www.nature.com/scientificreports Correction: Author Correction OPEN Transcriptomic signature of drought response in pearl millet (Pennisetum glaucum (L.) and Received: 4 September 2017 development of web-genomic Accepted: 4 February 2018 Published: xx xx xxxx resources 1 2 2 1 1 Sarika Jaiswal , Tushar J. Antala , M. K. Mandavia , Meenu Chopra , Rahul Singh Jasrotia , 2 2 1 1 2 Rukam S. Tomar , Jashminkumar Kheni , U. B. Angadi , M. A. Iquebal , B. A. Golakia , Anil 1 1 Rai & Dinesh Kumar Pearl millet, (Pennisetum glaucum L.), an efficient (C4) crop of arid/semi-arid regions is known for hardiness. Crop is valuable for bio-fortification combating malnutrition and diabetes, higher caloric value and wider climatic resilience. Limited studies are done in pot-based experiments for drought response at gene-expression level, but field-based experiment mimicking drought by withdrawal of irrigation is still warranted. We report de novo assembly-based transcriptomic signature of drought response induced by irrigation withdrawal in pearl millet. We found 19983 differentially expressed genes, 7595 transcription factors, gene regulatory network having 45 hub genes controlling drought response. We report 34652 putative markers (4192 simple sequence repeats, 12111 SNPs and 6249 InDels). Study reveals role of purine and tryptophan metabolism in ABA accumulation mediating abiotic response in which MAPK acts as major intracellular signal sensing drought. Results were validated by qPCR of 13 randomly selected genes. We report the first web-based genomic resource (http://webtom. cabgrid.res.in/pmdtdb/) which can be used for candidate genes-based SNP discovery programs and trait-based association studies. Looking at climatic change, nutritional and pharmaceutical importance of this crop, present investigation has immense value in understanding drought response in field condition. This is important in germplasm management and improvement in endeavour of pearl millet productivity. Pearl millet, (Pennisetum glaucum L.) cereal crop is well known for its hardiness and grown in arid and semi-arid tropical regions of Asia and Africa. It is a short duration annual, cross pollinated, C4 panicoid plant having dip- loid (n = 7) genome with draft assembly genome size ~1.79 Gb . Millet is the most important crop for biofortifica- tion which is highly relevant in combating global micronutrient malnutrition. Millet being medium GI (Glycemic Index) cereal, is diabetic friendly . Looking at pearl millet’s highest productivity (3.3 tonnes per hectare) and global average productivity (0.83 tonnes per hectare), it can be deduced that this promising crop has genome plasticity to increase its productivity at least by four fold . Pearl millet has 11.8 g of proteins providing 363 Kcal of energy relatively higher that rice or wheat . Attributes of pearl millet like rich in nutrients, high energy, less starch, high fibre (1.2 g/100 g, predominantly insoluble), and higher α-amylase activity (8–15 times) than wheat, make pearl millet globally relevant crop produce . Millet is also used as fodder. Being C4 plant, it is efficient biomass producer even with less water requirement making it further attractive crop especially in regime of climate change. Pearl millet is also used in making breads Centre for Agricultural Bioinformatics (CABin), ICAR-Indian Agricultural Statistics Research Institute, New Delhi, India. Department of Biochemistry and Biotechnology, Junagadh Agricultural University, Junagadh, Gujarat, India. Sarika Jaiswal and Tushar J. Antala contributed equally to this work. Correspondence and requests for materials should be addressed to D.K. (email: email@example.com) SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 1 www.nature.com/scientificreports/ and cookies, for ethanol production in industries, used as poultry and animal feed and biofuel crop . Since it is rich in antioxidants, it is used in nutraceutical industry . In a study projecting climate change till 2099, it is indicated that there will be extreme increase in drought selectively in Western Hemisphere (major part of Eurasia and Africa) but there will be contrarily high moist regions, from Alaska to Scandinavia . Adversely, drought ae ff cted region also covers major pearl millet growing area decreasing the productivity at least by 25% . In such geoclimatic situation, improved millet variety with fur- ther drought tolerance would be a preferred crop due to its high photosynthetic efficiency with low transpiration as C4 plant . Limited number of Quantitative Trait Loci (QTLs) and genomic resources are reported in pearl millet. The reported genomic resources of pearl millet are in the form of simple sequence repeats (SSRs) , expressed 9,13 14–17 18 19 sequence tags (EST) , EST-SSR , ISSR and SCAR . Gene-based single nucleotide polymorphism (SNP) 20 21 and Conserved-Intron Scanning Primer (CISP) marker and DArT markers . None of these are available in the form of web-based genomic resources. Looking at genome size, these markers are too less and moreover till now there is no trait specific genic region marker discovery using transcriptomic approach. Approaches like marker-trait associations, genomic selection, genome sequence and genotyping-by-sequencing are still war- ranted . Genomic resources in pearl millet and management of abiotic and biotic stress are required to have insights of functional genes enabling crop improvement by molecular breeding approaches . Earlier work were based on heat treatment in phytotran and chemically induced drought using hyperosmotic polyethylene glycol (PEG) for induction of drought. All these were based on subtractive hybridization and cDNA (complementary Deoxyribonucleic Acid) library approach thus yielding very limited number of differentially expressed transcripts (<500) . Moreover, tissues selected in previous experiments were confined to leaves only. For water and nutrient intake, root systems are most critical thus they should also be included for better holistic view at physiological and molecular level. Moreover, root senses and responds to drought first and mediates stress signals for root biomass adjustments, anatomical alterations, and physiological acclimations thus tran- scriptomic investigation of root tissue is much more required . Since the effect of drought on root and leaf varies significantly in millets , thus specific tissue approach is imperative for identification of pathway and marker discovery. None of these experiments were based on with- drawal of experiment which truly mimics the actual drought condition of field. Transcriptomic approach of a single crop genotype with drought treatment has been reported successfully in tea plant deciphering major pathways regulating drought tolerance . Such single genotype-based transcriptomic analysis has been successfully used to delineate major physiological response against drought in tomato and cas- 31 32 sava also. In case of field crop like soybean, water deficit response has been found variety/ accession specific . Since single genotypes-based experiment has been found more holistic in understanding the basic physiological mechanism operating at species level, thus such experiments are needed in investigation of drought response in pearl millet species also. Transcriptome database is available for large number of crops but no such database has been developed for orphan crop like pearl millet to be used as genomic resources in crop improvement research. Since whole genome of the pearl millet has recently been available , thus development of such web resources can be done advantageously with SNP discovery by comparing with reference sequence along with genome anno- tation analysis. Such genic region putative markers (SSRs, SNPs and InDels) discovery can be rapid and cost effective which can be used in drought trait improvement program in future for better productivity of pearl millet. The present study aims at identification of differentially expressed genes in leaf and root tissue of millet in response to drought induced by withdrawal of irrigation in the field. It also aims at identification of transcription factors (TFs), genic region putative markers viz., SSRs, SNPs and Indels (Insertions and Deletions), gene regula- tory network (GRN) having hub genes and development of web-genomic resources. Material and Methods Seeds sowing in green house and drought treatment with recording of soil parameters. Drought tolerant pearl millet J-2454 variety was sown in greenhouse during summer season of the year 2014-2015 at Agriculture Farm of Junagarh Agriculture University, Gujarat, India (21.5222° N, 70.4579° E, 107 meter above mean sea altitude). Seeds were sown in 2 kg polythene plastic bag under small greenhouse and polythene bag fill with equal weight soil mixture of sand, vermicompost and FYM in ratio of (40:40:20) and 25 to 30 seeds sown per polythene bag with three replication of one genotype to comparative study with control and water stress (or water withhold). Soil parameters, namely, average pH, electrical conductivity (EC), Maximum water holding capacity were recorded. Parameters were also recorded for water used in irrigation. During experiment in greenhouse condition day and night temperature and relative humidity (RH) were recorded. Seedlings were maintained by thinning post 10 days aer s ft owing (DAS). Drought conditions were created by withdrawal of irrigation for 6 days aer 23r ft d day of th sowing of millet. Leaf and root tissues of pearl millet were collected aer ft 6 days of withdrawal of irrigation (i.e., 29 days aer ft sowing) from control and drought treated plants. Withdrawal of irrigation at different time point can be an artificially created drought best suited by gene expression prolin fi g in field crop . In control plant, regular irriga- tion interval of alternate day was maintained. Major physiological information can be seen at transcriptomic level on 29th day of sowing as reported in other millet crop species . Sampling at 29th day of sowing were as per the leaf and root tissues (two sets each) were collected from control and drought induced plants for transcriptomic studies. Tissues were frozen in liquid nitrogen and stored at −80 °C. Plant tissue collection and RNA extraction. In order to minimize variability across samples, sample pooling approach was followed by taking ten biological replicates of both tissues, viz., root and leaf . Total RNA was isolated from these tissues under control and drought condition using the standard protocol of TRIZOL RNA SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 2 www.nature.com/scientificreports/ Replication R1 R2 R3 Average ± SD Soil pH, EC and maximum water holding capacity (MWHC %) Soil – pH 7.36 7.38 7.37 7.37 ± 0.01 Soil – EC 1.19 (ms) 1.18 (ms) 1.19 (ms) 1.18 ± 0.01 (ms) MWHC (%) 29.90 30.10 30.45 30.15 ± 0.28 Water pH and EC Water – pH 7.20 6.92 7.10 7.07 ± 0.14 Water – EC 0.36 (ms) 0.37 (ms) 0.36 (ms) 0.36 ± 0.01 (ms) Table 1. Soil Parameters and Weather Information. Trinity Mira CAP3 CLC Total size of contigs (bp) 46259620 86542001 39881482 50,938,923 No. of contigs 95017 287045 133171 235,000 Longest contig 7397 5091 3328 5097 Shortest Contig 40 201 45 15 Mean contig size 487 301 299 217 N50 contig length 949 583 335 259 Table 2. Summary of de novo assembly of Pennisetum glaucum L. isolation . It was further purified with Magnetic Oligo (dT) beads in accordance to the manufacturer’s instruc- tions (Life Technologies, Grand Island, NY). Pre-processing, de novo assembly and identification of differentially expressed genes (DEGs). The single-end Illumina reads were generated using root and leaf RNA of pearl millet genotype J-2454 having approximately 5678956 million and 6463411 million single-ends reads under normal and drought stress condi- tion. The raw reads were pre-processed to remove any adaptor contamination using trimmomatic software with parameters read length ≤36, poor quality ≤3 and HEADCROP:10 bases. These pre-processed data were used for transcriptome assembly along with the identification of differentially expressed genes and other analysis, tran- scription factor identification and putative genic markers’ prediction. The different combinations used were root control vs. root under drought (RC, RT), leaf control vs. leaf under drought (LC, LT), root control vs. leaf control (RC, LC) and root under drought vs. leaf under drought (RT, LT). The pre-processed high-quality reads were assembled using Trinity platform . The abundance estimation for transcripts obtained was performed using ‘RNA-Seq by Expectation-Maximization (RSEM) . For the above mentioned transcriptome datasets, differentially expressed genes were identified using edgeR package of Bioconductor. The significant DEGs were obtained with stringent parameters taking fold change value as two and FDR (False Discovery Rate) <0.05 . Functional Annotation of transcriptome assembly. The sequence similarity search was conducted against the National Center for Biotechnology Information (NCBI) non redundant protein (Nr) database, and −3 42 Swiss-Prot protein database using the BLASTx algorithm specifying E-values of less than 10 . BLAST2GO was used for Gene Ontology (GO) categorization and functional enrichment pathway analysis. Transcription factors (TFs) and cofactors play very important roles in the expression of genes. Differentially expressed genes from all the combinations, i.e. (RC, RT), (LC, LT), (RC, LC) and (RT, LT) were considered for identification of TFs against PlantTFDB 3.0 with threshold e-value 1e-3 . MicroRNA (MiRNA) binding site prediction. PsRNATarget server was used for prediction of microRNA targets against all the mature microRNAs of Poaceae family viz., Aegilops tauschii, Brachypodium distachyon, Elaeis guineensis, Festuca arundinacea, Hordeum vulgare, Oryza sativa, Sorghum bicolor, Saccharum officinarum, Saccharum sp., Triticum aestivum, Triticum turgidum, Zea mays. We used all the 4 sets of comparison, viz., (RC, RT), (LC, LT), (RC, LC) and (RT, LT) to predict miRNA targets. Gene Regulatory Network Analysis. The highly differentially expressed genes with fold value ≥8 from up and down regulated gene were considered for the gene interaction network. Network were visualized and carried out for further analysis using Cytoscape (version 3.2.1) which is an open source platform for visualizing complex networks. ARACNE (Algorithm for the Reconstruction of Accurate Cellular Networks), a novel algo- rithm, specifically designed to scale up to the complexity of regulatory networks operating in the living cells was used. Network Analyzer plug-in was used to analyse the network centrality. The plug-in computes specific node centrality parameters and describing the network topology. Hub genes of complex networks were also obtained according to analysis of degree, betweenness and stress. The genes at the top of degree, betweenness and stress distribution were defined as hub genes. SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 3 www.nature.com/scientificreports/ Differential expressed genes in 4 sets of root and leaf tissues comparison Combinations Up-Regulated Down-Regulated (RC, RT) 1919 (2–13) 2874 (2–12) (LC, LT) 1626 (2–12) 782 (2–11) (RC, LC) 2977 (2–11) 4443 (2–13) (RT, LT) 3043 (2–12) 2319 (2–13) Table 3. Number of up- and down-regulated differentially expressed genes in various sets (values in parenthesis are the their minimum and maximum logFC values). Figure 1. Venn diagram showing shared and unique DEGs of millet root and leaf transcriptome leaf transcriptome. Identification of SNP and SSR in Pearl Millet transcriptome. For identification of putative genic SSRs from both assembled transcripts and differentially expressed transcripts, MISA (Microstellite Analysis) tool was used. Further, Primer3 was used for designing primers with parameters like annealing temperature (Tm) min: 57 °C, optimal: 60 °C, and maximum: 63 °C, primer size min: 15, optimal: 18, and maximum: 28 oligo-nucleotides. Variant discovery (SNP and InDels) was done by both approaches namely, comparison of transcripts with de novo transcriptome assembly of genotypes J-2454 and also with available reference sequence of millet (genotype 23D2B1-P1-P5). For reference sequence based SNP discovery, genomic data of pearl millet Cenchrus americanus was downloaded from NCBI (https://www.ncbi.nlm.nih.gov/assembly/GCA_002174835.1/). Circos tool was used for generation of circular map of variants . Reads were mapped using BWA-0.7.5a (Burrows-Wheeler Aligner) and Samtools . The SNPs were identified at read depth (d) ≥8 and quality depth (Q) ≥20 . Using the SAMtools program “vcfutils”, SNP sites were further filtered, based on the criteria of 90 bp on both sides of the SNP in the alignment to ensure it in exon . Validation and Expression Analysis by qRT-PCR. e fir Th st strand cDNA was synthesized from an ali- quot of total RNA for each sample using RevertAid First Strand cDNA Synthesis Kit (Genetix, USA) and served as template for qRT-PCR (Quantitative Real Time Polymerase Chain Reaction). For quantitative PCR, 13 transcripts (6 for leaves and 7 for root) were randomly selected for primer designing using Primer 3 sowa ft re . The qRT-PCR was performed using QuantiFast SYBR Green PCR Master Mix (Genetix, USA) on ABI-7300 Real-Time PCR detection system, (Applied Biosystem) using standard 40 cycles along with melt curve step. Housekeeping gene, actin was used as endogenous reference for normalization. To obtain linear relationship, PCR conditions were optimized for each set of gene. Finally, differential gene expression were computed in terms of ΔΔCT fold change value . Web-genomic resource development. Pearl millet transcriptome database (PMDTDb) catalogues the information related to assembled contigs or transcripts, DEGs, the pathways in which these are involved, detailed SSR markers, and variants such SNPs and indels and miRNAs. It has three-tier architecture, i.e., client tier, middle tier and database tier. Web pages are developed for browsing the database along with the queries by user in client tier. All the information regarding contigs, markers, variants etc. are arranged in tables in various tables in MySQL in the database tier. For execution and fetching of user’s query, scripting is done in PHP (Hypertext Preprocessor) in the middle tier. The pearl millet web-resources is available at http://webtom.cabgrid.res.in/pmdtdb/. Standard of Reporting Resource Identification. Germplasm resource used in the studies are completely disclosed by name of variety. SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 4 www.nature.com/scientificreports/ Figure 2. Pathway classification of the top 30 Common pathways in all the four sets of comparison. Gene Nomenclature. HGNC is followed by default. Availability of Supporting Data. e t Th ranscriptome dataset of the study used in this article are available in the NCBI repository with following accessions and is kept at hold till the publication. These would be made public aer p ft ublication. Bioproject: PRJNA385901 Biosamples: SAMN06920424, SAMN06920426, SAMN06920432, SAMN06920433 SRA accession number: SRR5839373, SRR5839374, SRR5839375, SRR5839376 Since data is generated in the studies and kept in public domain, thus there is no violation of Fort Lauderdale and Toronto agreements. Results and Discussion Greenhouse conditions and soil parameters. Soil and weather parameters were recorded. Irrigation withdrawal method was used successfully to induce drought. Average pH and EC of soil mixture were found to be-7.37 ± 0.01 and-1.18 ± 0.01 (ms), respectively. Maximum water holding capacity of soil mixture was found to be 30.15 ± 0.28%. Average pH and EC of water used in irrigation were found to be −7.07 ± 0.14 and −0.36 ± 0.01 (ms), respectively. Maximum day temperature (36–37 °C) and minimum night temperature (24–25 °C) were recorded along with day (84–86%) and night (49–51%) humidity. Other soil parameters and weather information are given in the Table 1. Pre-processing and de novo assembly of transcriptomic data. Transcriptome data was generated successfully from both set of tissue samples, viz., leaf and root. A total of 12142367 SE reads were generated representing 2272632 (root, control), 3360164 (root, treated), 3406324 (leaf, control) and 3103247 (leaf, treated) reads. Aer d ft ata cleaning and quality assessment at Q ≥ 25 for control samples and Q ≥35 for treated samples, a final dataset was obtained. A total of 7563927 SE reads were represented by 1980048 (root, control), 2154770 (root, treated), 1216328 (leaf, control) and 2212781 (leaf, treated) reads. De novo transcriptome assembly using different assemblers viz., Trinity, MIRA, Cap3 and CLC revealed trinity to be the best one (N50 = 949, assembly size ~46.26 MB) and was considered for further analysis (Table 2). Identification of Differentially Expressed Genes (DEGs). e a Th nalysis was conducted on four datasets, i.e., root control vs. root under drought (RC, RT), leaf control vs. leaf under drought (LC, LT), root control vs. leaf control (RC, LC) and root under drought vs. leaf under drought (RT, LT). The calculated read counts for a feature or gene was normalized to reads per kilobase million. A total of 4793, 2408, 7420 and 5362 DEGs were found in 4 sets of comparison, viz., (RC, RT), (LC, LT), (RC, LC) and (RT, LT), respectively in response to drought stress with FDR and corrected P-values of less than 0.05 (Additional file 1). Number of differentially expressed gene with their minimum and maximum logFC (Logarithm Fold Change) values are reported in Table 3. Shared and unique DEGs are depicted in the Venn diagram (Fig. 1) which shows that root is having more unique differential expressed genes (1444) with respect to leaf (695) in response to drought. A total of 106 genes were found common in all the 4 sets of comparison viz., (RC, RT), (LC, LT), (RC, LC) and (RT, LT). SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 5 www.nature.com/scientificreports/ Figure 3. Venn diagram of common miRNAs. Hub Gene Description/Function Reference Status XP_004974121 cytochrome p450 78a3-like (6 plus) It mediates senescence associated processes in drought Up XP_009379995 galacturonosyltransferase 8 (9 plus) It plays role in root tip growth in response to drought Up DEX1 is associated with calcium signalling pathway and XP_004981208 ac091247_15 dex1 protein (low) Down low expression (DR) of this in rots inhibits signalling XP_004967558 outer envelope pore protein 16- ABA induced expression of OEP16 protein regulates Up chloroplastic-like(9 Plus) shuttling amino acids Scarecrow (SCR) TF is one of the major regulators of plant XP_002442368 scarecrow-like protein 9 (9 plus) Up cellular network in stress mediated by Gibberellic acid XP_002437817 eukaryotic translation initiation factor Involved in maintenance of homeostasis in water stress Up 1a (Negative) Involved in recovery of metabolic activity aer o ft xidative EEQ. 24508 serine acetyltransferase (UR/10 Plus) Up inhibition in root tissue XP_004970236 protein disulfide isomerase (PDI) It has been reported to be associated in leaf biomass and Up (UR /7 plus) leaf size under dehydration stress XP_004956826 probable ccr4-associated factor 1 It plays major role in deadenylation reaction involved in Down homolog 7 (DR/9 minus) abiotic stress associated with microtubules XP_004984154 serine threonine-protein kinase sapk1- This MAPK family gene is involved in osmosensory signal Up like (UR/9plus) transduction pathways in osmotic stress Serine-Threonine Kinase SAPK1 (Also Known as JNK) Table 4. Description of root hub genes of millet. After quantification of expressions of each transcript for all the four sets of data, heatmap of differentially expressed transcripts were plotted along with MA plot and Volcano plot generated by edgeR (Supplementary Figure 1). Expression values in terms of Fragments Per Kilobase of transcript per Million mapped reads (FPKM) were log2 transformed followed by median-centered by transcript. The red dots in the plots represent true differ - entially expressed genes. Functional Annotation of transcriptome assembly. As per Kyoto Encyclopedia of Genes and Genomes (KEGG) database, from a total of 4793, 2408, 7420 and 5362 DEGs obtained from (RC, RT), (LC, LT), (RC, LC) and (RT, LT) respectively, having their corresponding enzyme commission numbers were assigned to 121, 98, 126 and 117 KEGG pathways (Additional file 2). Out of these, 89 pathways were found common to all the four sets. The pathways most represented by contigs were purine and thiamine metabolism, biosynthesis of anti- biotics, starch and sucrose metabolism, aminobenzoate degradation, glycine, serine and threonine metabolism and phenylpropanoid biosynthesis. Our analysis of top 30 common pathways reveals that leaf tissue are showing less metabolic activity with respect to root. This reflects that root has more DEGs than leaf for energy production and production of metabolites in response to drought for survival (Fig. 2). Higher purine metabolism observed in KEGG analysis is due to role of purine metabolism in Abscisic acid (ABA) accumulation mediating abiotic stress pathway of plant defence . KEGG pathway analysis fur- ther reveals that, tryptophan metabolism was conspicuously high in both leaf and root tissues in response to drought. Abiotic stress in crop leads to accumulation of proline along with other amino acids. This is because of tryptophan’s multifaceted role as osmolyte, ion transport regulation, stomatal control, detoxification reactions and redox-homeostasis . This pathway analysis also reveals higher aminobenzoate degradation in both the tis- sues. Such higher degradation has been reported in abiotic stress of other crop like soybean . This degradation increases the proline concentration in cell to protect cell from water deficit or abiotic stress. In case of drought SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 6 www.nature.com/scientificreports/ Figure 4. Root Gene Network Analysis: Subnetwork of important Hub gene DEGs. AFW87112 serine-threonine kinase receptor- It mediates ABA dependent pathway of abiotic stress response Up associated protein which is major intracellular signal transduction Stress-induced transcription factor nac1 plays role in abiotic AGU13503 stress-induced transcription factor nac1 stress (drought and salt) by modulating ABA mediated Up pathway It mediates accumulation of proline in response to drought XP_004977017 (kda proline-rich) Down and salinity NP_001148485 26 s proteasome regulatory particle It mediates pathways having antioxidant, photosynthetic and Up triple-a atpase subunit4 oxidative phosphorylation activities It modulates ABA biosynthesis and signaling pathways in XP_004960921 ndr1 hin1-like protein 2 Down abiotic stress It mediates activity of proton pumps for translocating H + into XP_004973848 tonoplast dicarboxylate transporter-like the vacuoles, thus responsible for accumulation of ions and Up solutes in response to drought It represses biochemical activity associated with XP_004981510 cullin-4-like isoform x2 Down photomorphogenesis and flowering time under drought stress This gene family has number of protein repeats which XP_004973957 wd-40 repeat family expressed mediates plant secondary metabolism in response to abiotic Up stimulus Achilleol is a type of terpene (secondary metabolite) produced XP_004951923 achilleol b synthase-like Up by crop in response to drought in foliar tissue to protect leaves Table 5. Description of leaf hub genes of millet. resilience by rehydration there is both down regulation of proline biosynthetic pathway enzymes as well as upreg- ulation of proline degrading enzymes . Functional classification: GO annotation. GO classification of millet gene lists from (RC, RT), (LC, LT), (RC, LC) and (RT, LT) were also carried out. A total of 45 (20 BP, 13 MF and 12 CC) GO terms were assigned for (RC, RT), 40 (18 BP, 11 MF and 11 CC) for (LC, LT), and 43 (20 BP, 11 MF and 12 CC) for (RC, LC) and 50 (20 BP, 12 MF and 18 CC) for (RT, LT) (Supplementary Figure 2A–D). This GO classification reveals that under response of drought, root tissue has higher expression of signalling ((RC, RT): 239; (LC, LT): 115) which is due to role of root in sensing the drought first (rather than leaf ) and mediating various signalling pathway of abiotic stress . More growth activities in root with respect to leaf ((RC, RT): 60; (LC, LT): 28) is an adaptive response of plant where more root growth is expected for more absorption of water and slow growth of leaf is expected for water retention and energy balance in response to drought . Higher abundance of immunity related transcripts SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 7 www.nature.com/scientificreports/ Figure 5. Leaf Gene Network Analysis: Subnetwork of important Hub gene DEGs. De novo assembly (RC, RT) (LC, LT) (RC, LC) (RT, LT) Total no. of sequences examined 95017 4793 2408 7420 5344 Total no. of identified SSRs 4192 385 244 618 508 No. of SSR containing sequences 3891 345 212 547 452 No. of sequences containing more than 1 SSR 270 32 26 62 47 No. of SSRs present in compound formation 126 16 13 23 20 Mono 1019 87 57 132 116 Di 831 81 49 138 122 Tri 2164 200 129 320 249 Tetra 146 16 8 21 20 Penta 26 1 1 5 1 Hexa 6 0 0 2 0 Table 6. Detailed s tatistics of identified SSRs. ((RC, RT):52; (LC, LT): 34) in root is due to more defensive role of root in drought stress especially for pathways involved in detoxification and scavenging of free radicals . Similarly, regarding molecular function, under drought response, root tissues delineated higher catalytic activity ((RC, RT):1617; (LC, LT):952). Drought increases catabolic activity to produce and accumulate osmolytes like sugars, polyols, betaines and proline . Similarly, higher transporter activity ((RC, RT):228; (LC, LT):119) 60,61 in root is due to increase in nitrate, sodium and potassium transport activity under water deficiency stress . Drought also increases the antioxidant activity in root ((RC, RT):29; (LC, LT):15) for protection of tissues from free radicals . Under the cellular component, the response of root and leaf against drought showed that cell ((RC, RT): 2035; (LC, LT): 1272), organelle ((RC, RT): 1707; (LC, LT): 1080) and membrane ((RC, RT): 816; (LC, LT): 524) is higher for root. Again, this is due to higher cellular activity of root in response to drought. Such cellular activity involves cell organelles like mitochondria and glycosomes for catabolic metabolism. Higher membrane activity is inevitable for such cellular and catabolic activity . RAB gene reported to mediate polar root growth in response to drought stress in common bean was found with highest magnitude among the upregulated genes in root. Similarly the role of other observed DEGs like serine acetyltransferase in sulfur assimilation pathway , DHN9 (dehydrin 9) in membrane stabilizion, heat-shock gene (HSP17.8) and dehydrin 3 (DHN3) are involved in drought tolerance by controlling stomatal closure through controlling carbon metabolism . We also observed differential expression of genes involved in energy balance and anti-oxidant activities which are already reported in drought response by various crops like SRP (stress responsive proteins) and DHN5 gene, controlling osmotic stress in wheat , peroxidase controlling Reactive Oxygen Species (ROS) and antioxidant activities detoxifying. In fact SRP gene controls various other stress responsive genes . ROS concentration is increased in response to drought which may lead to cellular damage but gets detoxified due to antioxidant activities of peroxidase gene . In drought response, there is change in energy balance and metabolic pattern. Genes controlling these activ- ities were found differentially expressed in our dataset, for example, LEA (Late Embryogenesis Abundant) gene SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 8 www.nature.com/scientificreports/ Chromosome # Root 1 2697 2 3114 3 2846 4 1652 5 2692 6 2312 7 1959 Unplaced accessions 1088 Table 7. Chromosome wise distributions of Variants in Pearl millet reference genome. Figure 6. Circular map to depict chromosome-wise SNP distribution among millet genotypes. reported to be associated with salt stress , ATP (Adenosine Triphosphate) citrate synthase reported to control energy balance in drought , aspartate kinase-homoserine dehydrogenase reported to control tricarboxylic acid 69 70 (TCA) cycle , glycolysis and Krebs cycle controlled by NADP dependent malic enzyme gene . High affinity of nitrate transport uptake has been reported in drought treated roots and the same was observed in differential expression of nitrate transporter gene . Transcription Factors identification. We obtained a total of 1757, 1056, 2826 and 1957 transcription factors in 4793, 2408, 7420 and 5362 DEGs from the ((RC, RT), (LC, LT), (RC, LC) and (RT, LT) sets, respectively (Additional File 3). We found 52 TFs common among 4 sets of root and leaf tissues comparison aer r ft emoval of duplicates. These TFs can be used in drought and heat tolerance. Some of our TFs are already reported in other 71 72 crops along with its association with abiotic stress, for example, WRKY in wheat and NAC1 in barley . A total of 7595 differentially expressed transcription factors have been found having binding site in 5022 transcripts (after removal of duplicate transcripts). We observed more number of TF with respect to number SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 9 www.nature.com/scientificreports/ Figure 7. qRT-PCR analysis of randomly selected transcripts. 73 74 of DEGs. Potential reason for this could be due to one gene having multiple TFs , overlapping of sequences and computational stringency . TFs and miRNAs are two regulatory circuits coordinating transcriptional and post-transcriptional control of targeted genes . MiRNA binding site prediction. A total of 267, 90, 827 and 445 mature miRNAs which targeted die ff r - entially expressed genes from 4 sets of comparison, viz., (RC, RT), (LC, LT), (RC, LC) and (RT, LT), respectively were predicted (Additional file 4). Aer r ft emoval of duplicates, we get 177, 82, 416 and 242 miRNAs and 61, 37, 105 and 73 target sequences for (RC, RT), (LC, LT), (RC, LC) and (RT, LT), respectively. We found 7 miRNAs viz., ssp-miR444a; osa-miR169d; ssp-miR444b.2; bdi-miR169l; osa-miR444f; osa-miR414; hvu-miR169 common in all the four sets (Fig. 3). This is due to wide conservation of miRNA having diverse functions in seedling growth and also in response to abiotic stress . Few miRNAs may have multiple targets of different genes in the regulatory networks . Thus, this is obviously expected with at least in some extensively conserved miRNA we may get but still they may have diverse functions. miR444a is reported to mediate drought stress for its adaptation in wheat. It is also known to interact with MADS-box transcription factors, which is associated with stress response . Besides, miR444 responded to 80 81 drought stress in Dongxiang wild rice . miR169d plays role in fighting drought stress in cotton as well as sor- 82 83 ghum . Also, miR169 family responds differently to drought in plants . Gene Regulatory Network Analysis. In root GRN analysis, our dataset reveals response of drought by major intracellular signal transduction mediated by Serine Threonine Protein (STP) kinase which are actually Mitogen Activated Protein Kinases (MAPK) . Drought triggers low expression of this gene to lower MAP Kinase activity. High affinity potential transporter gene was found with lower expression in drought. This genes acts as a sensor for drought in root and also controls uptake of potassium. This gene has been reported to be downregu- lated in root to balance potassium translocation . Drought leads to high meristematic activity in root mediated by lower expression of O methyltransferase zrp4 . Similar low expression of this gene was found in root in response to drought. In drought, ROS gets accumulated in root tip and NADH controls its meristematic activity through ABA pathway by energy balance . This gene was also found differentially regulated in root in response to drought. The details of root GRN hub genes is given in Table 4. Also the leaf subnetwork of important hub DEGs are shown in Fig. 4. In leaf GRN analysis, our dataset reveals Dehydrin gene family is well known as candidate genes associated with drought tolerance in crop. Among these gene family, in our dataset, we found differential expression of genes viz., dehydrin DHN1 and dehydrin COR410-like along with isoform of dehydrin COR410. Dehydrin is known to play role in drought response for crucial protective functions of root tissue. They are produced in response to ABA pathway mediated abiotic stress . This gene family has consensus amino acid seq KIKEKLPG which is reported to be associated with differential expression along with its various isoform in response to drought . The gene, late embryogenesis abundant protein (LEA) is also reported to be a candidate gene of drought having larger gene family. In our dataset, we found up-regulation of late embryogenesis abundant protein, group 3-like isoform X2, late embryogenesis abundant protein D-34, late embryogenesis abundant protein 3, late embry- ogenesis abundant protein Lea5. This gene family is reported to play protective role in cells under drought . Two larger gene families, viz., dehydrin and LEA were found differentially expressed in our dataset with its isoform. Similar observation has been reported in durum wheat in desiccation stress . The details of leaf GRN hub genes is given in Table 5. Also the leaf subnetwork of important hub DEGs are shown in Fig. 5. SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 10 www.nature.com/scientificreports/ Figure 8. Web interface of PMDTDb showing search option for variants, transcripts expression profile and pathways, DEGs and miRNA targets. Identification of genic-SSR in Pearl Millet transcriptome. A total of 95017 transcripts were used for SSR mining and 4192 SSRs were obtained. e Th motif type and their relative abundance are shown in Table 6. Maximum abundance (%) was of trinucleotides repeats (51.62) followed by mononucleotides (24.3), dinucleo- tides (19.82), tetranucleotides (3.48), pentanucleotides (0.62) and hexanucleotides (0.14). SSRs were also mined from 4 sets of root and leaf tissues and found 385, 244, 618 and 508 markers in (RC, RT), (LC, LT), (RC, LC) and (RT, LT) respectively (Table 6). These putative SSRs markers can be validated by genotyping them in various cultivars. These putative SSRs markers can be validated by genotyping them in various cultivars. Out of 4192 genic region SSRs, we could successfully design primers for 2828 for de novo full assembly (Additional file 5). Our reported SSR can be further used as functional domain marker in millet variety improvement program especially with respect to drought tolerance. Such genic region based SSR markers from leaf associated with drought has been reported in mulberry plant . Such genic region SSR markers have an advantage over genomic region, viz., transferability, a priory information about gene itself with known functionality. All these are desirable in crop improvement program . SNP and InDel identification. SNPs and INDels were discovered successfully by both approaches. Transcriptome based approach using a stringent pipeline, we identified 9318 total variants, having 5587 single nucleotide polymorphisms (SNPs) and 3736 InDels using the SAMtools sowa ft re (Additional Files 6). In reference based SNP discovery, a total of 18360 SNPs and InDels were obtained. Chromosome wise SNP distribution shows highest SNP over chromosome 2 and lowest number of SNP over chromosome 4 (Table 7). Circular map was also generated to depict chromosome-wise SNP distribution among two genotypes of millet (Fig. 6). More SNPs were found by reference based method than transcriptome based. This is obviously expected as transcriptome based SNP represents intra-genotype variation due to presence of heterozygotes only whereas inter-genotype variation based SNP discovery includes SNPs obtained by alignment of sequences between two genotypes from through- out the genome. This has led to discovery of higher number of SNPs by reference based method. Such alternative alignment has been reported as an efficient method of SNP discovery using two genotypes . Putative SNPs in pearl millet transcriptome could be due to millet being predominantly protogynous crop, having high cross pollination resulting into very high heterozygosity also . Being field crop, pearl millet shows high genetic variability within a single open pollinated variety contributing SNP discovery in transcriptome based SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 11 www.nature.com/scientificreports/ approach. If the crop has large ee ff ctive population size and its wind-pollinated reproductive trategy, then there is further increase in heterozygosity yielding these putative SNPs . Besides this, the potential source of SNP could be variation between each of the two sets of 10 plantlets pooled for RNA extraction for transcriptomic data gen- eration. SNPs detected in silico may be either true SNP showing allelic polymorphisms or it may be gene dupli- cation or paralogous or homologous sequence variations . Further observed variants (SNP and InDels) might be due to various other factors such as alignment ambiguity and undetected paralogs . Transcriptome based puta- tive SNPs can be further validated by Sanger sequencing followed by detection of double peak in chromatogram using SNP discovery by heterozygote approach . These discovered informative SNPs will be of immense use in designing of various throughput genotyping assay using their respective chromosomal location and genomic co-ordinates over reference sequence. Genic region SNP discovery has been used in various crop trait improvement programs like cold tolerance and dor- 100 101 102 103 mancy in wheat, RIL for mapping in Brassica , rust resistance in switchgrass , cold tolerance in sorghum and blister rust in white pine . Such genic region SSRs and SNPs from candidate genes and hub genes of GRN are valuable genomic resource for eQTL discovery, high-density mapping and trait improvement in various crops 105 106 like common bean and chickpea . Validation and Expression Analysis by qRT-PCR. Relative gene expression value obtained by qRT-PCR analysis of all the 13 genes (6 leaf, 7 root) were in correspondence with computed log fold change value of DEGs (Additional File 7, Fig. 7). Web-genomic resource development. Drought associated web genomic resource of pearl millet, PMDTDb (http://webtom.cabgrid.res.in/pmdtdb/) has been developed using transcriptome data. It catalogues assembled contigs or transcripts, 19983 DEGs, 7596 transcription factors and a total of 34652 genic region puta- tive markers (SSR markers, SNPs and InDels). e Th o fl wchart for its usage is shown in Fig. 8. For other species like 107 108 foxtail millet, similar genomic resources with molecular markers and transcription factors database has been developed. For another species finger millet (Eleusine coracana (L.) Gaertn.), such transcriptome sequence has been reported to provides insights into drought tolerance and nutraceutical properties . Based on the present study, we report the first web-based genomic resources of pearl millet. These resources can be used for further in candidate genes-based SNP discovery programs and trait-based association studies in drought improvement. Potential application of millet genomic resources. For varietal improvement, traditional breeding pro- gram can be supplemented with molecular breeding approach by using genomic resources . Our web-genomic resource is having 17856 SNPs (mined from two millet genotypes) which can be used in variety improvement. Such SNP discovery using even single genotype has been reported in forage crop like Artemisia . Since there is no report of SNP discovery in candidate genes associated with drought in millet thus further research can be done by targeted sequencing of the reported genomic resources in large number of varieties and populations. Such approach can reduce the time and cost required for resequencing of large genotypes without prioritizing the candidate genes involved. Similar use of DEG-based SNP discovery has been reported in other crops like wheat 94,95 102 for abiotic stress tolerance , Switchgrass for biotic stress tolerance . Candidate genes depicted in GRN can be a preferred source of SNP discovery required for future association studies. Further, such transcriptome-based approach has been reported for eQTL discovery in development of high-density map like Brassica rapa . Our web-based genomic resources is also having 4192 SSRs along with ready to use primers for genotyp- ing can be used in variety identification and improvement program. Similar transcriptome-based SSR has been 112 113 114 115 116 used in various crops like barley , S. tuberosum , sugarcane , capsicum , eggplant and differentiation of Basmati rice from non-Basmati and also for DUS (Distinctness, Uniformity and Stability) testing for varietal 118,119 identification . Our enlisted SSR can be further used in MAS programme of millet improvement as reported 120 121 122 in sorghum , tagging stem rust resistance gene Sr35 in wheat , Saltol QTLs in rice . Genomic resources of miRNA and its target in PMDTDb can be further used in research. It can be useful for both knowledge discovery (mechanism/regulation of drought responsive genes) and application oriented research especially for variety improvement. Since in finger millet, it has been reported that drought tolerance can be increased by use of gene silencing of drought associated miRNA thus our enlisted predicted miRNA can be used for similar work . It has been widely reported that miRNA can be used to enhance crop yield along with increased tolerance to biotic and abiotic stress. Further, genome editing tool like CRISPR-cas9 can be used to con- trol expression of miRNA , thus there is greater scope for further research on enlisted miRNAs/genes by genetic modification for crop improvement. Such genome editing approach has been reported to be very promising in reducing anti-nutrients in millet, thus making it further enriched cereal . Our enlisted TF genes can be used for future SNP discovery for traits improvement of crop. Similar approach has been reported in oil plant for regulation of dwarsm b fi y negative regulation of DELLA protein . In case of Prunus, traits like the flowering, fruit quality, and biotic and abiotic stress resistance have been found regulated with TF . Conclusion Present work reports root and leaf transcriptomic signature of drought induced by irrigation withdrawal in pearl millet. We found 19983 DEGs, 7596 TFs and GRN having 45 genes controlling drought response. A total of 34652 genic region putative markers viz., 4192 SSRs, 12111 SNPs and 6249 InDels are reported. Validation of gene expression by 13 randomly selected genes was in correspondence with computed FPKM values. We report major candidate genes such as LEA, Dhn, ATP-citrate synthase family, peroxidase, stress responsive protein and Aspartate Kinase-Homoserine Dehydrogenase genes. Enlisted candidate genes can be used for further SNP dis- covery programs and association studies. Looking at climatic change and nutritional and pharmaceutical value of SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 12 www.nature.com/scientificreports/ this crop as well as its genetic potential of resilience, present investigation is of immense value in understanding drought response of millet in field condition. 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SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 15 www.nature.com/scientificreports/ Acknowledgements Authors are thankful to Indian Council of Agricultural Research, Ministry of Agriculture and Farmers’ Welfare, Govt. of India for providing financial assistance in the form of CABin grant (F. no. Agril. Edn.4-1/2013-A&P) as well as Advanced Super Computing Hub for Omics Knowledge in Agriculture (ASHOKA) facility at ICAR-IASRI, New Delhi, India. Author Contributions D.K., R.S.T., M.K.M. and M.A.I. conceived theme of the study and designed experiments. R.S.T., T.J.A., J.K. and M.K.M. collected samples and generated data. M.C., R.S.J., S.J., M.A.I. and U.B.A. did the computational analysis and development of web resources of generated data. S.J., T.J.A., M.C., R.S.J., M.A.I. and D.K. drae ft d the manuscript. D.K., M.A.I., S.J., A.R., M.K.M., B.A.G. and R.S.T. edited the manuscript. All co-authors read and approved the final manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-21560-1. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 SCIeNTIfIC RePo R TS | (2018) 8:3382 | DOI:10.1038/s41598-018-21560-1 16
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