Graphical genotyping as a method to map Ny (o,n)sto and Gpa5 using a reference panel of tetraploid potato cultivars.

KEY MESSAGE: The method of graphical genotyping is applied to a panel of tetraploid potato cultivars to visualize haplotype sharing. The method allowed to map genes involved in virus and nematode resistance. The physical coordinates of the amount of linkage drag surrounding these genes are easily interpretable. Graphical genotyping is a visually attractive and easily interpretable method to represent genetic marker data. In this paper, the method is extended from diploids to a panel of tetraploid potato cultivars. Application of filters to select a subset of SNPs allows one to visualize haplotype sharing between individuals that also share a specific locus. The method is illustrated with cultivars resistant to Potato virus Y (PVY), while simultaneously selecting for the absence of the SNPs in susceptible clones. SNP data will then merge into an image which displays the coordinates of a distal genomic region on the northern arm of chromosome 11 where a specific haplotype is introgressed from the wild potato species S. stoloniferum (CPC 2093) carrying a gene (Ny (o,n)sto ) conferring resistance to two PVY strains, PVYO and PVYNTN. Graphical genotyping was also successful in showing the haplotypes on chromosome 12 carrying Ry-f sto , another resistance gene derived from S. stoloniferum conferring broad-spectrum resistance to PVY, as well as chromosome 5 haplotypes from S. vernei, with the Gpa5 locus involved in resistance against Globodera pallida cyst nematodes. The image also shows shortening of linkage drag by meiotic recombination of the introgression segment in more recent breeding material. Identity-by-descent was found to be a requirement for using graphical genotyping, which is proposed as a non-statistical alternative method for gene discovery, as compared with genome-wide association studies. The potential and limitations of the method are discussed.

Development and analysis of a 20K SNP array for potato (Solanum tuberosum): an insight into the breeding history.

KEY MESSAGE: A 20K SNP array was developed and a comprehensive set of tetraploid cultivar was genotyped. This allowed us to identify footprints of the breeding history in contemporary breeding material such as identification of introgression segments, selection and founder signatures. A non-redundant subset of 15,138 previously identified SNPs and 4454 SNPs originating from the SolCAP project were combined into a 20k Infinium SNP array for genotyping a total of 569 potato genotypes. In this study we describe how this SNP array (encoded SolSTW array) was designed and analysed with fitTetra, software designed for autotetraploids. Genotypes from different countries and market segments, complemented with historic cultivars and important progenitors, were genotyped. This comprehensive set of genotypes combined with the deliberate inclusion of a large proportion of SNPs with a low minor allele frequency allowed us to distinguish genetic variation contributed by introgression breeding. This "new" (post 1945) genetic variation is located on specific chromosomal regions and enables the identification of SNP markers linked to R-genes. In addition, when the genetic composition of modern cultivars was compared with cultivars released before 1945, it appears that 96% of the genetic variants present in those ancestral cultivars remains polymorphic in modern cultivars. Hence, genetic erosion is almost absent in potato. Finally, we studied population genetic processes shaping the genetic composition of the modern European potato including drift, selection and founder effects. This resulted in the identification of major founders contributing to contemporary germplasm.

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 (π = 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.

Identification of alleles of carotenoid pathway genes important for zeaxanthin accumulation in potato tubers.

We have investigated the genetics and molecular biology of orange flesh colour in potato (Solanum tuberosum L.). To this end the natural diversity in three genes of the carotenoid pathway was assessed by SNP analyses. Association analysis was performed between SNP haplotypes and flesh colour phenotypes in diploid and tetraploid potato genotypes. We observed that among eleven beta-carotene hydroxylase 2 (Chy2) alleles only one dominant allele has 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 all (diploid) genotypes with orange tuber flesh were homozygous for one specific Zep allele. This Zep allele showed a reduced level of expression. The complete genomic sequence of the recessive Zep allele, including the promoter, was determined, and compared with the sequence of other Zep alleles. The most striking difference was the presence of a non-LTR retrotransposon sequence in intron 1 of the recessive Zep allele, which was absent in all other Zep alleles investigated. We hypothesise that the presence of this large sequence in intron 1 caused the lower expression level, resulting in reduced Zep activity and accumulation of zeaxanthin. Only genotypes combining presence of the dominant Chy2 allele with homozygosity for the recessive Zep allele produced orange-fleshed tubers that accumulated large amounts of zeaxanthin.

From QTL to candidate gene: genetical genomics of simple and complex traits in potato using a pooling strategy.

BACKGROUND: Utilization of the natural genetic variation in traditional breeding programs remains a major challenge in crop plants. The identification of candidate genes underlying, or associated with, phenotypic trait QTLs is desired for effective marker assisted breeding. With the advent of high throughput -omics technologies, screening of entire populations for association of gene expression with targeted traits is becoming feasible but remains costly. Here we present the identification of novel candidate genes for different potato tuber quality traits by employing a pooling approach reducing the number of hybridizations needed. Extreme genotypes for a quantitative trait are collected and the RNA from contrasting bulks is then profiled with the aim of finding differentially expressed genes. RESULTS: We have successfully implemented the pooling strategy for potato quality traits and identified candidate genes associated with potato tuber flesh color and tuber cooking type. Elevated expression level of a dominant allele of the beta-carotene hydroxylase (bch) gene was associated with yellow flesh color through mapping of the gene under a major QTL for flesh color on chromosome 3. For a second trait, a candidate gene with homology to a tyrosine-lysine rich protein (TLRP) was identified based on allele specificity of the probe on the microarray. TLRP was mapped on chromosome 9 in close proximity to a QTL for potato cooking type strengthening its significance as a candidate gene. Furthermore, we have performed a profiling experiment targeting a polygenic trait, by pooling individual genotypes based both on phenotypic and marker data, allowing the identification of candidate genes associated with the two different linkage groups. CONCLUSIONS: A pooling approach for RNA-profiling with the aim of identifying novel candidate genes associated with tuber quality traits was successfully implemented. The identified candidate genes for tuber flesh color (bch) and cooking type (tlrp) can provide useful markers for breeding schemes in the future. Strengths and limitations of the approach are discussed.

Metabolism of carotenoids and apocarotenoids during ripening of raspberry fruit.

Carotenoids are important lipophilic antioxidants in fruits. Apocarotenoids such as alpha-ionone and beta-ionone, which are breakdown products of carotenoids, are important for the flavor characteristics of raspberry fruit, and have also been suggested to have beneficial effects on human health. Raspberry is one of the few fruits where fruit ripening is accompanied by the massive production of apocarotenoids. In this paper, changes in levels of carotenoids and apocarotenoids during raspberry fruit ripening are described. In addition, the isolation and characterization of a gene encoding a carotenoid cleavage dioxygenase (CCD), which putatively mediates the degradation of carotenoids to apocarotenoids during raspberry fruit ripening, is reported. Such information helps us to better understand how these compounds are produced in plants and may also enable us to develop novel strategies for improved apocarotenoid production in fruits or indeed, alternative production systems.