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    Serine hydroxymethyltransferase6 is involved in growth and resistance against pathogens via ethylene and lignin production in Arabidopsis
    (John Wiley & Sons, 2024) Singh, Pooja; Kumari, Aprajita; Khaladhar, Vemula Chandra; Singh, Namrata; Pathak, Pradeep Kumar; Kumar, Vinod; Kumar, Ritika Jantu; Jain, Priyanka; Thakur, Jitendra K.; Fernie, Alisdair R.; Bauwe, Hermann; Raghavendra, A.S.; Gupta, Kapuganti Jagadis
    Photorespiratory serine hydroxymethyltransferases (SHMTs) are important enzymes of cellular one-carbon metabolism. In this study, we investigated the potential role of SHMT6 in Arabidopsis thaliana. We found that SHMT6 is localized in the nucleus and expressed in different tissues during development. Interestingly SHMT6 is inducible in response to avirulent, virulent Pseudomonas syringae and to Fusarium oxysporum infection. Overexpression of SHMT6 leads to larger flowers, siliques, seeds, roots, and consequently an enhanced overall biomass. This enhanced growth was accompanied by increased stomatal conductance and photosynthetic capacity as well as ATP, protein, and chlorophyll levels. By contrast, a shmt6 knockout mutant displayed reduced growth. When challenged with Pseudomonas syringae pv tomato (Pst) DC3000 expressing AvrRpm1, SHMT6 overexpression lines displayed a clear hypersensitive response which was characterized by enhanced electrolyte leakage and reduced bacterial growth. In response to virulent Pst DC3000, the shmt6 mutant developed severe disease symptoms and becomes very susceptible, whereas SHMT6 overexpression lines showed enhanced resistance with increased expression of defense pathway associated genes. In response to Fusarium oxysporum, overexpression lines showed a reduction in symptoms. Moreover, SHMT6 overexpression lead to enhanced production of ethylene and lignin, which are important components of the defense response. Collectively, our data revealed that SHMT6 plays an important role in development and defense against pathogens.
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    Petal abscission in roses is associated with the activation of a truncated version of the animal PDCD4 homologue, RbPCD1
    (Elsevier B.V., 2019) Singh, Priya; Singh, Amar Pal; Tripathi, Siddharth Kaushal; Kumar, Vinod; Sane, Aniruddha P.
    Abscission is a developmental process that leads to shedding of organs not needed by the plant. Apart from wallhydrolysis, the cells of the abscission zone (AZ) are also believed to undergo programmed cell death (PCD). Weshow that ethylene-induced petal abscission inRosa bourbonianais accompanied with the activation ofRbPCD1(PROGRAMMED CELL DEATH LIKE 1) encoding a protein of 78 amino acids. Its expression increases duringnatural and ethylene-induced petal abscission. Its transcription in most tissues is up-regulated by ethylene.RbPCD1 shows similarity to the N-terminal domain of animal PDCD4 (PROGRAMMED CELL DEATH PROTEIN 4)proteins that are activated during apoptosis and function as transcriptional and translational repressors. RbPCD1resides in the nucleus and cytoplasm and acts as a transcriptional repressor. Constitutive expression ofRbPCD1intransgenic Arabidopsis is seedling lethal. Heat-induced expression ofRbPCD1under the soybean heat-shockpromoter affects leaf function, inflorescence development, silique formation, seed yield and reduces survival.Nuclear localization of RbPCD1 is necessary for manifestation of its effects. RbPCD1 may be necessary to mediatesome of the ethylene-induced changes during abscission and senescence in specific tissues.
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    Nitric oxide accelerates germination via the regulation of respiration in chickpea
    (Oxford University Press, 2019) Pandey, Sonika; Kumari, Aprajita; Shree, Manu; Kumar, Vinod; Singh, Pooja; Bharadwaj, Chellapilla; Loake, Gary J.; Parida, Swarup K.; Masakapalli, Shyam Kumar; Gupta, Kapuganti Jagadis
    Seed germination is crucial for the plant life cycle. We investigated the role of nitric oxide (NO) in two chickpea varieties that differ in germination capacity: Kabuli, which has a low rate of germination and germinates slowly, and Desi, which shows improved germination properties. Desi produced more NO than Kabuli and had lower respiratory rates. As a result of the high respiration rates, Kabuli had higher levels of reactive oxygen species (ROS). Treatment with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) reduced respiration in Kabuli and decreased ROS levels, resulting in accelerated germination rates. These findings suggest that NO plays a key role in the germination of Kabuli. SNAP increased the levels of transcripts encoding enzymes involved in carbohydrate metabolism and the cell cycle. Moreover, the levels of amino acids and organic acids were increased in Kabuli as a result of SNAP treatment. 1 H-nuclear magnetic resonance analysis revealed that Kabuli has a higher capacity for glucose oxidation than Desi. An observed SNAP-induced increase in 13C incorporation into soluble alanine may result from enhanced oxidation of exogenous [13C]glucose via glycolysis and the pentose phosphate pathway. A homozygous hybrid that originated from a recombinant inbred line population of a cross between Desi and Kabuli germinated faster and had increased NO levels and a reduced accumulation of ROS compared with Kabuli. Taken together, these findings demonstrate the importance of NO in chickpea germination via the control of respiration and ROS accumulation.
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    Genetic dissection of seed-iron and zinc concentrations in chickpea
    (Nature Publishing Group, 2016) Upadhyaya, Hari D.; Bajaj, Deepak; Das, Shouvik; Kumar, Vinod; Gowda, C. L. L.; Sharma, Shivali; Tyagi, Akhilesh K.; Parida, Swarup K.
    he SNP-based high-resolution QTL mapping mapped eight major genomic regions harbouring robust QTLs governing seed-Fe and Zn concentrations (39.4% combined phenotypic variation explained/PVE) on six chromosomes of an intra-specific high-density genetic linkage map (1.56 cM map-density). 24620 SNPs discovered from genome-wide GBS (genotyping-by-sequencing) and 13 known cloned Fe and Zn contents-related chickpea gene-orthologs were genotyped in a structured population of 92 sequenced desi and kabuli accessions. The large-scale 16591 SNP genotyping- and phenotyping-based GWAS (genome-wide association study) identified 16 genomic loci/genes associated (29% combined PVE) with seed-Fe and Zn concentrations. Of these, 11 trait-associated SNPs in the genes linked tightly with eight QTLs were validated by QTL mapping. The seed-specific expression, including pronounced differential-regulation of 16 trait-associated genes particularly in accessions/mapping individuals with contrasting level of seed-Fe and Zn contents was apparent. Collectively, the aforementioned rapid integrated genomic strategy led to delineate novel functional non-synonymous and regulatory SNP allelic-variants from 16 known/candidate genes, including three strong trait-associated genes (encoding late embryogenesis abundant and yellow stripe-like 1 protein, and vacuolar protein sorting-associated protein) and eight major QTLs regulating seed-Fe and Zn concentrations in chickpea. These essential inputs thus have potential to be deployed in marker-assisted genetic enhancement for developing nutritionally-rich iron/zinc-biofortified chickpea cultivars.
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    Genome-wide scans for delineation of candidate genes regulating seed-protein content in chickpea
    (Frontiers Media S.A., 2016) Upadhyaya, Hari D.; Bajaj, Deepak; Narnoliya, Laxmi; Das, Shouvik; Kumar, Vinod; Gowda, C. L. L.; Sharma, Shivali; Tyagi, Akhilesh K.; Parida, Swarup K.
    Identification of potential genes/alleles governing complex seed-protein content (SPC) is essential in marker-assisted breeding for quality trait improvement of chickpea. Henceforth, the present study utilized an integrated genomics-assisted breeding strategy encompassing trait association analysis, selective genotyping in traditional bi-parental mapping population and differential expression profiling for the first-time to understand the complex genetic architecture of quantitative SPC trait in chickpea. For GWAS (genome-wide association study), high-throughput genotyping information of 16376 genome-based SNPs (single nucleotide polymorphism) discovered from a structured population of 336 sequenced desi and kabuli accessions [with 150–200 kb LD (linkage disequilibrium) decay] was utilized. This led to identification of seven most effective genomic loci (genes) associated [10–20% with 41% combined PVE (phenotypic variation explained)] with SPC trait in chickpea. Regardless of the diverse desi and kabuli genetic backgrounds, a comparable level of association potential of the identified seven genomic loci with SPC trait was observed. Five SPC-associated genes were validated successfully in parental accessions and homozygous individuals of an intra-specific desi RIL (recombinant inbred line) mapping population (ICC 12299 × ICC 4958) by selective genotyping. The seed-specific expression, including differential up-regulation (>four fold) of six SPC-associated genes particularly in accessions, parents and homozygous individuals of the aforementioned mapping population with a high level of contrasting SPC (21–22%) was evident. Collectively, the integrated genomic approach delineated diverse naturally occurring novel functional SNP allelic variants in six potential candidate genes regulating SPC trait in chickpea. Of these, a non-synonymous SNP allele-carrying zinc finger transcription factor gene exhibiting strong association with SPC trait was found to be the most promising in chickpea. The informative functionally relevant molecular tags scaled-down essentially have potential to accelerate marker-assisted genetic improvement by developing nutritionally rich chickpea cultivars with enhanced SPC.
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    Identification of candidate genes for dissecting complex branch number trait in chickpea
    (Elsevier B.V., 2016) Bajaj, Deepak; Upadhyaya, Hari D.; Das, Shouvik; Kumar, Vinod; Gowda, C.L.L.; Sharma, Shivali; Tyagi, Akhilesh K.; Parida, Swarup K.
    The present study exploited integrated genomics-assisted breeding strategy for genetic dissection of complex branch number quantitative trait in chickpea. Candidate gene-based association analysis in a branch number association panel was performed by utilizing the genotyping data of 401 SNP allelic variants mined from 27 known cloned branch number gene orthologs of chickpea. The genome-wide association study (GWAS) integrating both genome-wide GBS- (4556 SNPs) and candidate gene-based genotyping information of 4957 SNPs in a structured population of 60 sequenced desi and kabuli accessions (with 350–400 kb LD decay), detected 11 significant genomic loci (genes) associated (41% combined PVE) with branch number in chickpea. Of these, seven branch number-associated genes were further validated successfully in two inter (ICC 4958 × ICC 17160)- and intra (ICC 12299 × ICC 8261)-specific mapping populations. The axillary meristem and shoot apical meristem-specific expression, including differential up- and down-regulation (4–5 fold) of the validated seven branch number-associated genes especially in high branch number as compared to the low branch number-containing parental accessions and homozygous individuals of two aforesaid mapping populations was apparent. Collectively, this combinatorial genomic approach delineated diverse naturally occurring novel functional SNP allelic variants in seven potential known/candidate genes [PIN1 (PIN-FORMED protein 1), TB1 (teosinte branched 1), BA1/LAX1 (BARREN STALK1/LIKE AUXIN1), GRAS8 (gibberellic acid insensitive/GAI, Repressor of ga13/RGA and Scarecrow8/SCR8), ERF (ethylene-responsive element-binding factor), MAX2 (more axillary growth 2) and lipase] governing chickpea branch number. The useful information generated from this study have potential to expedite marker-assisted genetic enhancement by developing high-yielding cultivars with more number of productive (pods and seeds) branches in chickpea.
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    A genome-scale integrated approach aids in genetic dissection of complex flowering time trait in chickpea
    (Springer, 2015) Upadhyaya, Hari D.; Bajaj, Deepak; Das, Shouvik; Saxena, Maneesha S.; Badoni, Saurabh; Kumar, Vinod; Tripathi, Shailesh; Gowda, C. L. L.; Sharma, Shivali; Tyagi, Akhilesh K.; Parida, Swarup K.
    A combinatorial approach of candidate gene-based association analysis and genome-wide association study (GWAS) integrated with QTL mapping, differential gene expression profiling and molecular haplotyping was deployed in the present study for quantitative dissection of complex flowering time trait in chickpea. Candidate gene-based association mapping in a flowering time association panel (92 diverse desi and kabuli accessions) was performed by employing the genotyping information of 5724 SNPs discovered from 82 known flowering chickpea gene orthologs of Arabidopsis and legumes as well as 832 gene-encoding transcripts that are differentially expressed during flower development in chickpea. GWAS using both genome-wide GBS- and candidate gene-based genotyping data of 30,129 SNPs in a structured population of 92 sequenced accessions (with 200-250 kb LD decay) detected eight maximum effect genomic SNP loci (genes) associated (34 % combined PVE) with flowering time. Six flowering time-associated major genomic loci harbouring five robust QTLs mapped on a high-resolution intra-specific genetic linkage map were validated (11.6-27.3 % PVE at 5.4-11.7 LOD) further by traditional QTL mapping. The flower-specific expression, including differential up- and down-regulation (>three folds) of eight flowering time-associated genes (including six genes validated by QTL mapping) especially in early flowering than late flowering contrasting chickpea accessions/mapping individuals during flower development was evident. The gene haplotype-based LD mapping discovered diverse novel natural allelic variants and haplotypes in eight genes with high trait association potential (41 % combined PVE) for flowering time differentiation in cultivated and wild chickpea. Taken together, eight potential known/candidate flowering time-regulating genes [efl1 (early flowering 1), FLD (Flowering locus D), GI (GIGANTEA), Myb (Myeloblastosis), SFH3 (SEC14-like 3), bZIP (basic-leucine zipper), bHLH (basic helix-loop-helix) and SBP (SQUAMOSA promoter binding protein)], including novel markers, QTLs, alleles and haplotypes delineated by aforesaid genome-wide integrated approach have potential for marker-assisted genetic improvement and unravelling the domestication pattern of flowering time in chickpea.
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    Ultra-high density intra-specific genetic linkage maps accelerate identification of functionally relevant molecular tags governing important agronomic traits in chickpea
    (Nature Publishing Group, 2015) Kujur, Alice; Upadhyaya, Hari D.; Shree, Tanima; Bajaj, Deepak; Das, Shouvik; Saxena, Maneesha S.; Badoni, Saurabh; Kumar, Vinod; Tripathi, Shailesh; Gowda, C. L. L.; Sharma, Shivali; Singh, Sube; Tyagi, Akhilesh K.; Parida, Swarup K.
    We discovered 26785 and 16573 high-quality SNPs differentiating two parental genotypes of a RIL mapping population using reference desi and kabuli genome-based GBS assay. Of these, 3625 and 2177 SNPs have been integrated into eight desi and kabuli chromosomes, respectively in order to construct ultra-high density (0.20-0.37 cM) intra-specific chickpea genetic linkage maps. One of these constructed high-resolution genetic map has potential to identify 33 major genomic regions harbouring 35 robust QTLs (PVE: 17.9-39.7%) associated with three agronomic traits, which were mapped within <1 cM mean marker intervals on desi chromosomes. The extended LD (linkage disequilibrium) decay (~15 cM) in chromosomes of genetic maps have encouraged us to use a rapid integrated approach (comparative QTL mapping, QTL-region specific haplotype/LD-based trait association analysis, expression profiling and gene haplotype-based association mapping) rather than a traditional QTL map-based cloning method to narrow-down one major seed weight (SW) robust QTL region. It delineated favourable natural allelic variants and superior haplotype-containing one seed-specific candidate embryo defective gene regulating SW in chickpea. The ultra-high-resolution genetic maps, QTLs/genes and alleles/haplotypes-related genomic information generated and integrated strategy for rapid QTL/gene identification developed have potential to expedite genomics-assisted breeding applications in crop plants, including chickpea for their genetic enhancement.
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    A genome-wide SNP scan accelerates trait-regulatory genomic loci identification in chickpea
    (Nature Publishing Group, 2015) Kujur, Alice; Bajaj, Deepak; Upadhyaya, Hari D.; Das, Shouvik; Ranjan, Rajeev; Shree, Tanima; Saxena, Maneesha S.; Badoni, Saurabh; Kumar, Vinod; Tripathi, Shailesh; Gowda, C.L.L.; Sharma, Shivali; Singh, Sube; Tyagi, Akhilesh K.; Parida, Swarup K.
    We identified 44844 high-quality SNPs by sequencing 92 diverse chickpea accessions belonging to a seed and pod trait-specific association panel using reference genome- and de novo-based GBS (genotyping-by-sequencing) assays. A GWAS (genome-wide association study) in an association panel of 211, including the 92 sequenced accessions, identified 22 major genomic loci showing significant association (explaining 23–47% phenotypic variation) with pod and seed number/plant and 100-seed weight. Eighteen trait-regulatory major genomic loci underlying 13 robust QTLs were validated and mapped on an intra-specific genetic linkage map by QTL mapping. A combinatorial approach of GWAS, QTL mapping and gene haplotype-specific LD mapping and transcript profiling uncovered one superior haplotype and favourable natural allelic variants in the upstream regulatory region of a CesA-type cellulose synthase (Ca_Kabuli_CesA3) gene regulating high pod and seed number/plant (explaining 47% phenotypic variation) in chickpea. The up-regulation of this superior gene haplotype correlated with increased transcript expression of Ca_Kabuli_CesA3 gene in the pollen and pod of high pod/seed number accession, resulting in higher cellulose accumulation for normal pollen and pollen tube growth. A rapid combinatorial genome-wide SNP genotyping-based approach has potential to dissect complex quantitative agronomic traits and delineate trait-regulatory genomic loci (candidate genes) for genetic enhancement in crop plants, including chickpea.
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    Employing genome- wide SNP discovery and genotyping strategy to extrapolate the natural allelic diversity and domestication patterns in chickpea
    (Frontiers Media S.A., 2015) Kujur, Alice; Bajaj, Deepak; Upadhyaya, Hari D.; Das, Shouvik; Ranjan, Rajeev; Shree, Tanima; Saxena, Maneesha S.; Badoni, Saurabh; Kumar, Vinod; Tripathi, Shailesh; Gowda, C. L. L.; Sharma, Shivali; Singh, Sube; Tyagi, Akhilesh K.; Parida, Swarup K.
    The genome-wide discovery and high-throughput genotyping of SNPs in chickpea natural germplasm lines is indispensable to extrapolate their natural allelic diversity, domestication, and linkage disequilibrium (LD) patterns leading to the genetic enhancement of this vital legume crop. We discovered 44,844 high-quality SNPs by sequencing of 93 diverse cultivated desi, kabuli, and wild chickpea accessions using reference genome- and de novo-based GBS (genotyping-by-sequencing) assays that were physically mapped across eight chromosomes of desi and kabuli. Of these, 22,542 SNPs were structurally annotated in different coding and non-coding sequence components of genes. Genes with 3296 non-synonymous and 269 regulatory SNPs could functionally differentiate accessions based on their contrasting agronomic traits. A high experimental validation success rate (92%) and reproducibility (100%) along with strong sensitivity (93-96%) and specificity (99%) of GBS-based SNPs was observed. This infers the robustness of GBS as a high-throughput assay for rapid large-scale mining and genotyping of genome-wide SNPs in chickpea with sub-optimal use of resources. With 23,798 genome-wide SNPs, a relatively high intra-specific polymorphic potential (49.5%) and broader molecular diversity (13-89%)/functional allelic diversity (18-77%) was apparent among 93 chickpea accessions, suggesting their tremendous applicability in rapid selection of desirable diverse accessions/inter-specific hybrids in chickpea crossbred varietal improvement program. The genome-wide SNPs revealed complex admixed domestication pattern, extensive LD estimates (0.54-0.68) and extended LD decay (400-500 kb) in a structured population inclusive of 93 accessions. These findings reflect the utility of our identified SNPs for subsequent genome-wide association study (GWAS) and selective sweep-based domestication trait dissection analysis to identify potential genomic loci (gene-associated targets) specifically regulating important complex quantitative agronomic traits in chickpea. The numerous informative genome-wide SNPs, natural allelic diversity-led domestication pattern, and LD-based information generated in our study have got multidimensional applicability with respect to chickpea genomics-assisted breeding.