Search

MotivationThe 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.ResultsHere, 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., Bioinformatics

On a global scale, potato is an important crop for both consumption and the starch industry. At present, the starch industry is keen to create economic value and to reduce its impact on the environment by valorising all resources in potatoes. One way to contribute towards this goal is to develop new potato varieties with elite traits, i.e. a high level of tuber protein content and a reduced level of fibre hydration. Improving these traits is however challenging due to their complex nature. Therefore, a better understanding of the genetics and biological processes underlying tuber protein content and fibre hydration are relevant. The objectives of this study were to shed light on the genetic and molecular architectures of tuber protein content and fibre hydration and to pinpoint key factors (i.e. biological processes, molecular structures, genes and alleles) that are involved in modulation of these traits.Chapter 1 provides a description of the potato crop and the starch industry. We present protein and fibre as key resources, where improvement of their quantity and quality will lead to a better valorisation of the potato crop. The principles of potato genetics, potato breeding and modern tools for trait improvement are described.In Chapter 2 we studied the genetics of tuber protein content in a panel of tetraploid potato varieties. We estimated a moderate level of trait heritability, identified marker-trait associations (QTLs), haplotypes and candidate genes. Our findings showed that alleles of StCDF1 were associated with tuber protein content. The results provide resources for genomics-enabled breeding.In Chapter 3 we performed a multi-allelic QTL analysis of tuber protein content in a large bi-parental population of tetraploid potato. We estimated a moderate level of trait heritability and identified QTLs. The alleles underlying the QTLs provided both positive and negative effects on the level of tuber protein content. Our results showed that tuber protein content

Polyploid organisms possess more than two copies of their core genome and therefore contain k>2 haplotypes for each set of ordered genomic variants. Polyploidy occurs often within the plant kingdom, among others in important corps such as potato (k=4) and wheat (k=6). Current sequencing technologies enable us to read the DNA and detect genomic variants, but cannot distinguish between the copies of the genome, each inherited from one of the parents. To detect inheritance patterns in populations, it is necessary to know the haplotypes, as alleles that are in linkage over the same chromosome tend to be inherited together. In this work, we develop mathematical optimisation algorithms to indirectly estimate haplotypes by looking into overlaps between the sequence reads of an individual, as well as into the expected inheritance of the alleles in a population. These algorithm deal with sequencing errors and random variations in the counts of reads observed from each haplotype. These methods are therefore of high importance for studying the genetics of polyploid crops.

Many of our most important crop species are polyploid – an unusual phenomenon whereby each chromosome is present in multiple copies (more than the usual two copies). The most common such arrangement is tetraploidy, where each chromosome is present four times. Plant species can tolerate this condition quite well (the same cannot be said of animals or humans). In fact, polyploidy can confer certain advantages such as larger fruits and flowers, seedless fruits (useful for fruit growers) or improved tolerance to environmental stresses. However, carrying multiple copies of each chromosome complicates things, particularly when crop breeders would like to use DNA information to help inform selection decisions. This PhD project looked at how DNA information of polyploids should be best analysed, developing methods and new software tools to achieve this. We analysed DNA information from polyploid crops such as potato, rose and chrysanthemum, yielding many novel insights and important results.

Background Recent advances in ~omics technologies such as transcriptomics, metabolomics and proteomics along with genotypic profiling have permitted the genetic dissection of complex traits such as quality traits in non-model species. To get more insight into the genetic factors underlying variation in quality traits related to carbohydrate and starch metabolism and cold sweetening, we determined the protein content and composition in potato tubers using 2D–gel electrophoresis in a diploid potato mapping population. Upon analyzing we made sure that the proteins from the patatin family were excluded to ensure a better representation of the other proteins. Results We subsequently performed pQTL analyses for all other proteins with a sufficient representation in the population and established a relationship between proteins and 26 potato tuber quality traits (e.g. flesh colour, enzymatic discoloration) by co-localization on the genetic map and a direct correlation study of protein abundances and phenotypic traits. Over 1643 unique protein spots were detected in total over the two harvests. We were able to map pQTLs for over 300 different protein spots some of which co-localized with traits such as starch content and cold sweetening. pQTLs were observed on every chromosome although not evenly distributed over the chromosomes. The largest number of pQTLs was found for chromosome 8 and the lowest for chromosome number 10. For some 20 protein spots multiple QTLs were observed. Conclusions From this analysis, hotspot areas for protein QTLs were identified on chromosomes three, five, eight and nine. The hotspot on chromosome 3 coincided with a QTL previously identified for total protein content and had more than 23 pQTLs in the region from 70 to 80 cM. Some of the co-localizing protein spots associated with some of the most interesting tuber quality traits were identified, albeit far less than we had anticipated at the onset of the experiments.

Polyploid organisms carry more than two copies of each chromosome, a condition rarely tolerated in animals but which occurs relatively frequently in the plant kingdom. One of the principal challenges faced by polyploid organisms is to evolve stable meiotic mechanisms to faithfully transmit genetic information to the next generation upon which the study of inheritance is based. In this review we look at the tools available to the research community to better understand polyploid inheritance, many of which have only recently been developed. Most of these tools are intended for experimental populations (rather than natural populations), facilitating genomics-assisted crop improvement and plant breeding. This is hardly surprising given that a large proportion of domesticatedplant species are polyploid. We focus on three main areas: (1) polyploid genotyping; (2) genetic and physical mapping; and (3) quantitative trait analysis and genomic selection. We also briefly review some miscellaneous topics such as the mode of inheritance and the availability of polyploid simulation software. The current polyploid analytic toolbox includes software for assigning marker genotypes (and in particular, estimating the dosage of marker alleles in the heterozygous condition), establishing chromosome-scale linkage phase among marker alleles, constructing (short-range) haplotypes, generating linkage maps, performing genome-wide association studies (GWAS) and quantitative trait locus (QTL) analyses, and simulating polyploid populations. These tools can alsohelp elucidate the mode of inheritance (disomic, polysomic or a mixture of bothas in segmental allopolyploids) or reveal whether double reduction and multivalent chromosomal pairing occur. An increasing number of polyploids (or associated diploids) are being sequenced, leading to publicly available reference genome assemblies. Much work remains in order to keep pace with developments in genomic technologies. However, such technologies als

Rose, as many other important ornamental, vegetable and field crops, is polyploid. This poses constraints in genetic analyses due to the occurrence of multiple alleles at marker and trait loci and the existence of multiple allele dosages. Developments in marker discovery (next generation sequencing), detection (SNP arrays) and analysis (software for dosage scoring) now make it feasible to develop high-density molecular marker maps for the homologous chromosomes in tetraploids separately, and thus perform QTL analysis in tetraploids. We developed a SNP array for rose to develop genetic maps in tetraploid garden roses and cut roses, which are to be used for inheritance studies and genetic mapping. Here we have indicated the general strategy followed for developing a SNP array and for scoring and using the SNP data generated, and elaborated on the activities undertaken to use the WagRhSNP Axiom array in rose. The array design is not proprietary but can be used by all researchers working in rose

In order to develop a versatile and large SNP array for rose, we set out to mine ESTs from diverse sets of rose germplasm. For this RNA-Seq libraries containing about 700 million reads were generated from tetraploid cut and garden roses using Illumina paired-end sequencing, and from diploid Rosa multiflora using 454 sequencing. Separate de novo assemblies were performed in order to identify single nucleotide polymorphisms (SNPs) within and between rose varieties. SNPs among tetraploid roses were selected for constructing a genotyping array that can be employed for genetic mapping and marker-trait association discovery in breeding programs based on tetraploid germplasm, both from cut roses and from garden roses. In total 68,893 SNPs were included on the WagRhSNP Axiom array. Next, an orthology-guided assembly was performed for the construction of a non-redundant rose transcriptome database. A total of 21,740 transcripts had significant hits with orthologous genes in the strawberry (Fragaria vesca L.) genome. Of these 13,390 appeared to contain the full-length coding regions. This newly established transcriptome resource adds considerably to the currently available sequence resources for the Rosaceae family in general and the genus Rosa in particular.

Understanding the genetic basis of plant development in potato requires a proper characterization of plant morphology over time. Parameters related to different aging stages can be used to describe the developmental processes. It is attractive to map these traits simultaneously in a QTL analysis; because the power to detect a QTL will often be improved and it will be easier to identify pleiotropic QTLs. We included complex, agronomic traits together with plant development parameters in a multi-trait QTL analysis. First, the results of our analysis led to coherent insight into the genetic architecture of complex traits in potato. Secondly, QTL for parameters related to plant development were identified. Thirdly, pleiotropic regions for various types of traits were identified. Emergence, number of main stems, number of tubers and yield were explained by 9, 5, 4 and 6 QTL, respectively. These traits were measured once during the growing season. The genetic control of flowering, senescence and plant height, which were measured at regular time intervals, was explained by 9, 10 and 12 QTL, respectively. Genetic relationships between aboveground and belowground traits in potato were observed in 14 pleiotropic QTL. Some of our results suggest the presence of QTL-by-Environment interactions. Therefore, additional studies comparing development under different photoperiods are required to investigate the plasticity of the crop.

The genetic basis of seed germination and seedling vigour is largely unknown in Brassica species. We performed a study to evaluate the genetic basis of these important traits in a B. rapa doubled haploid population from a cross of a yellow-seeded oil-type yellow sarson and a black-seeded vegetable-type pak choi. We identified 26 QTL regions across all 10 linkage groups for traits related to seed weight, seed germination and seedling vigour under non-stress and salt stress conditions illustrating the polygenic nature of these traits. QTLs for multiple traits co-localized and we identified eight hotspots for quantitative trait loci (QTL) of seed weight, seed germination, and root and shoot lengths. A QTL hotspot for seed germination on A02 mapped at the B. rapa FLOWERING LOCUS C (BrFLC2). Another hotspot on A05 with salt stress specific QTLs co-located with the B. rapa FATTY ACID DESATURASE 2 (BrFAD2) locus. Epistatic interactions were observed between QTL hotspots for seed germination on A02 and A10 and with a salt tolerance QTL on A05. These results contribute to the understanding of the genetics of seed quality and seeding vigour in B. rapa and can offer tools for Brassica breeding.

Background Global trade has ensured that the ornamental horticulture continues to grow worldwide, with rose hybrids being the most economically important genus (Rosa x hybrida). Due to changes in global trade and an increase in energy costs the ornamental industry has seen a shift in the production and sale of flowers from the US and Europe alone to production in Africa and Latin America. As Kenya is a major exporter of roses to Europe we studied the genetic variation and heritability of specific morphological traits in a tetraploid population grown in the Netherlands and in Kenya. The aim was to estimate genotype by environment interaction (G???E) and to investigate the implications of (G???E) for rose breeding. Results A tetraploid rose population (K5) from a cross between two tetraploid parents was field tested over two seasons in the Netherlands (summer and winter) and two locations in Kenya (Nairobi and Njoro). Ten traits were compared per genotype across the four environments. There were differences in trait association across the four environments showing that the traits were partially influenced by the environment.The traits that had a low ratio of ?2 ge/?2 g also showed a high value for heritability. For the traits number of petals, prickles on petioles, prickles on stems the interaction is minimal. For the traits chlorophyll content, stem width and side shoots we observed a much higher interaction ratio of 0.83, 1.43 and 3.13 respectively. The trait number of petals had the highest heritability of 0.96 and the lowest ?2 ge/?2 g ratio (0.08). The trait number of side shoots (SS) with the lowest heritability (0.40) also had the highest ?2 ge/?2 g ratio of 3.13. Conclusion Results attained by this experiment showed that we have different magnitudes of non-crossover G???E interactions. For the traits number of petals, prickles on stems and prickles on petioles with a low interaction and high heritability, selection can be done at any of the environments. Thus

This paper describes the progress that has been made since the draft genome sequence of potato has been obtained and the analyses that need to be done to make further progress. Although sequencing has become less expensive and read lengths have increased, making optimal use of the information obtained is still difficult, certainly in the tetraploid potato crop. Major challenges in potato genomics are standardized genome assembly and haplotype analysis. Sequencing methods need to be improved further to achieve precision breeding. With the current new generation sequencing technology, the focus in potato breeding will shift from phenotype improvement to genotype improvement. In this respect, it is essential to realize that different alleles of the same gene can lead to different phenotypes depending on the genetic background and that there is significant epistatic interaction between different alleles. Genome-wide association studies will gain statistical power when binary single nucleotide polymorphism (SNP) data can be replaced with multi-allelic haplotype data. Binary SNP can be distributed across the many different alleles per locus or may be haplotype-specific, and potentially tag specific alleles which clearly differ in their contribution to a certain trait value. Assembling reads from the same linkage phase proved to allow constructing sufficiently long haplotype tracts to ensure their uniqueness. Combining large phenotyping data sets with modern approaches to sequencing and haplotype analysis and proper software will allow the efficiency of potato breeding to increase.

Chemical cues are considered to be the most important cues for mosquitoes to find their hosts and humans can be ranked for attractiveness to mosquitoes based on the chemical cues they emit. Human leukocyte antigen (HLA) genes are considered to be involved in the regulation of human body odor and may therefore affect human attractiveness to mosquitoes, and hence, affect the force of malaria transmission. In the present study the correlations between HLA profiles, human skin volatiles and human attractiveness to the malaria mosquito Anopheles gambiae Giles sensu stricto were examined. Skin emanations of 48 volunteers were collected by rubbing a foot over glass beads. Previously the attractiveness of these emanations to An. gambiae was determined. In this study, the chemical composition of these emanations was determined by gas chromatography–mass spectroscopy (GC–MS) and blood samples of all volunteers were taken for HLA analysis. Hierarchical cluster analysis (HCA), partial least squares discriminant analysis (PLS-DA), Fisher’s exact test and random forest regression were used to test for correlations between individuals classified as either highly or poorly attractive to mosquitoes and their HLA profile and volatile composition. HLA profiling suggests that people carrying HLA gene Cw*07 are more attractive to mosquitoes. GC–MS revealed that limonene, 2-phenylethanol and 2-ethyl-1-hexanol were associated with individuals that were poorly attractive to An.gambiae and lactic acid, 2-methylbutanoic acid, tetradecanoic acid and octanal with individuals that were highly attractive. Such compounds offer potential for disruption of mosquito behavior in malaria intervention programs.

Brassica seeds are important as basic units of plant growth and sources of vegetable oil. Seed development is regulated by many dynamic metabolic processes controlled by complex networks of spatially and temporally expressed genes. We conducted a global microarray gene co-expression analysis by measuring transcript abundance of developing seeds from two diverse B. rapa morphotypes: a pak choi (leafy-type) and a yellow sarson (oil-type), and two of their doubled haploid (DH) progenies, (1) to study the timing of metabolic processes in developing seeds, (2) to explore the major transcriptional differences in developing seeds of the two morphotypes, and (3) to identify the optimum stage for a genetical genomics study in B. rapa seed.

Background: While the genetics of diploid inheritance are well studied and software for linkage mapping, haplotyping and QTL analysis are available, for tetraploids the available tools are limited. In order to develop such tools it would be helpful if simulated populations based on a variety of models of the tetraploid meiosis would be available. Results: Here we present PedigreeSim, a software package that simulates meiosis in both diploid and tetraploid species and uses this to simulate pedigrees and cross populations. For tetraploids a variety of models can be used, including both bivalent and quadrivalent formation, varying degrees of preferential pairing of hom(oe)ologous chromosomes, different quadrivalent configurations and more. Simulation of quadrivalent meiosis results as expected in double reduction and recombination between more than two hom(oe) ologous chromosomes. The results are shown to match theoretical predictions. Conclusions: This is the first simulation software that implements all features of meiosis in tetraploids. It allows to generate data for tetraploid and diploid populations, and to investigate different models of tetraploid meiosis. The software and manual are available from http://www.plantbreeding.nl/UK/software_pedigreeSim.html and as Additional files 1, 2, 3 and 4 with this publication.

Genetic analysis in tetraploid rose is complex: inheritance of genes could be disomic, keeping the two original diploid genomes separate but it could also be tetrasomic, permitting the four homologous chromosomes to pair and recombine during meiosis, and including the possibility of double reduction. Marker-assisted breeding (MAB) is particularly suited for traits such as disease resistance since disease resistance assays are laborious, time and money consuming, but we need to extend the number of markers for better coverage of the genome before being able to find associations and apply MAB. Our goal was to produce hundreds of single nucleotide polymorphism (SNP) markers to provide the coverage needed. Disease resistance, more specifically powdery mildew resistance is our primary target because chemical pest control will be increasingly limited. Molecular markers could provide a good tool to facilitate selection in an earlier stage of a breeding program. We investigated the inheritance of powdery mildew resistance in an F1 population originating from a cross between two tetraploid genotypes, P540 and P867, differing in powdery mildew resistance. SNP marker development started with Next Generation Sequencing (Illumina GAII) of cDNA obtained from RNA isolated from flowers at three developmental stages for each parent. We identified a large number of putative single nucleotide polymorphisms (SNPs) between and within both parents. SNPs that were expected to segregate as simplex × nulliplex and duplex × nulliplex markers were selected for genotyping the entire F1 population using the KASP platform (KBioscience). For a pilot study on a limited number of SNPs we defined specific selection criteria resulting in a set of 20 SNPs for testing on the population. It was found that selecting the SNPs according to the ratio of the number of reads from the Illumina sequencing data did not guarantee the appropriate segregation ratios in the progeny. Continued research will focus on

The study of quantitative trait’s expression over time helps to understand developmental processes which occur in the course of the growing season. Temperature and other environmental factors play an important role. The dynamics of haulm senescence was observed in a diploid potato mapping population in two consecutive years (2004 and 2005) under field conditions in Finland. The available time series data were used in a smoothed generalized linear model to characterize curves describing the senescence development in terms of its onset, mean and maximum progression rate and inflection point. These characteristics together with the individual time points were used in a Quantitative trait loci (QTL) analysis. Although QTLs occurring early in the senescence process coincided with QTLs for onset of senescence, the analysis of the time points made it difficult to study senescence as a continuous trait. Characteristics estimated from the senescence curve allowed us to study it as a developmental process and provide a meaningful biological interpretation to the results. Stable QTLs in the two experimental years were identified for progression rate and year-specific QTLs were detected for onset of senescence and inflection point. Specific interactions between loci controlling senescence development were also found. Epistatic interaction between QTLs on chromosomes 4, 5 and 7 were detected in 2004 and pleiotopic effects of QTLs on chromosomes 3 and 4 were observed in 2005.

Tetraploid hybrid tea roses (Rosa hybrida) represent most of the commercial cultivars of cut roses and form the basis for breeding programmes. Due to intensive interspecific hybridizations, modern cut roses are complex tetraploids for which the mode of inheritance is not exactly known. The segregation patterns of molecular markers in a tetraploid mapping population of 184 genotypes, an F1 progeny from a cross of two heterozygous parents, were investigated for disomic and tetrasomic inheritance. The possible occurrence of double reduction was studied as well. We can exclude disomic inheritance, but while our observations are more in line with a tetrasomic inheritance, we cannot exclude that there is a mixture of both inheritance modes. Two novel parental tetraploid linkage maps were constructed using markers known from literature, combined with newly generated markers. Comparison with the integrated consensus diploid map (ICM) of Spiller et al. (Theor Appl Genet 122:489–500, 2010) allowed assigning numbers to each of the linkage groups of both maps and including small linkage groups. So far, the possibility of using marker-assisted selection in breeding of tetraploid cut roses and of other species with a tetrasomic or partly tetrasomic inheritance, is still limited due to the difficulties in establishing marker-trait associations. We used these tetraploid linkage maps to determine associations between markers, two morphological traits and powdery mildew resistance. The knowledge on inheritance and marker-trait associations in tetraploid cut roses will be of direct use to cut rose breeding.

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in total twenty lily cultivars representing Lilium longiflorum, Oriental × Trumpet and Longiflorum × Asiatic hybrids were maintained on a medium with 8.3 µM picloram (PIC). In this study, they were tested for their regeneration potential by transferring them onto a regeneration medium supplemented with 0.4 µM PIC and 0.044 µM 6-benzyladenine. Regeneration was obtained in all cultivars examined and the percentage varied from zero to 89 % in the 36 callus lines. Regeneration frequency was significantly influenced by the genotype (cultivar). Subculturing the calli every 4 weeks by refreshing the regeneration medium contributed positively to bulblet formation, when compared to an eight week subculture frequency. It was found that the regeneration ability generally decreased with an increasing age of the callus cultures for all cultivars. The origin of the callus (style or filament) did not lead to significant differences in regeneration frequency, but there was an interaction between callus origin and genotype. Calli of eight randomly chosen cultivars were co-cultivated with Agrobacterium tumefaciens strain AGL0 carrying binary vectors with the gus gene as reporter and putative transgenic plants were produced. GUS histochemical assays demonstrated transient and stable expression of the gus gene in both calli and regenerated lily plants. Transient expression frequencies ranged from 0.3 to 20.6 % while stable transformation was much lower, only 1.4 % as the maximum

With the recent advances in high throughput profiling techniques the amount of genetic and phenotypic data available has increased dramatically. Although many genetic diversity studies combine morphological and genetic data, metabolite profiling has yet to be integrated into these studies. For our study we selected 168 accessions representing the different morphotypes and geographic origins of Brassica rapa. Metabolite profiling was performed on all plants of this collection in the youngest expanded leaves, 5 weeks after transplanting and the same material was used for molecular marker profiling. During the same season a year later, 26 morphological characteristics were measured on plants that had been vernalized in the seedling stage. The number of groups and composition following a hierarchical clustering with molecular markers was highly correlated to the groups based on morphological traits (r = 0.420) and metabolic profiles (r = 0.476). To reveal the admixture levels in B. rapa, comparison with the results of the programme STRUCTURE was needed to obtain information on population substructure. To analyze 5546 metabolite (LC–MS) signals the groups identified with STRUCTURE were used for random forests classification. When comparing the random forests and STRUCTURE membership probabilities 86% of the accessions were allocated into the same subgroup. Our findings indicate that if extensive phenotypic data (metabolites) are available, classification based on this type of data is very comparable to genetic classification. These multivariate types of data and methodological approaches are valuable for the selection of accessions to study the genetics of selected traits and for genetic improvement programs, and additionally provide information on the evolution of the different morphotypes in B. rapa

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed “mesosynteny” is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and m

Background Association mapping is a statistical approach combining phenotypic traits and genetic diversity in natural populations with the goal of correlating the variation present at phenotypic and allelic levels. It is essential to separate the true effect of genetic variation from other confounding factors, such as adaptation to different uses and geographical locations. The rapid availability of large datasets makes it necessary to explore statistical methods that can be computationally less intensive and more flexible for data exploration. Methodology/Principal Findings A core collection of 168 Brassica rapa accessions of different morphotypes and origins was explored to find genetic association between markers and metabolites: tocopherols, carotenoids, chlorophylls and folate. A widely used linear model with modifications to account for population structure and kinship was followed for association mapping. In addition, a machine learning algorithm called Random Forest (RF) was used as a comparison. Comparison of results across methods resulted in the selection of a set of significant markers as promising candidates for further work. This set of markers associated to the metabolites can potentially be applied for the selection of genotypes with elevated levels of these metabolites. Conclusions/Significance The incorporation of the kinship correction into the association model did not reduce the number of significantly associated markers. However incorporation of the STRUCTURE correction (Q matrix) in the linear regression model greatly reduced the number of significantly associated markers. Additionally, our results demonstrate that RF is an interesting complementary method with added value in association studies in plants, which is illustrated by the overlap in markers identified using RF and a linear mixed model with correction for kinship and population structure. Several markers that were selected in RF and in the models with correction for kinship, but not

The African malaria mosquito Anopheles gambiae sensu stricto continues to play an important role in malaria transmission, which is aggravated by its high degree of anthropophily, making it among the foremost vectors of this disease. In the current study we set out to unravel the strong association between this mosquito species and human beings, as it is determined by odorant cues derived from the human skin. Microbial communities on the skin play key roles in the production of human body odour. We demonstrate that the composition of the skin microbiota affects the degree of attractiveness of human beings to this mosquito species. Bacterial plate counts and 16S rRNA sequencing revealed that individuals that are highly attractive to An. gambiae s.s. have a significantly higher abundance, but lower diversity of bacteria on their skin than individuals that are poorly attractive. Bacterial genera that are correlated with the relative degree of attractiveness to mosquitoes were identified. The discovery of the connection between skin microbial populations and attractiveness to mosquitoes may lead to the development of new mosquito attractants and personalized methods for protection against vectors of malaria and other infectious diseases

The genotype by environment interaction study of broccoli, amongst others, demonstrates that traits of a cultivar are sometimes ranked differently when grown in an organic production system compared to a conventional system. This has strong implications for breeding strategies. The breeders interviewed acknowledged that more attention on abiotic and biotic stress resistance in a broccoli breeding programme is needed which is in accordance with the farmers' varietal requirements. The first findings of the field trials show that cultivar performance is influenced by season and region, and differences in treatment (organic versus conventional management). The field trials showed that there are cultivars with broad adaptation such as "Green Goliath". These cultivars performed across locations, seasons and treatments within the sub-top group, however, organic farmers would benefit more from cultivars specifically adapted to their region and season. The trial results showed a wide range of glucosinolate levels. Glucoraphanin was very genotype dependent, while glucobrassicin and neoglucobrassicin were more influenced by abiotic and biotic environmental factors. Therefore, there are opportunities for nutritional performance enhancement under organic conditions which would provide an added value to the product quality with respect to human and plant health. Further elaboration of the dataset will contribute to the design of regional breeding strategies for improved broccoli cultivars for the organic market.

Background - The simulation of the data for the QTLMAS 2009 Workshop is described. Objective was to simulate observations from a growth curve which was influenced by a number of QTL. Results - The data consisted of markers, phenotypes and pedigree. Genotypes of 453 markers, distributed over 5 chromosomes of 1 Morgan each, were simulated for 2,025 individuals. From those, 25 individuals were parents of the other 2,000 individuals. The 25 parents were genetically related. Phenotypes were simulated according to a logistic growth curve and were made available for 1,000 of the 2,000 offspring individuals. The logistic growth curve was specified by three parameters. Each parameter was influenced by six Quantitative Trait Loci (QTL), positioned at the five chromosomes. For each parameter, one QTL had a large effect and five QTL had small effects. Variance of large QTL was five times the variance of small QTL. Simulated data was made available at http://www.qtlmas2009.wur.nl/UK/Dataset

Compound identification and annotation in (untargeted) metabolomics experiments based on accurate mass require the highest possible accuracy of the mass determination. Experimental LC/TOF-MS platforms equipped with a time-to-digital converter (TDC) give the best mass estimate for those mass signals with an intensity similar to that of the lock-mass used for internal calibration. However, they systematically underestimate the mass obtained at higher signal intensity and overestimate it at low signal intensities compared to that of the lock-mass. To compensate for these effects, specific tools are required for correction and automation of accurate mass calculations from LC/MS signals. Here, we present a computational procedure for the derivation of an intensity-dependent mass correction function. The chromatographic mass signals for a set of known compounds present in a large number of samples were reconstructed over consecutive scans for each sample. It was found that the mass error is a linear function of the logarithm of the signal intensity adjusted to the associated lock-mass intensity. When applied to all mass data points, the correction function reduced the mass error for the majority of the tested compounds to ¿1 ppm over a wide range of signal intensities. The mass correction function has been implemented in a Python 2.4 script, which accepts raw data in NetCDF format as input, corrects the detected masses and returns the corrected NetCDF files for subsequent (automated) processing, such as mass signal alignment and database searching

A glucocorticoid-regulated BBM protein (35S:BBM-GR) was used in combination with microarray analysis to identify genes directly activated by BBM. We employed the system described by (Lloyd et al., 1994) in which dexamethasone (DEX) and cycloheximide (CHX) are applied together to respectively, induce nuclear localization of the BBM-GR protein and prevent translation of the primary targets mRNAs. In this way it is possible to identify direct targets of a transcriptional activator by comparing gene expression profiles between DEX+CHX-treated transgenic and wild-type tissues. The ability of the 35S:BBM GR construct to induce somatic embryogenesis in Arabidopsis seedlings was determined by phenotypic observation of 35S:BBM GR seeds germinated and grown in the presence of 10 µM dexamethasone (DEX). As in 35S:BBM seedlings, we observed somatic embryo formation on the cotyledons, first leaves and shoot meristem of DEX-treated 35S:BBM GR seedlings. We identified a set of 20 genes (including BBM itself) and our analysis indicates that BBM directly activates a signaling pathway comprising transcription factors and other signaling molecules, but which does not initially include genes known to induce somatic embryogenesis, such as LEC1, LEC2 or WUS. The functions of the BBM target genes are unknown, however a number of them have recently been identified in microarray screens for meristem-expressed genes. The identification of BBM-interacting partners and downstream targets provides new tools for unraveling pathways related to plant cell growth and organogenesis.