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.

Exploring the alpha-gliadin locus: the 33-mer peptide with six overlapping coeliac disease epitopes in Triticum aestivum is derived from a subgroup of Aegilops tauschii.

Most alpha-gliadin genes of the Gli-D2 locus on the D genome of hexaploid bread wheat (Triticum aestivum) encode for proteins with epitopes that can trigger coeliac disease (CD), and several contain a 33-mer peptide with six partly overlapping copies of three epitopes, which is regarded as a remarkably potent T-cell stimulator. To increase genetic diversity in the D genome, synthetic hexaploid wheat lines are being made by hybridising accessions of Triticum turgidum (AB genome) and Aegilops tauschii (the progenitor of the D genome). The diversity of alpha-gliadins in A. tauschii has not been studied extensively. We analysed the alpha-gliadin transcriptome of 51 A. tauschii accessions representative of the diversity in A. tauschii. We extracted RNA from developing seeds and performed 454 amplicon sequencing of the first part of the alpha-gliadin genes. The expression profile of allelic variants of the alpha-gliadins was different between accessions, and also between accessions of the Western and Eastern clades of A. tauschii. Generally, both clades expressed many allelic variants not found in bread wheat. In contrast to earlier studies, we detected the 33-mer peptide in some A. tauschii accessions, indicating that it was introduced along with the D genome into bread wheat. In these accessions, transcripts with the 33-mer peptide were present at lower frequencies than in bread wheat varieties. In most A. tauschii accessions, however, the alpha-gliadins do not contain the epitope, and this may be exploited, through synthetic hexaploid wheats, to breed bread wheat varieties with fewer or no coeliac disease epitopes.

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.

Detection of ploidy and chromosomal aberrations in commercial oil palm using high-throughput SNP markers.

MAIN CONCLUSION: Karyotyping using high-density genome-wide SNP markers identified various chromosomal aberrations in oil palm (Elaeis guineensis Jacq.) with supporting evidence from the 2C DNA content measurements (determined using FCM) and chromosome counts. Oil palm produces a quarter of the world's total vegetable oil. In line with its global importance, an initiative to sequence the oil palm genome was carried out successfully, producing huge amounts of sequence information, allowing SNP discovery. High-capacity SNP genotyping platforms have been widely used for marker-trait association studies in oil palm. Besides genotyping, a SNP array is also an attractive tool for understanding aberrations in chromosome inheritance. Exploiting this, the present study utilized chromosome-wide SNP allelic distributions to determine the ploidy composition of over 1,000 oil palms from a commercial F1 family, including 197 derived from twin-embryo seeds. Our method consisted of an inspection of the allelic intensity ratio using SNP markers. For palms with a shifted or abnormal distribution ratio, the SNP allelic frequencies were plotted along the pseudo-chromosomes. This method proved to be efficient in identifying whole genome duplication (triploids) and aneuploidy. We also detected several loss of heterozygosity regions which may indicate small chromosomal deletions and/or inheritance of identical by descent regions from both parents. The SNP analysis was validated by flow cytometry and chromosome counts. The triploids were all derived from twin-embryo seeds. This is the first report on the efficiency and reliability of SNP array data for karyotyping oil palm chromosomes, as an alternative to the conventional cytogenetic technique. Information on the ploidy composition and chromosomal structural variation can help to better understand the genetic makeup of samples and lead to a more robust interpretation of the genomic data in marker-trait association analyses.

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.

Analysis of allelic variants of RhMLO genes in rose and functional studies on susceptibility to powdery mildew related to clade V homologs.

KEY MESSAGE: Rose has 19 MLO genes. Of these, RhMLO1 and RhMLO2 were shown to be required for powdery mildew infection, which suggests their potential as susceptibility targets towards disease resistance. Powdery mildew, caused by Podosphaera pannosa, is one of the most serious and widespread fungal diseases for roses, especially in greenhouse-grown cut roses. It has been shown that certain MLO genes are involved in powdery mildew susceptibility and that loss of function in these genes in various crops leads to broad-spectrum, long-lasting resistance against this fungal disease. For this reason, these MLO genes are called susceptibility genes. We carried out a genome-wide identification of the MLO gene family in the Rosa chinensis genome, and screened for allelic variants among 22 accessions from seven different Rosa species using re-sequencing and transcriptome data. We identified 19 MLO genes in rose, of which four are candidate genes for functional homologs in clade V, which is the clade containing all dicot MLO susceptibility genes. We detected a total of 198 different allelic variants in the set of Rosa species and accessions, corresponding to 5-15 different alleles for each of the genes. Some diploid Rosa species shared alleles with tetraploid rose cultivars, consistent with the notion that diploid species have contributed to the formation of tetraploid roses. Among the four RhMLO genes in clade V, we demonstrated using expression study, virus-induced gene silencing as well as transient RNAi silencing that two of them, RhMLO1 and RhMLO2, are required for infection by P. pannosa and suggest their potential as susceptibility targets for powdery mildew resistance breeding in rose.

Genome editing of polyploid crops: prospects, achievements and bottlenecks.

Plant breeding aims to develop improved crop varieties. Many crops have a polyploid and often highly heterozygous genome, which may make breeding of polyploid crops a real challenge. The efficiency of traditional breeding based on crossing and selection has been improved by using marker-assisted selection (MAS), and MAS is also being applied in polyploid crops, which helps e.g. for introgression breeding. However, methods such as random mutation breeding are difficult to apply in polyploid crops because there are multiple homoeologous copies (alleles) of each gene. Genome editing technology has revolutionized mutagenesis as it enables precisely selecting targets. The genome editing tool CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as all alleles and/or copies of a gene can be targeted at once. Even multiple genes, each with multiple alleles, may be targeted simultaneously. In addition to targeted mutagenesis, targeted replacement of undesirable alleles by desired ones may become a promising application of genome editing for the improvement of polyploid crops, in the near future. Several examples of the application of genome editing for targeted mutagenesis are described here for a range of polyploid crops, and achievements and bottlenecks are highlighted.

Outlook for coeliac disease patients: towards bread wheat with hypoimmunogenic gluten by gene editing of α- and γ-gliadin gene families.

BACKGROUND: Wheat grains contain gluten proteins, which harbour immunogenic epitopes that trigger Coeliac disease in 1-2% of the human population. Wheat varieties or accessions containing only safe gluten have not been identified and conventional breeding alone struggles to achieve such a goal, as the epitopes occur in gluten proteins encoded by five multigene families, these genes are partly located in tandem arrays, and bread wheat is allohexaploid. Gluten immunogenicity can be reduced by modification or deletion of epitopes. Mutagenesis technologies, including CRISPR/Cas9, provide a route to obtain bread wheat containing gluten proteins with fewer immunogenic epitopes. RESULTS: In this study, we analysed the genetic diversity of over 600 α- and γ-gliadin gene sequences to design six sgRNA sequences on relatively conserved domains that we identified near coeliac disease epitopes. They were combined in four CRISPR/Cas9 constructs to target the α- or γ-gliadins, or both simultaneously, in the hexaploid bread wheat cultivar Fielder. We compared the results with those obtained with random mutagenesis in cultivar Paragon by γ-irradiation. For this, Acid-PAGE was used to identify T1 grains with altered gliadin protein profiles compared to the wild-type endosperm. We first optimised the interpretation of Acid-PAGE gels using Chinese Spring deletion lines. We then analysed the changes generated in 360 Paragon γ-irradiated lines and in 117 Fielder CRISPR/Cas9 lines. Similar gliadin profile alterations, with missing protein bands, could be observed in grains produced by both methods. CONCLUSIONS: The results demonstrate the feasibility and efficacy of using CRISPR/Cas9 to simultaneously edit multiple genes in the large α- and γ-gliadin gene families in polyploid bread wheat. Additional methods, generating genomics and proteomics data, will be necessary to determine the exact nature of the mutations generated with both methods.

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.

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.

Satellite DNA in Paphiopedilum subgenus Parvisepalum as revealed by high-throughput sequencing and fluorescent in situ hybridization.

BACKGROUND: Satellite DNA is a rapidly diverging, largely repetitive DNA component of many eukaryotic genomes. Here we analyse the evolutionary dynamics of a satellite DNA repeat in the genomes of a group of Asian subtropical lady slipper orchids (Paphiopedilum subgenus Parvisepalum and representative species in the other subgenera/sections across the genus). A new satellite repeat in Paphiopedilum subgenus Parvisepalum, SatA, was identified and characterized using the RepeatExplorer pipeline in HiSeq Illumina reads from P. armeniacum (2n = 26). Reconstructed monomers were used to design a satellite-specific fluorescent in situ hybridization (FISH) probe. The data were also analysed within a phylogenetic framework built using the internal transcribed spacer (ITS) sequences of 45S nuclear ribosomal DNA. RESULTS: SatA comprises c. 14.5% of the P. armeniacum genome and is specific to subgenus Parvisepalum. It is composed of four primary monomers that range from 230 to 359 bp and contains multiple inverted repeat regions with hairpin-loop motifs. A new karyotype of P. vietnamense (2n = 28) is presented and shows that the chromosome number in subgenus Parvisepalum is not conserved at 2n = 26, as previously reported. The physical locations of SatA sequences were visualised on the chromosomes of all seven Paphiopedilum species of subgenus Parvisepalum (2n = 26-28), together with the 5S and 45S rDNA loci using FISH. The SatA repeats were predominantly localisedin the centromeric, peri-centromeric and sub-telocentric chromosome regions, but the exact distribution pattern was species-specific. CONCLUSIONS: We conclude that the newly discovered, highly abundant and rapidly evolving satellite sequence SatA is specific to Paphiopedilum subgenus Parvisepalum. SatA and rDNA chromosomal distributions are characteristic of species, and comparisons between species reveal that the distribution patterns generate a strong phylogenetic signal. We also conclude that the ancestral chromosome number of subgenus Parvisepalum and indeed of all Paphiopedilum could be either 2n = 26 or 28, if P. vietnamense is sister to all species in the subgenus as suggested by the ITS data.

The domestication and evolutionary ecology of apples.

The cultivated apple is a major fruit crop in temperate zones. Its wild relatives, distributed across temperate Eurasia and growing in diverse habitats, represent potentially useful sources of diversity for apple breeding. We review here the most recent findings on the genetics and ecology of apple domestication and its impact on wild apples. Genetic analyses have revealed a Central Asian origin for cultivated apple, together with an unexpectedly large secondary contribution from the European crabapple. Wild apple species display strong population structures and high levels of introgression from domesticated apple, and this may threaten their genetic integrity. Recent research has revealed a major role of hybridization in the domestication of the cultivated apple and has highlighted the value of apple as an ideal model for unraveling adaptive diversification processes in perennial fruit crops. We discuss the implications of this knowledge for apple breeding and for the conservation of wild apples.

Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere.

KEY MESSAGE: Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.

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.

New insight into the history of domesticated apple: secondary contribution of the European wild apple to the genome of cultivated varieties.

The apple is the most common and culturally important fruit crop of temperate areas. The elucidation of its origin and domestication history is therefore of great interest. The wild Central Asian species Malus sieversii has previously been identified as the main contributor to the genome of the cultivated apple (Malus domestica), on the basis of morphological, molecular, and historical evidence. The possible contribution of other wild species present along the Silk Route running from Asia to Western Europe remains a matter of debate, particularly with respect to the contribution of the European wild apple. We used microsatellite markers and an unprecedented large sampling of five Malus species throughout Eurasia (839 accessions from China to Spain) to show that multiple species have contributed to the genetic makeup of domesticated apples. The wild European crabapple M. sylvestris, in particular, was a major secondary contributor. Bidirectional gene flow between the domesticated apple and the European crabapple resulted in the current M. domestica being genetically more closely related to this species than to its Central Asian progenitor, M. sieversii. We found no evidence of a domestication bottleneck or clonal population structure in apples, despite the use of vegetative propagation by grafting. We show that the evolution of domesticated apples occurred over a long time period and involved more than one wild species. Our results support the view that self-incompatibility, a long lifespan, and cultural practices such as selection from open-pollinated seeds have facilitated introgression from wild relatives and the maintenance of genetic variation during domestication. This combination of processes may account for the diversification of several long-lived perennial crops, yielding domestication patterns different from those observed for annual species.

New insights into domestication of carrot from root transcriptome analyses.

BACKGROUND: Understanding the molecular basis of domestication can provide insights into the processes of rapid evolution and crop improvement. Here we demonstrated the processes of carrot domestication and identified genes under selection based on transcriptome analyses. RESULTS: The root transcriptomes of widely differing cultivated and wild carrots were sequenced. A method accounting for sequencing errors was introduced to optimize SNP (single nucleotide polymorphism) discovery. 11,369 SNPs were identified. Of these, 622 (out of 1000 tested SNPs) were validated and used to genotype a large set of cultivated carrot, wild carrot and other wild Daucus carota subspecies, primarily of European origin. Phylogenetic analysis indicated that eastern carrot may originate from Western Asia and western carrot may be selected from eastern carrot. Different wild D. carota subspecies may have contributed to the domestication of cultivated carrot. Genetic diversity was significantly reduced in western cultivars, probably through bottlenecks and selection. However, a high proportion of genetic diversity (more than 85% of the genetic diversity in wild populations) is currently retained in western cultivars. Model simulation indicated high and asymmetric gene flow from wild to cultivated carrots, spontaneously and/or by introgression breeding. Nevertheless, high genetic differentiation exists between cultivated and wild carrots (Fst = 0.295) showing the strong effects of selection. Expression patterns differed radically for some genes between cultivated and wild carrot roots which may be related to changes in root traits. The up-regulation of water-channel-protein gene expression in cultivars might be involved in changing water content and transport in roots. The activated expression of carotenoid-binding-protein genes in cultivars could be related to the high carotenoid accumulation in roots. The silencing of allergen-protein-like genes in cultivated carrot roots suggested strong human selection to reduce allergy. These results suggest that regulatory changes of gene expressions may have played a predominant role in domestication. CONCLUSIONS: Western carrots may originate from eastern carrots. The reduction in genetic diversity in western cultivars due to domestication bottleneck/selection may have been offset by introgression from wild carrot. Differential gene expression patterns between cultivated and wild carrot roots may be a signature of strong selection for favorable cultivation traits.

Quantitative and qualitative differences in celiac disease epitopes among durum wheat varieties identified through deep RNA-amplicon sequencing.

BACKGROUND: Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Various peptides with three major T cell epitopes involved in CD are derived from alpha-gliadin fraction of gluten. Alpha-gliadins are encoded by a large multigene family and amino acid variation in the CD epitopes is known to influence the immunogenicity of individual gene family members. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and thus to accurately quantify the overall CD epitope load of a given wheat variety. Such quantification is indispensable for correct selection of wheat varieties with low potential to cause CD. RESULTS: A 454 RNA-amplicon sequencing method was developed for alpha-gliadin transcripts encompassing the three major CD epitopes and their variants. The method was used to screen developing grains on plants of 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 'unique deduced protein fragments' of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes. CONCLUSIONS: The dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will be useful as a reference in proteomics analysis regarding the immunogenic potential of mature wheat grains.

Community genetics in the time of next-generation molecular technologies.

Understanding the interactions of co-occurring species within and across trophic levels provides key information needed for understanding the ecological and evolutionary processes that underlie biological diversity. As genetics has only recently been integrated into the study of community-level interactions, the time is right for a critical evaluation of potential new, gene-based approaches to studying communities. Next-generation molecular techniques, used in parallel with field-based observations and manipulative experiments across spatio-temporal gradients, are key to expanding our understanding of community-level processes. Here, we introduce a variety of '-omics' tools, with recent studies of plant-insect herbivores and of ectomycorrhizal systems providing detailed examples of how next-generation approaches can revolutionize our understanding of interspecific interactions. We suggest ways that novel technologies may convert community genetics from a field that relies on correlative inference to one that reveals causal mechanisms of genetic co-variation and adaptations within communities.

AFLP-based population structure analysis as a means to validate the complex taxonomy of dogroses (Rosa section Caninae).

Within the genus Rosa numerous species have been described. Circumscription of the dogrose section Caninae is straightforward, but the delineation of species and subsections within this section is less clear, partly due to hybridisation between species. We have investigated the extent to which DNA marker-based information of wild populations corroborates present-day dogrose taxonomy and hypotheses about the origination of taxa. Sampling was conducted in a transect across Europe, collecting over 900 specimens of all encountered dogrose taxa. For comparison, we also included more than 200 samples of species belonging to other sections. Two lines of statistical analyses were used to investigate the genetic structure based on AFLP data: (1) an unstructured model with principal coordinate analysis and hierarchical clustering, and (2) a model with a superimposed taxonomic structure based on analysis of genetic diversity using a novel approach combining assignment tests with canonical discriminant analysis. Support was found for five of the seven subsections, whereas R. balsamica apparently belongs to subsection Caninae thus omitting the need for recognising subsection Tomentellae. For R. stylosa, a hybridogenic origin with a non-dogrose section member has been suggested, and it can be treated either as a separate subsection or within subsection Caninae. Within the subsection Rubigineae, a species cluster with low support for the taxa R. micrantha, R. rubiginosa and the putatively hybridogenous R. gremlii was identified. Similarly, several species in the subsection Caninae overlapped considerably, and are best regarded as one common species complex. This population genetic approach provides a general method to validate the taxonomic system in complex and polyploid taxa.