GRADUATE AND POSTDOCTORAL STUDIES FINAL ORAL EXAMINATION

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1 GRADUATE AND POSTDOCTORAL STUDIES MCGILL UNIVERSITY FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF DHANANJAY DHOKANE DEPARTMENT OF PLANT SCIENCE Identification of fusarium head blight resistance genes in the wheat QTL-Fhb2 using forward and reverse genetics Tuesday, May 30 th, pm MS2-022 McGill University, Macdonald Campus COMMITTEE: Dr. Jan Franklin Adamowski (Pro-Dean) (Bioresource Engineering) Dr. Don Smith (Chair) (Plant Science) Dr. Ajjamada Kushalappa (Supervisor) (Plant Science) Dr. Philippe Seguin (Internal Examiner) (Plant Science) Dr. Suha Jabaji (Internal Member) (Plant Science) Dr. Jennifer Ronholm (External Member) (Food Sci. & Agricultural Chemistry) Dr. Josephine Nalbantoglu, Dean of Graduate and Postdoctoral Studies Members of the Faculty and Graduate Students Are invited to attend

2 ABSTRACT Fusarium head blight (FHB) is one of the most destructive diseases of wheat worldwide. FHB not only cause serious losses in yield but also significantly detoriates the quality of grains by contaminating them with health hazardous mycotoxins. Plant resistance is considered as the most sustainable and effective strategy to manage FHB and to reduce the accumulation of mycotoxins. However, lack of effective and precise phenotyping methods and limited understanding of the genetics of FHB resistance has hindered the development of resistant cultivars. Resistance to FHB in wheat is quantitative, therefore more than 100 QTL have been identified and mapped on all chromosomes in wheat. QTL-Fhb2 is the second major QTL that confers high levels of resistance to the spread of Fusarium graminearum (Fg) within a spike through the rachis. However, the genes underlying the QTL and the mechanisms of resistance are still unelucidated. The phenotyping method was improved by developing a qpcr based protocol to precisely quantify the levels of FHB resistance among genotypes, including bleaching symptoms, following single floret inoculation under greenhouse conditions. We used Fg Tri6 gene specific primers to identify the copy number of Tri6 gene in the samples collected after Fg inoculation. More the copy number of Tri6 correlates to higher Fg biomass and more the susceptibility of the genotype to FHB. Based on the protocol developed, we report that the qpcr based method is more sensitive and precise in discriminating genotypes varying in their levels to FHB resistance compared to disease severity analysis. Further, recombinant inbred lines (RILs), carrying resistant (R-RIL) and susceptible (S-RIL) alleles of QTL-Fhb2 were subjected to metabolome and transcriptome profiling following Fg and distilled water (mock) inoculation, to identify candidate genes localized within the QTL-Fhb2. Integrating metabolomic and transcriptomic datasets, we have identified six candidate genes localized within the QTL-Fhb2. The candidate genes localized within the QTL are, 4-coumarate: CoA ligase (4CL), callose synthase (CS), basic Helix Loop Helix (bhlh041) transcription factor, glutathione S-transferase (GST), ABC transporter-4 (ABC4) and cinnamyl alcohol dehydrogenase (CAD). Based on these findings, we report that the QTL-Fhb2 confers FHB resistance through combined effect of cell wall reinforcement and DON detoxification. Furthermore, a Ta4CL3 gene localized within the QTL-Fhb2 was functionally characterized using virus induced gene silencing and the resistance functions were proved by analyzing disease severity, fungal biomass and metabolic profiles of silenced and non-silenced control plants. Taken together, we report that Ta4CL3 contributes to resistance against the spread of Fg within a spike by reinforcing the cell walls, through the deposition of hydroxycinnamic acid amides, lignin and lignans.

3 CURRICULUM VITAE UNIVERSITY EDUCATION 2014-Present Doctor of Philosophy - Plant Science Department of Plant Science, McGill University Master of Science - Biotechnology (Tamil Nadu Agricultural University, Coimbatore, India) Bachelor of Science - Agricultural Biotechnology (College of Agricultural Biotechnology, Loni, India) EMPLOYMENT Teaching Assistant (Plant Breeding) Teaching Assistant (Organism-I) Teaching Assistant (Plant Pathology) Greenhouse Assistant Laboratory Assistant McGill university, Canada Technical Sales Executive Greenvalue bioproducts Pvt. Ltd, India till date AWARDS Mobility Research Award, McGill University to attend hands on training workshop on CRISPR/Cas9 based genome editing organized by Thermo fisher Scientific at Carlsbad, USA, Nov Graduate Research Enhancement and Travel Award, Department of Plant Science, McGill University to attend 8 th Canadian workshop on Fusarium head blight and 3 rd Canadian wheat symposium at Ottawa, Oct Travel award by Canadian Association for Plant Biotechnology to attend Plant Biotech conference at Kingston, July Graduate Research Enhancement and Travel Award, Department of Plant Science, McGill University to attend Plant Biotech conference at Kingston, June 2016.

4 Fredrick Dimmock Memorial Scholarship, McGill University, Oct Innovagrains Research Award, Jan Executive Committee Student Member of Canadian Association for Plant Biotechnology, Graduate Excellence Award, Department of Plant Sciences, McGill University 2014, 2015, Indian Council of Agricultural Research - International Fellowship for Ph.D studies, Jawaharlal Nehru Fellowship for pursuing Masters in Biotechnology by Department of Biotechnology, Government of India, PUBLICATIONS Dhokane, D., Kalenahalli, Y., Kushalappa, A., McCartney, C., coumarate CoA: ligase-3 (Ta4CL3) localized within wheat QTL-Fhb2 contributes to resistance against Fusarium graminearum. Submitted to Planta. Kumar, A., Mosa, K., Ji, L., Kage, U., Dhokane, D., Karre, S., Madalageri, D., Pathania, N., Metabolomics assisted biotechnological interventions for developing plant-based functional foods and nutraceuticals. Critical Reviews in Food Science and Nutrition. Kalenahalli, Y., Dhokane, D., Kushalappa, A., Felipe, S., Rodriguez, E., and Mosquera, T StWRKY8 transcription factor regulates benzylisoquinoline alkaloid pathway in potato conferring resistance to late blight. Plant Science. Dhokane D., Karre S., McCartney C., Kushalappa, A., Integrated metabolo-transcriptomics reveals fusarium head blight candidate resistance genes in wheat. Plos One. Karre, S., Kumar, A., Dhokane, D., Kushalappa, A., 2016 Metabolotranscriptome profiling of barley reveals induction of a novel chitin elicitor receptor kinase gene (HvCERK1) conferring resistance against Fusarium graminearum. Plant Molecular Biology. Kage, U., Hukkeri, S., Dhokane, D., Kumar, A., Karre, S., Kushalappa, A., Genome editing with CRISPR/Cas9 and its applications in crop plant improvement. Submitted to Critical Reviews in Biotechnology. Kumar, A., Karre, S., Dhokane, D., Kage, U., Hukkeri, S., Kushalappa, A., Real-time quantitative PCR based method for the quantification of fungal

5 biomass to discriminate quantitative resistance in barley and wheat to fusarium head blight. Journal of Cereal Science. Kage, U., Kumar, A., Dhokane, D., Karre, S., Kushalappa, A., Functional molecular markers in crop improvement. Critical Reviews in Biotechnology. Kumar, A., Kage, U., Mosa, M., Dhokane, D., Editorial, Metabolomics: a novel tool to bridge phenome to genome under changing climate to ensure food security. Journal of Medicinal and Aromatic Plants.