Roeland E. Voorrips

QualitySNP: a pipeline for detecting single nucleotide polymorphisms and insertions/deletions in EST data from diploid and polyploid species

BackgroundSingle nucleotide polymorphisms (SNPs) are important tools in studying complex genetic traits and genome evolution. Computational strategies for SNP discovery make use of the large number of sequences present in public databases (in most cases as expressed sequence tags (ESTs)) and are considered to be faster and more cost-effective than experimental procedures. A major challenge in computational SNP discovery is distinguishing allelic variation from sequence variation between paralogous sequences, in addition to recognizing sequencing errors. For the majority of the public EST sequences, trace or quality files are lacking which makes detection of reliable SNPs even more difficult because it has to rely on sequence comparisons only.ResultsWe have developed a new algorithm to detect reliable SNPs and insertions/deletions (indels) in EST data, both with and without quality files. Implemented in a pipeline called QualitySNP, it uses three filters for the identification of reliable SNPs. Filter 1 screens for all potential SNPs and identifies variation between or within genotypes. Filter 2 is the core filter that uses a haplotype-based strategy to detect reliable SNPs. Clusters with potential paralogs as well as false SNPs caused by sequencing errors are identified. Filter 3 screens SNPs by calculating a confidence score, based upon sequence redundancy and quality. Non-synonymous SNPs are subsequently identified by detecting open reading frames of consensus sequences (contigs) with SNPs. The pipeline includes a data storage and retrieval system for haplotypes, SNPs and alignments. QualitySNPs versatility is demonstrated by the identification of SNPs in EST datasets from potato, chicken and humans.ConclusionQualitySNP is an efficient tool for SNP detection, storage and retrieval in diploid as well as polyploid species. It is available for running on Linux or UNIX systems. The program, test data, and user manual are available at http://www.bioinformatics.nl/tools/snpweb/ and as Additional files.

Plasmodiophora brassicae: aspects of pathogenesis and resistance inBrassica oleracea

SummaryClubroot is one of the most damaging diseases inBrassica oleracea crops world-wide. The pathogenicity ofPlasmodiophora brassicae is highly variable between as well as within field populations. Several sources of resistance to clubroot have been identified inB. oleracea. Generally, resistance tends to inherit partly as a recessive, partly as an additive trait, and appears to be controlled by few major genes. Progress in the understanding of the inheritance of resistance is being made through the use of single-spore isolates of the pathogen, and the use of molecular markers for resistance genes.

Bayesian analysis of complex traits in pedigreed plant populations

A Bayesian approach to analyze complex traits is presented that can help plant eneticists and breeders in exploiting the marker and phenotypic data on pedigreed populations as available from ongoing breeding programs. The statistical model for the quantitative trait may include non-genetic and genetic components. The latter component can be divided into QTL on known marker linkage groups, major genes and a polygenic component. The full probability model, prior assumptions on model variables are presented and criterion for model selection and posterior inferences are given. Simulated data on a known pedigreed population structure of the EU project HiDRAS was used to illustrate the use of the Bayesian approach to analyze complex traits. It was shown that estimates for QTL parameters were more accurate when non-genetic factors were included in the model and when a polygenic component was included when not all linkage groups were analyzed simultaneously. The Bayesian approach has been implemented into the software package FlexQTL and allows plant breeders explore their pedigreed populations for segregating QTL alleles that are relevant in their breeding program.

Genotype calling in tetraploid species from bi-allelic marker data using mixture models

BackgroundAutomated genotype calling in tetraploid species was until recently not possible, which hampered genetic analysis. Modern genotyping assays often produce two signals, one for each allele of a bi-allelic marker. While ample software is available to obtain genotypes (homozygous for either allele, or heterozygous) for diploid species from these signals, such software is not available for tetraploid species which may be scored as five alternative genotypes (aaaa, baaa, bbaa, bbba and bbbb; nulliplex to quadruplex).ResultsWe present a novel algorithm, implemented in the R package fitTetra, to assign genotypes for bi-allelic markers to tetraploid samples from genotyping assays that produce intensity signals for both alleles. The algorithm is based on the fitting of several mixture models with five components, one for each of the five possible genotypes. The models have different numbers of parameters specifying the relation between the five component means, and some of them impose a constraint on the mixing proportions to conform to Hardy-Weinberg equilibrium (HWE) ratios. The software rejects markers that do not allow a reliable genotyping for the majority of the samples, and it assigns a missing score to samples that cannot be scored into one of the five possible genotypes with sufficient confidence.ConclusionsWe have validated the software with data of a collection of 224 potato varieties assayed with an Illumina GoldenGate™ 384 SNP array and shown that all SNPs with informative ratio distributions are fitted. Almost all fitted models appear to be correct based on visual inspection and comparison with diploid samples. When the collection of potato varieties is analyzed as if it were a population, almost all markers seem to be in Hardy-Weinberg equilibrium. The R package fitTetra is freely available under the GNU Public License from http://www.plantbreeding.wur.nl/UK/software_fitTetra.html and as Additional files with this article.

Responses of Brassica oleracea cultivars to infestation by the aphid Brevicoryne brassicae: an ecological and molecular approach

Intraspecific variation in resistance or susceptibility to herbivorous insects has been widely studied through bioassays. However, few studies have combined this with a full transcriptomic analysis. Here, we take such an approach to study the interaction between the aphid Brevicoryne brassicae and four white cabbage (Brassica oleracea var. capitata) cultivars. Both under glasshouse and field conditions, two of the cultivars clearly supported a faster aphid population development than the other two, indicating that aphid population development was largely independent of the environmental conditions. Genome-wide transcriptomic analysis using 70-mer oligonucleotide microarrays based on the Arabidopsis thaliana genome showed that only a small number of genes were differentially regulated, and that this regulation was highly cultivar specific. The temporal pattern in the expression behaviour of two B. brassicae-responsive genes in all four cultivars together with targeted studies employing A. thaliana knockout mutants revealed a possible role for a trypsin-and-protease inhibitor in defence against B. brassicae. Conversely, a xyloglucan endotransglucosylase seemed to have no effect on aphid performance. Overall, this study shows clear intraspecific variation in B. brassicae susceptibility among B. oleracea cultivars under glasshouse and field conditions that can be partly explained by certain differences in induced transcriptional changes.

Genotypic variation in genome-wide transcription profiles induced by insect feeding: Brassica oleracea--Pieris rapae interactions.

BackgroundTranscriptional profiling after herbivore attack reveals, at the molecular level, how plants respond to this type of biotic stress. Comparing herbivore-induced transcriptional responses of plants with different phenotypes provides insight into plant defense mechanisms. Here, we compare the global gene expression patterns induced by Pieris rapae caterpillar attack in two white cabbage (Brassica oleracea var. capitata) cultivars. The two cultivars are shown to differ in their level of direct defense against caterpillar feeding. Because Brassica full genome microarrays are not yet available, 70-mer oligonucleotide microarrays based on the Arabidopsis thaliana genome were used for this non-model plant.ResultsThe transcriptional responses of the two cultivars differed in timing as characterized by changes in their expression pattern after 24, 48 and 72 hours of caterpillar feeding. In addition, they also differed qualitatively. Surprisingly, of all genes induced at any time point, only one third was induced in both cultivars. Analyses of transcriptional responses after jasmonate treatment revealed that the difference in timing did not hold for the response to this phytohormone. Additionally, comparisons between Pieris rapae- and jasmonate-induced transcriptional responses showed that Pieris rapae induced more jasmonate-independent than jasmonate-dependent genes.ConclusionThe present study clearly shows that global transcriptional responses in two cultivars of the same plant species in response to insect feeding can differ dramatically. Several of these differences involve genes that are known to have an impact on Pieris rapae performance and probably underlie different mechanisms of direct defense, present in the cultivars.

QTL mapping of anthracnose (Colletotrichum spp.) resistance in a cross between Capsicum annuum and C. chinense

Anthracnose fruit rot is an economically important disease that affects pepper production in Indonesia. Strong resistance to two causal pathogens, Colletotrichum gloeosporioides and C. capsici, was found in an accession of Capsicum chinense. The inheritance of this resistance was studied in an F2 population derived from a cross of this accession with an Indonesian hot pepper variety (Capsicum annuum) using a quantitative trait locus (QTL) mapping approach. In laboratory tests where ripe fruits were artificially inoculated with either C. gloeosporioides or C. capsici, three resistance-related traits were scored: the infection frequency, the true lesion diameter (averaged over all lesions that actually developed), and the overall lesion diameter (averaged over all inoculation points, including those that did not develop lesions). One main QTL was identified with highly significant and large effects on all three traits after inoculation with C. gloeosporioides and on true lesion diameter after inoculation with C. capsici. Three other QTL with smaller effects were found for overall lesion diameter and true lesion diameter after inoculation with C. gloeosporioides, two of which also had an effect on infection frequency. Interestingly, the resistant parent carried a susceptible allele for a QTL for all three traits that was closely linked to the main QTL. The results with C. capsici were based on less observations and therefore less informative. Although the main QTL was shown to have an effect on true lesion diameter after inoculation with C. capsici, no significant QTL were identified for overall lesion diameter or infection frequency.

Large-scale identification of polymorphic microsatellites using an in silico approach

BackgroundSimple Sequence Repeat (SSR) or microsatellite markers are valuable for genetic research. Experimental methods to develop SSR markers are laborious, time consuming and expensive. In silico approaches have become a practicable and relatively inexpensive alternative during the last decade, although testing putative SSR markers still is time consuming and expensive. In many species only a relatively small percentage of SSR markers turn out to be polymorphic. This is particularly true for markers derived from expressed sequence tags (ESTs). In EST databases a large redundancy of sequences is present, which may contain information on length-polymorphisms in the SSR they contain, and whether they have been derived from heterozygotes or from different genotypes. Up to now, although a number of programs have been developed to identify SSRs in EST sequences, no software can detect putatively polymorphic SSRs.ResultsWe have developed PolySSR, a new pipeline to identify polymorphic SSRs rather than just SSRs. Sequence information is obtained from public EST databases derived from heterozygous individuals and/or at least two different genotypes. The pipeline includes PCR-primer design for the putatively polymorphic SSR markers, taking into account Single Nucleotide Polymorphisms (SNPs) in the flanking regions, thereby improving the success rate of the potential markers. A large number of polymorphic SSRs were identified using publicly available EST sequences of potato, tomato, rice, Arabidopsis, Brassica and chicken.The SSRs obtained were divided into long and short based on the number of times the motif was repeated. Surprisingly, the frequency of polymorphic SSRs was much higher in the short SSRs.ConclusionPolySSR is a very effective tool to identify polymorphic SSRs. Using PolySSR, several hundred putative markers were developed and stored in a searchable database. Validation experiments showed that almost all markers that were indicated as putatively polymorphic by polySSR were indeed polymorphic. This greatly improves the efficiency of marker development, especially in species where there are low levels of polymorphism, like tomato. When combined with the new sequencing technologies PolySSR will have a big impact on the development of polymorphic SSRs in any species.PolySSR and the polymorphic SSR marker database are available from http://www.bioinformatics.nl/tools/polyssr/.

Pedimap: Software for the Visualization of Genetic and Phenotypic Data in Pedigrees

Pedimap is a user-friendly software tool for visualizing phenotypic and genotypic data for related individuals linked in pedigrees. Genetic data can include marker scores, Identity-by-Descent probabilities, and marker linkage map positions, allowing the visualization of haplotypes through lineages. The pedigrees can accommodate all types of inheritance, including selfing, cloning, and repeated backcrossing, and all ploidy levels are supported. Visual association of the genetic data with phenotypic data simplifies the exploration of large data sets, thereby improving breeding decision making. Data are imported from text files; in addition data exchange with other software packages (FlexQTLTM and GenomeStudioTM) is possible. Instructions for use and an executable version compatible with the Windows platform are available for free from http://www.plantbreeding.wur.nl/UK/software_pedimap.html.

Genetic linkage of QTLs for late blight resistance and foliage maturity type in six related potato progenies

A set of test crosses of diploid potatoes was used to identify QTLs for foliage resistance against Phytophthora infestans and QTLs for foliage maturity type, and to assess their genetic relationship. The most important locus for both traits was found on chromosome 5 near marker GP21: the allele of marker GP21 that is associated with resistance to late blight is also associated with late foliage maturity. An additional QTL with a small effect on foliage maturity type was identified on chromosome 3, and additional QTLs for late blight resistance were found on chromosomes 3 and 10. Another QTL was detected on chromosome 7 when resistance was adjusted for the effect of foliage maturity type. The additional QTLs for resistance against P. infestans on chromosomes 3 and 10 seem to be independent of foliage maturity type and are not affected by epistatic effects of the locus on chromosome 5. The effects of the additional QTLs for resistance are small, but early maturing genotypes that necessarily have the allele for susceptibility for late blight on chromosome 5 may benefit from the resistance that is provided by these QTLs on chromosomes 3 and 10.