Botany - Research Publications
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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 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 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 KumarItem Genetic diversity and population structure of Melia azedarach in North-Western Plains of India(Springer Verlag, 2016) Thakur, Sapna; Choudhary, Shruti; Singh, Amandeep; Ahmad, Kamal; Sharma, Gagan; Majeed, Aasim; Bhardwaj, PankajKey message: Genetic structure amongM. azedarachpopulations was detected and two subpopulations were present among them. A significant ?isolation by distance? was found inM. azedarachpopulation in North-Western Plains of India. Abstract: Melia azedarach is an important forest tree with pharmaceutical, insecticidal, pesticidal, and commercial significance. It is a good reforestation tree because of its fast growth and drought hardy nature. Genetic variation in a species allows itself to adapt, evolve and respond to environmental stress. It provides the basis for survival of a species and critically influences its evolutionary potential. Assessment of genetic diversity is necessary for improvement and conservation of a species. For this, microsatellite markers are of particular interest given the attributes like co-dominance, reproducibility, hyper variability and abundance throughout the genome. In the present study, we analyzed the genetic diversity and population structure of M. azedarach, an ecologically imperative species growing in the North-Western Plains of India. We developed 43 microsatellite markers, of which 20 were subsequently employed for analysis of diversity and population structure among 33 populations encompassing 318 genotypes representing North-Western Plains of India. A moderate level of diversity (Na?=?5.1, Ho?=?0.506, He?=?0.712, I?=?1.386) was assessed. The highest value of ?K estimated using STRUCTURE indicated 2 subpopulations (K?=?2). AMOVA exhibited 73?% variation within populations and 12?% variation was found among regions. Significant positive correlation between geographical and genetic distance was found (Rxy?=?0.365, P?=?0.010). The present study lays a foundation on a better understanding of genetic dynamics of the species and reveals its diversity and population structure in North-Western Plains of India. ? 2016, Springer-Verlag Berlin Heidelberg.Item Development and characterization of genomic microsatellite markers in Tinospora cordifolia(Springer India, 2017) Gargi, Madhuranjana; Thakur, Sapna; Anand, Shashi Shekhar; Choudhary, Shruti; Bhardwaj, Pankaj; Gargi, M.; Thakur, S.; Anand, S.S.; Choudhary, S.; Bhardwaj, P.Item Identification and cross-species amplification of microsatellite markers derived from expressed sequence data of rose species(Springer India, 2015) Sharma, Ram Kumar; Chaudhary, Anil; Sharma, Himanshu; Bhardwaj, Pankaj; Sharma, Vikas; Kumar, Rahul; Ahuja, Paramveer SinghGenic SSR markers derived from public expressed sequence tags (ESTs) data are valuable and cost effective marker resources for genome mapping and diversity studies. Owing to their derivation from the transcribed regions which often have putative functions, these markers can be easily associated with desired trait. In the present study, 19 novel SSR markers were identified from 450 non redundant unigenes derived from 3,726 public ESTs of two rose species. Among SSRs, tri-repeats (61.3?%) were most abundant followed by di-repeat (29?%). Newly identified EST-SSR markers recorded significant homology with the known/putative proteins of Arabidopsis thaliana. The cross transferability to 12 rose species ranged from 63.2 to 100?%. Novel SSR loci found to be moderately to highly polymorphic with locus wise average number of alleles and polymorphism information content (PIC) were 4.1 and 0.33, respectively. Cloning and sequencing of EST-SSR size variant amplicons of marker locus Rches12 revealed that the variation in the number of SSR repeat-units was the main source of fragment polymorphism. The high polymorphic potential coupled with high cross-transferability rate demonstrates wider applicability of novel SSR markers in genetic diversity, genome mapping and evolutionary studies in various rose species. ? 2014, Society for Plant Biochemistry and Biotechnology.Item Development and characterization of genomic microsatellite markers in Prosopis cineraria(Elsevier B.V., 2017) Anand, Shashi Shekhar; Thakur, Sapna; Gargi, Madhuranjana; Choudhary, Shruti; Bhardwaj, Pankaj; Anand, S.S.; Thakur, S.; Gargi, M.; Choudhary, S.; Bhardwaj, P.Characterization of genetic diversity is a must for exploring the genetic resources for plant development and improvement. Prosopis cineraria is ecologically imperative species known for its innumerable biological benefits. Since there is a lack of genetic resources for the species, so it is crucial to unravel the population dynamics which will be very effective in plant improvement and conservation strategies. Of the 41 genomic microsatellite markers designed from (AG)n enriched library, 24 were subsequently employed for characterization on 30 genotypes of Indian arid region. A total of 93 alleles with an average 3.875 could be amplified by tested primer pairs. The average observed and expected heterozygosity was 0.5139 and 0.5786, respectively with 23 primer pairs showing significant deviations from Hardy-Weinberg equilibrium. Polymorphic information content average to 0.5102 and the overall polymorphism level was found to be 93.27%. STRUCTURE analysis and DARwin exhibited the presence of 4 clusters among 30 genotypes. ? 2017 The Authors