellular organismal development, developmental process, and photosynthesis were downregulated by Cd treatment. Furthermore, 616 membrane transport proteins were found to be significantly differentially expressed. Cd-related transporters such as metal transporter (Nramp1), metal tolerance protein (MTPC2, MTP11), cadmium-transporting ATPase, and plant cadmium resistance protein (PCR2, PCR6) were upregulated whereas cadmium/zinc-transporting ATPase (HMA2, HMA3, HMA4), high-affinity calcium antiporter (CAX1), and iron transport protein (IRT1) were downregulated by Cd treatment. A total of 332 different gene-networks affected by Cd stress were identified using KAAS analysis. Various plant hormones signaling cascades were modulated suggesting their role in Cd stress tolerance. The regulation overview using MapMan analysis also revealed gene expression related to plant hormones, calcium regulation, and MAP kinases were altered under Cd stress.Item Exploring microRNA profiles for circadian clock and flowering development regulation in Himalayan Rhododendron(Elsevier, 2018) Choudhary, Shruti; Thakur, Sapna; Majeed, Aasim; Bhardwaj, PankajmiRNA is a non-coding, yet crucial entity in remodeling the genetic architecture. Rhododendron arboreum of Himalayas grows and even flower under fluctuating climate. sRNA from leaves of vegetative and reproductive periods was sequenced to elucidate its seasonal associations. Conserved (256) and novel (210) miRNAs and their precursors were located based on homology with plant databases and transcriptome of the species. 27,139 predicted targets were involved with metabolism, reproduction, and response to abiotic stimuli. A comparative analysis showed differential expression of 198 miRNAs with season-specific abundance of 103 miRNAs. Specific isoforms of 11 miRNA families exhibited a temporal expression and targeted different genes implying a complex regulation. The variable miRNA expression among the tissues of different conditions can be associated with the adaptability of the species, which will prove essential for further study on miRNAs mediating seasonal response. Moreover, exogenous cues also mediate phase transition via networking of flowering pathways and their components. In this context, 18 known families and 77 novel miRNAs modulating 117 genes crucial in circadian entrainment were filtered. A negative correlation was obtained between the expression of 18 of these miRNAs and their targets when tested through quantitative-PCR. It highlighted the role of miRNA-target pairs in perceiving environmental variabilities and monitoring flowering growth. Furthermore, a phylogenetic clustering was performed, which supported the lineage-specific evolution and function of putative miR156 sequence in the species. This documentation of genome-wide profiling of miRNA, their targets, and expression will enhance the understanding of developmental and climate-tolerance strategies in high-altitude trees.Item Identification and characterization of novel UniGene-derived microsatellite markers in Podophyllum hexandrum (Berberidaceae).(2013) Nag, Akshay; Bhardwaj, Pankaj; Ahuja, Paramvir Singh.; Sharma, Ram Kumar