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And Studying P. Capsici and Phytophthora Hybrids in Peru University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 8-2008 Generating Genetic Resources for Phytophthora capsici (L.) and Studying P. capsici and Phytophthora Hybrids in Peru Oscar Pietro Hurtado-Gonzales University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Earth Sciences Commons Recommended Citation Hurtado-Gonzales, Oscar Pietro, "Generating Genetic Resources for Phytophthora capsici (L.) and Studying P. capsici and Phytophthora Hybrids in Peru. " PhD diss., University of Tennessee, 2008. https://trace.tennessee.edu/utk_graddiss/455 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Oscar Pietro Hurtado-Gonzales entitled "Generating Genetic Resources for Phytophthora capsici (L.) and Studying P. capsici and Phytophthora Hybrids in Peru." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Plants, Soils, and Insects. Kurt H. Lamour, Major Professor We have read this dissertation and recommend its acceptance: Feng Chen, John K. Moulton, Beth Mullin Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a thesis written by Oscar Pietro Hurtado-Gonzales entitled “Generating genetic resources for Phytophthora capsici (L.) and studying P. capsici and Phytophthora hybrids in Peru.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Plant, Soils and Insects. ___________________________ Kurt H. Lamour, Major Professor We have read this dissertation and recommend its acceptance: _________________________ Feng Chen _________________________ John K. Moulton _________________________ Beth Mullin _________________________ Bonnie H. Ownley Accepted for the Council: ___________________________ Carolyn R. Hodges, Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) Generating genetic resources for Phytophthora capsici (L.) and studying P. capsici and Phytophthora hybrids in Peru A Dissertation Presented for the Degree Of Doctor Of Philosophy University Of Tennessee, Knoxville Oscar Pietro Hurtado-Gonzales August 2008 Acknowledgments I would like to thank God for the gift of Life. I would like to thank to my family (far and near) because they are a source of inspiration and constant motivation to move forward. I want to thanks my wife Sarah Hendricks for her moral support and proof reading of this document. I would like to express my gratitude to my advisor Dr. Kurt Lamour for giving me the great opportunity to work under his mentoring and for all the good lessons to become a better researcher and a better human being. I would like to thank to the members of my committee Dr. Feng Chen, Dr. John Moulton, Dr. Bonnie Ownley, and Dr. Beth Mullin for the availability and support throughout my years at UT. I would like to extend my acknowledgments to all faculty, students, and departmental staff from the Department of Entomology and Plant Pathology who have made my days in the school more enjoyable. ii In memory of my beloved Mother Cristina iii Abstract The genus Phytophthora includes more than 90 described species infecting over 1000 plant species. Population studies were conducted to investigate the survival and spread of P. capsici in the Peruvian coastal region. A total of 227 P. capsici isolates, recovered at widely distant localities from 2005-2007, were fingerprinted with AFLPs and SNP genotyping. A clonal population (PcPE-1) represented by 221 isolates was found to be distributed throughout the country. Atypical isolates of P. nicotianae were isolated from loquat trees in Peru and nuclear (internal transcribed spacer [ITS], the phenol acid carboxylase gene, and AFLPs) and mitochondrial genotyping (cytochrome oxidase gene [coxI]) identified this species as a hybrid between P. nicotianae and P. cactorum. A comparison of five Phytophthora hybrid isolates from Peru and Taiwan (also infecting loquat trees) suggested that isolates from Peru likely originated from a single hybridization event and that the two isolates from Taiwan originated through different hybridization events. The generation of genetic resources for the study of complex genetic traits in P. capsici was initiated by studying its inbreeding up to the sixth generation. A total of 692 oospore-derived isolates were fingerprinted and a subset was characterized for pathogenicity in cucumber and jalapeno fruits and for segregation of the mating type. The traits tested revealed no-Mendelian segregation, and apomixis were observed to be more prevalent (100%) in deep (fifth generation) inbreeding crosses. Inbreeding was measured by studying iv the segregation of 20 AFLP markers, which indicated a loss of heterozygosity of ~75% by the sixth generation. The seminal cross from this study was used as a mapping population (F1) for generating a genetic linkage framework with 189 AFLP and 18 SNP markers. A total of 18 linkage groups were produced for each parental isolate using 65 and 42 markers for CBS121657 and CBS121656 isolates respectively covering 409 cM. SNP markers FL5 and FL6 were used for estimating the genome size of P. capsici and precision of the genome assembly. In order to conduct functional studies in P. capsici, we tested the efficacy of the polyethylene glycol mediated transformation. We regenerated up to 30 antibiotic resistant isolates and 53% of them were stable after three months of subculturing. v Table of contents Chapter One. Introduction........................................................................................... 1 LITERATURE CITED ........................................................................................... 9 Chapter Two. Survival and spread of Phytophthora capsici in coastal Peru ............................................................................................................... 12 ABSTRACT.......................................................................................................... 13 INTRODUCTION ................................................................................................ 13 MATERIALS AND METHODS.......................................................................... 15 Isolate recovery and sampling scheme...................................................................... 15 DNA isolation and AFLP analyses........................................................................... 17 SNP genotyping ........................................................................................................ 18 RESULTS ............................................................................................................. 19 AFLP genotyping and mating type ........................................................................... 19 SNP and genotyping ................................................................................................. 19 DISCUSSION....................................................................................................... 20 LITERATURE CITED ......................................................................................... 25 APPENDIX 2........................................................................................................ 34 Tables........................................................................................................................ 34 Figures....................................................................................................................... 37 Chapter Three. Molecular comparison of natural hybrids of Phytophthora nicotianae and P. cactorum infecting loquat trees in Peru and Taiwan ............................................................................................................... 41 ABSTRACT.......................................................................................................... 42 INTRODUCTION ................................................................................................ 42 MATERIALS AND METHODS.......................................................................... 44 Isolate recovery and maintenance............................................................................. 44 Phytophthora cactorum x P. nicotianae crosses....................................................... 45 DNA isolation and AFLP fingerprinting .................................................................. 46 Hi-resolution DNA melting analysis of mitochondrial DNA ................................... 46 ITS and nuclear gene sequencing ............................................................................. 47 RESULTS ............................................................................................................. 48 AFLP
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