Association mapping in tetraploid potato

The results of a four year project within the Centre for BioSystems Genomics (www.cbsg.nl), entitled “Association mapping and family genotyping in potato” are described in this thesis. This project was intended to investigate whether a recently emerged methodology, association mapping, could provide the means to improve potato breeding efficiency. In an attempt to answer this research question a set of potato cultivars representative for the commercial potato germplasm was selected. In total 240 cultivars and progenitor clones were chosen. In a later stage this set was expanded with 190 recent breeds contributed by five participating breeding companies which resulted in a total of 430 genotypes. In a pilot experiment, the results of which are reported in Chapter 2, a subset of 220 of the abovementioned 240 cultivars and progenitor clones was used. Phenotypic data was retrieved through contributions of the participating breeding companies and represented summary statistics of recent observations for a number of traits across years and locations, calculated following company specific procedures. With AFLP marker data, in the form of normalised log-transformed band intensities, obtained from five well-known primer combinations, the extent of linkage disequilibrium (LD), using the r2 statistic, was estimated. Population structure within the set of 220 cultivars was analysed by deploying a clustering approach. No apparent, nor statistically supported population structure was revealed and the LD seemed to decay below the threshold of 0.1 at a genetic distance of about 3cM with this set of marker data. Furthermore, marker-trait associations were investigated by fitting single marker regression models for phenotypic traits on marker band intensities with and without correction for population structure. Population structure correction was performed in a straightforward way by incorporating a design matrix into the model assuming that each breeding company represented a different breeding germplasm pool. The potential of association mapping in tetraploid potato has been demonstrated in this pilot experiment, because existing phenotypic data, a modest number of AFLP markers, and a relatively straightforward statistical analysis allowed identification of interesting associations for a number of agro-morphological and quality traits. These promising results encouraged us to engage into an encompassing genome-wide association mapping study in potato. Two association mapping panels were compiled. One panel comprising 205 genotypes, all of which were also present in the set used for the pilot experiment, and another panel containing in total 299 genotypes including the entire set of 190 recent breeds together with a series of standard cultivars, about 100 of which are in common with the first panel. Phenotypic data for the association panel with 205 genotypes were obtained in a field trial performed in 2006 in Wageningen at two locations with two replicates. We will refer to this set as the “2006 field trial”. Phenotypic data for the other panel with 299 genotypes was contributed by the five participating breeding companies and consisted of multi-year-multi-location data obtained during generations of clonal selection. The 2006 data were nicely balanced, because the trial was designed in that way. The historical breeding dataset was highly unbalanced. Analysis of these two differing phenotypic datasets was performed to deliver insight in variance components for the genotypic main effects and the genotype by environment interaction (GEI), besides estimated genotype main effects across environments. Both phenotypic datasets were analysed separately within a mixed model framework including terms for GEI. In Chapter 3 we describe both phenotypic datasets by comparing variance components, heritabilities (=repeatabilities), intra-dataset relationships and inter-dataset relationships. Broader aspects related to phenotypic datasets and their analysis are discussed as well. To retrieve information about hidden population structure and genetic relatedness, and to estimate the extent of LD in potato germplasm, we used marker information generated with 41 AFLP primer combinations and 53 microsatellite loci on a collection of 430 genotypes. These 430 genotypes contain all genotypes present in the two association mapping panels introduced before plus a few extra genotypes to increase potato germplasm coverage. Two methods were used: a Bayesian approach and a distance-based clustering approach. Chapter 4 describes the results of this exercise. Both strategies revealed a weak level of structure in our material. Groups were detected which complied with criteria such as their intended market segment, as well as groups differing in their year of first registration on a national list. Linkage disequilibrium, using the r2 statistic, appeared to decay below the threshold of 0.1 across linkage groups at a genetic distance of about 5cM on average. The results described in Chapter 4 are promising for association mapping research in potato. The odds are reasonable that useful marker-trait associations can be detected and that the potential mapping resolution will suffice for detection of QTL in an association mapping context. In Chapter 5 a comprehensive genome-wide association mapping study is presented. The adjusted genotypic means obtained from two association mapping panels as a result of phenotypic analysis performed in Chapter 3 were combined with marker information in two association mapping models. Marker information consisted of normalised log-transformed band intensities of 41 AFLP primer combinations and allele dosage information from 53 microsatellites. A baseline model without correction for population structure and a more advanced model with correction for population structure and genetic relatedness were applied. Population structure and genetic relatedness were estimated using available marker information. Interesting QTL could be identified for 19 agro-morphological and quality traits. The observed QTL partly confirm previous studies e.g. for tuber shape and frying colour, but also new QTL have been detected e.g. for after baking darkening and enzymatic browning. In the final chapter, the general discussion, results of preceding chapters are evaluated and their implications for research as well as breeding are discussed. <br/

Phenotypic analyses of multi-environment data for two diverse tetraploid potato collections: comparing an academic panel with an industrial panel

Phenotypic analyses of two different association panels of tetraploid potato cultivars are presented. Association panels are sets of variously related genotypes assembled for association analysis purposes. The aims of this research were to inspect, analyse and compare two phenotypic data sets, a first step in association mapping analysis. A first panel of 205 contemporary and historical cultivars, selected to represent the commercial potato germplasm pool, was evaluated in two trials in 2006, one on sandy soil and the other on clay soil, both with two replications. It was called the academic panel. Data for the second panel with 299 genotypes were compiled from contributions from five breeding companies and included 66 locations and 18 years. Each of the participating breeding companies contributed data from their clonal selection programmes for 38 advanced breeding clones and a series of standard cultivars. It was called the industrial panel. Variance components for genotypic main effects and genotype-by-environment interactions were calculated, and estimates for the random genotypic main effects were produced. The genotypic main effects for 19 agro-morphological and quality traits were used to study trait by trait correlations within each panel. In addition, for the genotypes shared by both panels, the correlation of genetic main effects between the panels was investigated. The heritability of all traits was high and no large differences were observed between panels. Coefficients of trait variation were highly correlated (r¿=¿0.9) for both panels and trait by trait correlations in both panels showed highly similar patterns. These results demonstrate that a single-year balanced field trial as well as using breeders’ records yields robust phenotypic information that can be used in a genome-wide association study. Issues related to data management and definition of traits are discussed

A Next-Generation Sequencing Method for Genotyping-by-Sequencing of Highly Heterozygous Autotetraploid Potato

Assessment of genomic DNA sequence variation and genotype calling in autotetraploids implies the ability to distinguish among five possible alternative allele copy number states. This study demonstrates the accuracy of genotyping-by-sequencing (GBS) of a large collection of autotetraploid potato cultivars using next-generation sequencing. It is still costly to reach sufficient read depths on a genome wide scale, across the cultivated gene pool. Therefore, we enriched cultivar-specific DNA sequencing libraries using an in-solution hybridisation method (SureSelect). This complexity reduction allowed to confine our study to 807 target genes distributed across the genomes of 83 tetraploid cultivars and one reference (DM 1–3 511). Indexed sequencing libraries were paired-end sequenced in 7 pools of 12 samples using Illumina HiSeq2000. After filtering and processing the raw sequence data, 12.4 Gigabases of high-quality sequence data was obtained, which mapped to 2.1 Mb of the potato reference genome, with a median average read depth of 63× per cultivar. We detected 129,156 sequence variants and genotyped the allele copy number of each variant for every cultivar. In this cultivar panel a variant density of 1 SNP/24 bp in exons and 1 SNP/15 bp in introns was obtained. The average minor allele frequency (MAF) of a variant was 0.14. Potato germplasm displayed a large number of relatively rare variants and/or haplotypes, with 61% of the variants having a MAF below 0.05. A very high average nucleotide diversity (p = 0.0107) was observed. Nucleotide diversity varied among potato chromosomes. Several genes under selection were identified. Genotyping-by-sequencing results, with allele copy number estimates, were validated with a KASP genotyping assay. This validation showed that read depths of ~60–80× can be used as a lower boundary for reliable assessment of allele copy number of sequence variants in autotetraploids. Genotypic data were associated with traits, and alleles strongly influencing maturity and flesh colour were identified

Phenotypic analyses of multi-environment data for two diverse tetraploid potato collections: comparing an academic panel with an industrial panel

Phenotypic analyses of two different association panels of tetraploid potato cultivars are presented. Association panels are sets of variously related genotypes assembled for association analysis purposes. The aims of this research were to inspect, analyse and compare two phenotypic data sets, a first step in association mapping analysis. A first panel of 205 contemporary and historical cultivars, selected to represent the commercial potato germplasm pool, was evaluated in two trials in 2006, one on sandy soil and the other on clay soil, both with two replications. It was called the academic panel. Data for the second panel with 299 genotypes were compiled from contributions from five breeding companies and included 66 locations and 18 years. Each of the participating breeding companies contributed data from their clonal selection programmes for 38 advanced breeding clones and a series of standard cultivars. It was called the industrial panel. Variance components for genotypic main effects and genotype-by-environment interactions were calculated, and estimates for the random genotypic main effects were produced. The genotypic main effects for 19 agro-morphological and quality traits were used to study trait by trait correlations within each panel. In addition, for the genotypes shared by both panels, the correlation of genetic main effects between the panels was investigated. The heritability of all traits was high and no large differences were observed between panels. Coefficients of trait variation were highly correlated (r¿=¿0.9) for both panels and trait by trait correlations in both panels showed highly similar patterns. These results demonstrate that a single-year balanced field trial as well as using breeders’ records yields robust phenotypic information that can be used in a genome-wide association study. Issues related to data management and definition of traits are discussed