Development of a 135K SNP genotyping array for Actinidia arguta and its applications for genetic mapping and QTL analysis in kiwifruit.

Kiwifruit (Actinidia spp) is a woody, perennial and deciduous vine. In this genus, there are multiple ploidy levels but the main cultivated cultivars are polyploid. Despite the availability of many genomic resources in kiwifruit, SNP genotyping is still a challenge given these different levels of polyploidy. Recent advances in SNP array technologies have offered a high-throughput genotyping platform for genome-wide DNA polymorphisms. In this study, we developed a high-density SNP genotyping array to facilitate genetic studies and breeding applications in kiwifruit. SNP discovery was performed by genome-wide DNA sequencing of 40 kiwifruit genotypes. The identified SNPs were stringently filtered for sequence quality, predicted conversion performance and distribution over the available Actinidia chinensis genome. A total of 134 729 unique SNPs were put on the array. The array was evaluated by genotyping 400 kiwifruit individuals. We performed a multidimensional scaling analysis to assess the diversity of kiwifruit germplasm, showing that the array was effective to distinguish kiwifruit accessions. Using a tetraploid F1 population, we constructed an integrated linkage map covering 3060.9 cM across 29 linkage groups and performed QTL analysis for the sex locus that has been identified on Linkage Group 3 (LG3) in Actinidia arguta. Finally, our dataset presented evidence of tetrasomic inheritance with partial preferential pairing in A. arguta. In conclusion, we developed and evaluated a 135K SNP genotyping array for kiwifruit. It has the advantage of a comprehensive design that can be an effective tool in genetic studies and breeding applications in this high-value crop.

PolyHaplotyper: haplotyping in polyploids based on bi-allelic marker dosage data.

BACKGROUND: For genetic analyses, multi-allelic markers have an advantage over bi-allelic markers like SNPs (single nucleotide polymorphisms) in that they carry more information about the genetic constitution of individuals. This is especially the case in polyploids, where individuals carry more than two alleles at each locus. Haploblocks are multi-allelic markers that can be derived by phasing sets of closely-linked SNP markers. Phased haploblocks, similarly to other multi-allelic markers, will therefore be advantageous in genetic tasks like linkage mapping, QTL mapping and genome-wide association studies. RESULTS: We present a new method to reconstruct haplotypes from SNP dosages derived from genotyping arrays, which is applicable to polyploids. This method is implemented in the software package PolyHaplotyper. In contrast to existing packages for polyploids it makes use of full-sib families among the samples to guide the haplotyping process. We show that in this situation it is much more accurate than other available software, using experimental hexaploid data and simulated tetraploid data. CONCLUSIONS: Our method and the software package PolyHaplotyper in which it is implemented extend the available tools for haplotyping in polyploids. They perform especially well in situations where one or more full-sib families are present.

Multiallelic models for QTL mapping in diverse polyploid populations.

Quantitative trait locus (QTL) analysis allows to identify regions responsible for a trait and to associate alleles with their effect on phenotypes. When using biallelic markers to find these QTL regions, two alleles per QTL are modelled. This assumption might be close to reality in specific biparental crosses but is unrealistic in situations where broader genetic diversity is studied. Diversity panels used in genome-wide association studies or multi-parental populations can easily harbour multiple QTL alleles at each locus, more so in the case of polyploids that carry more than two alleles per individual. In such situations a multiallelic model would be closer to reality, allowing for different genetic effects for each potential allele in the population. To obtain such multiallelic markers we propose the usage of haplotypes, concatenations of nearby SNPs. We developed "mpQTL" an R package that can perform a QTL analysis at any ploidy level under biallelic and multiallelic models, depending on the marker type given. We tested the effect of genetic diversity on the power and accuracy difference between bi-allelic and multiallelic models using a set of simulated multiparental autotetraploid, outbreeding populations. Multiallelic models had higher detection power and were more precise than biallelic, SNP-based models, particularly when genetic diversity was higher. This confirms that moving to multi-allelic QTL models can lead to improved detection and characterization of QTLs. KEY MESSAGE: QTL detection in populations with more than two functional QTL alleles (which is likely in multiparental and/or polyploid populations) is more powerful when using multiallelic models, rather than biallelic models.

Detecting quantitative trait loci and exploring chromosomal pairing in autopolyploids using polyqtlR.

MOTIVATION: The investigation of quantitative trait loci (QTL) is an essential component in our understanding of how organisms vary phenotypically. However, many important crop species are polyploid (carrying more than two copies of each chromosome), requiring specialized tools for such analyses. Moreover, deciphering meiotic processes at higher ploidy levels is not straightforward, but is necessary to understand the reproductive dynamics of these species, or uncover potential barriers to their genetic improvement. RESULTS: Here, we present polyqtlR, a novel software tool to facilitate such analyses in (auto)polyploid crops. It performs QTL interval mapping in F1 populations of outcrossing polyploids of any ploidy level using identity-by-descent probabilities. The allelic composition of discovered QTL can be explored, enabling favourable alleles to be identified and tracked in the population. Visualization tools within the package facilitate this process, and options to include genetic co-factors and experimental factors are included. Detailed information on polyploid meiosis including prediction of multivalent pairing structures, detection of preferential chromosomal pairing and location of double reduction events can be performed. AVAILABILITYAND IMPLEMENTATION: polyqtlR is freely available from the Comprehensive R Archive Network (CRAN) at http://cran.r-project.org/package=polyqtlR. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genetic dissection of morphological variation in rosette leaves and leafy heads in cabbage (Brassica oleracea var. capitata).

KEY MESSAGE: Correlations between morphological traits of cabbage rosette leaves and heads were found. Genome-wide association studies of these traits identified 50 robust quantitative trait loci in multiple years. Half of these loci affect both organs. Cabbage (Brassica oleracea var. capitata) is an economically important vegetable crop cultivated worldwide. Cabbage plants go through four vegetative stages: seedling, rosette, folding and heading. Rosette leaves are the largest leaves of cabbage plants and provide most of the energy needed to produce the leafy head. To understand the relationship and the genetic basis of leaf development and leafy head formation, 308 cabbage accessions were scored for rosette leaf and head traits in three-year field trials. Significant correlations were found between morphological traits of rosette leaves and heads, namely leaf area with the head area, height and width, and leaf width with the head area and head height, when heads were harvested at a fixed number of days after sowing. Fifty robust quantitative trait loci (QTLs) for rosette leaf and head traits distributed over all nine chromosomes were identified with genome-wide association studies. All these 50 loci were identified in multiple years and generally affect multiple traits. Twenty-five of the QTL were associated with both rosette leaf and leafy head traits. We discuss thirteen candidate genes identified in these QTL that are expressed in heading leaves, with an annotation related to auxin and other phytohormones, leaf development, and leaf polarity that likely play a role in leafy head development or rosette leaf expansion.

Using probabilistic genotypes in linkage analysis of polyploids.

KEY MESSAGE: In polyploids, linkage mapping is carried out using genotyping with discrete dosage scores. Here, we use probabilistic genotypes and we validate it for the construction of polyploid linkage maps. Marker genotypes are generally called as discrete values: homozygous versus heterozygous in the case of diploids, or an integer allele dosage in the case of polyploids. Software for linkage map construction and/or QTL analysis usually relies on such discrete genotypes. However, it may not always be possible, or desirable, to assign definite values to genotype observations in the presence of uncertainty in the genotype calling. Here, we present an approach that uses probabilistic marker dosages for linkage map construction in polyploids. We compare our method to an approach based on discrete dosages, using simulated SNP array and sequence reads data with varying levels of data quality. We validate our approach using experimental data from a potato (Solanum tuberosum L.) SNP array applied to an F1 mapping population. In comparison to the approach based on discrete dosages, we mapped an additional 562 markers. All but three of these were mapped to the expected chromosome and marker position. For the remaining three markers, no physical position was known. The use of dosage probabilities is of particular relevance for map construction in polyploids using sequencing data, as these often result in a higher level of uncertainty regarding allele dosage.

Fine mapping of a thrips resistance QTL in Capsicum and the role of diterpene glycosides in the underlying mechanism.

KEY MESSAGE: A major thrips resistance QTL in Capsicum was fine-mapped to a region of 0.4 Mbp, and a multidisciplinary approach has been used to study putative underlying mechanisms. Resistance to thrips is an important trait for pepper growers. These insects can cause extensive damage to fruits, flowers and leaves on field and greenhouse grown plants worldwide. Two independent studies in Capsicum identified diterpene glycosides as metabolites that are correlated with thrips resistance. In this study, we fine-mapped a previously defined thrips resistance QTL on chromosome 6, to a region of 0.4 Mbp harbouring 15 genes. Two of these 15 candidate genes showed differences in gene expression upon thrips induction, when comparing plants carrying the resistance allele in homozygous state to plants with the susceptibility allele in homozygous state for the QTL region. Three genes, including the two genes that showed difference in gene expression, contained a SNP that was predicted to lead to changes in protein structure. Therefore, these three genes, i.e. an acid phosphatase 1 (APS1), an organic cation/carnitine transporter 7 (OCT7) and an uncharacterized locus LOC107874801, are the most likely candidates for playing a role in thrips resistance and are a first step in elucidating the genetic basis of thrips resistance in Capsicum. In addition, we show that the diterpene glycoside profiles did not differ between plants with the resistance and susceptibility allele for the chromosome 6 QTL, suggesting that these compounds do not play a role in the resistance conferred by the genes located in the major thrips resistance QTL studied.

Aphid resistance in Capsicum maps to a locus containing LRR-RLK gene analogues.

KEY MESSAGE: A QTL for aphid resistance on pepper chromosome 2 was identified and validated. This QTL affects aphid survival and reproduction, and was fine mapped to a locus containing LRR-RLK analogues. Myzus persicae is one of the most threatening insect pests that adversely affects pepper (Capsicum) cultivation. Resistance to aphids was previously identified in Capsicum baccatum. This study aimed at elucidating the genetics of aphid resistance in C. baccatum. A QTL analysis was carried out for M. persicae resistance in an F2 population derived from an intraspecific cross between a highly resistant plant and a susceptible plant. Survival and reproduction were used as resistance parameters. Interval mapping detected two QTLs affecting aphid survival (Rmpas-1) and reproduction (Rmprp-1), respectively, both localized in the same area and sharing the same top marker on chromosome 2. Use of this marker as co-factor in multiple-QTL mapping analysis revealed a second, minor QTL (Rmprp-2) only affecting aphid reproduction, on chromosome 4. Fine mapping confirmed the effects of Rmpas-1 and Rmprp-1 and narrowed the major QTL Rmprp-1 down to a genomic region of 96 kb which is predicted to encode four analogues of resistance genes of the receptor-like kinase family containing a leucine-rich repeat domain (LRR-RLKs). This work provides not only initial information for breeding aphid-resistant pepper varieties, but also forms the basis for future molecular analysis of gene(s) involved in aphid resistance.

FitTetra 2.0 - improved genotype calling for tetraploids with multiple population and parental data support.

BACKGROUND: Genetic studies in tetraploids are lagging behind in comparison with studies of diploids as the complex genetics of tetraploids require much more elaborated computational methodologies. Recent advancements in development of molecular techniques and computational tools facilitate new methods for automated, high-throughput genotype calling in tetraploid species. We report on the upgrade of the widely-used fitTetra software aiming to improve its accuracy, which to date is hampered by technical artefacts in the data. RESULTS: Our upgrade of the fitTetra package is designed for a more accurate modelling of complex collections of samples. The package fits a mixture model where some parameters of the model are estimated separately for each sub-collection. When a full-sib family is analyzed, we use parental genotypes to predict the expected segregation in terms of allele dosages in the offspring. More accurate modelling and use of parental data increases the accuracy of dosage calling. We tested the package on data obtained with an Affymetrix Axiom 60 k array and compared its performance with the original version and the recently published ClusterCall tool, showing that at least 20% more SNPs could be called with our updated. CONCLUSION: Our updated software package shows clearly improved performance in genotype calling accuracy. Estimation of mixing proportions of the underlying dosage distributions is separated for full-sib families (where mixture proportions can be estimated from the parental dosages and inheritance model) and unstructured populations (where they are based on the assumption of Hardy-Weinberg equilibrium). Additionally, as the distributions of signal ratios of the dosage classes can be assumed to be the same for all populations, including parental data for some subpopulations helps to improve fitting other populations as well. The R package fitTetra 2.0 is freely available under the GNU Public License as Additional file with this article.

polymapR-linkage analysis and genetic map construction from F1 populations of outcrossing polyploids.

Motivation: Polyploid species carry more than two copies of each chromosome, a condition found in many of the world's most important crops. Genetic mapping in polyploids is more complex than in diploid species, resulting in a lack of available software tools. These are needed if we are to realize all the opportunities offered by modern genotyping platforms for genetic research and breeding in polyploid crops. Results: polymapR is an R package for genetic linkage analysis and integrated genetic map construction from bi-parental populations of outcrossing autopolyploids. It can currently analyse triploid, tetraploid and hexaploid marker datasets and is applicable to various crops including potato, leek, alfalfa, blueberry, chrysanthemum, sweet potato or kiwifruit. It can detect, estimate and correct for preferential chromosome pairing, and has been tested on high-density marker datasets from potato, rose and chrysanthemum, generating high-density integrated linkage maps in all of these crops. Availability and implementation: polymapR is freely available under the general public license from the Comprehensive R Archive Network (CRAN) at http://cran.r-project.org/package=polymapR. Supplementary information: Supplementary data are available at Bioinformatics online.

QTL mapping of insect resistance components of Solanum galapagense.

KEY MESSAGE: QTLs for insect resistance parameters, trichome type IV development, and more than 200 non-volatile metabolites, including 76 acyl sugars, all co-locate at the end of Chromosome 2 of Solanum galapagense. Host plant resistance is gaining importance as more and more insecticides are being banned due to environmental concerns. In tomato, resistance towards insects is found in wild relatives and has been attributed to the presence of glandular trichomes and their specific phytochemical composition. In this paper, we describe the results from a large-scale QTL mapping of data from whitefly resistance tests, trichome phenotyping and a comprehensive metabolomics analysis in a recombinant inbred line population derived from a cross between the cultivated Solanum lycopersicum and the wild relative S. galapagense, which is resistant to a range of pest insects. One major QTL (Wf-1) was found to govern the resistance against two different whitefly species. This QTL co-localizes with QTLs for the presence of trichomes type IV and V, as well as all 76 acyl sugars detected and about 150 other non-volatile phytochemicals, including methyl esters of the flavonols myricetin and quercetin. Based on these results, we hypothesize that Wf-1 is regulating the formation of glandular trichome type IV on the leaf epidermis, enabling the production and accumulation of bioactive metabolites in this type of trichomes.

Partial preferential chromosome pairing is genotype dependent in tetraploid rose.

It has long been recognised that polyploid species do not always neatly fall into the categories of auto- or allopolyploid, leading to the term 'segmental allopolyploid' to describe everything in between. The meiotic behaviour of such intermediate species is not fully understood, nor is there consensus as to how to model their inheritance patterns. In this study we used a tetraploid cut rose (Rosa hybrida) population, genotyped using the 68K WagRhSNP array, to construct an ultra-high-density linkage map of all homologous chromosomes using methods previously developed for autotetraploids. Using the predicted bivalent configurations in this population we quantified differences in pairing behaviour among and along homologous chromosomes, leading us to correct our estimates of recombination frequency to account for this behaviour. This resulted in the re-mapping of 25 695 SNP markers across all homologues of the seven rose chromosomes, tailored to the pairing behaviour of each chromosome in each parent. We confirmed the inferred differences in pairing behaviour among chromosomes by examining repulsion-phase linkage estimates, which also carry information about preferential pairing and recombination. Currently, the closest sequenced relative to rose is Fragaria vesca. Aligning the integrated ultra-dense rose map with the strawberry genome sequence provided a detailed picture of the synteny, confirming overall co-linearity but also revealing new genomic rearrangements. Our results suggest that pairing affinities may vary along chromosome arms, which broadens our current understanding of segmental allopolyploidy.

Reduced phloem uptake of Myzus persicae on an aphid resistant pepper accession.

BACKGROUND: The green peach aphid (GPA), Myzus persicae, is economically one of the most threatening pests in pepper cultivation, which not only causes direct damage but also transmits many viruses. Breeding aphid resistant pepper varieties is a promising and environmentally friendly method to control aphid populations in the field and in the greenhouse. Until now, no strong sources of resistance against the GPA have been identified. Therefore the main aims of this study were to identify pepper materials with a good level of resistance to GPA and to elucidate possible resistance mechanisms. RESULTS: We screened 74 pepper accessions from different geographical areas for resistance to M. persicae. After four rounds of evaluation we identified one Capsicum baccatum accession (PB2013071) as highly resistant to M. persicae, while the accessions PB2013062 and PB2012022 showed intermediate resistance. The resistance of PB2013071 resulted in a severely reduced uptake of phloem compared to the susceptible accession, as determined by Electrical Penetration Graph (EPG) studies. Feeding of M. persicae induced the expression of callose synthase genes and resulted in callose deposition in the sieve elements in resistant, but not in susceptible plants. CONCLUSIONS: Three aphid resistant pepper accessions were identified, which will be important for breeding aphid resistant pepper varieties in the future. The most resistant accession PB2013071 showed phloem-based resistance against aphid infestation.

Multi-environment QTL analysis of plant and flower morphological traits in tetraploid rose.

KEY MESSAGE: Rose morphological traits such as prickles or petal number are influenced by a few key QTL which were detected across different growing environments-necessary for genomics-assisted selection in non-target environments. Rose, one of the world's most-loved and commercially important ornamental plants, is predominantly tetraploid, possessing four rather than two copies of each chromosome. This condition complicates genetic analysis, and so the majority of previous genetic studies in rose have been performed at the diploid level. However, there may be advantages to performing genetic analyses at the tetraploid level, not least because this is the ploidy level of most breeding germplasm. Here, we apply recently developed methods for quantitative trait loci (QTL) detection in a segregating tetraploid rose population (F1 = 151) to unravel the genetic control of a number of key morphological traits. These traits were measured both in the Netherlands and Kenya. Since ornamental plant breeding and selection are increasingly being performed at locations other than the production sites, environment-neutral QTL are required to maximise the effectiveness of breeding programmes. We detected a number of robust, multi-environment QTL for such traits as stem and petiole prickles, petal number and stem length that were localised on the recently developed high-density SNP linkage map for rose. Our work explores the complex genetic architecture of these important morphological traits at the tetraploid level, while helping to advance the methods for marker-trait exploration in polyploid species.

Conclusive evidence for hexasomic inheritance in chrysanthemum based on analysis of a 183 k SNP array.

BACKGROUND: Cultivated chrysanthemum is an outcrossing hexaploid (2n = 6× = 54) with a disputed mode of inheritance. In this paper, we present a single nucleotide polymorphism (SNP) selection pipeline that was used to design an Affymetrix Axiom array with 183 k SNPs from RNA sequencing data (1). With this array, we genotyped four bi-parental populations (with sizes of 405, 53, 76 and 37 offspring plants respectively), and a cultivar panel of 63 genotypes. Further, we present a method for dosage scoring in hexaploids from signal intensities of the array based on mixture models (2) and validation of selection steps in the SNP selection pipeline (3). The resulting genotypic data is used to draw conclusions on the mode of inheritance in chrysanthemum (4), and to make an inference on allelic expression bias (5). RESULTS: With use of the mixture model approach, we successfully called the dosage of 73,936 out of 183,130 SNPs (40.4%) that segregated in any of the bi-parental populations. To investigate the mode of inheritance, we analysed markers that segregated in the large bi-parental population (n = 405). Analysis of segregation of duplex x nulliplex SNPs resulted in evidence for genome-wide hexasomic inheritance. This evidence was substantiated by the absence of strong linkage between markers in repulsion, which indicated absence of full disomic inheritance. We present the success rate of SNP discovery out of RNA sequencing data as affected by different selection steps, among which SNP coverage over genotypes and use of different types of sequence read mapping software. Genomic dosage highly correlated with relative allele coverage from the RNA sequencing data, indicating that most alleles are expressed according to their genomic dosage. CONCLUSIONS: The large population, genotyped with a very large number of markers, is a unique framework for extensive genetic analyses in hexaploid chrysanthemum. As starting point, we show conclusive evidence for genome-wide hexasomic inheritance.

An ultra-dense integrated linkage map for hexaploid chrysanthemum enables multi-allelic QTL analysis.

KEY MESSAGE: We constructed the first integrated genetic linkage map in a polysomic hexaploid. This enabled us to estimate inheritance of parental haplotypes in the offspring and detect multi-allelic QTL. Construction and use of linkage maps are challenging in hexaploids with polysomic inheritance. Full map integration requires calculations of recombination frequency between markers with complex segregation types. In addition, detection of QTL in hexaploids requires information on all six alleles at one locus for each individual. We describe a method that we used to construct a fully integrated linkage map for chrysanthemum (Chrysanthemum × morifolium, 2n = 6x = 54). A bi-parental F1 population of 406 individuals was genotyped with an 183,000 SNP genotyping array. The resulting linkage map consisted of 30,312 segregating SNP markers of all possible marker dosage types, representing nine chromosomal linkage groups and 107 out of 108 expected homologues. Synteny with lettuce (Lactuca sativa) showed local colinearity. Overall, it was high enough to number the chrysanthemum chromosomal linkage groups according to those in lettuce. We used the integrated and phased linkage map to reconstruct inheritance of parental haplotypes in the F1 population. Estimated probabilities for the parental haplotypes were used for multi-allelic QTL analyses on four traits with different underlying genetic architectures. This resulted in the identification of major QTL that were affected by multiple alleles having a differential effect on the phenotype. The presented linkage map sets a standard for future genetic mapping analyses in chrysanthemum and closely related species. Moreover, the described methods are a major step forward for linkage mapping and QTL analysis in hexaploids.

Evaluation of LD decay and various LD-decay estimators in simulated and SNP-array data of tetraploid potato.

KEY MESSAGE: The number of SNPs required for QTL discovery is justified by the distance at which linkage disequilibrium has decayed. Simulations and real potato SNP data showed how to estimate and interpret LD decay. The magnitude of linkage disequilibrium (LD) and its decay with genetic distance determine the resolution of association mapping, and are useful for assessing the desired numbers of SNPs on arrays. To study LD and LD decay in tetraploid potato, we simulated autotetraploid genotypes and used it to explore the dependence on: (1) the number of haplotypes in the population (the amount of genetic variation) and (2) the percentage of haplotype specific SNPs (hs-SNPs). Several estimators for short-range LD were explored, such as the average r 2, median r 2, and other percentiles of r 2 (80, 90, and 95 %). For LD decay, we looked at LD½,90, the distance at which the short-range LD is halved when using the 90 % percentile of r 2 at short range, as estimator for LD. Simulations showed that the performance of various estimators for LD decay strongly depended on the number of haplotypes, although the real value of LD decay was not influenced very much by this number. The estimator LD½,90 was chosen to evaluate LD decay in 537 tetraploid varieties. LD½,90 values were 1.5 Mb for varieties released before 1945 and 0.6 Mb in varieties released after 2005. LD½,90 values within three different subpopulations ranged from 0.7 to 0.9 Mb. LD½,90 was 2.5 Mb for introgressed regions, indicating large haplotype blocks. In pericentromeric heterochromatin, LD decay was negligible. This study demonstrates that several related factors influencing LD decay could be disentangled, that no universal approach can be suggested, and that the estimation of LD decay has to be performed with great care and knowledge of the sampled material.

Integrating haplotype-specific linkage maps in tetraploid species using SNP markers.

KEY MESSAGE: Linkage mapping can help unravel the complexities of polyploid genomes. Here, we integrate haplotype-specific linkage maps in autotetraploid potato and explore the possibilities for mapping in other polyploid species. High-density linkage mapping in autopolyploid species has become possible in recent years given the increasing number of molecular markers now available through modern genotyping platforms. Such maps along with larger experimental populations are needed before we can obtain sufficient accuracy to make marker-trait association studies useful in practice. Here, we describe a method to create genetic linkage maps for an autotetraploid species with large numbers of markers and apply it to an F1 population of tetraploid potato (Solanum tuberosum L.) of 235 individuals genotyped using a 20K SNP array. SNP intensity values were converted to allele dosages after which we calculated pairwise maximum likelihood estimates of recombination frequencies between all marker segregation types under the assumption of random bivalent pairing. These estimates were used in the clustering of markers into linkage groups and their subsequent ordering into 96 homologue maps. The homologue maps were integrated per chromosome, resulting in a total map length of 1061 cM from 6910 markers covering all 12 potato chromosomes. We examined the questions of marker phasing and binning and propose optimal strategies for both. We also investigated the effect of quadrivalent formation and preferential pairing on recombination frequency estimation and marker phasing, which is of great relevance not only for potato but also for genetic studies in other tetraploid species for which the meiotic pairing behaviour is less well understood.

Population structure and genome-wide association analysis for frost tolerance in oat using continuous SNP array signal intensity ratios.

KEY MESSAGE: Infinium SNP data analysed as continuous intensity ratios enabled associating genotypic and phenotypic data from heterogeneous oat samples, showing that association mapping for frost tolerance is a feasible option. Oat is sensitive to freezing temperatures, which restricts the cultivation of fall-sown or winter oats to regions with milder winters. Fall-sown oats have a longer growth cycle, mature earlier, and have a higher productivity than spring-sown oats, therefore improving frost tolerance is an important goal in oat breeding. Our aim was to test the effectiveness of a Genome-Wide Association Study (GWAS) for mapping QTLs related to frost tolerance, using an approach that tolerates continuously distributed signals from SNPs in bulked samples from heterogeneous accessions. A collection of 138 European oat accessions, including landraces, old and modern varieties from 27 countries was genotyped using the Infinium 6K SNP array. The SNP data were analyzed as continuous intensity ratios, rather than converting them into discrete values by genotype calling. PCA and Ward's clustering of genetic similarities revealed the presence of two main groups of accessions, which roughly corresponded to Continental Europe and Mediterranean/Atlantic Europe, although a total of eight subgroups can be distinguished. The accessions were phenotyped for frost tolerance under controlled conditions by measuring fluorescence quantum yield of photosystem II after a freezing stress. GWAS were performed by a linear mixed model approach, comparing different corrections for population structure. All models detected three robust QTLs, two of which co-mapped with QTLs identified earlier in bi-parental mapping populations. The approach used in the present work shows that SNP array data of heterogeneous hexaploid oat samples can be successfully used to determine genetic similarities and to map associations to quantitative phenotypic traits.