Discovery and genotyping of existing and induced DNA sequence variation in potato

In this thesis natural and induced DNA sequence diversity in potato (Solanum tuberosum) for use in marker-trait analysis and potato breeding is assessed. The study addresses the challenges of reliable, high-throughput identification and genotyping of sequence variants in existing tetraploid potato cultivar panels using traditional Sanger sequencing and next-generation massively parallel sequencing (MPS), and the application of this knowledge in the form of genetic markers. Furthermore, it explores the efficiency of ethyl methanesulphonate (EMS) mutagenesis combined with high resolution melting (HRM) DNA screening to induce and discover novel sequence variants in potato genotypes. Discovery and genotyping of sequence diversity in outcrossing autotetraploid species like potato is complex. In autotetraploid species, genotyping implies the quantitative identification of five alternative allele copy number states. In Chapter 1, several methodologies to identify and genotype DNA sequence variants, and the application of these sequence variants is discussed. This chapter provides an introduction to genotyping-by-sequencing (GBS) and the determination of allele copy number. In Chapter 2 the sequence diversity in three genes of the carotenoid pathway is assessed in diploid and tetraploid potato genotypes using direct Sanger sequencing. To investigate the genetics and molecular biology of orange and yellow flesh colour in potato, association analysis between SNP haplotypes and flesh colour phenotypes was performed, and the inheritance and gene expression of associated alleles was studied. We observed among eleven beta-carotene hydroxylase 2 (CHY2) alleles one dominant allele with a major effect, changing white into yellow flesh colour. In contrast, none of the lycopene epsilon cyclase (LCYe) alleles seemed to have a large effect on flesh colour. Analysis of zeaxanthin epoxidase (ZEP) alleles showed that a recessive allele with a non-LTR retrotransposon sequence in intron 1 reduced the expression level of the ZEP gene and caused accumulation of zeaxanthin. Genotypes combining presence of the dominant CHY2 allele with homozygosity for the recessive ZEP allele produced orange-fleshed tubers that accumulate large amounts of zeaxanthin. Sanger amplicon sequencing was applied in Chapter 3 to evaluate the sequence diversity in α-Glucan Water Dikinase (StGWD), a candidate gene underlying a QTL involved in potato starch phosphate content. Sanger sequences of two StGWD amplicons from a global collection of 398 commercial cultivars and progenitor lines were used to identify 16 unique haplotypes. By assigning tag SNPs to these haplotypes and by determining the allele copy number of identified sequence variants, we inferred the four-allele genetic composition for almost all cultivars assayed at this locus. This allowed genetic diversity parameters like the average number of different alleles present in a single cultivar (Ai=3.1) and the average intra-individual heterozygosity (Ho=0.765) to be estimated for this locus. Pedigree analysis confirmed that the identified haplotypes are identical by descent (IBD) and offered insight in the breeding history of elite potato germplasm. Haplotype association analysis led to the identification of two StGWD alleles causing altered starch phosphate content, which was further verified in diploid and tetraploid mapping populations containing the relevant alleles. One of these alleles (Allel H) increases the fraction of starch that is phosphorylated, while the other one (Allele A) decreases it. To scale up the discovery and genotyping of sequence variants, and to make it more whole-genome oriented, Chapter 4 reports on massively parallel sequencing (MPS) of approximately 800 genes scattered over the potato genome and resequenced in 83 tetraploid potato cultivars and a monoploid reference accession. We show that by combining MPS with genome complexity reduction and indexed sequencing, sufficient read depth for GBS can be achieved for reliable discovery and genotyping of sequence variants in individual tetraploid potato genotypes. With a custom designed, SureSelect enrichment library, 1.44 Mb of DNA sequence was targeted. The genes targeted were mainly single-copy genes, selected based on putative gene functions in both primary and secondary metabolic pathways, potato quality traits and biotic and abiotic stresses, and included a large set of conserved orthologous sequence genes (COSII) useful for genetic anchoring and phylogenetic studies. The indexed and enriched DNA libraries were sequenced on a Illumina HiSeq. After filtering and processing the raw sequence data, 12.4 Gb of high-quality sequence data was mapped to the potato genome, covering 2.1 Mb of the genome sequence with a median average read depth of 63× per cultivar. We detected over 129,000 sequence variants in these data and determined allele copy number of the variants in individual potato samples. The accuracy of the sequence-based allele copy number estimates was verified by a low-density SNP genotyping assay. This showed that for reliable genotyping a read count-based genotype quality score is best applied and a read depth of 80× is recommended for determining allele copy number in autotetraploid potato. Average nucleotide diversity (π=10.7×10-3 genome-wide, ≈1 variant/93 bp between two random alleles) varied along the twelve potato chromosomes, and individual genes under selection were identified. As an example for application of GBS for genome-wide association analysis (GWAS), the identified sequence variants and genotype data were tested in a marker-trait association analysis with plant maturity and tuber flesh colour. This led to the identification of alleles accounting for significant phenotypic variation in these traits. In Chapter 5 we applied the chemical mutagen EMS to diploid potato by two different treatments, a pollen and a seed treatment. We screened the resulting populations for novel mutations using HRM analysis. A pollen treatment with EMS dissolved in a sucrose solution was found to induce mutations only at a low frequency (only one mutation discovered after screening >2.7 Mb of sequence). In planta selection of the most vital mutagenized pollen seems to have lowered the mutation density to a frequency that is not suitable for reverse genetics studies. Treatment of potato seeds with EMS on the other hand provided a high density of novel mutations (1 mutation/65 kb), discovered in the M1 generation. In contrast to most EMS mutagenesis studies, we directly screened the M1 generation of the seed-treated population. In six candidate genes involved in potato starch and frying quality traits, 65 novel sequence variants were discovered. In all six genes, missense mutations that are predicted to damage protein function were discovered, and for four genes five premature stop codon mutations were identified. We attempted to stabilize and transfer 27 putatively interesting mutations to the M2 and M3 generation for further evaluation. Genetically stable M2 and M3 plants have been generated for 10 (37%) of these mutations. The estimated density of M1 mutations that are transferable to the M2 generation (one “accessible” mutation/118-176 kb) is higher than the mutation densities obtained in most other plant species, for which the M2 generation has been screened. The results of this chapter thus demonstrate that screening the M1 generation offers a good alternative to the commonly applied M2 screening for the rapid generation of novel genetic variation at a high density, without too much complication in recovering mutations in the M2 generation. In the concluding Chapter 6, results of preceding chapters are evaluated, and the prospects of the findings for potato research and breeding are discussed.