Bread Wheat Genome Research Articles

Transferability and Polymorphism Between Group 7 Chromosome Specific Simple Sequence Repeat (SSR) Markers of Bread Wheat and Its Related Non-Progenitor Aegilops Species

ABSTRACTSimple sequence repeat (SSR) markers are highly efficient for genetic mapping and molecular breeding in crop plants. Chromosomes 7S and 7U of related non-progenitor Aegilops species possess superior genetic systems for high grain iron and zinc content. This study was undertaken to conduct a comprehensive analysis of transferability and polymorphism among group-7 anchored SSR markers of bread wheat (Triticum aestivum L.) to Aegilops species for wheat improvement. The study revealed 77% transferability of group 7-specific SSR markers of bread wheat to 7U/7S chromosomes of Aegilops species. More than 80% of the 7D specific markers were found to be transferable with a high level of polymorphism. The transferability to 7S and 7U genome suggested higher similarity between bread wheat genome(s) and S genome as compared with U genome of Aegilops species. These polymorphic markers are highly informative, robust and cost-effective for molecular breeding and could be further utilized for identification of introgression/transfer(s) of 7S/7U chromosomal fragments in bread wheat genome through wide hybridization.

CDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing.

The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.

Identification and molecular characterization of the nicotianamine synthase gene family in bread wheat.

SummaryNicotianamine (NA) is a non‐protein amino acid involved in fundamental aspects of metal uptake, transport and homeostasis in all plants and constitutes the biosynthetic precursor of mugineic acid family phytosiderophores (MAs) in graminaceous plant species. Nicotianamine synthase (NAS) genes, which encode enzymes that synthesize NA from S‐adenosyl‐L‐methionine (SAM), are differentially regulated by iron (Fe) status in most plant species and plant genomes have been found to contain anywhere from 1 to 9 NAS genes. This study describes the identification of 21 NAS genes in the hexaploid bread wheat (Triticum aestivum L.) genome and their phylogenetic classification into two distinct clades. The TaNAS genes are highly expressed during germination, seedling growth and reproductive development. Fourteen of the clade I NAS genes were up‐regulated in root tissues under conditions of Fe deficiency. Protein sequence analyses revealed the presence of endocytosis motifs in all of the wheat NAS proteins as well as chloroplast, mitochondrial and secretory transit peptide signals in four proteins. These results greatly expand our knowledge of NAS gene families in graminaceous plant species as well as the genetics underlying Fe nutrition in bread wheat.

Development of new SSR markers for homoeologous WFZP gene loci based on the study of the structure and location of microsatellites in gene-rich regions of chromosomes 2AS, 2BS, and 2DS in bread wheat

Microsatellites, or simple sequence repeats, are widely distributed in eukaryotic genomes, including plant genomes. The peculiarities of the structure and location of the microsatellite loci determine their potential as molecular genetic markers and can influence the assumed function of microsatellites in important biological processes. The identification and study of the distribution of microsatellite loci in gene-rich genome regions of the bread wheat and the development (based on them) of new microsatellite markers are of practical interest and are important for the study of the organization of the bread wheat genome. The sequences of BAC clones that contain the homoeologous WFZP genes of the bread wheat (Triticum aestivum L.) controlling the development of the ear were the basis for the identification and localization of microsatellite loci in gene-rich regions of the 2AS, 2BS, and 2DS chromosomes. Diand trinucleotide microsatellite repeats are the most widespread in the studied sequences. The AG and GA/TC motifs prevail among the dinucleotide motifs; the dinucleotide repeats are found in noncoding gene regions, mobile elements, and nonannotated DNA sequences. Most of the trinucleotide repeats are associated with mobile genetic elements. It was found that homoeologous microsatellite loci are located either in the genes or in the nonannotated DNA sequences. The comparison of the structure of homoeologous loci demonstrated that the divergence in them is associated both with a change in the number of repeats and with nucleotide substitutions. The new microsatellite markers, which are colocalized in the genetic maps with the WFZP-A-B-D genes and can be used for marking these genes in molecular genetic studies and in breeding controlled by markers, were developed.

Transfer of useful variability of high grain iron and zinc from Aegilops kotschyi into wheat through seed irradiation approach

Purpose To transfer the 2S chromosomal fragment(s) of Aegilops kotschyi (2Sk) into the bread wheat genome which could lead to the biofortification of wheat with high grain iron and zinc content.Materials and methods Wheat-Ae. kotschyi 2A/2Sk substitution lines with high grain iron and zinc content were used to transfer the gene/loci for high grain Fe and Zn content into wheat using seed irradiation approach.Results Bread wheat plants derived from 40 krad-irradiated seeds showed the presence of univalents and multivalents during meiotic metaphase-I. Genomic in situ hybridization analysis of seed irradiation hybrid F2 seedlings showed several terminal and interstitial signals indicated the introgression of Ae. kotschyi chromosome segments. This proves the efficacy of seed radiation hybrid approach in gene transfer experiments. All the radiation-treated hybrid plants with high grain Fe and Zn content were analyzed with wheat group 2 chromosome-specific polymorphic simple sequence repeat markers to identify the introgression of small alien chromosome fragment(s).Conclusion Radiation-induced hybrids showed more than 65% increase in grain iron and 54% increase in Zn contents with better harvest index than the elite wheat cultivar WL711 indicating effective and compensating translocations of 2Sk fragments into wheat genome.

Effects of limited introgressions from Triticum timopheevii Tausch. into the genome of bread wheat (Triticum aestivum L.) on physiological and biochemical traits under normal watering and drought

Effects of limited introgressions from Triticum timopheevii Tausch. into the genome of bread wheat (Triticum aestivum L.) on physiological and biochemical traits under normal watering and drought

Development of new SSR markers for homoeologous WFZP loci based on the study of structure and location of microsatellites in gene-rich regions of chromosomes 2AS , 2BS, 2DS

Microsatellites, or simple sequence repeats (SSRs), are ubiquitous in genomes of eukaryotes, including plant genomes. The structure and location of SSR loci determine their potential as molecular genetic markers and may have impact on the potential function of microsatellites in important biological processes. Identification and study of the distribution of SSR loci in gene-rich regions of the bread wheat genome and development of novel SSR markers based on these data are of practical interest, being important for the study of bread wheat genome organization. Bread wheat BACclone sequences containing homoeologous WFZP genes that control spikelet development served as the base for the identification and localization of SSR loci in generich regions of chromosomes 2AS, 2BS, and 2DS. It was found that di- and trinucleotide motifs were predominant. The most common dinucleotide motifs were AG and GA/TC. They were distributed in noncoding regions of genes, transposable elements (TEs) and unannotated sequences. Most identified trinucleotide motifs were associated with transposable elements. Homoeologous SSR loci were found in either genes or unannotated sequences. Comparison of these loci showed that the divergence in their structure was caused both by changes in repeat number and nucleotide substitutions. New SSR markers were developed and mapped. On the genetic maps of chromosomes 2A, 2B и 2D, they collocated with the WFZP-A-B-D genes. Thus, they can be used for gene tagging in molecular research and in marker-assisted selection.

Radiation hybrid maps of the D-genome of Aegilops tauschii and their application in sequence assembly of large and complex plant genomes.

BackgroundThe large and complex genome of bread wheat (Triticum aestivum L., ~17 Gb) requires high resolution genome maps with saturated marker scaffolds to anchor and orient BAC contigs/ sequence scaffolds for whole genome assembly. Radiation hybrid (RH) mapping has proven to be an excellent tool for the development of such maps for it offers much higher and more uniform marker resolution across the length of the chromosome compared to genetic mapping and does not require marker polymorphism per se, as it is based on presence (retention) vs. absence (deletion) marker assay.MethodsIn this study, a 178 line RH panel was genotyped with SSRs and DArT markers to develop the first high resolution RH maps of the entire D-genome of Ae. tauschii accession AL8/78. To confirm map order accuracy, the AL8/78-RH maps were compared with:1) a DArT consensus genetic map constructed using more than 100 bi-parental populations, 2) a RH map of the D-genome of reference hexaploid wheat ’Chinese Spring’, and 3) two SNP-based genetic maps, one with anchored D-genome BAC contigs and another with anchored D-genome sequence scaffolds. Using marker sequences, the RH maps were also anchored with a BAC contig based physical map and draft sequence of the D-genome of Ae. tauschii.ResultsA total of 609 markers were mapped to 503 unique positions on the seven D-genome chromosomes, with a total map length of 14,706.7 cR. The average distance between any two marker loci was 29.2 cR which corresponds to 2.1 cM or 9.8 Mb. The average mapping resolution across the D-genome was estimated to be 0.34 Mb (Mb/cR) or 0.07 cM (cM/cR). The RH maps showed almost perfect agreement with several published maps with regard to chromosome assignments of markers. The mean rank correlations between the position of markers on AL8/78 maps and the four published maps, ranged from 0.75 to 0.92, suggesting a good agreement in marker order. With 609 mapped markers, a total of 2481 deletions for the whole D-genome were detected with an average deletion size of 42.0 Mb. A total of 520 markers were anchored to 216 Ae. tauschii sequence scaffolds, 116 of which were not anchored earlier to the D-genome.ConclusionThis study reports the development of first high resolution RH maps for the D-genome of Ae. tauschii accession AL8/78, which were then used for the anchoring of unassigned sequence scaffolds. This study demonstrates how RH mapping, which offered high and uniform resolution across the length of the chromosome, can facilitate the complete sequence assembly of the large and complex plant genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2030-2) contains supplementary material, which is available to authorized users.

The physical map of wheat chromosome 5DS revealed gene duplications and small rearrangements.

BackgroundThe substantially large bread wheat genome, organized into highly similar three sub-genomes, renders genomic research challenging. The construction of BAC-based physical maps of individual chromosomes reduces the complexity of this allohexaploid genome, enables elucidation of gene space and evolutionary relationships, provides tools for map-based cloning, and serves as a framework for reference sequencing efforts. In this study, we constructed the first comprehensive physical map of wheat chromosome arm 5DS, thereby exploring its gene space organization and evolution.ResultsThe physical map of 5DS was comprised of 164 contigs, of which 45 were organized into 21 supercontigs, covering 176 Mb with an N50 value of 2,173 kb. Fifty-eight of the contigs were larger than 1 Mb, with the largest contig spanning 6,649 kb. A total of 1,864 molecular markers were assigned to the map at a density of 10.5 markers/Mb, anchoring 100 of the 120 contigs (>5 clones) that constitute ~95 % of the cumulative length of the map. Ordering of 80 contigs along the deletion bins of chromosome arm 5DS revealed small-scale breaks in syntenic blocks. Analysis of the gene space of 5DS suggested an increasing gradient of genes organized in islands towards the telomere, with the highest gene density of 5.17 genes/Mb in the 0.67-0.78 deletion bin, 1.4 to 1.6 times that of all other bins.ConclusionsHere, we provide a chromosome-specific view into the organization and evolution of the D genome of bread wheat, in comparison to one of its ancestors, revealing recent genome rearrangements. The high-quality physical map constructed in this study paves the way for the assembly of a reference sequence, from which breeding efforts will greatly benefit.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1641-y) contains supplementary material, which is available to authorized users.

Chloroplast phylogeny of Triticum/Aegilops species is not incongruent with an ancient homoploid hybrid origin of the ancestor of the bread wheat D‐genome

In a recent Letter to New Phytologist Li et al. (2015) presented a re-evaluation of our recent finding from a phylogenomics study that the bread wheat D-genome lineage is probably of homoploid hybrid origin (Marcussen et al., 2014). The authors claim to present evidence from chloroplast phylogeny and gene tree topologies that necessitate additional hybridization events compared to the phylogeny proposed by us. Unfortunately Li et al.'s (2015) conclusions appear to be confounded by differences in nomenclatures used to describe the major Triticum/Aegilops clades (Fig. 1) and a misunderstanding concerning the temporal dimension of our phylogenetic model (i.e. when hybridization happened) (Fig. 1). In our original paper we presented phylogenomic data suggesting that one or more basal hybridization(s) between the ancestor of the A-genome and the ancestor of the B-genome in modern bread wheat gave rise to an ancestor of the bread wheat D-genome lineage 5–6 million years ago (Fig. 1). We used the terms ‘A-genome lineage’, ‘B-genome lineage’, and ‘D-genome lineage’ to refer to the three deep lineages encompassing all extant species in the Triticum/Aegilops clade (Fig. 1 in this Letter; Figs 2, 3, and S6 and their captions in Marcussen et al., 2014). This informal nomenclature was chosen because Marcussen et al. (2014) was part of a series of papers on the hexaploid bread wheat genome constituents (i.e. A, B and D) (International Wheat Genome Sequencing Consortium (IWGSC), 2014; Pfeifer et al., 2014) in addition to the lack of a phylogenetic infrageneric classification of Triticum/Aegilops. Thus, our use of ‘D-genome lineage’ in Marcussen et al. (2014) refers to the entire lineage containing T. aestivum-D, Ae. tauschii, and Ae. sharonensis (Fig. 1). This is not synonymous with how Li et al. (2015) define the ‘D-genome’ according to the haplome nomenclature of wheats (Fig. 1 and summarized in Table 1 of Li et al., 2015). Hence, in their re-evaluation of our hypothesis Li et al. (2015) consider the D-lineage as encompassing Aegilops tauschii only, while Ae. sharonensis is considered to belong to an S*+M clade (Fig. 1). One sentence from the Li et al. (2015) Letter that highlights this misunderstanding is: ‘Marcussen et al. (2014) proposed that A. tauschii originated from homoploid hybridization between A. speltoides and the ancestor of modern T. urartu/T. monococcum.’ This is a factual error. Marcussen et al. (2014) never claimed that Ae. tauschii evolved through a homoploid hybridization. What we proposed was that a homoploid hybridization gave rise to an ancestor of all D+S*+M clade species (including Ae. tauschii and Ae. sharonensis) (Fig. 1). In addition, Li et al. (2015) seem to have overlooked the timescale of our phylogenetic analyses. We report on an ancient hybridization (illustrated clearly in Fig. 2b in Marcussen et al., 2014) several million years before Aegilops tauschii and Ae. sharonensis existed as species. The hybrid history of the D-genome lineage (as Marcussen et al., 2014 refers to) is therefore not restricted to Ae. tauschii but encompasses the entire D+S*+M clade. The very same chloroplast phylogeny that Li et al. (2015) use to re-evaluate our conclusions is therefore completely congruent with our proposed phylogenetic network for wheats. Finally, we fully agree with Li et al. (2015) that the evolutionary history of Tritium/Aegilops is likely to be even more complex than we currently understand it to be. Judging from the young age of the Triticeae and the numerous reports of suspected reticulations, sequencing and analysis of additional diploid and polyploid Triticum/Aegilops genomes will most likely provide evidence for additional hybridization events not yet detected.

Proteomic profiling of 16 cereal grains and the application of targeted proteomics to detect wheat contamination.

Global proteomic analysis utilizing SDS-PAGE, Western blotting and LC-MS/MS of total protein and gluten-enriched extracts derived from 16 economically important cereals was undertaken, providing a foundation for the development of MS-based quantitative methodologies that would enable the detection of wheat contamination in foods. The number of proteins identified in each grain correlated with the number of entries in publicly available databases, highlighting the importance of continued advances in genome sequencing to facilitate accurate protein identification. Subsequently, candidate wheat-specific peptide markers were evaluated by multiple-reaction monitoring MS. The selected markers were unique to wheat, yet present in a wide range of wheat varieties that represent up to 80% of the bread wheat genome. The final analytical method was rapid (15 min) and robust (CV < 10%), showed linearity (R(2) > 0.98) spanning over 3 orders of magnitude, and was highly selective and sensitive with detection down to 15 mg/kg in intentionally contaminated soy flour. Furthermore, application of this technology revealed wheat contamination in commercially sourced flours, including rye, millet, oats, sorghum, buckwheat and three varieties of soy.

Deep transcriptome sequencing provides new insights into the structural and functional organization of the wheat genome.

BackgroundBecause of its size, allohexaploid nature, and high repeat content, the bread wheat genome is a good model to study the impact of the genome structure on gene organization, function, and regulation. However, because of the lack of a reference genome sequence, such studies have long been hampered and our knowledge of the wheat gene space is still limited. The access to the reference sequence of the wheat chromosome 3B provided us with an opportunity to study the wheat transcriptome and its relationships to genome and gene structure at a level that has never been reached before.ResultsBy combining this sequence with RNA-seq data, we construct a fine transcriptome map of the chromosome 3B. More than 8,800 transcription sites are identified, that are distributed throughout the entire chromosome. Expression level, expression breadth, alternative splicing as well as several structural features of genes, including transcript length, number of exons, and cumulative intron length are investigated. Our analysis reveals a non-monotonic relationship between gene expression and structure and leads to the hypothesis that gene structure is determined by its function, whereas gene expression is subject to energetic cost. Moreover, we observe a recombination-based partitioning at the gene structure and function level.ConclusionsOur analysis provides new insights into the relationships between gene and genome structure and function. It reveals mechanisms conserved with other plant species as well as superimposed evolutionary forces that shaped the wheat gene space, likely participating in wheat adaptation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0601-9) contains supplementary material, which is available to authorized users.

A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome.

Polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible to construct a mapping population.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0582-8) contains supplementary material, which is available to authorized users.

New insights into the wheat chromosome 4D structure and virtual gene order, revealed by survey pyrosequencing

Survey sequencing of the bread wheat (Triticum aestivum L.) genome (AABBDD) has been approached through different strategies delivering important information. However, the current wheat sequence knowledge is not complete. The aim of our study is to provide different and complementary set of data for chromosome 4D. A survey sequence was obtained by pyrosequencing of flow-sorted 4DS (7.2×) and 4DL (4.1×) arms. Single ends (SE) and long mate pairs (LMP) reads were assembled into contigs (223Mb) and scaffolds (65Mb) that were aligned to Aegilops tauschii draft genome (DD), anchoring 34Mb to chromosome 4. Scaffolds annotation rendered 822 gene models. A virtual gene order comprising 1973 wheat orthologous gene loci and 381 wheat gene models was built. This order was largely consistent with the scaffold order determined based on a published high density map from the Ae. tauschii chromosome 4, using bin-mapped 4D ESTs as a common reference. The virtual order showed a higher collinearity with homeologous 4B compared to 4A. Additionally, a virtual map was constructed and ∼5700 genes (∼2200 on 4DS and ∼3500 on 4DL) predicted. The sequence and virtual order obtained here using the 454 platform were compared with the Illumina one used by the IWGSC, giving complementary information.

Organization and evolution of transposable elements along the bread wheat chromosome 3B.

BackgroundThe 17 Gb bread wheat genome has massively expanded through the proliferation of transposable elements (TEs) and two recent rounds of polyploidization. The assembly of a 774 Mb reference sequence of wheat chromosome 3B provided us with the opportunity to explore the impact of TEs on the complex wheat genome structure and evolution at a resolution and scale not reached so far.ResultsWe develop an automated workflow, CLARI-TE, for TE modeling in complex genomes. We delineate precisely 56,488 intact and 196,391 fragmented TEs along the 3B pseudomolecule, accounting for 85% of the sequence, and reconstruct 30,199 nested insertions. TEs have been mostly silent for the last one million years, and the 3B chromosome has been shaped by a succession of bursts that occurred between 1 to 3 million years ago. Accelerated TE elimination in the high-recombination distal regions is a driving force towards chromosome partitioning. CACTAs overrepresented in the high-recombination distal regions are significantly associated with recently duplicated genes. In addition, we identify 140 CACTA-mediated gene capture events with 17 genes potentially created by exon shuffling and show that 19 captured genes are transcribed and under selection pressure, suggesting the important role of CACTAs in the recent wheat adaptation.ConclusionAccurate TE modeling uncovers the dynamics of TEs in a highly complex and polyploid genome. It provides novel insights into chromosome partitioning and highlights the role of CACTA transposons in the high level of gene duplication in wheat.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0546-4) contains supplementary material, which is available to authorized users.

Next-generation sequencing of flow-sorted wheat chromosome 5D reveals lineage-specific translocations and widespread gene duplications.

BackgroundThe ~17 Gb hexaploid bread wheat genome is a high priority and a major technical challenge for genomic studies. In particular, the D sub-genome is relatively lacking in genetic diversity, making it both difficult to map genetically, and a target for introgression of agriculturally useful traits. Elucidating its sequence and structure will therefore facilitate wheat breeding and crop improvement.ResultsWe generated shotgun sequences from each arm of flow-sorted Triticum aestivum chromosome 5D using 454 FLX Titanium technology, giving 1.34× and 1.61× coverage of the short (5DS) and long (5DL) arms of the chromosome respectively. By a combination of sequence similarity and assembly-based methods, ~74% of the sequence reads were classified as repetitive elements, and coding sequence models of 1314 (5DS) and 2975 (5DL) genes were generated. The order of conserved genes in syntenic regions of previously sequenced grass genomes were integrated with physical and genetic map positions of 518 wheat markers to establish a virtual gene order for chromosome 5D.ConclusionsThe virtual gene order revealed a large-scale chromosomal rearrangement in the peri-centromeric region of 5DL, and a concentration of non-syntenic genes in the telomeric region of 5DS. Although our data support the large-scale conservation of Triticeae chromosome structure, they also suggest that some regions are evolving rapidly through frequent gene duplications and translocations.Sequence accessionsEBI European Nucleotide Archive, Study no. ERP002330Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1080) contains supplementary material, which is available to authorized users.

A high-density, SNP-based consensus map of tetraploid wheat as a bridge to integrate durum and bread wheat genomics and breeding.

Consensus linkage maps are important tools in crop genomics. We have assembled a high-density tetraploid wheat consensus map by integrating 13 data sets from independent biparental populations involving durum wheat cultivars (Triticum turgidum ssp.durum), cultivated emmer (T.turgidum ssp.dicoccum) and their ancestor (wild emmer, T.turgidum ssp.dicoccoides). The consensus map harboured 30144 markers (including 26626 SNPs and 791 SSRs) half of which were present in at least two component maps. The final map spanned 2631cM of all 14 durum wheat chromosomes and, differently from the individual component maps, all markers fell within the 14 linkage groups. Marker density per genetic distance unit peaked at centromeric regions, likely due to a combination of low recombination rate in the centromeric regions and even gene distribution along the chromosomes. Comparisons with bread wheat indicated fewer regions with recombination suppression, making this consensus map valuable for mapping in the A and B genomes of both durum and bread wheat. Sequence similarity analysis allowed us to relate mapped gene-derived SNPs to chromosome-specific transcripts. Dense patterns of homeologous relationships have been established between the A- and B-genome maps and between nonsyntenic homeologous chromosome regions as well, the latter tracing to ancient translocation events. The gene-based homeologous relationships are valuable to infer the map location of homeologs of target loci/QTLs. Because most SNP and SSR markers were previously mapped in bread wheat, this consensus map will facilitate a more effective integration and exploitation of genes and QTL for wheat breeding purposes.

Using genic sequence capture in combination with a syntenic pseudo genome to map a deletion mutant in a wheat species.

Mapping-by-sequencing analyses have largely required a complete reference sequence and employed whole genome re-sequencing. In species such as wheat, no finished genome reference sequence is available. Additionally, because of its large genome size (17 Gb), re-sequencing at sufficient depth of coverage is not practical. Here, we extend the utility of mapping by sequencing, developing a bespoke pipeline and algorithm to map an early-flowering locus in einkorn wheat (Triticum monococcum L.) that is closely related to the bread wheat genome A progenitor. We have developed a genomic enrichment approach using the gene-rich regions of hexaploid bread wheat to design a 110-Mbp NimbleGen SeqCap EZ in solution capture probe set, representing the majority of genes in wheat. Here, we use the capture probe set to enrich and sequence an F2 mapping population of the mutant. The mutant locus was identified in T. monococcum, which lacks a complete genome reference sequence, by mapping the enriched data set onto pseudo-chromosomes derived from the capture probe target sequence, with a long-range order of genes based on synteny of wheat with Brachypodium distachyon. Using this approach we are able to map the region and identify a set of deleted genes within the interval.

Identification and characterization of more than 4 million intervarietal SNPs across the group 7 chromosomes of bread wheat.

Despite being a major international crop, our understanding of the wheat genome is relatively poor due to its large size and complexity. To gain a greater understanding of wheat genome diversity, we have identified single nucleotide polymorphisms between 16 Australian bread wheat varieties. Whole-genome shotgun Illumina paired read sequence data were mapped to the draft assemblies of chromosomes 7A, 7B and 7D to identify more than 4 million intervarietal SNPs. SNP density varied between the three genomes, with much greater density observed on the A and B genomes than the D genome. This variation may be a result of substantial gene flow from the tetraploid Triticum turgidum, which possesses A and B genomes, during early co-cultivation of tetraploid and hexaploid wheat. In addition, we examined SNP density variation along the chromosome syntenic builds and identified genes in low-density regions which may have been selected during domestication and breeding. This study highlights the impact of evolution and breeding on the bread wheat genome and provides a substantial resource for trait association and crop improvement. All SNP data are publically available on a generic genome browser GBrowse at www.wheatgenome.info.

Utilization of deletion bins to anchor and order sequences along the wheat 7B chromosome.

A total of 3,671 sequence contigs and scaffolds were mapped to deletion bins on wheat chromosome 7B providing a foundation for developing high-resolution integrated physical map for this chromosome. Bread wheat (Triticum aestivum L.) has a large, complex and highly repetitive genome which is challenging to assemble into high quality pseudo-chromosomes. As part of the international effort to sequence the hexaploid bread wheat genome by the international wheat genome sequencing consortium (IWGSC) we are focused on assembling a reference sequence for chromosome 7B. The successful completion of the reference chromosome sequence is highly dependent on the integration of genetic and physical maps. To aid the integration of these two types of maps, we have constructed a high-density deletion bin map of chromosome 7B. Using the 270 K Nimblegen comparative genomic hybridization (CGH) array on a set of cv. Chinese spring deletion lines, a total of 3,671 sequence contigs and scaffolds (~7.8 % of chromosome 7B physical length) were mapped into nine deletion bins. Our method of genotyping deletions on chromosome 7B relied on a model-based clustering algorithm (Mclust) to accurately predict the presence or absence of a given genomic sequence in a deletion line. The bin mapping results were validated using three different approaches, viz. (a) PCR-based amplification of randomly selected bin mapped sequences (b) comparison with previously mapped ESTs and (c) comparison with a 7B genetic map developed in the present study. Validation of the bin mapping results suggested a high accuracy of the assignment of 7B sequence contigs and scaffolds to the 7B deletion bins.