Publications of NIPGR Scientists

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Subject: search.filters.subject.Functional genomics

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Now showing 1 - 7 of 7
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    A CRISPR-Cas9 library to target putative redundant gene sets facilitates their functional exploration in grain development in rice
    (Springer Nature Publishing AG, 2025) Yadav, Banita; Sardar, Shaswati; Yadav, Anil; Kumari, Annapurna; Gautam, Mohini; Mandlik, Rushil; Arora, Simran; Kumar, Shailesh; Jewaria, Pawan Kumar; Sonah, Humira; Deshmukh, Rupesh; Chinnusamy, Viswanathan; Ram, Hasthi
    Advent of CRISPR-Cas9 library approach has revolutionized the field of high throughput targeted mutagenesis in plants. By identifying an sgRNA spacer that can target multiple paralogous genes in a genome, higher-order knockout plants can be developed. Using this concept, we developed ten CRISPR-Cas9 pool libraries and generated higher-order knockout plants in rice. Towards this, firstly we identified genome-wide sets of genes which are co-expressed and have high sequence similarity and can be targeted by a single sgRNA. Based on the expression pattern, these genes were divided into ten groups, and subsequently ten CRISPR-Cas9 plasmid libraries were developed. One such library designed against seed-expressed genes was transformed into rice and higher-order knockout plants were developed. Genotyping revealed that around 90% T0 plants had editing, and among the edited plants majority of them were higher-order knockouts. Phenotypic analysis in the next generation discovered functions of several seed specific genes in grain length, width, number and 100-grain weight. By analyzing single and double mutants for two Agenet domain-containing proteins, we have discovered an epistatic interaction between them for grain development. Further application of our approach will help to uncover hidden functions of the targeted genes and accelerate functional genomics research in rice. The CRISPR-Cas9 library is a useful approach to generate higher-order knockout mutants and identify functions of the targeted genes in rice.
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    Global gene regulatory network underlying miR165a in Arabidopsis shoot apical meristem
    (Springer Nature Publishing AG, 2023) Sinha, Sonali; Sahadevan, Sudeep; Ohno, Carolyn; Ram, Hasthi; Heisler, Marcus G
    Arabidopsis microRNA165a (miR165a) targets Class III Homeodomain Leucine-Zipper (HD-ZIPIII) transcription factors to regulate various aspects of plant development and stress response. Overexpression of miR165a mimics the loss-of-function phenotype of HD-ZIPIII genes and leading to ectopic organ formation, shoot apical meristem (SAM) termination, loss of leaf polarity, and defective vasculature development. However, the molecular mechanisms underlying these phenotypes remain unresolved. Here, we over-expressed miR165a in a dexamethasone inducible manner and identifed diferentially expressed genes in the SAM through RNA-Seq. Simultaneously, using multichannel FACS combined with RNA-Seq approach, we characterized global transcriptome patterns in miR165a expressing cell-types compared to HD-ZIPIII expressing cell-types and other cell-types in SAM. By integrating our results we identifed sets of genes which are up-regulated by miR165a as well have enriched expression in miR165a cell-types, and vice-versa. Known plant development related genes such as HD-ZIPIII and their targets LITTLE ZIPPERs, Like AUXIN RESISTANT 2, BEL1-like homeodomain 6, ROTUNDIFOLIA like 16 were found to be down-regulated. Among the up-regulated genes, GIBBERELLIN 2-OXIDASEs, various elemental transporters (YSL3, ZIFL1, SULTR), and other transporter genes were prominent. Thus, the genes identifed in this study help to unravel the molecular mechanism of miR165a and HD-ZIPIII regulated plant development and stress response.
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    Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: a rapid and efficient method for reverse genetics studies
    (BioMed Central Ltd, 2018) Aggarwal, Pooja Rani; Nag, Papri; Choudhary, Pooja; Chakraborty, Niranjan; Chakraborty, Subhra
    Background: Chickpea (Cicer arietinum L.), an important legume crop is one of the major source of dietary protein. Developing an efcient and reproducible transformation method is imperative to expedite functional genomics studies in this crop. Here, we present an optimized and detailed procedure for Agrobacterium rhizogenes-mediated root transformation of chickpea. Results: Transformation positive roots were obtained on selection medium after two weeks of A. rhizogenes inoculation. Expression of green fuorescent protein further confrmed the success of transformation. We demonstrate that our method adequately transforms chickpea roots at early developmental stage with high efciency. In addition, root transformation was found to be genotype-independent and the efcacy of our protocol was highest in two (Annigiri and JG-62) of the seven tested chickpea genotypes. Next, we present the functional analysis of chickpea hairy roots by expressing Arabidopsis TRANSPARENT TESTA 2 (AtTT2) gene involved in proanthocyanidins biosynthesis. Overexpression of AtTT2 enhanced the level of proanthocyanidins in hairy roots that led to the decreased colonization of fungal pathogen, Fusarium oxysporum. Furthermore, the induction of transgenic roots does not afect functional studies involving infection of roots by fungal pathogen. Conclusions: Transgenic roots expressing genes of interest will be useful in downstream functional characterization using reverse genetics studies. It requires 1 day to perform the root transformation protocol described in this study and the roots expressing transgene can be maintained for 3–4 weeks, providing sufcient time for further functional studies. Overall, the current methodology will greatly facilitate the functional genomics analyses of candidate genes in root-rhizosphere interaction in this recalcitrant but economically important legume crop.
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    Enhancing crop yield by optimizing plant developmental features
    (The Company of Biologists, 2016) Mathan, Jyotirmaya; Bhattacharya, Juhi; Ranjan, Aashish
    A number of plant features and traits, such as overall plant architecture, leaf structure and morphological features, vascular architecture and flowering time are important determinants of photosynthetic efficiency and hence the overall performance of crop plants. The optimization of such developmental traits thus has great potential to increase biomass and crop yield. Here, we provide a comprehensive review of these developmental traits in crop plants, summarizing their genetic regulation and highlighting the potential of manipulating these traits for crop improvement. We also briefly review the effects of domestication on the developmental features of crop plants. Finally, we discuss the potential of functional genomics-based approaches to optimize plant developmental traits to increase yield.
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    Expanding frontiers in plant transcriptomics in aid of functional genomics and molecular breeding
    (John Wiley & Sons Ltd, 2014) Agarwal, Pinky; Parida, Swarup K.; Mahto, Arunima; Das, Sweta; Mathew, Iny Elizebeth; Malik, Naveen; Tyagi, Akhilesh K.
    The transcript pool of a plant part, under any given condition, is a collection of mRNAs that will pave the way for a biochemical reaction of the plant to stimuli. Over the past decades, transcriptome study has advanced from Northern blotting to RNA sequencing (RNA-seq), through other techniques, of which real-time quantitative polymerase chain reaction (PCR) and microarray are the most significant ones. The questions being addressed by such studies have also matured from a solitary process to expression atlas and marker-assisted genetic enhancement. Not only genes and their networks involved in various developmental processes of plant parts have been elucidated, but also stress tolerant genes have been highlighted. The transcriptome of a plant with altered expression of a target gene has given information about the downstream genes. Marker information has been used for breeding improved varieties. Fortunately, the data generated by transcriptome analysis has been made freely available for ample utilization and comparison. The review discusses this wide variety of transcriptome data being generated in plants, which includes developmental stages, abiotic and biotic stress, effect of altered gene expression, as well as comparative transcriptomics, with a special emphasis on microarray and RNA-seq. Such data can be used to determine the regulatory gene networks, which can subsequently be utilized for generating improved plant varieties.
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    Recent advances in tomato functional genomics: utilization of VIGS
    (Springer, 2012) Sahu, Pranav Pankaj; Puranik, Swati; Khan, Moinuddin; Prasad, Manoj
    Tomato unquestionably occupies a significant position in world vegetable production owing to its world-wide consumption. The tomato genome sequencing efforts being recently concluded, it becomes more imperative to recognize important functional genes from this treasure of generated information for improving tomato yield. While much progress has been made in conventional tomato breeding, post-transcriptional gene silencing (PTGS) offers an alternative approach for advancement of tomato functional genomics. In particular, virus-induced gene silencing (VIGS) is increasingly being used as rapid, reliable, and lucrative screening strategy to elucidate gene function. In this review, we focus on the recent advancement made through exploiting the potential of this technique for manipulating different agronomically important traits in tomato by discussing several case studies.
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    Crop breeding prospects in the genomic era
    (Taylor & Francis Group, 2006) Kumar, Sushil; Prasad, Manoj
    Grain/cereal crop yields need to be increased in the range of 5t ha 1, to meet the challenge of international food requirements, in the near future. Post-green revolution, conventional plant breeding and related procedures had limited success and grain crop yields have stagnated. The developments in plant genomics offer new means for genetic improvement in crop plants, such as transgenesis and marker aided precise selection of rare recombinants. A strategy, based on the use of existing genomic information on the genetic control of drought tolerance in Arabidopsis thaliana, for breeding wheat for high yields and drought tolerance is exemplified. In future plant breeding, the importance of systems approach, involving multidisciplinary contributions, to the understanding of gene structure Xenvironment interactions in respect of the concerned genes at whole plant or crop level is emphasized.