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Item Identifying the mechanistic basis to nitrogen responsiveness in two contrasting Setaria italica accessions(Oxford University Press, 2024) Bandyopadhyay, Tirthankar; Maurya, Jyoti; Bentley, Alison R; Griffiths, Howard; Swarbreck, Stéphanie M; Prasad, ManojNitrogen (N) is a macronutrient limiting crop productivity with varied requirements across species and genotypes. Understanding the mechanistic basis of N responsiveness by comparing contrasting genotypes could inform the development and selection of varieties with lower N demands, or inform agronomic practices to sustain yields with lower N inputs. Given the established role of millets in ensuring climate-resilient food and nutrition security, we investigated the physiological and genetic basis of nitrogen responsiveness in foxtail millet (Setaria italica L.). We had previously identified genotypic variants linked to N responsiveness, and here, we dissect the mechanistic basis of the trait by examining the physiological and molecular behaviour of N responsive (NRp-SI58) and non-responsive (NNRp-SI114) accessions at high and low N. Under high N, NRp-SI58 allocates significantly more biomass to nodes, internodes and roots, more N to developing grains, and is more effective at remobilising flag leaf N compared to NNRp-SI114. Post anthesis flag leaf gene expression suggests that differences in N induce much higher transcript abundance in NNRp-SI114 than NRp-SI58, a large proportion of which are potentially regulated by APETALA2 (AP2) transcription factors. Overall, the study provides novel insights into the regulation and manipulation of N responsiveness in S. italica.Item An efficient Agrobacterium-mediated genetic transformation method for foxtail millet (Setaria italica L.)(Springer Nature Publishing AG, 2020) Sood, Priyanka; Singh, Roshan Kumar; Prasad, ManojFoxtail millet (Setaria italica L.) is a model crop to study C4 photosynthesis, abiotic stress tolerance, and bioenergy traits. Advances in molecular genetics and genomics had identified several potential genes in this crop that would serve as candidates for imparting climate-resilient traits in related millets, cereals, and biofuel crops. However, the lack of an efficient genetic transformation system has been impeding the functional characterization of these genes in foxtail millet per se. Given this, an easy and efficient regeneration and transformation protocol was optimized using mature seeds as a choicest explant. The suitability of secondary embryogenic calli over primary calli is underlined due to their high competence. The use of perfect combinations of plant growth regulators together with the ionic strength of organic and inorganics salts was found to influence regeneration and genetic transformation. We studied and optimized various crucial factors that affect the genetic transformation of foxtail millet calli using Agrobacterium tumefaciens-mediated approach. Secondary embryogenic calli and LBA44404 strain were found to be the best targets for transformation. The use of high sucrose and glucose, together with freshly prepared tobacco leaves extract, Silwet L-77 and acetosyringone, improved the efficiency of the genetic transformation of foxtail millet. Moreover, the use of an in vitro regeneration system with 84% callusing efficiency and 70–74% regeneration frequency led to a high recovery of transformants. Altogether, the present study reports a highly efficient (~ 27%) transformation system in foxtail millet that will expedite forward and reverse genetic studies in this important crop.Item Genome-wide association study of major agronomic traits in foxtail millet (Setaria italica L.) using ddRAD sequencing(Springer Nature, 2019) Jaiswal, Vandana; Gupta, Sarika; Gahlaut, Vijay; Muthamilarasan, Mehanathan; Bandyopadhyay, Tirthankar; Ramchiary, Nirala; Prasad, ManojFoxtail millet (Setaria italica), the second largest cultivated millet crop after pearl millet, is utilized for food and forage globally. Further, it is also considered as a model crop for studying agronomic, nutritional and biofuel traits. In the present study, a genome-wide association study (GWAS) was performed for ten important agronomic traits in 142 foxtail millet core eco-geographically diverse genotypes using 10 K SNPs developed through GBS-ddRAD approach. Number of SNPs on individual chromosome ranged from 844 (chromosome 5) to 2153 (chromosome 8) with an average SNP frequency of 25.9 per Mb. The pairwise linkage disequilibrium (LD) estimated using the squared-allele frequency correlations was found to decay rapidly with the genetic distance of 177 Kb. However, for individual chromosome, LD decay distance ranged from 76 Kb (chromosome 6) to 357 Kb (chromosome 4). GWAS identified 81 MTAs (marker-trait associations) for ten traits across the genome. High confidence MTAs for three important agronomic traits including FLW (flag leaf width), GY (grain yield) and TGW (thousand-grain weight) were identified. Significant pyramiding effect of identified MTAs further supplemented its importance in breeding programs. Desirable alleles and superior genotypes identified in the present study may prove valuable for foxtail millet improvement through marker-assisted selection.Item Global analysis of WRKY transcription factor superfamily in Setaria identifies potential candidates involved in abiotic stress signaling(Frontiers Media S.A., 2015) Muthamilarasan, Mehanathan; Bonthala, Venkata S.; Khandelwal, Rohit; Jaishankar, Jananee; Shweta, Shweta; Nawaz, Kashif; Prasad, ManojTranscription factors (TFs) are major players in stress signaling and constitute an integral part of signaling networks. Among the major TFs, WRKY proteins play pivotal roles in regulation of transcriptional reprogramming associated with stress responses. In view of this, genome- and transcriptome-wide identification of WRKY TF family was performed in the C4model plants, Setaria italica (SiWRKY) and S. viridis (SvWRKY), respectively. The study identified 105 SiWRKY and 44 SvWRKY proteins that were computationally analyzed for their physicochemical properties. Sequence alignment and phylogenetic analysis classified these proteins into three major groups, namely I, II, and III with majority of WRKY proteins belonging to group II (53 SiWRKY and 23 SvWRKY), followed by group III (39 SiWRKY and 11 SvWRKY) and group I (10 SiWRKY and 6 SvWRKY). Group II proteins were further classified into 5 subgroups (IIa to IIe) based on their phylogeny. Domain analysis showed the presence of WRKY motif and zinc finger-like structures in these proteins along with additional domains in a few proteins. All SiWRKY genes were physically mapped on the S. italica genome and their duplication analysis revealed that 10 and 8 gene pairs underwent tandem and segmental duplications, respectively. Comparative mapping of SiWRKY and SvWRKY genes in related C4 panicoid genomes demonstrated the orthologous relationships between these genomes. In silico expression analysis of SiWRKY and SvWRKY genes showed their differential expression patterns in different tissues and stress conditions. Expression profiling of candidate SiWRKY genes in response to stress (dehydration and salinity) and hormone treatments (abscisic acid, salicylic acid, and methyl jasmonate) suggested the putative involvement of SiWRKY066 and SiWRKY082 in stress and hormone signaling. These genes could be potential candidates for further characterization to delineate their functional roles in abiotic stress signaling.Item Unraveling 14-3-3 proteins in C4 panicoids with emphasis on model plant Setaria italica reveals phosphorylation-dependent subcellular localization of RS splicing factor(PLOS, 2015) Kumar, Karunesh; Muthamilarasan, Mehanathan; Bonthala, Venkata Suresh; Roy, Riti; Prasad, Manoj14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3), sorghum (Sb14-3-3) and maize (Zm14-3-3), respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A) in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize, which provides interesting information on their gene structure, protein domains, phylogenetic and evolutionary relationships, and expression patterns during abiotic stresses and hormonal treatments, which could be useful in choosing candidate members for further functional characterization. In addition, demonstration of interaction between Si14-3-3 and SiRSZ21A provides novel clues on the involvement of 14-3-3 proteins in the splicing events.Item FmMDb: A versatile database of foxtail millet markers for millets and bioenergy grasses research(PLOS, 2013) Suresh B, Venkata; Muthamilarasan, Mehanathan; Misra, Gopal; Prasad, ManojThe prominent attributes of foxtail millet (Setaria italica L.) including its small genome size, short life cycle, inbreeding nature, and phylogenetic proximity to various biofuel crops have made this crop an excellent model system to investigate various aspects of architectural, evolutionary and physiological significances in Panicoid bioenergy grasses. After release of its whole genome sequence, large-scale genomic resources in terms of molecular markers were generated for the improvement of both foxtail millet and its related species. Hence it is now essential to congregate, curate and make available these genomic resources for the benefit of researchers and breeders working towards crop improvement. In view of this, we have constructed the Foxtail millet Marker Database (FmMDb; http://www.nipgr.res.in/foxtail.html), a comprehensive online database for information retrieval, visualization and management of large-scale marker datasets with unrestricted public access. FmMDb is the first database which provides complete marker information to the plant science community attempting to produce elite cultivars of millet and bioenergy grass species, thus addressing global food insecurity.Item Comprehensive genome-wide survey, genomic constitution and expression profiling of the NAC transcription factor family in foxtail millet (Setaria italica L.)(PLOS, 2013) Puranik, Swati; Sahu, Pranav Pankaj; Mandal, Sambhu Nath; Suresh B., Venkata; Parida, Swarup K.; Prasad, ManojThe NAC proteins represent a major plant-specific transcription factor family that has established enormously diverse roles in various plant processes. Aided by the availability of complete genomes, several members of this family have been identified in Arabidopsis, rice, soybean and poplar. However, no comprehensive investigation has been presented for the recently sequenced, naturally stress tolerant crop, Setaria italica (foxtail millet) that is famed as a model crop for bioenergy research. In this study, we identified 147 putative NAC domain-encoding genes from foxtail millet by systematic sequence analysis and physically mapped them onto nine chromosomes. Genomic organization suggested that inter-chromosomal duplications may have been responsible for expansion of this gene family in foxtail millet. Phylogenetically, they were arranged into 11 distinct sub-families (I-XI), with duplicated genes fitting into one cluster and possessing conserved motif compositions. Comparative mapping with other grass species revealed some orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of genes. The evolutionary significance as duplication and divergence of NAC genes based on their amino acid substitution rates was understood. Expression profiling against various stresses and phytohormones provides novel insights into specific and/or overlapping expression patterns of SiNAC genes, which may be responsible for functional divergence among individual members in this crop. Further, we performed structure modeling and molecular simulation of a stress-responsive protein, SiNAC128, proffering an initial framework for understanding its molecular function. Taken together, this genome-wide identification and expression profiling unlocks new avenues for systematic functional analysis of novel NAC gene family candidates which may be applied for improvising stress adaption in plants.Item Development of eSSR-markers in Setaria italica and their applicability in studying genetic diversity, cross-transferability and comparative mapping in millet and non-millet species(PLOS, 2013) Kumari, Kajal; Muthamilarasan, Mehanathan; Misra, Gopal; Gupta, Sarika; Subramanian, Alagesan; Parida, Swarup K.; Chattopadhyay, Debasis; Prasad, ManojFoxtail millet (Setariaitalica L.) is a tractable experimental model crop for studying functional genomics of millets and bioenergy grasses. But the limited availability of genomic resources, particularly expressed sequence-based genic markers is significantly impeding its genetic improvement. Considering this, we attempted to develop EST-derived-SSR (eSSR) markers and utilize them in germplasm characterization, cross-genera transferability and in silico comparative mapping. From 66,027 foxtail millet EST sequences 24,828 non-redundant ESTs were deduced, representing ~16 Mb, which revealed 534 (~2%) eSSRs in 495 SSR containing ESTs at a frequency of 1/30 kb. A total of 447 pp were successfully designed, of which 327 were mapped physically onto nine chromosomes. About 106 selected primer pairs representing the foxtail millet genome showed high-level of cross-genera amplification at an average of ~88% in eight millets and four non-millet species. Broad range of genetic diversity (0.02-0.65) obtained in constructed phylogenetic tree using 40 eSSR markers demonstrated its utility in germplasm characterizations and phylogenetics. Comparative mapping of physically mapped eSSR markers showed considerable proportion of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (~68%), maize (~61%) and rice (~42%) chromosomes. Synteny analysis of eSSRs of foxtail millet, rice, maize and sorghum suggested the nested chromosome fusion frequently observed in grass genomes. Thus, for the first time we had generated large-scale eSSR markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species.Item Reference genes for quantitative real-time PCR analysis in the model plant foxtail millet (Setaria italica L.) subjected to abiotic stress conditions(Springer, 2013) Kumar, Karunesh; Muthamilarasan, Mehanathan; Prasad, ManojReference genes are standards for quantifying gene expression through quantitative real-time PCR (qRT-PCR); however, the variation observed in their expression levels is the major hindrance towards realising their effective use. Hence, a systematic validation of reference genes is required to ensure proper normalization. However, no such study has been conducted in foxtail millet [Setaria italica (L.)], which has recently emerged as a model crop for genetic and genomic studies. In the present study, 8 commonly used reference genes were evaluated, including 18S ribosomal RNA, elongation factor-1α, Actin2, alpha tubulin, beta tubulin, translation factor, RNA polymerase II and adenine phosphoribosyl transferase. Expression stability of candidate internal control genes was investigated under salinity and dehydration treatments. The results obtained suggested a wide range of Ct values and variable expression of all reference genes. geNorm and NormFinder analysis had revealed that Act2 and RNA POL II are suitable reference genes for salinity stress-related studies and EF-1α and RNA POL II are appropriate internal controls for dehydration stress-related expression analyses. These qualified reference genes has also been validated for relative quantification of 14-3-3 expression analysis which demonstrated their applicability. Thus, this is the first report on selection and validation of superior reference genes for qRT-PCR in foxtail millet under different abiotic stress conditions.Item The DNA-binding activity of an AP2 protein is involved in transcriptional regulation of a stress-responsive gene, SiWD40, in foxtail millet(Elsevier, 2012) Mishra, Awdhesh Kumar; Puranik, Swati; Bahadur, Ranjit Prasad; Prasad, ManojA differentially expressed transcript, encoding a putative WD protein (Setaria italica WD40; SiWD40), was identified in foxtail millet. Tertiary structure modeling revealed that its C-terminus possesses eight blade β-propeller architecture. Its N-terminal has three α-helices and two 310-helices and was highly induced by different abiotic stresses. The SiWD40:GFP fusion protein was nuclear localized. Promoter analysis showed the presence of many cis-acting elements, including two dehydration responsive elements (DRE). A stress-responsive SiAP2 domain containing protein could specifically bind to these elements in the SiWD40 promoter. Thus, for the first time, we report that DREs probably regulate expression of SiWD40 during envi- ronmental stress. Molecular docking analysis revealed that the circumference of the β‐propeller structure was involved in an interaction with a SiCullin4 protein, supporting the adaptability of SiWD40 to act as a scaf- fold. Our study thus provides a vital clue for near future research on the stress-regulation of WD proteins.