TRANSCRIPTOME ANALYSIS AND IDENTIFICATION OF DEFENCE RELATED GENES IN RESPONSE TO DOWNY MILDEW (Peronospora plantaginis) INFECTION IN ISABGOL (Plantago ovata Forsk)

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dc.contributor.advisorShukla, Y. M.
dc.contributor.authorKUMAR, VINAY
dc.date.accessioned2018-05-16T05:20:07Z
dc.date.available2018-05-16T05:20:07Z
dc.date.issued2013
dc.description.abstractIsabgol (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 isabgolen_US
dc.identifier.urihttp://krishikosh.egranth.ac.in/handle/1/5810046210
dc.keywordsTRANSCRIPTOME ANALYSIS, IDENTIFICATION OF DEFENCE RELATED GENES, RESPONSE TO DOWNY MILDEW (Peronospora plantaginis), INFECTION IN ISABGOL (Plantago ovata Forsk)en_US
dc.language.isoenen_US
dc.publisherAAU, Ananden_US
dc.research.problemTRANSCRIPTOME ANALYSIS AND IDENTIFICATION OF DEFENCE RELATED GENES IN RESPONSE TO DOWNY MILDEW (Peronospora plantaginis) INFECTION IN ISABGOL (Plantago ovata Forsk)en_US
dc.subAgricultural Biotechnologyen_US
dc.subjectPLANT MOLECULAR BIOLOGY & BIOTECHNOLOGYen_US
dc.subjectANALYSISen_US
dc.themeTRANSCRIPTOME ANALYSIS AND IDENTIFICATION OF DEFENCE RELATED GENES IN RESPONSE TO DOWNY MILDEW (Peronospora plantaginis) INFECTION IN ISABGOL (Plantago ovata Forsk)en_US
dc.these.typePh.Den_US
dc.titleTRANSCRIPTOME ANALYSIS AND IDENTIFICATION OF DEFENCE RELATED GENES IN RESPONSE TO DOWNY MILDEW (Peronospora plantaginis) INFECTION IN ISABGOL (Plantago ovata Forsk)en_US
dc.typeThesisen_US
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