Dr. R. S. TomarDHAWALE RAMESH NARAYANRAO10101190062023-12-152023-12-152022-10https://krishikosh.egranth.ac.in/handle/1/5810203386Little millet (Panicum sumatrense), having relatively diploid genome (2n=4x=36) with unknown genome size. It is one among the minor millets grown to a limited extent all over India up to altitudes of 2100 m. This crop is resistant to adverse agro-climatic conditions. The complex carbohydrates, phenolic compounds, antioxidants help to prevent metabolic disorders like diabetes, cancer, obesity etc., Although minor millets were superior to other cereals with many nutritional benefits due to its proximate composition of protein, fat, carbohydrates, ash, moisture, and energy, their utilization was limited because of low palatability, the coarseness of grain, and lack of diversified food preparations. Hence evaluation of the nutritional quality of millets would help to understand, diversifying the usage of millets in ensuring food, nutritional security in the ever-changing modern world. Keeping theses benefits in the mind present study was undertaken to find out putative genes and metabolites responsible for drought tolerance. The little millet genotype OLM 203/Tarini tolerant to drought was given in control and stressed condition in pots. 23 days old regularly watered seedling (control) and 32 days old water stressed seedlings were utilized for present experiment. In the current study, transcriptome analysis identified key genes regulating drought tolerance viz, in all 04-sample, number of reads varied 582,326 to 1,302,251 and average read length 497.13 to 589.32 bp. After trimming, there was decrease in number of reads, average read length and trimmed percentage which is varied between 569,157 to 1,265,909, 501.4 to 588.85, 90.74 to 97.74 % respectively. As well as, in all four samples number of total raw reads and average read length were 3,188,195 and 531.64 respectively and after trimming by CLC-GWB v20.0 number of raw reads decreased to 3,048,100 trimmed reads, trimmed read length 532.60, and trimmed percentage 95.24 %. Clean reads were obtained after quality control and they were subsequently used for De novo assembly and expression analysis. The generated draft assembly was 214498057 bp was approximately percentage of the total genome size of little millet is unknown. The maximum and minimum contig size was 40533 and 400bp in leaf and 398 base pairs in root respectively. However, the average size of contigs was 1138.04 bp and N50 was 1642 bp. Total de nono assembly were generated assembled reads of 270248626 to 214498057 with GC (%) was 48.39 from four samples with a hit length of 13112251 to 12759055 with GC (%) was 46.99. Differential expression analysis, total number of genes in comparison of 23 days leaf vs 32 days leaf (treated) were observed 7264, and in comparison of 23 days root vs 32 days root (treated) were extracted 7606. In expression profiling of 134 important DEGs, up or down regulated 20 important drought tolerance or resistance responsive genes in control and treated root of OLM-203 genotype was observed such as, in leaf XB34, and RS193 (up-regulated genes), WLIM1 (down regulated genes). In case of roots, SKI35 (up-regulated genes) while, MPK6 and TCMOp1 (down regulated genes). However, in leaf, up-regulated top 10 important drought tolerance or resistance responsive genes (LHW, CML25, PLP2, OBE1, SKI35p1, ATESY, SKI35p2, C3H7, PSAH1, PAP14) and in root MPK6, SPT41, THIC, LRX6, LRX5, COQ4, DMP5, LRX5, IAA6 and SERL2 genes were found to be switched on. Among these top 10 genes excluding SPT41, THIC (taking part in transcription process) genes takes part in drought tolerance in the genotype. There are top twenty commonly expressed drought tolerant or resistance genes in leaf and root of OLM-203 genotype viz., AX22A, BOB2, C3H32, C71B8, CAHC, CAL, CCS1, DGDG1, FLA2, GL52, GMPP2, HAK18, IAA6, IDL2, M3K1, NAC81, OHP1, PRX2C, SCP44 and TPR3. These all genes are responsive for positive regulator of stress signaling and drought tolerance mechanism. There are 36 up-regulated and 21 down-regulated serine transcripts were expressed in root and leaf of OLM-203 genotype of little millet respectively. ‘SKI35’ (Log₂ FC=7.66) up-regulated and ‘ALA2’ (Log₂ FC= -5.82) down-regulated genes are serine regulated differential expression genes in root of OLM-203 genotype. ‘NPRT1’ (Log₂ FC= 2.89) up-regulated and ‘KN7F’ (Log₂ FC= -4.11) down regulated genes are serine regulated differential expression genes in leaf of OLM-203 genotype. A perusal of dendrogram was clearly indicated that 134 drought tolerance genes were considered and three genes IAA6, ALA2, and HP301 among 134 genes played a key role during the drought or stress tolerance of the genotype. Most GO gene annotations of leaf samples were categorized as having ‘biological process’ (35%), followed by ‘cellular component (19%) and ‘molecular function’ (46%). Within the top represented category of ‘cellular process (125 contigs)’ were enriched, as was ‘metabolic process’ (117 contigs) and the next most enriched ortholog-biosynthesis of ‘cellular component’ (150) found in this category. Most GO gene annotations of root samples were categorized as having ‘biological process’ (573 contigs), followed by ‘molecular function (401 contigs) and ‘cellular components (166 contigs)’. In root samples, 1140 genes were found which are most enriched with biological process, molecular function and cellular component, whereas in leaf samples 670 genes most enriched ortholog-biological process, molecular function and cellular component. Enzyme commission (EC) numbers were assigned for control leaf vs treated leaf with 105 enzyme codes for 692 unique sequences, whereas in case of control root vs treated root with 105 enzyme codes for 699 unique sequences. Among all the leaf samples, the GO level distribution graph showed 1270 total annotations with mean level 6.036%, and having standard deviation 2.509, whereas in root samples containing 1617 total annotations with mean level 5.947%, and having standard deviation 2.516. Several putative functions were identified functional domains such as zinc finger, drought tolerance, serine related proteins. The confirmation results of repeats obtained by STRING 10.0 shown in MCL clustering of repeats depicted the 11 gene clustered with the specific repeats obtained from STRING 10.0. Six protein sequences (genes) viz., IPR019734 Tetratricopeptide, IPR013105 Tetratricopeptide, IPR018108 Mitochondrial subs, IPR019734 Tetratricopeptide string MCL clusters, IPR013105 Tetratricopeptide string MCL clusters, IPR002885 Pentatricopeptide string MCL clusters deciphered the role of its genes were regulated by their 11 genes cluster jointly. While, Venn diagram showing a total of 134 genes were found commonly shared and unique DEGs in leaf and root and 27 up-regulated genes were found commonly in leaf and root. There are 18 enzymes were analyzed and showed 44 KEGG pathways were found to be regulated. There were unique and common sequences regulate the 53 pathways. There are 35 unique and common KEGG pathways regulated by 58 enzymes of OLM-203 genotype. Total 147 KEGG pathways were commonly operated among control and treated samples of root and leaf. There are 15 and 35 number of unique and common KEGG pathways regulated by specific enzyme and sequences in root and leaf of genotype respectively up to 32 days of the genotype. Using the NIST library for the metabolomics study, metabolites were identified as sugars, sugar alcohols, sugar acids, fatty acids, and others such as dicarboxylic acid, diterpene alcohol, organic acid, and sugar amine. The detailed of total number of sixty (60) metabolites produced in both samples. In the mean decrease accuracy, metabolites were linolenic acid, mannonic acid, hexadecanoic acid, pentonic acid, glucopyranosid, glucohexodialdose, and malic acid were present in higher concentrations in the OLM-203 genotype. In treated samples, glucose, threonine, acetamide, serine, oleic acid, fructose glucose oxime, and hydroquinone were intensively observed, while in control sample, linolenic acid, mannonic acid, hexadecanoic acid, pentonic acid, glucopyranosid, glucohexodialdose, and malic acid were observed in OLM-203. Sugar also acts as a signaling molecule and helps to modulate the plant’s growth, development, and response to multiple stresses. Mannose was found to be accumulated in the leaf of OLM-203 (Tarini) in stress conditions; similar findings were reported earlier in the drought-tolerant little millet. Genomic and metabolomics changes were observed during drought treatment in tolerant genotype ‘OLM-203/Tarini’. It was observed that the typical increase of stress-related genes and metabolites. Therefore, it can sum-up that ‘OLM-203/Tarini’ genotype was well tolerated genotype and functions was normal even under water stress, although the exact complex mechanism of tolerance remains to be investigated. There were 128 EST-SSR identified which serves as a resource of high quality transcripts for gene discovery and development of functional markers associated with water stress tolerant. In the present study, sequence collection represents a major transcriptomics resource for little millet genotype and the large number of genetic markers predicted should contribute to future research thrust.EnglishThesis