The Pennsylvania State University The Graduate School Intercollege Graduate Degree Program in Genetics GENETIC CHARACTERIZATION AND MAPPING OF LATE BLIGHT RESISTANCE GENES IN THE WILD TOMATO ACCESSIONS PI 163245 AND PI 224710 A Dissertation in Genetics by Erik William Ohlson © 2015 Erik William Ohlson Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2015 ii The dissertation of Erik William Ohlson was reviewed and approved* by the following: Majid R. Foolad Professor of Plant Genetics Dissertation Advisor David R. Huff Professor of Turfgrass Breeding and Genetics Chair of Committee Surinder Chopra Professor of Maize Genetics Beth K. Gugino Associate Professor of Vegetable Pathology Timothy W. McNellis Associate Professor of Plant Pathology Yinong Yang Associate Professor of Plant Pathology Robert F. Paulson Professor of Veterinary and Biomedical Sciences Chair of the Intercollege Graduate Degree Program in Genetics *Signatures are on file in the Graduate School. iii ABSTRACT Late blight (LB), caused by the oomycete Phytophthora infestans (Mont.) de Bary is one of the most destructive diseases of tomato and potato worldwide. Development of fungicide resistant and more aggressive P. infestans clonal lineages has emphasized the importance of discovering and incorporating new genetic resistance in tomato cultivars. Although the cultivated tomato, Solanum lycopersicum L., contains limited genetic diversity, several related wild species of tomato are suitable for identification of new desirable traits. Previously, 67 S. pimpinellifolium accessions were screened for LB resistance in field, greenhouse and detached leaflet trials and 12 accessions with strong resistance to LB were identified. In this dissertation, two resistant accessions, PI 163245 and PI 224710, were selected for further genetic characterization. PI 163245 and PI 224710 were each hybridized with a LB susceptible tomato breeding line, Fla. 8059, and F1 progeny were self-fertilized to develop F2 populations. Large F2 populations were grown and screened for LB resistance under greenhouse conditions, and the most resistant and most susceptible individuals in each F2 population were retained for conducting heritability studies as well as identifying and mapping of resistance loci. To characterize the genetic basis of resistance in the two accessions, estimates of 2 heritability (h ) were obtained based on F2:F3 parent-offspring (P:O) correlation analyses. 2 An additional estimate of h was obtained based on F3:F4 generations when using the 2 2 accession PI 163245. Estimates of h were moderately-high for both PI 163245 (h F2:F3 = 2 2 0.78, h F3:F4 = 0.94) and PI 224710 (h F2:F3 = 0.87). The heritable nature of the resistance suggested that PI 163245 and PI 224710 were potentially viable for breeding LB resistance in tomato, and that mapping of LB resistance loci was warranted. iv To discover SNP markers for genetic mapping studies, reduced representation libraries (RRLs) for each of PI 163245, PI 224710 and Fla. 8059 were constructed and sequenced. Comparisons of accessions PI 163245 and PI 224710 with breeding line Fla. 8059 resulted in the identification of 33,385 and 20,894 single nucleotide polymorphisms (SNPs), respectively. The most resistant (n = 39) and susceptible (n = 35) F2 individuals in the PI 163245 mapping population were genotyped with 233 SNP markers, which were distributed throughout the genome. This selective genotyping approach identified four genomic intervals (quantitative trait loci, QTLs) on chromosomes 2, 3, 10, and 11 associated with LB resistance. Similarly, the most resistant (n = 40) and susceptible (n = 40) F2 individuals in the PI 224710 mapping population were genotyped with 144 SNPs, and LB resistance QTLs were identified on chromosomes 1, 2, 10, and 12. Resistance QTLs on chromosomes 2 and 10 co-localized with two previously identified LB resistance genes, qPh2.1 and Ph-2 respectively, however fine mapping and cloning is necessary to determine how these QTLs correspond to previously identified LB resistance genes. The remaining resistance QTLs did not appear to correspond to known LB resistance genes or QTLs in tomato, and thus are likely unique to this study. Efforts to fine map these resistance QTLs and incorporate them into elite tomato breeding lines are currently in progress. v TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................ ix LIST OF TABLES ......................................................................................................... xi Chapter 1 Introduction ........................................................................................................ 1 Tomato ............................................................................................................................ 1 Tomato as a model organism ....................................................................................... 3 The tomato genome ..................................................................................................... 4 Late blight ....................................................................................................................... 8 Phytophthora infestans ................................................................................................ 9 Phytophthora infestans genome ................................................................................ 11 Late blight management ............................................................................................ 11 Late blight resistance in potato .................................................................................. 12 Late blight resistance in tomato ................................................................................. 16 Dissertation research background ................................................................................. 19 Research Objectives ...................................................................................................... 19 Literature Cited ............................................................................................................. 23 Chapter 2 Genetic characterization and heritability of late blight resistance conferred by the Solanum pimpinellifolium accession PI 163245 ......................................................... 32 Abstract ......................................................................................................................... 32 Introduction ................................................................................................................... 33 Materials and Methods .................................................................................................. 39 Plant Material ............................................................................................................ 39 Inoculum preparation ................................................................................................. 40 F2 disease evaluation ................................................................................................. 41 F3 disease evaluations ................................................................................................ 42 F4 disease evaluations ................................................................................................ 43 Data analysis .............................................................................................................. 43 Results ........................................................................................................................... 45 Disease response of parental, F1, and control lines ................................................... 45 F2 disease response .................................................................................................... 46 F2:F3 parent-offspring correlation analyses ............................................................... 47 F3:F4 parent-offspring correlation analyses ............................................................... 53 Realized heritability ................................................................................................... 55 vi Number of resistance loci .......................................................................................... 56 Discussion ..................................................................................................................... 56 Literature Cited ............................................................................................................. 60 Chapter 3 Genetic characterization and heritability of late blight resistance conferred by the Solanum pimpinellifolium accession PI 224710 ......................................................... 64 Abstract ......................................................................................................................... 64 Introduction ................................................................................................................... 65 Materials and Methods .................................................................................................. 70 Plant Material ............................................................................................................ 70 Inoculum preparation ................................................................................................. 71 F2 disease evaluation ................................................................................................