Discovery and Genotyping of Existing and Induced DNA Sequence Variation in Potato Jan Uitdewilligen Thesis committee Thesis supervisor Prof. dr. R.G.F. Visser Professor of Plant Breeding Wageningen University Thesis co-supervisors Dr. ir. H.J. van Eck Assistant professor, Laboratory of Plant Breeding Wageningen University Dr. ir. A.M.A. Wolters Scientist, Laboratory of Plant Breeding Wageningen University Other members Prof. dr. F.A. van Eeuwijk, Wageningen University Prof. dr. M. Groenen, Wageningen University Prof. dr. M.E. Schranz, Wageningen University Dr. N.C. de Vetten, Averis Seeds, Valthermond This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences Discovery and Genotyping of Existing and Induced DNA Sequence Variation in Potato Jan Uitdewilligen Thesis submitted in fulfilment 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 18 September 2012 at 4 p.m. in the Aula. Jan G.A.M.L. Uitdewilligen Discovery and genotyping of existing and induced DNA sequence variation in potato, 166 pages. Thesis, Wageningen University, Wageningen, NL (2012) With references, with summaries in Dutch and English ISBN 978-94-6173-233-0 TABLE OF CONTENTS CHAPTER 1 – General Introduction .................................................................................................... 7 CHAPTER 2 – Identification of Alleles of Carotenoid Pathway Genes Important for Zeaxanthin Accumulation in Potato Tubers ........................................................ 19 CHAPTER 3 – Sequence Characterization of StGWD Haplotypes and the Genetics of Starch Phosphate Content in Tetraploid Potato .................................................. 41 CHAPTER 4 – A Next-Generation Sequencing Method for Genotyping-By-Sequencing of Highly Heterozygous Autotetraploid Potato ...................................................... 63 CHAPTER 5 – EMS-Induced Mutation Discovery in the M1 Generation of Potato as a Strategy for Reverse Genetics .............................................................................. 101 CHAPTER 6 – General Discussion ................................................................................................... 123 REFERENCES .................................................................................................................................. 141 SUMMARY .................................................................................................................................. 155 SAMENVATTING .................................................................................................................................. 159 DANKWOORD .................................................................................................................................. 163 OVER DE AUTEUR ................................................................................................................................. 165 CHAPTER 1 General Introduction General Introduction POTATO BREEDING Potato (Solanum tuberosum L.) is a nutritious food crop, contributing carbohydrates, important amino acids and vitamins to the human diet. The species is highly heterozygous, autotetraploid (2n=4x=48), and largely reproduces vegetatively. The early adaptation of cultivated potato in Europe and North America from landraces of South American Solanum tuberosum ‘Andigenum group’ and ‘Chilotanum Group’, has been accompanied by strong artificial selection for adaptation to long days (AMES and SPOONER 2008). In the 1840s, the late blight (Phytophthora infestans) disease caused the “Irish potato famine”, leading to subsequent mass selection for late blight resistance. This was coupled with continued selection for desirable agronomic characteristics such as vigor and yield and ultimately led to introgression of many disease resistance genes from wild species into the gene pool of European and North American potato cultivars. Current potato breeding activities are aimed at developing new high-yielding cultivars with pest and disease resistance while also meeting the increasing quality requirements of consumers and the potato processing industry. Most of the characteristics targeted for improvement are quantitative traits for which phenotypic variation depends on both genetic and environmental factors. The molecular basis of many of these traits is, however, poorly understood (HAMILTON et al. 2011). MOLECULAR MARKERS Identification and genotyping of DNA variants lies at the core of modern genetics and allows the exploration of important questions in population genetics, evolution and plant breeding (DAVEY et al. 2011). Molecular markers are a common tool for the development of saturated genetic and physical maps, genetic diversity analysis, genotype identification, gene isolation and the identification of loci controlling phenotypic traits, marker-assisted selection (MAS), and, more recently, genomic selection (GS). Two main types of molecular markers are fragment-based markers and the more recently developed sequence-based markers. Traditional fragment-based molecular markers Multiple approaches have been developed for the characterization of DNA variation in plants and to identify sequence polymorphisms using fragments of DNA. Until recently, amplified fragment length polymorphisms (AFLPs) or simple sequence repeats (SSRs) were the molecular markers of choice for DNA fingerprinting of plant genomes (ALLENDER and KING 2010; CASTILLO et al. 2010; D'HOOP et al. 2008). Both methods rely on gel-based identification of DNA fragment sizes after PCR amplification. These marker types have a number of inherent weaknesses. In AFLP and SSR-based genetic studies, all genotypes with the same SSR pattern or with the same AFLP fragment are usually considered to be identical for this marker locus. However, the internal DNA sequence composition of the AFLP or SSR marker fragment with a specific gel mobility and/or the chromosomal regions linked with a given marker fragment may exhibit significant sequence variation (GORT and VAN EEUWIJK 2012). In such cases the markers are identical by state (IBS) without being identical by descent (IBD). Furthermore, in marker-trait associations analysis, the value of a marker fragment depends on the strength of its linkage with the functional alleles of a target locus influencing phenotypic variation in the Page | 9 Chapter 1 target trait. Since SSR and AFLP markers are usually derived from non-functional, intergenic sequences, and since these markers occur at a relatively low frequency, their strong linkage with causal alleles is less likely. Sequence-based markers The underlying DNA sequence variants that define AFLP and SSR polymorphisms are sequence variants like single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs), and insertions and deletions (indels). These sequence variants are the basic units of genetic diversity and have additional value, relative to that of SSR and AFLP markers, in their ability to quantify genetic diversity and explain phenotypic variation. By generating distinct alleles, individual sequence variants, or multiple variants combined into distinct haplotypes, can be responsible for variation in specific traits or phenotypes. Sequence- based markers may also represent neutral variation that is useful for evaluating genetic diversity. Compared to multi-allelic SSR markers, an individual base change detected as a SNP is more likely to have occurred only once in evolutionary time. Owing to their greater utility in making evolutionary inferences, abundance, ease of high-throughput detection, and efficient automated genotyping, SNP markers are increasingly applied to study genetic diversity and phenotypic variation in plant breeding and basic genetics research (GRATTAPAGLIA et al. 2011; GRIFFIN et al. 2011; HUANG et al. 2010; LIJAVETZKY et al. 2007; RICKERT et al. 2003). SNPs as haplotype markers In practice, SNPs are mostly bi-allelic, even though in principle any of the four nucleotides can be present at any position in a stretch of DNA. This bi-allelic nature is due to the low frequency of mutations that lead to new SNPs. As a result, the chance for a second independent mutation that introduces a third allele at the same base position is low. Unlike for multi-allelic markers, diversity values (expected heterozygosity) of SNPs are therefore generally low. The relatively poor information content of individual SNP markers can, however, be enhanced by using haplotype markers rather than single marker scores. Haplotypes can be defined as common sets of markers in linkage phase at adjacent loci. These linked markers are further structured into haplotype blocks likely to be transmitted as a unit from generation to generation, assuming no recombination occurs (i.e., IBD). Each haplotype block contains only a few haplotypes. The minimal informative subset of SNPs associated with the haplotypes in a block are often referred to as the “haplotype tagging SNPs” or tag SNPs (GABRIEL et al. 2002; JOHNSON et al. 2001). Depending on the amount of sequence diversity, tag SNPs can be either pairwise-defined or multi-marker-defined, e.g. either a single tag SNP or a combination of tag SNPs identifies a single haplotype (DE BAKKER et al. 2005). Used as tag SNPs, bi-allelic SNP markers can be as informative as multi-allelic molecular markers. When inbred