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Open Access
TRANSCRIPTOME SEQUENCING AND METABOLOMIC CHARACTERIZATION OF BARNYARD MILLET (Echinochloa frumentacea L.) TO DISCOVER PUTATIVE GENES INVOLVED IN SPIKE DEVELOPMENT AND ITS NEUTRACEUTICAL PROPERTIES
(JAU,JUANAGDH, 2020-12) PADHIYAR SHITALBEN MALDEBHAI; Dr. R. S. Tomar
Barnyard millet comprises two different cultivated species, Japanese barnyard millet (Echinochloa esculenta L.) and Indian Barnyard millet (Echinochloa frumentacea L.) belongs to the plant family Poaceae.The other names of barnyard millet in Gujarati is moraiyo. Barnyard or sawa millet is the fastest growing crop of all millet, which can produce ripe grains within 45 days from the sowing time under optimal weather conditions. In addition to these agronomic advantages, the grains are valued for their high nutritional value and lower expense as compared to major cereals like rice, wheat, and maize. It is important minor millet in Japan, China, India and other South East Asian countries. Barnyard millet is a multi-purpose crop which is cultivated for food and fodder. Barnyard millet grains are a rich source of dietary fiber, iron, zinc, calcium, protein, carbohydrate, magnesium, fat, vitamins, some essential amino acids and, most notably, contains more micronutrients (iron and zinc) than other major cereals. Due to low glycemic index and high dietary fiber, it helps in preventing diabetes and cardio vascular disease with regular intake. Barnyard millet could be a good source of iron for vegetarians. Keeping these benefits in the mind present study was undertaken to find out candidate genes and metabolites responsible for Fe content in barnyard millet genotypes. In the current study, biochemical parameter (proximate and minerals) was collected out in 30 barnyard millet genotypes and result shown that total carbohydrate content was found in range between 53.51-73.98 %. The range for total protein (9.55- 11.99 %), crude fiber (11.14–16.64 %), moisture content (4.47-7.68 %), ash content (3.47 – 5.99 %), total fat content (3.38 - 6.95% ), energy (304.45–380.11 Kcal/mole), fatty acid profiling reveled Oleic acid content was observed higher (81.19 %) followed by Palmitic acid (69.33 %), Linolenic acid (50.96 %) and Linoleic acid (48.11 %), nitrogen (1.56-2.72 %), phosphorus (326.67-486 mg/100g), potassium (170-383 mg/100g), sodium (31.67-51 mg/100g), iron content (3.83-11.14 mg/100g), zinc (2.01- 3.88 mg/100g), manganese (1.33-2.85 mg/100g), calcium (35.83–209.18 mg/100g), copper (0.161-0.407 mg / 100g), magnesium (101.65 and 182.21 mg/100g) and boron (0.45-1.99 mg /100g) were observed in 30 barnyard millet genotypes. Two barnyard millet genotypes having high Fe (BAR-1433) and low Fe (BAR 1423) in their seeds were taken for transcriptome and metabolomic analysis during spike development stage. Total pool 30 HQ reads were considered for de novo assembly optimization using four standard assembler Trinity, Soapdenovo_trans, CLC and CAP3 assembler. The trimmed number of reads yielded a total of 138 million high-quality reads, which were assembled by trinity into 4,88,689 transcript. The trinity generated highest transcript with assembly size of 340 MB, N50 size of 1202 and N90 size of 611. CAP3 assembler was employed to reduce transcript redundancy and it generate 27,228 transcript with assembly size of 31.84 MB, N50 size of 1570 bp and N90 size of 723 bp. Moreover, more redundancy were removed by CD-HIT programme and assembler which generated 20,849 transcripts with size assembly of 24.42 MB, N50 size of 1604 bp and N90 size of 719 bp. Transcriptome analysis identified key genes regulating Fe accumulation like CIAO1, ISAM1 and UCRIA during different spike development stage. Further, Ferritin, ABC, Cytochrome, Metal, Zinc, Serine, Wall-associated and Cytochrome, Mitogen and Photosynthesis regulating differential gene also were identified which are also involved in iron content of Low Fe Vs High Fe barnyard millet genotype. Functional annotation was carried out for Gene Ontology (GO) enrichment and KEGG pathway enrichment from significant DEGs. Gene ontology (GO) and pathways analysis revealed that different metabolic pathway like glycolysis, purine and pyruvate metabolism based on expressed genes revealed the mechanism of minerals transportation. The resources generated in this study will facilitate discovery of new genes and further EST-SSR markers and thereby accelerate both genetic and functional genomic research in barnyard millet. Identified genes may be used for marker-assisted selection and breeding to develop minerals rich crops. The Comparison of transcript expression levels between transcriptome data and qRT-PCR depicted positive correlation. The validation through qRT-PCR was carried out using ACBL, ABA, RP8L, PDF1 and PHY which were among the up and down regulated transcripts in different stages of barnyard millet. Metabolome profiling of barnyard millet genotypes containing high Fe and low Fe was performed using GC-MS platform and results observed highest percentage of sugar and sugar alcohol (34%) followed by organic acid (26%), Amino acids, sterol, other compound (23%) and fatty acid (17%) in spike development stages of Low Fe Vs High Fe genotypes. Heat map revealed that during spike emergence the metabolic compounds like d-Ribose, D-Fructose, D-Glucose, Galactose, D-Turanose, Glucopyranose, D-Mannitol, Hexadecanoic acid, Docosanoic acid, alpha.- Glycerophosphoric acid and .beta.-Sitosterol were found high in high Fe genotype in comparison to Low Fe genotype. The hierarchical cluster analysis, revealed that high Fe five spike development stages shares close metabolite pool to each other and in low Fe four spike development stages also shares similarly metabolite pool to each other except spike emergence stage of Low Fe genotype. PCA showed that the expression patterns of the five developmental stages differed significantly.
Open Access
DEVELOPMENT AND VALIDATION OF EST DERTVED SSR MARKERS WITH FUNCTIONAL RELEVENCE TO BIOTIC AND ABIOTIC STRESS RESPONSES IN GROUNDNUT 1980
(JAU,JUNAGADH, 2015-03) Bosamia Tejas Chimanlal; Dr. Radhalcrishnan T.
Availability of polymorphic markers in a crop species is a prerequisite for genetic improvement through marker-assisted selection, construction of high density genetic linkage map and mapping of Quantitative Trait Loci (QTL). Simple sequence repeat (SSR) markers are widely employed in molecular breeding programmes and thus are needed to be developed and validated. Conventional methods for developing SSR markers are laborious and expensive. The alternative and cost effective way is to explore the existing public databases harboring abundant number of genomic and genie (expressed sequence tag, EST) sequences for the development of new SSR markers. The SSR markers developed from ESTs leverages functional aspect of transcripts and thus associated with its function. Insufficient number of polymorphic molecular markers is an obstacle for current molecular breeding research in groundnut (Arachis hypogaea L.), an important legume grain as well as oilseed cash crop of the semi-arid tropics. It becomes exceedingly essential to develop more number of polymorphic SSR markers for potential use in groundnut improvement. Therefore, the present study was carried out to develop EST-SSRs from publically available groundnut EST resources. In the present study, a total of 178490 EST sequences of Arachis hypogaea ^ere downloaded from NCBI public databases and pre-processed for assembling. In order to find novel non-redundant EST-SSR markers, 7170 publically available SSR primer sequences were subjected to sequence similarity search with the downloaded EST sequences. A total of 23696 (13.28%) very similar sequences were excluded and the remaining non-redundant sequences (154794) were further pre-processed for removai of low corapiexity sequences, poiy A and poly T tail and vector sequences. A total of 138628 high quality sequences were assembled using TGICL software, which yielded 16424 unigenes comprising of 13428 (81.76%) contigs and 2995 (18.24%) singletons with an average length of 857 bp and N50 value of 942 bases. The assembly by TGICL software reduced the redundancy by 82.21% A total of 2784 sequences were indentifled from unigenes which harbored 3373 SSR motifs The number of sequences harboring more than one SSR was 487 (17.49%) and 289 (10.38%) sequences comprised of compound SSRs. The avemge frequency of SSR was found to be one in 4.17 kb which was relatively higher frequency than previous reports in groundnut.
Open Access
DEVELOPMENT AND VALIDATION OF EST DERTVED SSR MARKERS WITH FUNCTIONAL RELEVENCE TO BIOTIC AND ABIOTIC STRESS RESPONSES IN GROUNDNUT 1982
(JAU, JUNAGADH, 2015-03) Bosamia Tejas Chimanlal; Dr. Radhalcrishnan T.
Availability of polymorphic markers in a crop species is a prerequisite for genetic improvement through marker-assisted selection, construction of high density genetic linkage map and mapping of Quantitative Trait Loci (QTL). Simple sequence repeat (SSR) markers are widely employed in molecular breeding programmes and thus are needed to be developed and validated. Conventional methods for developing SSR markers are laborious and expensive. The alternative and cost effective way is to explore the existing public databases harboring abundant number of genomic and genie (expressed sequence tag, EST) sequences for the development of new SSR markers. The SSR markers developed from ESTs leverages functional aspect of transcripts and thus associated with its function. Insufficient number of polymorphic molecular markers is an obstacle for current molecular breeding research in groundnut (Arachis hypogaea L.), an important legume grain as well as oilseed cash crop of the semi-arid tropics. It becomes exceedingly essential to develop more number of polymorphic SSR markers for potential use in groundnut improvement. Therefore, the present study was carried out to develop EST-SSRs from publically available groundnut EST resources. In the present study, a total of 178490 EST sequences of Arachis hypogaea ^ere downloaded from NCBI public databases and pre-processed for assembling. In order to find novel non-redundant EST-SSR markers, 7170 publically available SSR primer sequences were subjected to sequence similarity search with the downloaded EST sequences. A total of 23696 (13.28%) very similar sequences were excluded and the remaining non-redundant sequences (154794) were further pre-processed for removai of low corapiexity sequences, poiy A and poly T tail and vector sequences. A total of 138628 high quality sequences were assembled using TGICL software, which yielded 16424 unigenes comprising of 13428 (81.76%) contigs and 2995 (18.24%) singletons with an average length of 857 bp and N50 value of 942 bases. The assembly by TGICL software reduced the redundancy by 82.21% A total of 2784 sequences were indentifled from unigenes which harbored 3373 SSR motifs The number of sequences harboring more than one SSR was 487 (17.49%) and 289 (10.38%) sequences comprised of compound SSRs. The avemge frequency of SSR was found to be one in 4.17 kb which was relatively higher frequency than previous reports in groundnut. Regarding functional annotation, 2784 unigenes harboring SSR motifs were subjected to BIast2G0, an online tool for functional annotation. Among the 2784 un,genes, 2027 (72.81%) unigenes were annotated and assinned f . ontology (GO) temrs (4124) were ascribed to unigenes and categorizeriUrr components (391), molecular functions (1120) and hiol • , "^^I'tilar ntaximum homology of groundnut unigenes through aiyclne n,ax (48.2%) followed by Cto orterimm, (16.6o/„). and the sequences of rep^ ml^C rl'XT'''' motifs were categorized in to class I flonap tu ^ (^^-47%) SSR motifs in to short length range class II a' ^ <«»"%) SSR were the most predominant with 33.86% foUowel 77 "-ofifs Tetra-, penta- and hexa-nucleotide repeats meas L (27-51%). r ~AAT/ATT (4.80%) and 1^1" ^^<='ATG (fss^ assembly. The searrvi, ^ repeats wert^ i ~ -* '■ i. .N. ..z,Pnmers were designed hv BatchPrimer3 tool and 2456 • 2784 SSR com • • Pnmers were designed A ^'"^"8 sequences by 5>ica. A set of . 366 pr,mer pairs with functional relevance to biotic and abiotic stresses were selected, synthesized and screened for polymorphism in eleven genotypes which represents the parents of six mapping populations. A set of 339 (92.62%) primer pairs yielded scorable amplicons and 39 (10.66%) primer pairs showed polymorphism among eleven parental genotypes. The 339 primers detected 2 to 12 alleles with an average of 3.77 alleles per marker where as among the 39 polymorphic primers yielded 2 to 12 alleles with an average of 5.10 alleles per marker. The Polymorphic Information Content (PIC) value for these 39 polymorphic markers ranged form 0.028 to 0.375 with an average of 0.325 per marker. The polymorphism screening indicated that compound SSRs were the most predominantly polymorphic (26.09%) followed by di-nucleotide (13.34%) and tri-nucleotide (11.72%) motifs. Among the SSR motif repeats, di-nucleotide repeat SSR markers showed higher PIC value (average 0.357 per marker) followed by penta (average 0.335 per marker) and compound (average 0.332 per marker). The newly designed novel SSR markers have added to the repository of groundnut genomic resources utilizing almost all the available ESTs in public databases as on available till date. The EST-SSR markers showing polymorphism in the parental genotypes could be further screened for polymorphism on a panel of mapping population segregating for biotic or abiotic stress for finding markers linked with that trait. These EST-SSRs could also be effectively utilized for saturation of genetic map or transcript map, QTL mapping and marker-assisted selection for cultivated groundnut.
Open Access
BIOCHEMICAL AND PHYSIOLOGICAL ANALYSIS AND PROTEIN PROFILING IN WHEAT {Triticum aestivum L.) GENOTYPES UNDER HEAT STRESS 1939
(JAU, JUNAGADH, 2014-10) Hitesh Ranchhodbhai Raman!; Dr. M. K. Mandavia
The present experiment on "Biochemical and Physiological Analysis and Protein Profiling in Wheat {Triticum aestivum L.) Genotypes under Heat Stress" was conducted at Department of Biotechnology, Junagadh Agricultural University, Junagadh. The experiment-1 was carried out using fourteen wheat genotypes and four heat treatments using Factorial CRD design, where seeds were grown in germination bag filled with soil for 10 days. The seedlings were subjected to control and heat treatments at 35°C, 40°C and 45°C for four hour and samples were analysed for relative water content, membrane stability and injury, lipid peroxidation, hydrogen peroxide content and chlorophyll stability index. Heat tolerant genotype GW-190 genotype showed highest membrane stability and relative water content and lowest membrane injury, lipid peroxidation and hydrogen peroxide compared to other genotype so it was selected as best heat tolerant genotype. Heat susceptible genotype J-2010-11 showed lowest membrane stability (MS) and relative water content and highest membrane injury, lipid peroxidation and hydrogen peroxide content so it was selected as highly susceptible genotype. Above physiological and biochemical parameters may be used for screening the susceptible and tolerant wheat genotypes against heat stress. H2O2 and MS are more effective indicators for screening heat tolerant genotypes under stress condition. From results of the experiment-1, one heat tolerant (GW-190) and heat susceptible (J-2010-11) wheat genotypes were selected and the plants were subjected to two groups; control and heat treatments where 40°C and 45°C heat treatments given for 2 h and 4 h of duration and physiological, biochemical, antioxidant enzyme activities, Isoenzymes and Protein profiling by 2D electrophoresis analysis were performed. Relative water content and membrane stability were found to be higher in heat tolerant genotype GW-190 compared to heat susceptible genotype J-2010-11 at tillering and grain filling stages. As heat stress and duration of heat stress increased the relative water content and membrane stability of heat tolerant and heat susceptible genotypes were decreased at both the stages of wheat development. Compared to tillering stage, relative water content and membrane stability were found lower in grain filling stage because of increased temperature. Protein, Proline and glycine betaine, Glutathione reductase, Peroxidase and Superoxide dismutase acitivities were found to be higher in heat tolerant genotype GW-190 compared to heat susceptible genotype J-2010-11 at tillering and grain filling stages. As heat stress and duration of heat stress increased, the biochemical constitutes and antioxidant enzymes activities of heat tolerant and heat susceptible genotypes also increased at both the stages of wheat development. As well all the biochemical constitutes and all three antioxidant enzymes activities were found to be higher in tillering stage compared to grain filling stage. At tillering stage, in case of Peroxidase, band No. 5 (Rm= 0.289) and band No. 6 (Rm=0.495) were present only in heat tolerant genotype while it was absent in heat susceptible genotype. At grain filling stage, band No. 3 (Rm=0.160) was present only in heat tolerant genotype. In case of Superoxide dismutase at tillering stage all the bands were present in heat tolerant as well in heat susceptible genotypes. At graing filling stage, band No. 2 (Rm—0.072) was present only in heat tolerant genotype. So isoenzymes may be useful for screening the heat tolerant and heat susceptible genotypes. At tillering stage, more total protein spots (1207) were recorded compared to that of (972) spots at grain filling stage. Compared to control, in heat stress condition expression of spots were increased. This was not true for grain filling stage. At tillering stage, highest numbers of protein spots (207) were found at 45°C for 4h duration in heat tolerant genotype GW-190 while it was true (148) spots at 40°C for 2h duration in heat tolerant genotype GW-190 at grain filling stage. The protein spots showed the differential expression pattern in treated heat tolerant genotype might be responsible for the stronger heat tolerance. Scanning electron microscopy of wheat leaves showed that analysis of variance indicated significant differences for stomatal length existed among heat tolerant and susceptible genotype as well significant differences were found for stomatal width among heat tolerant and susceptible genotype. Total 12 Operon series RAPD primers were amplified to generate the 105 fragments. The percent polymorphism obtained for RAPD primers were ranged from 71.4% to 100% with an average value of 92.33% per primer. Subcluster A1 (b) of cluster-1 consisted of only one heat tolerant genotype J-2010-06 with more than 66% of similarity. Subcluster A2 of cluster-I consisted of only one heat susceptible genotype J-2010-13 similarity of more than 85%. Cluster-II consisted of two genotypes J-2010-05 and GW-190 sh " similarity of more than 85% that belongs to heat tolerant groups. These primers be used to screen the genotypes against heat stress.
Open Access
CONSTRUCTION OF LINKAGE MAPPING AND IDENTIFICATION OF QUANTITATIVE TRAIT LOCI (QTL) FOR EARLINESS, GRAIN SIZE AND LEAF RUST RESISTANCE IN BREAD WHEAT (Triticum aestivum L.) 2538
(JAU,JUNAGADH, 2018-06) Delvadiya Niravkumar A.; Dr. D. R. Mehta
Present experiment was carried out at the Biotechnology Laboratory of the Department of Genetics and Plant Breeding, J.A.U., Junagadh during the year 2014 to 2017. The experimental material comprised of P1, P2, F1, F2 and F2:3 generations of two wheat crosses viz., GW-11 X GW-322 and DL-788-2 X GW-322 especially for grain size related traits and earliness related traits, respectively to fulfill the objective of linkage and QTL mapping as well as to estimate gene effects using means of the five generations for grain yield and its components in bread wheat. Out of 200 SSR markers screened for parental polymorphism for grain size and related traits, about 23% of SSR markers showed good polymorphism between two parental lines. Out of 46 tests for calculated chi-square, 42 test markers do not deviate significantly from expected ratios revealing that observed data are in agreement with expected ratio of 1:2:1. The linkage map was constructed using software IciMapping v.4.1. Recombination frequencies were converted into map distance using Kosambi’s mapping function. The markers were grouped with minimum logarithm of the odds (LOD) of 3.0 with walking speed was set at 1.0 cM. Seven linkage groups with a total map length of 77.31 cM were constructed using data from 46 SSR marker loci for 74 F2 plants which ranged from minimum of 2.74 cM (LG2) to maximum of 26.89 cM (LG3). In case of earliness and related traits, out of 200 markers screened, only 11% of SSR markers showed good polymorphism between two parental lines. Out of 22 tests, all the test markers showed non-significant chi-square which revealed that observed data are agreement with expected ratio of 1:2:1 segregation ratio. Four linkage groups with a total map length of 267.12 cM were constructed using data from 22 marker loci for 74 F2 plants that ranged from minimum of 8.62 cM (LG4) to maximum of 126.56 cM (LG1). Genotypic data of F2 and phenotypic data of on 74 F2:3 lines were analyzed for identification of the main effect QTLs using the software ICIM-ADD mapping in QTL IciMappingV4.1. A linkage map of grain size related traits output data file was used for the construction of QTL mapping. A total six QTL had been identified for grain size and related traits, one each for 100-grain weight (LG3 at 13 cM, LOD 6.68, 42.20 PVE%); number of grain per main spike (NGPMS) (LG4 at 4 cM, LOD 3.28, 19.48 PVE%); grain ii yield per plant (GYPP) (LG5 at 14 cM, LOD 7.17, 36.25 PVE%); number of effective tillers per plant (NETPP) (LG7 at 3 cM, LOD 14.34, 59.17 PVE%); and two QTLs for grain weight per the main spike (GWPMS) (LG3 at 20 cM, LOD 4.64, 15.29 PVE% and LG6 at 11 cM, LOD 12.29, 58.03 PVE%). Similarly, earliness and related traits output data file of linkage map was used for the construction of QTL mapping. One QTL was identified for days to 50% flowering (LG1 at 58.0 cM, LOD 3.06, 18 PVE %) and two QTLs for days to maturity (LG1 at 21 cM, LOD 8.89, 31.51 PVE% and LG3 at 38 cM, LOD 12.83, 45.16 PVE%). Scaling tests C and D and joint scaling test showed non-significant chi-square and hence additive-dominance model was found adequate for grain yield per plant and 100-grain weight in the cross GW-11 x GW 322 and length of main spike in the cross DL 788-2 x GW 322. In three-parameter model, only mean (m) effect was found significant in above three cases besides significance of additive (d) and dominance (h) effects for length of main spike in cross DL 788-2 x GW 322. Significant chi-square in joint scaling test confirmed the results obtained in individual scaling test C and D in both the crosses (except above three cases) indicating the presence of epistasis. The five-parameter model revealed that in addition to the significance of m, (d) and (h) effects, both (i) and (l) were significant for number of grains per main spike and biological yield per plant in cross GW 11 x GW 322 and days to 50% flowering, plant height, number of spiklets per main spike, peduncle length of main spike days to maturity, number of grains per main spike, grain yield per plant and biological yield per plant in the cross DL 788-2 x GW 322. The hybrids showing epistasis had significant and positive dominance (h) effect for days to maturity and number of grains per main spike in both the two crosses and plant height and peduncle length of main spike in the cross DL 788-2 x GW 322. Wider phenotypic range and higher co-efficient of range was observed in F2 generation as compared to F2:3 generation for days to 50% flowering, plant height, number of grain per main spike, grain weight per main spike and biological yield per plant GW-11 X GW-322 as well as for plant height, number of grain per main spike, grain weight per main spike and harvest index in DL 788-2 x GW 322. High estimates of heritability (above 60%) coupled with high genetic advance as per cent of mean (above 20%) was observed in both F2 and F2:3 generations for plant height, number of effective tillers per plant and grain yield per plant in GW-11 X GW-322 as well as for plant height, number of effective tiller per plant, peduncle length of main spike, grain yield per plant, biological yield per plant, harvest index in DL 788-2 x GW 322. Key words: Linkage mapping, QTL mapping, SSR marker, Gene effects, Bread wheat
Open Access
IN SILICO IDENTIFICATION AND TARGET PREDICTION OF microRNAs AND THEIR EXPRESSION PROFILING IN Sesamum indicum L. 2524
(JAU,JUNAGADH, 2018-04) Joshi Halakben V.; Dr. M. K.Mandavia
Sesame (SesamumindicumL., 2n = 26), a member of the Pedaliaceae family, is one of the oldest oilseed crops. For its high oil content, it is known as the “queen of oilseeds”.Sesame seeds are important source of oil, protein, and carbohydrates.Sesame oil is used as an active ingredient in antiseptics, bactericides, viricides, disinfectants, moth repellants, and antitubercular agents because they contain natural antioxidants such as sesamin and sesamolin. MicroRNAs (miRNAs) represent a class of endogenous non-coding small RNAs that playimportant roles in multiple biological processes by degrading targeted mRNAs or repressingmRNA translation. Thousands of miRNAs have been identified in many plant species,whereas there is no report of miRNAs in S.indicumtill date. In present study, previously known plant miRNAs were BLASTedagainst the Expressed Sequence Tag (EST),Genomic Survey Sequence (GSS) and Whole Genome Sequencing (WGS) database of sesame genes.The aligned miRNA hits were further aligned to protein database and BLASTX was carried out to remove protein coding primary miRNAs. The non-coding precursor miRNAs were subjected to online Mfold server in order to predict their secondary structures. Numbers of filtering criteria like proper hairpin-loop structure, Minimum Folding Free Energy (MFE), %A+U content etc. were applied. A total of 124 potential miRNAs belonging to 28miRNAs familieswere detected after applying the range of filtering. The targets were predicted online by psRNATarget web server.320 uniquemiRNA:target pairs were subsequentlypredicted, most of which encode transcription factors or enzymes that participate in the regulationof development, growth, metabolism, and other physiological processes and stress response. In order to analyze expression level of the predicted miRNAs, qRT-PCR was applied to detect the stress related expression levels of 20 putativemiRNAs in four samples viz., control, Seedlings treated for drought stress, Seedlings treated for salinity stress and Seedlings treated withFusariumoxysporumf. sp. sesami pathogen.The relative expression specified which miRNAs were up/down regulated during stress. The targets for respective miRNAs were compared with the findings of in silicoanalysis and similarities were found. It could be suggestive of the validity of the computational methods. This study providessome important information about sesame pre-miRNAs, mature miRNAs, and miRNA targetgenes and these findings can be applied to future research of miRNA functions.
Open Access
MICRO RNA PROFILING, ANTIFUNGAL AND NANOFORMULATION CHARACTERIZATIONS OF MULTI STRESS TOLERANT Trichoderma FUSANTS FOR BIOCONTROL ACTIVITY AGAINST Sclerotium rolfsii Sacc. CAUSING STEM ROT IN GROUNDNUT 2522
(JAU,JUNAGADH, 2018-04) Ms. Darshna G. Hirpara; Dr. H. P. Gajera
The soil borne fungal disease contributed 30-80% of average yield losses in agricultural crops. Application of chemical fungicides as a chemical disease management against fungal disease is widely used in farming system. The chemical control produced environment and health hazards to human and also adversely affects the beneficial microorganisms in soil and ecosystem. Trichoderma is one of the most important filamentous fungi common in soil and root ecosystems and used as an effective biocontrol agent against phytopathogens. There is an emerging need for biocontrol Trichoderma strains that can tolerate chemical fungicides, which would be a prerequisite for their application in an integrated disease management strategy combining chemical and biological means of control. Protoplast fusion is an imperative tool to develop Trichoderma inter-fusants having desire traits through genetic manipulation. The study designed to develop a unique and diverse inter specific protoplast Trichoderma fusants from T. virens Tvs 12 (mycoparasitic) × T. koningii MTCC 796 (multistress tolerant) having a characteristic of multi stress tolerance (drought, salt and fungicides) with enhancing antagonistic activity against Sclerotium rolfsii causing stem rot in groundnut. Total 36 stable fusants were obtained and screened for mycoparasitism, fungicides [Mancozeb 75 WP (3000 ppm a.i.) + Thiram 75 SD (1000 ppm a.i.) + Tebuconazole 100 WP (500 ppm a.i.) + Carbendazim 50 WP (5 ppm a.i.)] and abiotic stress [11.9 % of PEG (MW 6000) (-0.2 MPa or -2 bar osmotic stress for drought tolerant) + 100 mM sodium chloride (NaCl) (-0.45 MPa or -4.5 bar water potential for salt tolerant)] tolerance. The results indicated that 20 homozygous progenies showing characteristic of either one parental strain and 14 heterozygous mutants depicting trait of both parental strains (i.e. mycoparasitic and multi-stress tolerant). Trichoderma fusants and parental strains were subjected to in vitro antagonism against Sclerotium rolfsii (phytopathogen of stem rot of groundnut) up to 12 days after inoculation. Novel concept of inhibition coefficient representing pathogen biology and biocontrol related biophysics of Trichoderma fusants were estimated using growth related key parameters. Results showed differential inhibition coefficient of tested pathogen and highest inhibition coefficient (92.88 %) of S. rolfsii was observed by inter-stable fusant Fu21 which also exhibited higher multi stress tolerant capacity. The codominant SSR molecular analysis revealed highest observed heterozygosity (0.544), coefficient of gene differentiation (0.526) and gene flow (0.387) by Fu21 indicating better genetic exploitation of parental strains into that fusant with good genetic purity. The study explained antagonist fusants microbiological, biochemical and molecular mechanism thoroughly to restrain fungal test pathogen S. rolfsii. Some antifungal secretome (31 metabolites: up-regulated), chitinase and β-1,3-glucanase was positive correlated with inhibition coefficient of test pathogen during in vitro antagonism. Indepth metabolome study (GC-MS) of antagonists demonstrated the biochemical behavior and production of different bioactive compounds during biological interactions. Identification and characterization of miRNAs, their targeted and novel genes, functional annotation and gene expression pattern are reported in this work, which might be enhanced to understand miRNAs regulatory mechanism during antagonistic activities of Trichoderma parental strains and derived fusants against S. rolfsii. The study might be elucidated cellular, molecular and biological functions of miRNA and designed biochemical pathway analysis for specific functions of these regulatory miRNA for biocontrol activity of best Trichoderma fusant (Fu21). Total 56 unique miRNA in FU21_CB (down expressed) and 66 unique miRNA in FU21_IB (up expressed), 14 miRNA found to be down expressed during interaction with pathogen S. rolfsii compared to normal growth (control) of antagonist FU21. Most of the conserved miRNA families were predicted to target transcription factor genes; this suggests that they may play a role in post transcriptional regulation and transcriptional networks. Other miRNAs were predicted to target genes involved in diverse physiological and metabolic processes, including the regulation of fungal metabolism, transport, cell growth and maintenance, and stress responses (biotic and abiotic). Four novel miRNAs (chi-mir-493, hme-miR-6309, efu-miR-9393 and mdo-miR-144) found to be induced in Fu21_IB during interactions and correlated their expression pattern with the pathway panel. The ggo-miR-320b responsible for down expression in FU21_IB and elevate the pathway for protein phosphorylation, response to stress, deoxyribonucleotide biosynthetic process, oxidation-reduction process. However, tcamiR- 3824 expressed in Fu21 during interaction (FU21_IB) and found against response to salt by activating T cell receptor signaling pathway and aminobenzoate degradation pathway. The potential bioformulation (Fu21) developed having microbial load 1.83 x 108 cfu.g-1talc. The bioformulation reduced the stem rot disease incidence about 85% in the field condition which is eco-friendly and cost-effective application. Nanobiotechnology has immense potentials in agricultural uprising, high reactivity, better bioavailability, bioactivity and the surface effect of nano-bioformulation for smart protection of fungal diseases in plants. A novel nanoparticles based green bioformulation prepared from diverse and potent Trichoderma fusant (Fu21) would be efficient, eco-friendly and cost effective remedies to control the stem rot infection in groundnut under adverse condition (climate change). The potent Fu21 was used for green synthesis of silver nanoparticles (NPs). The morphology and uniformity of NPs were investigated by using UV-visible spectrophotometer (λ max - 430 nm), particle size analyzer (62.6 nm), zeta potential analysis (51.2 mv), scanning electron microscopic (spherical shape). The interactions between protein and NPs were analyzed by fourier transform infrared spectroscopy. The bioefficacy of Fu21 based nanoformulations at minimum inhibitory concentration (20 μg Ag/ml green NPs) was tested for smart protection of S. rolfsii (stem rot) infection in groundnut. The results revealed that about 95% disease incidence reduced by green nanoformulation Fu21 under pathogen infestation compared to even better than fungicides treatment. Disease severity index during the entire crop growth was found to be minimum with green nano-formulation treatment (0.32) compared the pathogen infestation (1.34). The cost effective green nano-bioformulation product developed as organic input which is prime needs for promotion of organic cultivation under changing climate -drought, salt and fungicides. The output of research presented a new business model fitting into the criteria of green chemistry and sustainable agriculture.
Open Access
“ Genome and transcriptome sequencing of coriander (Coriandrum sativum L.) to reveal its genome architecture.”2508
(JAU,JUNAGADH, 2013) Tulsani Nilam J.; Dr. B. A. Golakiya
Coriander (Coriandrum sativum L.) is an important spice and medicinal plant having a prime position in flavoring food. It is a cross pollinated annual herb belongs to family Apiaceae. The basic chromosome number of the genus Coriandrum is x=11, and C. sativum L. is a diploid with 2n=22. The different parts of this plant contain monoterpenes, α-pinene, limpnene, γ-terpinene, p-cymene, borneol, citronellol, camphor, geraniol, coriandrin, dihydrocoriandrin, coriandrons A-E, flavonoids and essential oils. Linalool is the main volatile compound in coriander seeds. For the measurement of the genome size, flow cytometer (Accuri C6) was used, found approximately 1.5 Gb. In genome sequencing after completions of sequencing run the total raw sequence generated by Ion S5 sequencer was 18,343,973 and 83,491,312bp followed by first and second lane. Total raw data was 22.8 Gb. Raw data (Reads) generated from two different runs for coriander were assessed for quality check using FastQC. After trimming of reads average read length was 315 and 189 bp. The de novo assembly (using CLC genomic workbench V 8.2) yielded assembled reads of 81,261,231 and number of contigs was 175,275 Total 121 unspecified repeats were found using repeat masker along with transposable elements found in coriander genome. In our study unspecified elements were found BNINTMO BoSB6D, BoSB7A, BvL1, BvL1-2, CALYPSHAN2_I_MT, CAULIV1. Transposable elements like COP10_I_MT, COP12_I_MT, COP16_I_MT, Copia10-VV_LTR and also other were found as listed in table. In total 76069 with highest number of hexa nucleotides were found in Gujarat coriander 2 genome. The highest numbers of SSRs were hexa nucleotide about 23% and 18% hepta ii nucleotides contribution to all SSRs followed by penta nucleotide 16 % and 14% di and tetra nucleotides in respective genome. In transcriptome study raw data (reads) from sequencing (NGS) was assessed through FASTQC quality control tool in which all samples (leaf, flower and seed) having good quality sequence for further analysis. After trimming of raw reads, total of 759,382,622 in leaf 371,962,963 in flower and 362,392,162 in seed sample with a mean length of 104.32bp, 150.42bp and164.82bp in leaf, flower and in seed. The de novo assembly (using trinity) yielded assembled reads of 109116bp. Gene expression level was differentiated among three groups (Leaf, flower and seed). To study gene expression pattern and also validation of the RNA sequencing technique, 15 primers were selected based on their expression for quantitative RT PCR. The GOLD-like protein and shows up regulation in leaf while Fimbrin-like protein shows Down regulation in flower and leaf. In seed chaperone and transcription factor 22 genes are up regulate, while MADS-box protein was down regulated. During flower developmental stage transcription activator GLK1 was up regulated and MADS box transcription_factor was down regulated. Total 4232 SSRs were identified with Tm and GC % range of 56–62 °C and 40-70, respectively. Length of SSR primer is between 17-27 bp with 100-250bp of product size range. During this study SSR of Guj-Coriander 2 having total number 204 SSRs identified, validatation of the SSR primers in 14 different varieties of coriander were Gujarat coriander 1, Gujarat coriander 2, GDLC, Punjab Sugandham, Loca; Kalmi (Raj), PAU-150, Co-1, Co-2, Co-3, Rcr-435, Rcr-436, Rcr-446, Rcr-480, Rcr-728.Total of 204 primers were used. Out of 204 primer 88 primers are amplified a total 164 band. The largest amplicone of 1608 bp was amplified by SSR primer COR 3 and smallest fragment of 98 bp was found with SSR primer COR62. In the present study, the qualitative analysis of ethanolic extract of coriander was carried out using GC-MS for the identification of bioactive components. The finger prints of the compound were identified from The National Institute of Standard and Technology (NIST) library database. The result of GC-MS analysis of coriander revealed the existence of sugars, organic acids, Vitamins and other bioactive compounds.
Open Access
“ Genome and transcriptome sequencing of coriander (Coriandrum sativum L.) to reveal its genome architecture.”2508
(JAU,JUNAGADH) Tulsani Nilam J.; Dr. B. A. Golakiya
Coriander (Coriandrum sativum L.) is an important spice and medicinal plant having a prime position in flavoring food. It is a cross pollinated annual herb belongs to family Apiaceae. The basic chromosome number of the genus Coriandrum is x=11, and C. sativum L. is a diploid with 2n=22. The different parts of this plant contain monoterpenes, α-pinene, limpnene, γ-terpinene, p-cymene, borneol, citronellol, camphor, geraniol, coriandrin, dihydrocoriandrin, coriandrons A-E, flavonoids and essential oils. Linalool is the main volatile compound in coriander seeds. For the measurement of the genome size, flow cytometer (Accuri C6) was used, found approximately 1.5 Gb. In genome sequencing after completions of sequencing run the total raw sequence generated by Ion S5 sequencer was 18,343,973 and 83,491,312bp followed by first and second lane. Total raw data was 22.8 Gb. Raw data (Reads) generated from two different runs for coriander were assessed for quality check using FastQC. After trimming of reads average read length was 315 and 189 bp. The de novo assembly (using CLC genomic workbench V 8.2) yielded assembled reads of 81,261,231 and number of contigs was 175,275 Total 121 unspecified repeats were found using repeat masker along with transposable elements found in coriander genome. In our study unspecified elements were found BNINTMO BoSB6D, BoSB7A, BvL1, BvL1-2, CALYPSHAN2_I_MT, CAULIV1. Transposable elements like COP10_I_MT, COP12_I_MT, COP16_I_MT, Copia10-VV_LTR and also other were found as listed in table. In total 76069 with highest number of hexa nucleotides were found in Gujarat coriander 2 genome. The highest numbers of SSRs were hexa nucleotide about 23% and 18% hepta ii nucleotides contribution to all SSRs followed by penta nucleotide 16 % and 14% di and tetra nucleotides in respective genome. In transcriptome study raw data (reads) from sequencing (NGS) was assessed through FASTQC quality control tool in which all samples (leaf, flower and seed) having good quality sequence for further analysis. After trimming of raw reads, total of 759,382,622 in leaf 371,962,963 in flower and 362,392,162 in seed sample with a mean length of 104.32bp, 150.42bp and164.82bp in leaf, flower and in seed. The de novo assembly (using trinity) yielded assembled reads of 109116bp. Gene expression level was differentiated among three groups (Leaf, flower and seed). To study gene expression pattern and also validation of the RNA sequencing technique, 15 primers were selected based on their expression for quantitative RT PCR. The GOLD-like protein and shows up regulation in leaf while Fimbrin-like protein shows Down regulation in flower and leaf. In seed chaperone and transcription factor 22 genes are up regulate, while MADS-box protein was down regulated. During flower developmental stage transcription activator GLK1 was up regulated and MADS box transcription_factor was down regulated. Total 4232 SSRs were identified with Tm and GC % range of 56–62 °C and 40-70, respectively. Length of SSR primer is between 17-27 bp with 100-250bp of product size range. During this study SSR of Guj-Coriander 2 having total number 204 SSRs identified, validatation of the SSR primers in 14 different varieties of coriander were Gujarat coriander 1, Gujarat coriander 2, GDLC, Punjab Sugandham, Loca; Kalmi (Raj), PAU-150, Co-1, Co-2, Co-3, Rcr-435, Rcr-436, Rcr-446, Rcr-480, Rcr-728.Total of 204 primers were used. Out of 204 primer 88 primers are amplified a total 164 band. The largest amplicone of 1608 bp was amplified by SSR primer COR 3 and smallest fragment of 98 bp was found with SSR primer COR62. In the present study, the qualitative analysis of ethanolic extract of coriander was carried out using GC-MS for the identification of bioactive components. The finger prints of the compound were identified from The National Institute of Standard and Technology (NIST) library database. The result of GC-MS analysis of coriander revealed the existence of sugars, organic acids, Vitamins and other bioactive compounds.

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