Genetic and Physiological Quality of Tomato Seed and Seedlings Noorullah Khan Thesis committee Promotor Prof. Dr. H.J. Bouwmeester Professor of Plant Physiology Co-promotors Dr. H.W.M. Hilhorst Associate Professor, Laboratory of Plant Physiology Wageningen University Dr. W. Ligterink Researcher, Laboratory of Plant Physiology Wageningen University Other members Prof. Dr. M.E. Schranz, Wageningen University Prof. Dr. J.J.B. Keurentjes, University of Amsterdam / Wageningen University Dr. C.H. de Vos, Plant Research International, Wageningen Dr. P. Spoelstra, Incotec Holding B.V., Enkhuizen This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences Genetic and Physiological Quality of Tomato Seed and Seedlings Noorullah Khan Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Tuesday 3 September 2013 at 1.30 p.m. in the Aula. Noorullah Khan Genetic and Physiological Quality of Tomato Seed and Seedlings 249 pages PhD thesis, Wageningen University, Wageningen, NL (2013) With references, with summaries in English and Dutch ISBN 978-94-6173-647-5 CONTENTS Chapter 1 General introduction 7 Chapter 2 Natural Variation for Seedling Traits and their Link with 35 Seed Dimensions in Tomato Chapter 3 Seed Quality Phenotypes in a Recombinant Inbred 63 Population of an Interspecific Cross between Solanum lycopersicum x Solanum pimpinellifolium Chapter 4 Genetic Analysis of Whole Seed and Tissue-Specific 103 Food Reserves Reveals a Close Link between the Abundance of Seed Reserves and Seed and Seedling Biomass Chapter 5 Canonical Association Reveals a Strong Link between 143 Metabolic Signatures of Seed and Seedling Quality in a Recombinant Inbred Population of Tomato Chapter 6 Using Heterogeneous Inbred Families (HIFs) to Confirm 177 Natural Allelic Variation for Complex Seed and Seedling Phenotypes on Tomato Chromosomes 6 and 9 Chapter 7 General discussion 199 Summary 227 Samenvatting 233 Acknowledgements 239 Curriculum vitae 243 Publication list 245 Education statement 247 Chapter 1 General Introduction Seed Quality Seed quality is one of the most important factors to affect the success of a crop (Finch- Savage, 1995) and is thought to be associated with many interlinked physiological and genetic traits (Hilhorst and Koornneef, 2007; Hilhorst et al., 2010). The success of germination, growth and final yield of every crop depends to a large extent on the quality of the seeds used to grow the crop. Seed quality is a complex trait and is defined as “the viability and vigour attribute of a seed that enables the emergence and establishment of normal seedlings under a wide range of environments” (Khan et al., 2012). The practical definition of seed quality is determined by the end user and will, therefore, differ substantially, depending on the use of seeds as propagule or commodity. For a farmer or plant grower high quality seeds are those seeds that germinate to a high percentage and establish vigourous seedlings under a wide range of field conditions. On the other hand, high quality seeds for use in the food industry may be seeds with a high starch or oil content or oil seeds with a specific protein or fatty acid composition (Nesi et al., 2008). Seed quality (for propagation) is determined by a number of physiological processes related to important plant developmental events, such as embryogenesis, growth, stress-resistance and the transition from a seed to an autotrophic seedling (Ouyang et al., 2002; Spanò et al., 2007). Seed quality comprises many different attributes, including germination characteristics, dormancy, seed and seedling vigour, uniformity in seed size, normal embryo- and seedling morphology, storability, absence of mechanical damage, as well as the ability to develop into a normal and vigourous plant (Goodchild and Walker, 1971; Bewley, 1997; Delseny et al., 2001; Finch-Savage and Leubner-Metzger, 2006; El- Kassaby et al., 2008; Angelovici et al., 2010). Because of its complex nature, testing of seed quality is in many cases, at best, an ‘educated guess’ in order to predict subsequent behavior in the field (Powell and Basra, 2006). Therefore, seed producers have redefined the term ‘seed quality’ to include important attributes such as ‘usable plants’ and ‘seedling and crop establishment’. The attribute ‘usable plants’ is one of the major characteristics of seed quality used by seed producers and plant breeders (Ligterink et al., 2012). Seed quality is mainly acquired during seed development and maturation, and is drastically affected by interactions between the genome and the prevailing environmental conditions. This process is part of the normal adaptation of plants to a varying environment 7 Chapter 1 and is aimed at maximizing the possibility of successful offspring (Huang et al., 2010). As the ultimate performance of a seed is a function of the complex interaction between the genome and the environment, seed quality can be enhanced at all the different steps of the production process. Since it is difficult to influence the production environment, even under greenhouse conditions, plant breeders and seed companies try to acquire the best possible quality of seeds mainly by varying the time and method of harvest, and particularly by post-harvest treatments such as cleaning, sorting, coating and priming and by controlling the storage conditions. However, the genetic component of the interaction between the genome and the environment can be investigated and this variation in genetic adaptation may provide opportunities for plant breeders and seed companies to breed for better seed quality. Despite these opportunities, the genetic regulation of seed quality has hardly been investigated to be used in breeding programs. Although, a few studies have documented some quantitative trait loci (QTLs) associated with germination, storability and stress tolerance in Arabidopsis and tomato (Foolad et al., 2003; Clerkx et al., 2004), a systematic study of the genetics of seed quality is lacking. The present study seeks to discover integrative approaches that can facilitate the understanding of the underlying causes of the complex trait of seed quality. Our objective is to provide new methods for dissecting the genetic components of seed quality by integrating the physiology, genetics, genomics and metabolomics of seeds to identify loci, and subsequently genes, controlling seed quality traits in tomato. Important Seed Quality Attributes Seed size variation and its influence on seedling establishment Among others seed size and mass are important traits determining seed quality(Panthee et al., 2005), which in turn are the most variable traits in the plant kingdom (TeKrony and Egli, 1991; Orsi and Tanksley, 2009). Seed size is a key determinant of evolutionary fitness in plants and is a trait that often undergoes tremendous changes during crop domestication. Seed size is most often quantitatively inherited and seeds range in weight from less than 1 microgram in the Coral-root orchid (Corallorhiza maculate) to more than 10 kg in the Coco- de-mer palm (Lodoicea maldivica). This large variation in seed size can be observed not only among taxa, but also within taxa. For example, the genus Solanum contains a set of 9 cross- compatible species, closely related to tomato. Despite their close taxonomic affinities, these species show a 10-fold range in seed size, suggesting a rapid rate of evolutionary change. There is typically at least a 105 fold variation of seed mass between species within a single area (Westoby et al., 1992; Orsi and Tanksley, 2009). In addition to the variation in seed size among different species, many studies have emphasized that seed size varies significantly within the same species (Michaels et al., 1988) and between different populations and different mother plants and even between different seeds of the same 8 General Introduction mother plant. Nevertheless, this variation within species is very small compared to the range across species (Westoby et al., 1996). Many studies have interpreted seed size differences between species by reference to larger seed size being more adaptive under a variety of environmental hazards. However, experimental confirmation of the benefits of large seed size in relation to particular hazards is rare. More experiments are now being reported but a consistent picture has yet to emerge. The reason for this large variation in seed size is not clear. However, evolutionists and ecologists have long observed this great variation and suggested its importance in adaption to different environments (Metz, 1999). With respect to survival there are both risks and benefits for a species to have large or small seed size. Seed size is thought to have evolved as a compromise between producing numerous smaller seeds, each with small resources, and fewer larger seeds, each with more resources. Because seed size trades off with seed number due to limited availability of maternal resources, small seeded species clearly have the advantage in fecundity, but the countervailing advantage of large seeds appears to be their tolerance to stresses such as shade or drought that are present in some but not all regeneration sites (Smith and Fretwell, 1974; Westoby et al., 1992; Metz, 1999; Orsi and Tanksley, 2009; Muller-Landau, 2010). Most of the domesticated crops