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Open Access
PHYSIO-BIOCHEMICAL ANALYSIS OF TOMATO cv. GAT 5 FRUITS FOR SHELF LIFE IN RESPONSE TO EXOGENEOUS MELATONIN
(Department of Biotechnology B. A. College of Agriculture, Anand Agricultural University Anand, 2020) Tushar Ranchhodbhai Gadhavi; Dr. J. J. Dhruv
The present investigation was undertaken on tomato fruit (Solanum lycopersicumL.) in order to estimate biochemical characterization and molecular analysis. The tomato fruits and genotypes were received from Main vegetable research centre (MVRS), Anand Agricultural University, Anand during summer 2019-20. The main objective of the experiment was to study the Physio-biochemical property of tomato fruit one variety and characterization of ten tomato (Solanum lycopersicum L.) genotypes by molecular markers. The various physio-biochemical characters were analyzed at Biochemistry Department, A.A.U., Anand, while the molecular characterization was done at Biotechnology Department, A.A.U., Anand.
Open Access
MOLECULAR STUDIES FOR APHID [Lipaphis erysimi (Kalt.)] RESISTANCE IN ADVANCED GENERATION (F4) OF Brassica INTERSPECIFIC HYBRID GM-3 × PUSA SWARNIM
(DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY B. A. COLLEGE OF AGRICULTURE ANAND AGRICULTURAL UNIVERSITY ANAND, 2019) Desai Pallavi Kumari Onkarnath; Dr. Sasidharan. N
Rapeseed mustard is the third important oilseed crop in the world after soybean (Glycine max) and palm (Elaeis guineensis Jacq.) and globally, India account for 72 lakh hectare, 85.0 lakh tonnes (lt) and 1184 Kg per hectare of the total acreage , production and productivity respectively. Whereas Gujarat account for 1.90 lakh hectare , 3.06 lakh tonnes (lt) and 1611 Kg per hectare of the total acreage, production and productivity respectively. The mustard crop suffers from various biotic and abiotic stresses. Among the different pests and diseases affecting this crop, mustard aphid, Lipaphis erysimi (Kalt.) is the major limiting factor for qualitative as well as quantitative production of mustard, which infests the crops right from vegetative stage to pod stage and accounts for 27% to 69% of yield loss with 15% reduction in its oil content and 75% productivity losses. In this context, breeding for aphid resistance and identification of molecular markers for this trait could be a valuable approach for development of resistant genotype.
Open Access
Transcriptome based identification and level of expression study of candidate genes related to andrographolide content and their validation in Andrographis paniculata (Burm f.) Nees.
(DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY ANAND AGRICULTURAL UNIVERSITY ANAND, 2017) ANKITABAHEN ATULKUMAR PATEL; Dr. Y. M. Shukla
In heritage, India is blessed with many medicinal and aromatic plants. Natural and ayurvedic medicines were used by old people prepared from medicinal plants for treatment of diseases. Now a days also, the importance of medicinal plants remain as such due to their efficient way of curing and researchers focus more in the field of medicinal plants. The present investigation has paid attention on one of the important medicinal plants “Andrographis paniculata (Burm. f.) Nees.”, due to presence of its valuable secondary metabolite “Andrographolide”. The common name of Andrographis paniculata is kalmegh. The present study aimed to elucidate the expression of candidate genes associated with biosynthesis pathway leading to andrographolide synthesis in different genotypes of kalmegh at particular vegetative growth stage. The validation of expressed genes was also carried out.
Open Access
MAPPING QTLs FOR IRON AND ZINC CONCENTRATIONS IN RICE (ORYZA SATIVA L.)
(DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY ANAND AGRICULTURAL UNIVERSITY ANAND, 2017) DHARA K. SAVSANI; Dr. Y. M. Shukl
Rice (Oryza sativa L.) is nutritionally, one of the most important staple food crops for approximately half of the world population. It is rich in carbohydrate and the predominant dietary energy source for Asia, North and South America and Africa, re iterating its importance to human welfare.
Open Access
TRANSCRIPTOME ANALYSIS AND IDENTIFICATION OF DEFENCE RELATED GENES IN RESPONSE TO DOWNY MILDEW (Peronospora plantaginis) INFECTION IN ISABGOL (Plantago ovata Forsk)
(AAU, Anand, 2013) KUMAR, VINAY; Shukla, Y. M.
Isabgol (Plantago ovata Forsk), a member of Plantaginaceae family is a crucial medicinal emd industrial crop cultivated in India, having a good foreign exchange in world market. In spite of its immense important, it faces constraints in productivity due to the prevalence of numerous biotic and abiotic stresses, downy mildew disease caused by an obligate fvmgal pathogen, Peronospora plantaginis being a major one causing an estimated yield loss of up to 73 per cent. In order to identify the defence response involved in isabgol-downy mildew interaction, transcriptome analysis of downy mildew resistant (EC 124345) and susceptible (Niharika) genotypes under downy mildew stress as well as control conditions was executed through 454 GS FLX Titanium Pyrosequencing, yielding 600.81 Mb data with an average read length of 434 bp. De novo transcriptome assembly of four samples viz. resistant inoculated; control and susceptible inoculated; control was performed using CLC Genomics Workbench producing 7246, 7650, 7390 and 9278 unigenes respectively. The Guanine: cytosine (GC) content in isabgol transcriptome varied from 51-52%. The unigenes were annotated to identify putative gene fimctions with the different databases like non redundant protein database. KEGG and IntreProscan and the highest GO terms were obtained from UniprotKB databases. The homology of isabgol tmigenes with the model plant species revealed the highest hits with Vitis vinifera followed by Glycine max, Populus trichocarpa, Medicago truncatula and Arabidopsis thaliana. Gene Ontology enrichment analyses revealed the over and under representation of GO terms by the up and down regulation of associated genes respectively. The genes encoding various types of enzymes and/or protein associated with the defence or stress related responses included pathogenesis related (PR) proteins, such as (PR-9) peroxidase, (PR-5) thaumatin like proteins, (PR-3, 4, 8, 11) a group of chitinases or endochitinases, (PR-2), P- 1, 3-glucanases, (PR-10) ribonuclease, (PR-13) thiordne, (PR-6) proteinase inhibitors and (PR-12), resistance genes NBS-LRR, CC-NBS-LRR, ser/threonine receptors kinase, LRR repeat type, MLO and Cf9 types of R genes were also foxmd. Transcription factors families viz. WRKY, zinc finger, leucine zipper, chitin-inducible gibberellin-responsive protein 1-like, dna binding, g-boxbinding factor 4, homeobox leucine zipper protein, myb family, nac domain containing, ERF were identified from the isabgol downy mildew interaction. The pathogenesis and defence related genes identified in isabgoldowny mildew interaction were validated through Real time quantitative PCR. In order to identify the most stable reference genes during downy mildew pathogen infection, the expression stabilities of the reference genes viz. elongation factor a, actin, tubulin, glyceraldehyde 3 phosphate dehydrogenase and 60S ribosomal protein were examined by the five algorithms and EF1a and ACT 7 were found to be the most stable reference genes. A total of 18 PR proteins, defence related enzymes/genes were used for validation through Real time-qPCR. The expression of peroxidase, spermine synthase, chitinases, ADP ribosylation factors, mitogen activated protein kinase kinase, 4-coumarate ligase and resistance gene CC-NBS-LRR were prominentiy enhanced in resistant inoculated samples suggesting that the defence related genes were induced during isabgol downy mildew interaction. Photosynthesis related genes like chlorophyll a-b binding protein, 14-3-3 were down regulated during downy mildew disease. The transcriptome data was mined for EST-SSR and identified the first set of genie SSRs markers. A total of 1440, and 1824 microsatellite repeats were identified from the EC 124345 and Niharika genotypes, respectively. The most frequently occurring di-nucleotide motifs were AG/CT followed by GA/TC and CA/TG and among the tri- nucleotide motifs, AGC/CTG was abundantiy present closely followed by AAG/CTT. The tri-nucleotide motifs coding for serine was highest (12%) followed by leucine (9%), while the methionine were least present (1%). A set of 34 EST-SSR was selected for validation on Plantago ovata genotypes. Out of the 34 primers, 28 primers (82%) produced their specific amplicon and four primers viz. ECSSR 4, ECSSR 9, ECSSR 14, ECSSR 31 were polymorphic within P. ovata genotypes. For cross species transferability, 24 primers out of 28 (85%) produced amplification in any of the six species tested viz. Plantago coronopus, P. lanceolata, P. indica, P. arenaria, P. syllium and P. serrena. The transcriptome data would serve as a resource for genomic studies such as identification and isolation of genes of economically important traits and disease resistance in isabgol. The newly developed first set of EST-SSR markers could be useful in identification of gene based or trait linked SSR markers for improvement of isabgol though conventional or molecular breeding approaches like marker- aided selection (MAS). This study generated for the very first time genomic resources for a highly valued medicinal plant using high throughput next generation sequencing approach and is expected to accelerate the field of functional genomics of isabgol
Open Access
Transcriptome based identification of genes imparting bacterial leaf blight (Xanthomonas oryzae) resistance in rice (Oryza sativa L.)
(AAU, Anand, 2017) DESAI AMRUTA S.; Dr. Subhash N.
Rice is one of the most important staple foods for a larger part of the world’s population. It is a rich source of carbohydrates, energy, thiamine, pantothenic acid and folic acid. Because world-wide rice production has been severely affected by one of the major disease referred to as bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo), early efforts has been focusing on cloning and utilizing of disease resistance (R) genes in rice for resistance breeding. In the present study, thirty day old seedlings of BB resistant (IR 64) and susceptible (GR 11) genotypes were inoculated with and without BB (4 x l04 cells /ml). The seedlings were harvested 36 hours post inoculation for transcriptome sequencing employing the Miseq Illumina NGS sequencer after assaying the normal range for quantity and quality of total RNA, mRNA and cDNA. Transcriptome sequencing yielded 19311304 bp data with an average read length of 150 bp and constituted of 18440773 high quality reads which were assembled employing Trinity assembler for functional annotation. Trinity assembler gave assembly matrix (N50 of 2,453), proteome coverage and read alignment (90%) resulting in 30,000 contigs and 2,35,433 singletons/transcripts which were subjected to gene ontology (GO) following functional annotation. ii The maximum number of gene ontology was ascribed to cellular components, molecular function and biological processes showed that the maximum transcripts belonged to plastid, mitochondria and plasma membrane; nucleotide binding, kinase activity, DNA binding; cellular protein modification response to abiotic stress and biotic stress, respectively. Rice transcripts showed highest homology to Setaria italica and maximum blast hits with Hordeum vulgare. The maximum number of annotated transcripts was ascribed to transferase, hydrolase and oxidoreductase class of enzymes. The highly represented transcripts included disease resistance genes, pathogenesis related proteins, transcription factors (MYB, WRKY), signaling molecules (MAPK), enzymes of oxidative burst and LRR domain. These proteins were assumed to be involved in signaling networks induced in rice during BB infection. The transcripts were further subjected to confer their metabolic involvement through biochemical pathway through Kyoto encyclopedia of genes and genomes. Total 118 metabolic pathways were identified in rice transcriptome. Metabolic pathway enrichment ascribed the maximum number of transcripts in Glutathione metabolism leading to activation of defence responses called systemic acquired resistance (SAR). Metabolic pathway for purine metabolism, phenylalanine metabolism and starch and sucrose metabolism were prominently present. Transcriptomes of resistant and susceptible rice genotypes (with and without inoculum) were further screened for differential gene expression analysis by mapping individual reads to the assembly. Among the resistant and susceptible genotypes (with and without inoculum), 1314 transcripts were either up regulated or down regulated. The number of differentially expressed transcripts between the samples resistant inoculated and susceptible inoculated was 154. Further confirmation and validation was carried out through quantitative real time PCR. iii Relative quantification of defence related transcripts by quantitative real time PCR was performed using a-tubulin as endogeneous control. The relative expression of defence related genes associated with BB disease MYB, bZIP; defence related enzymes including lipoxygenase (LOX), phenylalanine ammonia lyase (PAL), respiratory burst oxidase (RBO), beta glucanase (BGLUC), Ribonuclease (RNase), Endo chitinase (CHI), signaling molecules, MAPK and CDPK showed up regulation in resistant inoculated than susceptible inoculated. Overall results of transcriptome study generated the information on involvement of MYB, WRKY, mitogen activated protein kinases, cellular ATPases, calcium dependent protein kinase and phenylalanine ammonia lyase transcription factors and the genes associated with resistance involved R genes homologous with the cell wall degrading enzymes like chitinase, 3-1,3 glucanases and metabolic pathway coding enymes polygalacturonase inhibiting proteins (PGIPs), cysteine proteases in the process of BB resistance. It could be inferred that defence responses associated with BB in rice were activated by hypersensitive response arising due to gene to gene interaction and systemic acquired resistance associated with pathogenesis related proteins, transcription factors, signaling molecules and enzyme related to glutathione pathway
Open Access
IN SILICO IDENTIFICATION OF microRNAs AND THEIR VALIDATION IN PEARL MILLET [Pennisetum glaucum L.]
(AAU, Anand, 2016) AMIT KUMAR; Dr. R. S. Fougat
The endogenous small non-coding functional microRNAs (miRNAs) are short in size, ranging from ~21 to 24 nucleotides in length and play a pivotal role in gene expression in plants and animals by negative regulation or silencing of genes either by cleavage or blocking of translation of homologous mRNA. Although various highthroughput and expensive techniques like forward genetics and direct cloning are employed to detect miRNAs in plants but comparative genomics complemented with novel bioinformatic tools paced the way for efficient and cost-effective identification of miRNAs through homologous sequence search with previously known miRNAs. Many miRNAs have been identified and investigated extensively in plant species with available sequences in NCBI database. However, only one miRNA has been identified in pearl millet and reported on PMRD. Peal millet is widely grown crop in arid and semi-arid regions of world hence this crop has great importance in dry land agriculture. In this study, an attempt was made to identify and characterize conserved miRNAs in pearl millet expressed sequence tag (EST) sequences; genome survey sequences (GSS) and nucleotide sequences using bioinformatics approach. For ii identification of novel miRNAs in pearl millet, a total 7240 known mature miRNAs of plant kingdom were searched for homology against 6014 unigene sequences resulting in identification of 14 potential miRNA candidates belonging to 11 different families. The majority of predicted pearl millet miRNAs were 21 nucleotides in length and they were found on both the 5′ and 3′ arms of the stem-loop hairpin secondary structure. The pearl millet pre-miRNAs varied in length from 60 to 109 nt. Five out of 14 predicted miRNAs were confirmed in young plants of pearl millet using quantitative real time polymerase chain reaction (qRT-PCR). The psRNATarget server predicts 34 potential target genes and their probable functions. Most of the pearl millet miRNA target gene involved in stress response, transcriptional regulation, signal transduction, DNA replication proteins, male sterility, metabolic enzymes, plant growth and development. The results of the present study will shed more light on the understanding of molecular mechanisms of miRNA in pearl millet which may aid in development of novel and precise techniques to understand some post-transcriptional gene silencing mechanism in response to stress tolerance
Open Access
GENDER IDENTIFICATION USING LOOP MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) IN PAPAYA (Carica papaya L.) var. Madhubindu
(AAU, Anand, 2016) RATHOD DIVYARAJSINH VIRENDRASINH; Dr. Subhash N.
Experiments were conducted to screen and validate suitable male specific markers in the identification of sex in papaya (cv. Madhubindu). From the reported markers W11, T12, Napf and CPSM 90 showed detectable results in sex determination and was used for further experimentation with routinely used PCR method as well as loop mediated isothermal amplification (LAMP). Using screened markers unknown samples were identified with 100% efficiency of the marker used in predicting the sex type. At the time of flowering all the predicted males as well as female were confirmed visually. Moreover, after the detection with PCR, identification of papaya DNA samples were carried out with turbidity method, fluorescence method (Calcein, SYBR green, HNB, EtBr dyes). Using four primers i.e. W11, T12, Napf and CPSM90, LAMP reaction was performed initially with confirmed male and female DNA samples. Among the various dyes used, detection with calcein dye was most revealing and intense fluorescence colour was obtained. Among various primers used T12 primer was found most prominent. With the use of T12 primer and calcein detection system, various experiments were conducted for optimization of reaction time, temperature, starting quantity of DNA and concentration of enzyme. Thus in this study 4 ii male specific markers were developed for sex identification using both PCR and loop mediated isothermal amplification (LAMP). LAMP reaction carried out by T12 primer using calcein dye showed promising results in terms of rapid reaction time (< 1h), isothermal condition (55°c), a high efficiency (20 ng of DNA required in reaction mixture) and concentration of enzyme (8 units of the enzyme).
Open Access
CHARACTERIZATION AND EXPRESSION STUDIES FOR IRON TRANSPORTER AND STORAGE GENES IN KODO MILLET (Paspalum scrobiculatum L.)
(AAU, Anand, 2016) PRAJAPATI VIJAYKUMAR ISHWARBHAI; Dr. J. G. Talati
Kodo millet is a balanced and staple food of tribal and economically poor section of the population. The grains of kodo millet possess excellent storage properties and are known for health benefits. It is highly nutritious food crop with higher fiber content along with quality protein and mineral composition, which can serve as excellent dietary source for these elements. Poor nutritional quality of cereals is the primary cause of disorders related to nutritional imbalance among the population having cereal-based diet, especially those belonging to underdeveloped or developing counties. The present study was carried out with an aim to study the relative importance of nutritional quality, molecular diversity existing in kodo millet and to study genes responsible for the iron transport and storage at three developmental stages (10 DAS, 20 DAS and 30 DAS). A wide variation for the micronutrient content was observed in grains of twenty kodo millet genotypes under study. Iron and zinc concentrations ranged from 17.1 to 66.4 ppm and 18.3 to 29.0 ppm respectively. The highest iron content was found to be in KAVT-13, whereas zinc in PSC-1. The observed values were higher than the average iron content present in major cereal crops such as rice, wheat, maize, etc. The protein content ranged from 6.86 to 10.36 %, which is equivalent to protein content of other cereals. The percent oil content of different genotypes varied from 1.60 to 3.13, carbohydrates 70.54 to 76.04 and starch percent content from 59.51 to 68.63. It also showed higher crude fiber content, ranging from 13.15 to 15.57%. Phenolic acids from defatted grains of kodo millet genotypes were analysed by UPLC method. Among all the phenolic acids, the salicylic acid was found to be highest in kodo millet. The phenolic acid content was found to differ from genotype to genotype. The highest content for different phenolic acids such as proto-catechuic acid (KAVT-25), p-Hydroxybenzoic acid (PSC-1), Chlorogenic acid (JK-48), Caffeic acid (CO-2), Syringic acid (KAVT-13), p-Coumaric acid (KAVT-13), Ferulic acid (DK-127), Vanilllic acid (KAVT-30), Sinapic acid (JK-41) and Cinnamic acid (KAVT-13) were observed in different genotypes. In SDS-PAGE protein profiling of kodo millet grains revealed 20.54 to 104.93 kDa protein. There is little variation in the SDS-PAGE electrophoretic patterns of the twenty genotypes of kodo millet. Iron content specific thirteen SSR markers were utilized in this study, which generated 302 scorable bands. The average number of alleles per locus was found to be 2.69. The highest PIC value of YS4 indicated that it would be a very useful SSR marker for diversity analysis of kodo millet genotypes. The candidate gene markers of NRAMP family, NRAMP1 and NRAMP3 can be useful to discriminate high and low iron-containing genotypes. The candidate gene specific markers viz., YS1, IRT1, ZIP3 and Ferritin were found to discriminate high iron genotypes and the marker YS12 for low iron containing genotypes. The relative gene expression profiling of seven iron transporter and storage related genes i.e., FRO2, IRT1, NAAT1, NAS2 and YSL2 were analyzed in roots, whereas gene NRAMP5 and Ferritin were analyzed in leaves of GK-2 (Low Fe) and GPUK-3 (High Fe) kodo millet varieties under three different stages viz., 10 DAS, 20 DAS and 30 DAS. Expression of two genes FRO2 and ITR1 which are part of strategy I iron transport, was found to be correlated to high grain Fe content in kodo millet. These genes are the part of strategy I for iron transport in plant. Expression of three genes viz., NAAT1, NAS2 and YSL2 (Strategy II iron transport) are significantly higher in GPUK-3 as compared to GK-2. It was possible due to deficient Fe content in the soil at later stages of development. The expression of NRAMP5 and ferritin in kodo millet showed differential expression in leaf at 10 to 30 days after sowing. This result indicates that kodo millet possess the Strategy II iron (Fe) uptake system in which Fe is absorbed by roots as Fe3+-phytosiderophore. However, in spite of being a Strategy II plant, kodo millet identifies Fe2+ transporter (Strategy I) genes viz., FRO2 and IRT1.

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