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  • ThesisItemOpen Access
    TRANSCRIPTOME AND HORMONE PROFILING OF SESAME (Sesamum indicum L.) DURING INTERACTION WITH Macrophomina phaseolina 3035
    (JAU, JUNAGADH, 2019-07) RADADIYA NIDHI GHANSHYAMBHA; B. A. Golakiya
    Sesame (Sesamum indicum L.; Pedaliaceae) is a diploid (2n = 26) dicotyledonous and one of the oldest oil seed crop grown widely in tropical and subtropical areas for its edible oil and food products. Beside of large land covered for cultivation of sesame there is a wide demand–supply gap as its production is constrained by various biotic and abiotic stresses which leads to less productivity. Biotic stresses such as diseases, insects, and pests affect sesame crops adversely causing substantial yield losses. Among the major diseases, charcoal rot caused by Macrophomina phaseolina affects severely at the all stages of the crop growth. In India, this disease incidence was recorded up to 50%. Keeping this constrain in mind present study was undertaken to understand the molecular responses of the host cell against a fungal infection and the putative role of regulatory players. In present study, two different genotypes GT-10 [resistant(R)] and RT-373 [susceptible(S)] were selected to perform transcriptome and four genotypes GT-10, Rama (resistant) and RT-373, AT-306 (susceptible) for biochemical characterization. Total 8 samples were collected at post flowering stage at four different post inoculation stages. Root samples of resistant and susceptible genotypes were collected at 0 hpi (hours post inoculation), 24 hpi, 48 hpi, 72 hpi. The mRNA was isolated from all the collected samples and was sequenced in IonS5 next generation sequencer. After completions of sequencing run the total raw sequence generated were assessed through FastQC quality control tool in which all 8 (eight) samples having good quality sequence were selected for further analysis. Trimming of raw reads yielded a total of 8443423, 8381018, 10143124, 11552102 reads in R1, R2, R3 and R4 respectively and 8713438, 8149237, 11265254, 9281491reads yielded in S1, S2, S3 and S4 respectively. This HQ data was mapped on M. phaseolina genome to remove possible contamination of fungus genome. Unmapped data was extracted and mapped on sesame genome which was downloaded from NCBI. Clear reads were used to carry out differential gene expression analysis. Differential expression analysis of both the genotypes yielded top 10 up and down regulated transcripts expressed at each PIS which were selected for the Gene Ontology enrichment analysis. GO terms like response to auxin (GO:0009733), integral component of membrane (GO:0016021), lipid metabolic process (GO:0006629) were found to be significantly over-represented in sequences having higher expression in GT-10. Moreover, glutathione transferase activity (GO:0004364), oxidoreductase activity (GO:0016491), alpha,alpha-trehalose-phosphate synthase (UDP-forming) activity (GO:0003825), oxidoreductase activity (GO:0016491), integral component of membrane (GO:0016021), beta-fructofuranosidase activity ii (GO:0004564), malate transport(GO:0015743) were found in up regulated sequence in GT-10 over the RT-373. In pathway enrichment analysis, common KEGG pathways like starch and sucrose metabolism and phenyl propanoid biosynthesis were enriched in up-regulated transcripts, however their fold expression was different in both the genotypes. Fatty acid degradation, Arachidonic acid metabolism, Glycerophospholipid metabolism, Tryptophan metabolism, Glyoxylate and dicarboxylate metabolism pathways were enriched in down regulated transcripts in susceptible genotype. Inositol phosphate metabolism, Ascorbate and aldarate metabolism and zeatin biosynthesis were enriched in up regulated transcripts of susceptible genotypes. The DEGs obtained from Deseq software were validated by qRT PCR. The expression patterns of the randomly selected DEGs in to RT-qPCR were in agreement with those obtained by the RNA-Seq, suggesting that the RNA-seq data reflected the real expression patterns of the sesame genes in the compatible interaction. The leaves and roots were analyzed at the interval of 24 hours up to 72 hours after pathogen inoculation. The β-1,3 glucanase and chitinase activities were increased up to 48 hpi thereafter decreased at 72 hpi. The activation of chitinase was more rapid and higher in plants of resistant genotypes than in susceptible genotypes. The results of phenol determination using LC-MS/MS reveals that in both the resistant genotypes, Rama and GT-10, vanillic acid, chlorogenic acid and salicylic acid were found to be higher in diseased condition which indicates these phenolics might play important role in defense mechanism. While in susceptible genotype (RT-373) syringic acid, catechin, coumaric acid, salicylic acid, ferulic acid and cinnamic acid were high in post inoculation stages. In AT-306 vanllic acid, catechin, gallic acid and cinnamic acid were increased during interaction with pathogen. In phytohormone profiling, the levels of IAA, Zeatin, ABA, ACC, JA and SA in the plant extracts were successfully quantified in MRM mode, but this was not possible for GA because of the low level of this phytohormone in plant tissues. At 48 hpi the level of IAA increased in resistant genotypes. The concentration of JA was found higher at 24 hpi in leaves and roots of both susceptible genotypes. No significant change in JA concentration was found in both resistant genotypes among all PIS. The level of SA increased at 48 hpi in all genotypes however its concentration varied among the all. The level of ACC increased in all genotypes after fungus inoculation in both the tissues. Zeatin slightly increased in leaves and root of GT-10 at 48 hpi. It drastically increased in root of Rama at 72 hpi.
  • ThesisItemOpen Access
    MAPPING AND VALIDATION OF SSR MARKERS ASSOCIATED WITH FOLIAR-FUNGAL DISEASE RESISTANCE QTLs AND DEVELOPMENT OF RILs IN GROUNDNUT (Arachis hypogaea L.) 2928
    (JAU, JUNAGADH, 2019-08) SUHAIL AHMAD A. HAKEEM; H. P. Gajera
    Groundnut (Arachis hypogaea L.) is an economically important oilseed crop which is widely cultivated in semi-arid tropic regions of Asia, Africa and America. Two devastating foliar-fungal diseases, viz., late leaf spot (LLS, Phaseoisariopsis personata) and rust (Puccinia arachidis), which often occurs together causes heavy losses along with adverse effects on the quality of kernel and fodder. The objective of the present study was to identify and map the biotic stress relevant candidate EST-SSR markers in the major QTL regions controlling resistance genes for LLS and rust diseases and validate the identified linked markers in diverse groundnut genotypes. The F2 mapping population consisting of 328 lines was developed from diverse genotypes i.e. GJG 17 and GPBD 4. In the parental polymorphism survey, 84 out of 1311 SSR markers were found polymorphic (which includes 900 newly developed EST-SSR at ICAR-DGR and SSRs from the previous reports). A total of 70 out of 84 markers were mapped on 14 linkage groups (LGs) covering a total map distance of 797.55 cM with an average inter-marker distance of 11.39 cM. In the bulk segregant analysis (BSA) four new EST-SSR markers namely DGR329, DGR508, DGR800 and DGR2409 were identified as putatively linked to LLS and rust resistance genomic region. A total of seven new EST-SSR markers, including newly identified 4 markers with BSA, were successfully mapped in the LLS and rust fine QTL region. Among them, DGR259 was found flanked to major LLS QTL. One common QTL for LLS and rust was identified in the map interval of 1.41 cM (between SSR_GO340445 and FRS72) on linkage group (LG-A03) which contributed 47.45% and 70.52% PVE variation, respectively. These two markers, SSR_GO340445 and FRS72, were tightly linked to LLS and rust resistance. Another major QTL for LLS was found on same linkage group (LG_A03), explained 29.06% PVE flanked by the marker DGR259 and FRS59 close to LLS and rust common QTL. The common QTL region identified for LLS and rust resistance carries one Resistance ABSTRACTgene (R gene) and five resistance-related genes which shown to have role in inducing a programmed cell death or hypersensitive response (HR) to combat pathogen attack. A total of 7 new biotic stress relevant EST-SSR markers (DGR259, DGR312, DGR329, DGR361, DGR508, DGR800 and DGR2409) were integrated in the major QTL hotspot region, where one of it flanking the major LLS QTL region and six markers showed tight association with major QTL region. Most EST-SSR markers were from the genes involved in the disease resistance. Initial validation carried out in selected genotypes showed that these markers were able to distinguish resistant genotypes more clearly and efficiently. Further validation of all the 24 SSR markers saturated in LLS and rust QTL region were validated in 177 diverse Indian genotypes. Of these, only 12 genotypes resistant to both LLS and rust carries resistant alleles of markers flanking the LLS and rust QTLs and eight more markers present in the surrounding region of these QTLs. Moreover, the new identified EST-SSR markers were validated in the introgression lines. All the markers were able to distinguish resistant introgression line from susceptible recurrent parent and susceptible genotypes. These markers could be used to introgress this fine QTL regions to improve LLS and rust resistance in the groundnut breeding program more efficiently by minimizing the risk of linkage drag. Furthermore, these markers will be of great help to the groundnut breeders for the selection of resistant genotype from the segregating population, advanced breeding lines and varieties even in the absence of a disease nursery or epiphytotics and will be useful in MAS under crop improvement program
  • ThesisItemOpen Access
    ISOLATION, CHARACTERIZATION AND IDENTIFICATION OF HALOPHILIC BACTERIA FROM THE SOILS OF COASTAL REGIONS OF SAURASHTRA 2920
    (JAU, JUNAGADH, 2019-07) LIKHINDRA REANG; S. B. BHATT
    Halophiles are extremophilic organisms that thrive in environments with very high salt concentrations and osmotic stress. The agricultural soils in coastal regions of Saurashtra lies in extreme proximity to the Arabian Sea and the diverse microbial communities inhabiting its coastal environments have not yet been explored. In this context, the present study was undertaken with an objective to isolate halophilic bacteria from the soils of coastal regions of Saurashtra, identify and characterize the isolates using cultural and biochemical tools, and study their qualitative and quantitative production of biotechnologically important enzymes, other valuable bioactive compounds and plant growth promoting characteristics and PCR based molecular screening of salt stress tolerant genes present in the isolates. In this present study, forty three halophilic bacterial strains were isolated from fifty soil samples collected from agricultural soils of coastal regions of Saurashtra. Morphological, physiological and biochemical characterization of these strains were studied by testing pH, NaCl and temperature tolerance, and the fifteen best performing isolates were selected as model halophilic bacteria for investigation of the above stated research objectives. The isolated strains were capable of growing in media with 5-25% NaCl concentrations, optimum pH and temperature of 6-8 and 35-45℃, respectively. The selected halophilic bacterial strains were divided into three groups on the basis of their halotolerance activity against various NaCl concentrations tested as moderate and extreme halophiles and moderate-extreme halotolerant bacteria. The isolates were identified by microscopic Gram’s staining as gram positive/negative and by scanning electron microscope as short to thin long rods and coccus shape bacteria. The isolates also exhibited some considerable antimicrobial activities against phytopathogenic bacteria and fungi tested and intrinsic antibiotic resistance activity against antibiotics such as Cloxacillin, Erythromycin, Penicillin-G, Amoxicillin, Ampicillin, Co Trimoxazole, Nitrofurantoin, Chloramphenicol and Vancomycin. The various biochemical tests such as starch, lipid and gelatin hydrolysis, methyl red, catalase, oxidase, nitrate reduction, utilization of glucose, lactose, arabinose, sorbitol, lysine, and production of chitinase, cellulase and protease enzymes were found to be positive for majority of the isolates. The isolates also showed promising heavy metal tolerance activity with 75-95% growth against various concentrations of seven heavy metals tested viz., cadmium, manganese, copper, cobalt, nickel, zinc and lead. The isolates tested for plant growth promoting properties showed IAA production ranging from 18.77 to 33.48 μg ml-1 , phosphate solubilization efficiency ranging from 41.80-183.60% on 3 DAI to 50.10-106.10% on 10 DAI. The potash solubilization efficiency of isolates ranged from 101.99-128.91% on 3 DAI to 180.42- 239.92% on 10 DAI. However, the zinc solubilization capacity was tested negative for all the isolates. The nitrogen fixation capacity of the isolates ranged from 0.170 to 0.480 g kg-1 of Jensen’s agar medium. On the other hand, only four and two out of fifteen isolates showed positive test of carotene and siderophore production, respectively. The siderophore type produced was further identified by tetrazolium test as hydroxymate type. Majority of the isolates also showed positive antineoplastic enzyme production activity. The EPS production of isolates ranged from 17.839 to 34.243 mg ml-1 while the production of compatible solutes viz., ectoine and glycine betaine ranged from 0.01 to 3.17 mg l-1 and 0.307 to 0.963 μ mol per g of cell dry weight, respectively. The PCR based molecular screening of nine salt stress tolerant genes showed the presence of five genes viz., proA, proJ, proH, ectABC and BADH1. The fifteen selected halophilic bacterial isolates studied were identified by phylogenetic analysis of sequences obtained from partial 16S rRNA gene sequencing. Eight out of ten isolates belonging to genus Halomonas were identified as H. pacifica and the other two as H. stenophila, while three out of four isolates belonging to genus Bacillus were identified as B. haynesii and the other one as B. licheniformis, and the remaining one isolate was identified as Oceanobacillus aidingensis
  • ThesisItemOpen Access
    GENOME AND TRANSCRIPTOME SEQUENCING OF KODOMILLET (Paspalum scrobiculatum L.) FOR THE DEVELOPMENT OF GENIC MARKER AND DIVERSITY ANALYSIS 2900
    (JAU, JUNAGADH, 2019-07) HUMBAL NEHALI BHAGAVANJIBHAI; R. S. Tomar
    Kodo millet, (Paspalum scrobiculatum L.) (2n=4x=40) also known as kodra, varagu, harka is an annual grain that is grown primarily in India. It is widely distributed in damp habitats across the tropics and subtropics of the world. In India, kodo millet occupies an area of 152.5 thousand hectares with the production and productivity of 41.2 thousand tones and 312 kg/ha, respectively. It matures in three to four months with yields varying from 250 to 1000 kg/ha and a potential yield of 2000 kg/ha. After completions of genome sequencing run, the total raw sequence generated was about 6.55 Gb, which includes 21,080,571 total reads of sequence data. The raw data, where quality checked using FastQC and the following data had a total sequence of 1,087,611 and 57,81,23,572bp. In the present work, an RNA-seq project for Paspalum scrobiculatum L. was initiated. Three RNA samples, including vegetative, Flowering and maturity development and ripening stages, were sequenced using the high-throughput Ion torrent sequencing technique. The number of raw reads in vegetative stage were 1,34,40,824 while in reproductive and maturity stage the reads were 1,66,35,843 and 97,43,629 respectively. The average read length at three different stage was 131.6bp, 137.6bp, and 148.8bp respectively. The SSR and EST-SSR primers were designed using BatchPrimer3 (version 1.0) interface modules. These SSR marker wills use to study of genetic diversity, genetic linkage mapping and evolution analysis. For the identification of SSRs four Major Guide Dr. R. S. Tomar Name of Student Humbal Nehali B. ii FASTA files of contigs were used belonging to genome and transcriptome. Total 30 SSRs were identified with Tm and GC % range of 58-62°C and 40-70 respectively. For validatation of these 30 SSR and EST-SSR primers, 27 different varieties of Kodomillet were taken among these 30 primer, only 17 primer were able to amplify the DNA. The PCR amplification of 27 different varieties yielded 24 bands. Genome sequencing also gave an idea about evolution of genetic structure and function as well as study of molecular phylogeny of individual species with the genome of evolutionary proximate species. It also provides data through which we can identify gene and functional elements of genome and give basis for annotation of complete plant genome. The transcriptome sequencing of kodomillet may help improve future genetic and genomics studies on the molecular mechanisms behind the chemical composition of the plant. RNA sequencing (RNA-seq) has become a powerful technology to profile transcriptomes due to its high-throughput, accuracy, and reproducibility. In plants, RNA-seq has accelerated the investigation of the complexity of gene transcription patterns, functional analyses and gene regulation networks. Due to the limited genomic sources for Paspalum scrobiculatum L. it is important to identify whole transcripts for complete gene expression profiling by RNA-seq. SSRs are new dataset provides the most comprehensive resource currently available for gene expression, gene discovery, and future genomic research on Paspalum scrobiculatum L.
  • ThesisItemOpen Access
    ISOLATION AND CHARACTERIZATION OF ENTOMOPATHOGENIC MICROBES FROM THE SOILS 2844
    (JAU, JUNAGADH, 2019-07) HIRAPARA KINJAL MUKESHBHAI; S.B. BHATT
    The present investigation on “Isolation and characterization of entomopathogenic microbes from the soils”, was carried out at Microbial cell, Department of Biotechnology, College of Agriculture, Junagadh Agricultural University, Junagadh (Gujarat) during the year 2018-19. Entomopathogenic microbes are natural enemies of insects and arachnids and contribute to the regulation of their host populations. In agriculture, the microbes have been observed to cause mortality in pest populations and several microbial species have been investigated for their potential as biological control agents. Therefore, knowledge of the community of natural enemies in the agroecosystem as well as the effect of the agronomical practices on these organisms is essential to use a conservation biological control strategy. In this context, the present study was undertaken with an objective to isolate entomopathogenic microbes from the soils of research station of Junagadh Agricultural University, identify and characterize the isolates using cultural and biochemical tools, and study their qualitative and quantitative production of biotechnologically important enzymes, siderophore test and plant growth promoting characteristics, antipesticidal activity to confirm entomopathogenic isolates and PCR based molecular identification to confirm the species of isolates. In this present study, seventy two bacterial and thirty five fungal strains were isolated from thirty five soil samples collected from agricultural soils of different research station. Morphological, physiological and biochemical characteristics of these strains were studied by optimizing their growth conditions such as pH and NaCl, and the fifteen best performing isolates were selected as best entomopathogenic microbes for investigation of the above stated research objectives. All the bacterial isolates were able to tolerate NaCl concentration ranging from 2 to 8 per cent and fungal isolates were able to tolerate Nacl concentration ranging from 2 to 6. Best growth of all the fungal isolates was observed at the pH 6 and bacterial isolates was observed at the pH 8. The isolates were identified by microscopic Gram’s staining as gram positive, negative and by scanning electron microscope as short to thin long rods and coccus shape bacteria and fungi showed septate hyphae, dense to disperse, flate and color strain. The isolates also exhibited some considerable antimicrobial activities against phytopathogenic bacteria, fungi and pest tested and intrinsic antibiotic resistance activity against antibiotics such as Ampicillin (AMP), Amoxicillin (AMX), Cloxacillin (COX), Erythromycin (E), Nitrofurantoin (NIT), Penicillin-G (P) etc. The various biochemical tests such as lipid and gelatin hydrolysis, Voges-Proskauer, catalase, oxidase, nitrate reduction, utilization of glucose, Citrate and production of chitinase, cellulase and protease enzymes were found to be positive for majority of the isolates. The quantitative chitinase, cellulase and protease enzyme activities produced by the isolates ranged from 0.303 to 0.827 U ml-1 , 0.778 to 2.714 U ml-1 and 0.550 to 0.990 U ml-1 respectively. The isolates tested for plant growth promoting properties showed phosphate solubilization efficiency ranging from 10.00-50.00% on 3 DAI to 80.00-160.00% on 11 DAI, and concentration of inorganic phosphorous released ranging from 215.02- 685.02 μg/ml on 3 DAI to 452.88-740.74 μg/ml on 11 DAI. The potash solubilization efficiency of isolates ranged from 11.11-70.00% on 3 DAI to 25.00-140.00% on 11 DAI and the zinc solubilization efficiency of isolates ranged from 9.09-80.00% on 3 DAI to 16.67-127.27% on 11 DAI. The nitrogen fixation capacity of the isolates ranged from 1.515 to 3.846 mg ‘N’/g of Jensen’s agar medium. On the other hand, eight out of fifteen isolates showed positive test of siderophore production. The siderophore type produced was further identified by tetrazolium test as hydroxymate type and quantification range of siderophore 51.8 to 155.2 µg ml-1 . The fifteen selected entomopathogenic bacterial and fungal isolates studied were identified by phylogenetic analysis of sequences obtained from partial 16S and 18S rRNA gene sequencing. five out of twelve bacterial isolates belongs to genus Bacillus while four isolates belongs to genus Pseudomonas and remaining three isolate belongs to genus Enterobacter and one out of three fungal isolates belongs to genus Verticillium while another one isolates belongs to genus Metarhizium and remaining one isolate belongs to genus Beauveria.
  • ThesisItemOpen Access
    GENOME AND TRANSCRIPTOME SEQUENCING OF BARNYARD MILLET (Echinochloa esculenta L.) FOR THE DEVELOPMENT OF GENIC SSR MARKER AND DIVERSITY ANALYSIS 2879
    (JAU,JUNAGADH, 2019-07) Meniya Vipul H.; Dr. R. S. Tomar
    Barnyard millet (Echinochloa esculenta L. ) is grown for human consumption as well as fodder. Barnyard millet is the second important small millet after finger millet having production and productivity 87 thousand tonnes and 857 kg/ha, respectively. Among small millets, it is the fastest growing millet and highly self-pollinated crop in to family Poaceae. Barnyard millets are high in fibre content, phosporous and calcium. The protein content in barnyard millet ranged from 11.1% to 13.9%. Barnyard millet grain contains about 65% carbohydrate, majority of which is in the form of non-starchy polysaccharide and dietary fibre. Barnyard has non-glutinous type of endosperm. The tannin content is at lowest level in barnyard millet (102.96 mg). Barnyard millet is highly suitable for commercial foods for diabetics, infants and pregnant women because of high iron content 38.5 to 42 ppm. Bardyard has low glycemic index and thus helps in type 2 diabetes, cardiovascular disease with regular intake of this millet. The draft genome sequencing of barnyard millet was carried out and found ion S5 next generation sequencing after preparation of DNA library of 400bp.The total raw sequence generated by Ion S5 sequencer was 2,12,17,622Mbp. Total raw data was 6.75 Gb. Raw data (Reads) generated for barnyard millet were assessed for quality check using Fast QC. After trimming of reads average read length was 318bp. The de novo assembly (using CLC genomic workbench V 8.2) yielded assembled reads of 59,67,79,933bp and number of contigs were 1,139,481. Similar to genome sequencing, transcriptome sequencing was also carried out for barnyard millet. Three RNA samples, for vegetative, reproductive and maturity stages, were sequenced using the high-throughput Ion torrent sequencing technique. The number of raw reads in vegetative stage were 1,39,65,364 while in reproductive and maturity stage the reads were 1,56,90,496 and 1,48,43,221 respectively. The average read length at three different stage were 127bp, 132bp, and 129bp. The SSR and EST-SSR primers were designed using BatchPrimer3 (version 1.0) interface modules. These SSR marker were used to study genetic diversity, For the identification of SSRs four FASTA files of contigs were used belonging to genome and transcriptome. Total 30 SSRs were identified with Tm and GC % range of 56-60°C and 40-70 respectively. For validation of these 30 SSR and EST-SSR primers, 30 different varieties of Barnyard millet were taken, Among 30 primer, 21 primer were able to amplify the DNA. The PCR amplification of 30 different varieties yielded 33 bands. While about genetic diversity, Genome sequencing also gave an idea about evolution of genetic structure and function as well as study of molecular phylogeny of individual species with the genome of evolutionary proximate species. It also provides data through which we can identify gene and functional elements of genome and give basis for annotation of complete plant genome. The transcriptome sequencing of barnyard millet may help improve future genetic and genomics studies on the molecular mechanisms behind the chemical composition of the plant. In barnyard millet, RNA-seq were accelerated the investigation of the complexity of gene transcription patterns, functional analyses and gene regulation networks. SSRs dataset generated, will provide the most comprehensive resource currently available for gene expression, gene discovery, and future genomic research on Echinochloa esculenta L. Dedicated
  • ThesisItemOpen Access
    BIOCHEMICAL AND MOLECULAR CHARACTERIZATION FOR AROMA IN RICE (Oryza sativa L.) GENOTYPES OF GUJARAT REGION 2991
    (JAU, JUNAGADH, 2019-10) PATIL RAHUL ISHWAR; A. G. Vala
    Aromatic rice is highly cherished in many countries of the world and commands premium prices at all levels of the global rice trade. Generally, aroma in rice is a result of numerous volatile and semi-volatile compounds. 2-acetyl-1-pyrroline (2-AP) is the major volatile compound responsible for its fragrance. The presence of aroma in aromatic rice is controlled by betaine aldehyde dehydrogenase 2.1 (badh2.1/ fgr) gene allele which results from an eight-base pair deletion and three single nucleotide polymorphisms (SNPs) in exon seven of Betaine aldehyde dehydrogenase 2 (Badh2) gene. This mutation is responsible for the introduction of premature stop codon which produce a truncated protein, this results in loss of function of the enzyme Betaine aldehyde dehydrogenase 2 (BADH2) leading to accumulation of 2-acetyl 1-pyrroline (2AP) in aromatic rice varieties. In the present study, the association of badh2.1 aromatic allele with aromatic compound (2-AP), total free amino acids and protein content was investigated in 20 rice Gujarat genotypes. For this purpose, Leaf aromatic tests was conducted for aroma evaluation, effect of free amino acids and protein content on the sensory quality were compared to determine the most important factors affecting the sensory quality. Quantification of 2-AP were carried out using HP-GCMS. Screening of badh2.1/ fgr gene allele was conducted using allele specific amplification (ASA) and qRT-PCR was performed to determine the expression levels of badh2.1 in the fragrant and non- fragrant rice genotypes of Gujarat. In addition, SSR profiling was employed to analyse the genetic diversity among aromatic and non-aromatic rice genotypes. Based on leaf aromatic test, four genotypes were detected for having strong aroma, three for moderate aroma; eight for slight aroma and five for no aroma. Total free amino acid was ranged from 1.67 to 0.97 mg/g and the protein content varied between 4.8 to 7.50 %. Free amino acids had positive correlations while protein contents showed negative correlation with the aroma and both were non-significant. It was observed that the amount of 2-AP were present in almost all genotypes except GAR-13 and Gurjari, both of these genotypes had no 2-AP. The range of 2-AP observed in between 0 - 202.09 ppb in rice samples. The highest concentration of 2-AP found in the genotype 4814 followed by the Pusa basmati (194.98 ppm). A total of 84 alleles were amplified with the SSR markers with an average of 6.46 alleles per locus. A total of 17 rare alleles were detected at eight loci, whereas 27 unique alleles were detected at 11 loci. Polymorphic information content (PIC) values for SSR ranged from 0.18 to 0.87 with an average of 0. 72. Principal Coordinate analysis (PCoA) with SSR markers showed that genotypes were uniformly distributed across the two axes with 32.50% of cumulative variation. Analysis of Molecular Variance (AMOVA) revealed that 97% of the total variation observed in this genotype came from within the populations, while 3% of the variation emanated among the populations. Cluster analysis based on Jaccard’s similarity coefficient using UPGMA, grouped all rice cultivars into two cluster. Matrix similarity coefficient ranged from 0.65 to 0.90. All the rice genotypes included in the study could be distinguished from each based upon the polymorphic bands generated by employing the primers. Screening for badh2.1 allele revels nine genotypes as homozygous aromatic (badh2.1/badh2.1), 11 were homozygous non-aromatic (BADH2/BADH2) and no heterozygous non-aromatic (BADH2/badh2.1). Five rice genotypes viz; 4815, 4814, 4820, 4819 and 4808 were found to be up regulated (↑) and the maximum up-regulation of badh2 gene was reported for the genotype 4808 (6.58). These genotypes also found as homozygous aromatic by ASA and has higher aroma score with higher quantity of 2-AP. Conversely, three genotypes from aromatic group viz; 4809, 4817, 4810 and five genotypes from non-aromatic group viz; GR-11, GR-12, GAR-13, 4816 and Gurjari were found to be down-regulated and hence, their aroma score as well as quantity of 2-AP were found lower with homozygous non-aromatic by ASA. In contrast to all, genotype 4813 was up-regulated and found as homozygous aromatic by ASA and slightly higher quantity of 2-AP but still has low aroma score. While, genotype 4821 was down-regulated (↓) in gene expression study and also categorised as homozygous non-aromatic by ASA but still show high aromatic by leaf aromatic test and has higher quantity of 2-AP. Overall, among the 20 genotyped and phenotyped; 15 genotypes were classified as aromatic by the presence of aroma in leaf aromatic tests, eight out of 15 carried badh2.1/fgr gene allele with high concentration of 2-AP with up-regulated expression of badh2.1/fgr gene. This suggests that badh2.1/fgr gene allele is the present in most of the Gujarat rice genotypes. These findings will contribute significantly in planning for effective rice breeding strategies especially in selection of appropriate parental materials for developing high yielding aromatic rice varieties in the Gujarat region.
  • ThesisItemOpen Access
    GENOME AND TRANSCRIPTOME SEQUENCING OF LITTLE MILLET (Panicum sumatrense Roth.) FOR THE DEVELOPMENT OF GENIC SSR MARKERS AND ITS VALIDATION 2984
    (jau.junagadh, 2019-09) BHUT NISHANT HIMMATBHAI; Dr. K. H. Dabhi
    Little millet is the small grained cereal belonging to family poaceae. It has 1.2% fat, 7.7% protein and 68.8% carbohydrates. It is used as bread or as rice roughage for animal and grain for poultry feeding. It is poor man food particularly in dry region of country where it is not possible to grow other food crop. Little millet has long history of cultivation of more than thousand years and grows in many states. Maharashtra is the first in case of little millet covering an area of 96 thousand hectares being eco-friendly crop. It is suitable for fragile and vulnerable ecosystems and regarded as preferred crops for sustainable and green agriculture. The draft genome sequencing of little millet was carried out on ion S5 next generation sequencing after preparation of DNA library of 400bp. The total raw sequence generated was about 5.86 Gb, which includes 1,97,68,054 Mbp of sequence data. The raw data, where quality checked using FastQC. After trimming of reads average read length was 247 bp. The de novo assembly (using CLC genomic workbench V 8.2) yielded assembled reads of 8,32,56,643 bp and number of contigs were 1,89,827. Similar to genome sequencing, transcriptome sequencing was also carried out for little millet. Three RNA samples, for vegetative, flowering and maturity stages, were sequenced using the high-throughput Ion torrent sequencing technique. The number of raw reads in vegetative stage were 1,14,58,128 while in flowering and maturity stage the reads were 1,05,48,834 and 76,22,224 respectively. The average read length at three different stage were 158bp, 150bp and 165bp. The SSR and EST-SSR primers were designed using BatchPrimer3 (version 1.0) interface modules. These SSR marker were used to study genetic diversity. For the identification of SSRs four FASTA files of contigs were used belonging to genome and transcriptome. Total 30 SSRs were identified with Tm and GC % range of 58-59°C and 40% respectively. For validation of these 30 SSR and EST-SSR primers, 26 different genotypes of little millet were taken Among 30 primers, 20 primers were able to amplify the DNA. The PCR amplification of 26 different genotypes yielded 38 bands. While about genetic diversity, genome sequencing also gave an idea about evolution of genetic structure and function as well as study of molecular phylogeny of individual species with the genome of evolutionary proximate species. It also provides data through which we can identify gene and functional elements of genome and give basis for annotation of complete plant genome. The transcriptome sequencing of little millet may help improve future genetic and genomics studies on the molecular mechanisms behind the chemical composition of the plant. In little millet, RNA-seq were accelerated the investigation of the complexity of gene transcription patterns, functional analyses and gene regulation networks. SSRs dataset generated, will provides the most comprehensive resource currently available for gene expression, gene discovery and future genomic research on Panicum sumatrense Roth.
  • ThesisItemOpen Access
    BIOCHEMICAL AND MOLECULAR CHARACTERIZATION FOR AROMA IN RICE (Oryza sativa L.) GENOTYPES OF GUJARAT REGION 2991
    (JAU, JUNAGADH, 2019-10) PATIL RAHUL ISHWAR; A. G. Vala
    Aromatic rice is highly cherished in many countries of the world and commands premium prices at all levels of the global rice trade. Generally, aroma in rice is a result of numerous volatile and semi-volatile compounds. 2-acetyl-1-pyrroline (2-AP) is the major volatile compound responsible for its fragrance. The presence of aroma in aromatic rice is controlled by betaine aldehyde dehydrogenase 2.1 (badh2.1/ fgr) gene allele which results from an eight-base pair deletion and three single nucleotide polymorphisms (SNPs) in exon seven of Betaine aldehyde dehydrogenase 2 (Badh2) gene. This mutation is responsible for the introduction of premature stop codon which produce a truncated protein, this results in loss of function of the enzyme Betaine aldehyde dehydrogenase 2 (BADH2) leading to accumulation of 2-acetyl 1-pyrroline (2AP) in aromatic rice varieties. In the present study, the association of badh2.1 aromatic allele with aromatic compound (2-AP), total free amino acids and protein content was investigated in 20 rice Gujarat genotypes. For this purpose, Leaf aromatic tests was conducted for aroma evaluation, effect of free amino acids and protein content on the sensory quality were compared to determine the most important factors affecting the sensory quality. Quantification of 2-AP were carried out using HP-GCMS. Screening of badh2.1/ fgr gene allele was conducted using allele specific amplification (ASA) and qRT-PCR was performed to determine the expression levels of badh2.1 in the fragrant and non- fragrant rice genotypes of Gujarat. In addition, SSR profiling was employed to analyse the genetic diversity among aromatic and non-aromatic rice genotypes. Based on leaf aromatic test, four genotypes were detected for having strong aroma, three for moderate aroma; eight for slight aroma and five for no aroma. Total free amino acid was ranged from 1.67 to 0.97 mg/g and the protein content varied between 4.8 to 7.50 %. Free amino acids had positive correlations while protein contents showed negative correlation with the aroma and both were non-significant. It was observed that the amount of 2-AP were present in almost all genotypes except GAR-13 and Gurjari, both of these genotypes had no 2-AP. The range of 2-AP observed in between 0 - 202.09 ppb in rice samples. The highest concentration of 2-AP found in the genotype 4814 followed by the Pusa basmati (194.98 ppm). A total of 84 alleles were amplified with the SSR markers with an average of 6.46 alleles per locus. A total of 17 rare alleles were detected at eight loci, whereas 27 unique alleles were detected at 11 loci. Polymorphic information content (PIC) values for SSR ranged from 0.18 to 0.87 with an average of 0. 72. Principal Coordinate analysis (PCoA) with SSR markers showed that genotypes were uniformly distributed across the two axes with 32.50% of cumulative variation. Analysis of Molecular Variance (AMOVA) revealed that 97% of the total variation observed in this genotype came from within the populations, while 3% of the variation emanated among the populations. Cluster analysis based on Jaccard’s similarity coefficient using UPGMA, grouped all rice cultivars into two cluster. Matrix similarity coefficient ranged from 0.65 to 0.90. All the rice genotypes included in the study could be distinguished from each based upon the polymorphic bands generated by employing the primers. Screening for badh2.1 allele revels nine genotypes as homozygous aromatic (badh2.1/badh2.1), 11 were homozygous non-aromatic (BADH2/BADH2) and no heterozygous non-aromatic (BADH2/badh2.1). Five rice genotypes viz; 4815, 4814, 4820, 4819 and 4808 were found to be up regulated (↑) and the maximum up-regulation of badh2 gene was reported for the genotype 4808 (6.58). These genotypes also found as homozygous aromatic by ASA and has higher aroma score with higher quantity of 2-AP. Conversely, three genotypes from aromatic group viz; 4809, 4817, 4810 and five genotypes from non-aromatic group viz; GR-11, GR-12, GAR-13, 4816 and Gurjari were found to be down-regulated and hence, their aroma score as well as quantity of 2-AP were found lower with homozygous non-aromatic by ASA. In contrast to all, genotype 4813 was up-regulated and found as homozygous aromatic by ASA and slightly higher quantity of 2-AP but still has low aroma score. While, genotype 4821 was down-regulated (↓) in gene expression study and also categorised as homozygous non-aromatic by ASA but still show high aromatic by leaf aromatic test and has higher quantity of 2-AP. Overall, among the 20 genotyped and phenotyped; 15 genotypes were classified as aromatic by the presence of aroma in leaf aromatic tests, eight out of 15 carried badh2.1/fgr gene allele with high concentration of 2-AP with up-regulated expression of badh2.1/fgr gene. This suggests that badh2.1/fgr gene allele is the present in most of the Gujarat rice genotypes. These findings will contribute significantly in planning for effective rice breeding strategies especially in selection of appropriate parental materials for developing high yielding aromatic rice varieties in the Gujarat region.