UNIVERSITY OF READING Genetic mapping and phenotyping plant characteristics, fruit quality and disease resistance traits in octoploid strawberry (Fragaria × ananassa) Thesis submitted for the degree of Doctor of Philosophy School of Biological Sciences Laima Antanaviciute February, 2016 Declaration I confirm that this is my own work and the use of all material from other sources has been properly and fully acknowledged. ……………………………………………………… Abstract The cultivated strawberry (Fragaria × ananassa Duch.) is the third most economically important fruit crop. In recent years the withdrawal of many fungicides and soil fumigants have made the sustainability and profitability of this crop more challenging. To overcome these challenges, plant breeders aim to improve upon existing cultivars and to release new ones with higher yield, better fruit quality and more disease resistance. Through Quantitative Trait Mapping, markers linked to genetic variants associated with traits of economic and agronomic importance can be identified through and molecular markers such as simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), can be used to improve plant breeding efficiency at the molecular level, which significantly reduces the breeding time and cost of phenotyping. In this thesis the following work is described: a correlation analysis of plant characteristics and fruit quality traits; the saturation of an existing SSR-based linkage map; the development of a high density consensus SNP-based octoploid strawberry linkage map, and the identification of quantitative trait loci (QTL) linked to two key plant attributes, fruit quality and powdery mildew resistance. In addition, the most closely linked SSR markers were identified and validated in a wider strawberry germplasm using firmness as an example study. Moreover, the physical locations of expansin genes and SNP markers associated with firmness QTLs were investigated. The purpose of this analysis was to find out if QTLs associated with fruit firmness overlapped the positions of expansins, genes known to be important in controlling fruit firmness. i Overall the results provide new insights into how complex traits are correlated in octoploid strawberry and valuable information about QTL location and relationships. The information generated will be important for future analyses, such as identification of genes linked to traits of importance, and novel molecular marker development for marker-assisted breeding. Finally, the data from this study will contribute towards genomic selection studies conducted in cultivated strawberry and other closely related rosaceous crops. ii Acknowledgements First of all, I would like to thank my supervisors, Professor Nick H. Battey at University of Reading, and Doctor Richard J. Harrison at East Malling Research, for their help in the revision of this thesis, support and encouragement during the three years of this project, which was based at East Malling Research, Kent. I wish to thank Dr Richard Harrison in particular for his invaluable advice and useful discussions in every aspect of this work, for guidance and assistance in analysing the data and help with the graphics and several software for the presentation of the data. I would like to express my sincere thanks to Dr Robert Vickerstaff (East Malling Research) for his help in analysing genotypic data observed from the IStraw90 SNP genotyping array. I am grateful to Izabela Zabinska, Antonio Gómez-Cortecero, César Marina-Montes, Vincent Colas, Judit Linka and Ana Mertinez-Crucis for their help in phenotyping strawberry plants. I would also like to thank members of the Genetics and Crop Improvement Department, particularly Adam Whitehouse, at East Malling Research for their support, assistance and personal communications during the research period. Special thanks to my wonderful partner John Fabule and my parents for their moral support and understanding. Finally, I thank The Agriculture and Horticulture Development Board for sponsoring Fragaria genetics and genomics projects at East Malling Research, and for the financial support for my studies at University of Reading. iii Table of Contents Abstract ---------------------------------------------------------------------------------------------- i Acknowledgements ------------------------------------------------------------------------------ iii List of Abbreviations --------------------------------------------------------------------------- viii Chapter 1 General introduction -------------------------------------------------------------- 1 1.1 The Rosaceae family ------------------------------------------------------------------ 2 1.1.1 Taxonomy of the Rosaceae -------------------------------------------------------- 2 1.1.2 The genus Fragaria ----------------------------------------------------------------- 3 1.1.3 Strawberry species, distribution and self-incompatibility --------------------- 5 1.1.4 Economic importance and strawberry production ------------------------------ 9 1.2 Strawberry breeding ---------------------------------------------------------------- 10 1.3 Molecular markers in plant breeding ------------------------------------------- 15 1.3.1 Marker types ------------------------------------------------------------------------ 15 1.3.2 Linkage mapping in strawberry -------------------------------------------------- 17 1.3.3 Marker-assisted selection --------------------------------------------------------- 19 1.4 Mapping quality traits in strawberry-------------------------------------------- 21 1.4.1 Multiple models for linking traits of interest in octoploid strawberry ------ 21 1.4.2 Next generation sequencing approaches to understand fruit quality traits - 22 1.5 Pathogens infecting cultivated strawberry ------------------------------------- 24 1.5.1 Diseases caused by different types of pathogens ------------------------------ 24 1.5.2 Difficulties in breeding disease resistance -------------------------------------- 28 1.5.3 Defence mechanisms against infections in strawberry ------------------------ 30 1.6 Genomic selection in plant breeding --------------------------------------------- 32 1.7 The aims of this investigation ----------------------------------------------------- 35 Chapter 2 The investigation of correlations between morphological and fruit quality traits using phenotypic and genotypic data in Fragaria × ananassa -------- 37 2.1 Introduction --------------------------------------------------------------------------- 38 2.2 Materials and methods -------------------------------------------------------------- 41 2.2.1 Plant material ----------------------------------------------------------------------- 41 2.2.2 Phenotypic data collection – plant characteristics ----------------------------- 42 2.2.3 Phenotypic data collection – fruit quality --------------------------------------- 45 2.2.4 Weather data ------------------------------------------------------------------------ 48 2.2.5 Statistical data analysis ------------------------------------------------------------ 49 2.3 Results ---------------------------------------------------------------------------------- 51 2.3.1 Phenotypic data analysis ---------------------------------------------------------- 51 2.3.2 Weather data analysis -------------------------------------------------------------- 59 iv 2.3.3 Correlation analysis of plant characteristics and fruit quality traits --------- 60 2.3.4 Clustering analysis of the phenotypic data ------------------------------------- 64 2.3.5 Broad-sense heritability ----------------------------------------------------------- 66 2.4 Discussion ------------------------------------------------------------------------------ 68 2.4.1 Phenotypic variation among parental genotypes and the progeny ---------- 68 2.4.2 Correlation analysis ---------------------------------------------------------------- 70 2.4.3 Heritability analysis ---------------------------------------------------------------- 72 2.5 Conclusions ---------------------------------------------------------------------------- 73 Chapter 3 The development of a high density SNP-based genetic linkage map of an octoploid strawberry mapping population --------------------------------------------- 74 3.1 Introduction --------------------------------------------------------------------------- 75 3.2 Materials and methods -------------------------------------------------------------- 80 3.2.1 Plant material and DNA extractions --------------------------------------------- 80 3.2.2 SSR marker selection for SSR-based genetic linkage map saturation ------ 81 3.2.3 PCR conditions --------------------------------------------------------------------- 82 3.2.4 Product visualization --------------------------------------------------------------- 84 3.2.5 SSR marker amplification and genetic linkage map development ---------- 86 3.2.6 The genotyping a 90 K Axiom® SNP array in octoploid strawberry ------- 87 3.2.7 90 K Axiom array data analysis -------------------------------------------------- 90 3.2.8 SNP-base map construction ------------------------------------------------------- 92 3.3 Results ---------------------------------------------------------------------------------- 93 3.3.1 Simple sequence repeat marker polymorphisms ------------------------------- 93 3.3.2 Novel SSR mapping and loci distribution -------------------------------------- 94 3.3.3 SNP marker amplification in ‘Redgauntlet’ × ‘Hapil’ progeny ----------- 106 3.3.4 SNP-based linkage map construction