Genomic sequencing of Thinopyrum elongatum chromosome arm 7EL, carrying fusarium head blight resistance, and characterization of its impact on the transcriptome of the introgressed line CS-7EL.

BACKGROUND: The tall wheatgrass species Thinopyrum elongatum carries a strong fusarium head blight (FHB) resistance locus located on the long arm of chromosome 7 (7EL) as well as resistance to leaf and stem rusts, all diseases with a significant impact on wheat production. Towards understanding the contribution of Th. elongatum 7EL to improvement of disease resistance in wheat, the genomic sequence of the 7EL fragment present in the wheat Chinese Spring (CS) telosomic addition line CS-7EL was determined and the contribution and impact of 7EL on the rachis transcriptome during FHB infection was compared between CS and CS-7EL. RESULTS: We assembled the Th. elongatum 7EL chromosome arm using a reference-guided approach. Combining this assembly with the available reference sequence for CS hexaploid wheat provided a reliable reference for interrogating the transcriptomic differences in response to infection conferred by the 7EL fragment. Comparison of the transcriptomes of rachis tissues from CS and CS-7EL showed expression of Th. elongatum transcripts as well as modulation of wheat transcript expression profiles in the CS-7EL line. Expression profiles at 4 days after infection with Fusarium graminearum, the causal agent of FHB, showed an increased in expression of genes associated with an effective defense response, in particular glucan endo-1,3-beta-glucosidases and chitinases, in the FHB-resistant line CS-7EL while there was a larger increase in differential expression for genes associated with the level of fungal infection in the FHB-susceptible line CS. One hundred and seven 7EL transcripts were expressed in the smallest 7EL region defined to carry FHB resistance. CONCLUSION: 7EL contributed to CS-7EL transcriptome by direct expression and through alteration of wheat transcript profiles. FHB resistance in CS-7EL was associated with transcriptome changes suggesting a more effective defense response. A list of candidate genes for the FHB resistance locus on 7EL has been established.

Dissecting the U, M, S and C genomes of wild relatives of bread wheat (Aegilops spp.) into chromosomes and exploring their synteny with wheat.

Goat grasses (Aegilops spp.) contributed to the evolution of bread wheat and are important sources of genes and alleles for modern wheat improvement. However, their use in alien introgression breeding is hindered by poor knowledge of their genome structure and a lack of molecular tools. The analysis of large and complex genomes may be simplified by dissecting them into single chromosomes via flow cytometric sorting. In some species this is not possible due to similarities in relative DNA content among chromosomes within a karyotype. This work describes the distribution of GAA and ACG microsatellite repeats on chromosomes of the U, M, S and C genomes of Aegilops, and the use of microsatellite probes to label the chromosomes in suspension by fluorescence in situ hybridization (FISHIS). Bivariate flow cytometric analysis of chromosome DAPI fluorescence and fluorescence of FITC-labelled microsatellites made it possible to discriminate all chromosomes and sort them with negligible contamination by other chromosomes. DNA of purified chromosomes was used as a template for polymerase chain reation (PCR) using Conserved Orthologous Set (COS) markers with known positions on wheat A, B and D genomes. Wheat-Aegilops macrosyntenic comparisons using COS markers revealed significant rearrangements in the U and C genomes, while the M and S genomes exhibited structure similar to wheat. Purified chromosome fractions provided an attractive resource to investigate the structure and evolution of the Aegilops genomes, and the COS markers assigned to Aegilops chromosomes will facilitate alien gene introgression into wheat.

Sequencing flow-sorted short arm of Haynaldia villosa chromosome 4V provides insights into its molecular structure and virtual gene order.

BACKGROUND: Haynaldia villosa (H. villosa) has been recognized as a species potentially useful for wheat improvement. The availability of its genomic sequences will boost its research and application. RESULTS: In this work, the short arm of H. villosa chromosome 4V (4VS) was sorted by flow cytometry and sequenced using Illumina platform. About 170.6 Mb assembled sequences were obtained. Further analysis showed that repetitive elements accounted for about 64.6% of 4VS, while the coding fraction, which is corresponding to 1977 annotated genes, represented 1.5% of the arm. The syntenic regions of the 4VS were searched and identified on wheat group 4 chromosomes 4AL, 4BS, 4DS, Brachypodium chromosomes 1 and 4, rice chromosomes 3 and 11, and sorghum chromosomes 1, 5 and 8. Based on genome-zipper analysis, a virtual gene order comprising 735 gene loci on 4VS genome was built by referring to the Brachypodium genome, which was relatively consistent with the scaffold order determined for Ae. tauschii chromosome 4D. The homologous alleles of several cloned genes on wheat group 4 chromosomes including Rht-1 gene were identified. CONCLUSIONS: The sequences provided valuable information for mapping and positional-cloning genes located on 4VS, such as the wheat yellow mosaic virus resistance gene Wss1. The work on 4VS provided detailed insights into the genome of H. villosa, and may also serve as a model for sequencing the remaining parts of H. villosa genome.

Multiple displacement amplification of the DNA from single flow-sorted plant chromosome.

A protocol is described for production of micrograms of DNA from single copies of flow-sorted plant chromosomes. Of 183 single copies of wheat chromosome 3B, 118 (64%) were successfully amplified. Sequencing DNA amplification products using an Illumina HiSeq 2000 system to 10× coverage and merging sequences from three separate amplifications resulted in 60% coverage of the chromosome 3B reference, entirely covering 30% of its genes. The merged sequences permitted de novo assembly of 19% of chromosome 3B genes, with 10% of genes contained in a single contig, and 39% of genes covered for at least 80% of their length. The chromosome-derived sequences allowed identification of missing genic sequences in the chromosome 3B reference and short sequences similar to 3B in survey sequences of other wheat chromosomes. These observations indicate that single-chromosome sequencing is suitable to identify genic sequences on particular chromosomes, to develop chromosome-specific DNA markers, to verify assignment of DNA sequence contigs to individual pseudomolecules, and to validate whole-genome assemblies. The protocol expands the potential of chromosome genomics, which may now be applied to any plant species from which chromosome samples suitable for flow cytometry can be prepared, and opens new avenues for studies on chromosome structural heterozygosity and haplotype phasing in plants.

Exploring the tertiary gene pool of bread wheat: sequence assembly and analysis of chromosome 5M(g) of Aegilops geniculata.

Next-generation sequencing (NGS) provides a powerful tool for the discovery of important genes and alleles in crop plants and their wild relatives. Despite great advances in NGS technologies, whole-genome shotgun sequencing is cost-prohibitive for species with complex genomes. An attractive option is to reduce genome complexity to a single chromosome prior to sequencing. This work describes a strategy for studying the genomes of distant wild relatives of wheat by isolating single chromosomes from addition or substitution lines, followed by chromosome sorting using flow cytometry and sequencing of chromosomal DNA by NGS technology. We flow-sorted chromosome 5M(g) from a wheat/Aegilops geniculata disomic substitution line [DS5M(g) (5D)] and sequenced it using an Illumina HiSeq 2000 system at approximately 50 × coverage. Paired-end sequences were assembled and used for structural and functional annotation. A total of 4236 genes were annotated on 5M(g) , in close agreement with the predicted number of genes on wheat chromosome 5D (4286). Single-gene FISH indicated no major chromosomal rearrangements between chromosomes 5M(g) and 5D. Comparing chromosome 5M(g) with model grass genomes identified synteny blocks in Brachypodium distachyon, rice (Oryza sativa), sorghum (Sorghum bicolor) and barley (Hordeum vulgare). Chromosome 5M(g) -specific SNPs and cytogenetic probe-based resources were developed and validated. Deletion bin-mapped and ordered 5M(g) SNP markers will be useful to track 5M-specific introgressions and translocations. This study provides a detailed sequence-based analysis of the composition of a chromosome from a distant wild relative of bread wheat, and opens up opportunities to develop genomic resources for wild germplasm to facilitate crop improvement.

BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes.

The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC-by-BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high-resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high-resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome-scale analysis of repetitive sequences and revealed a ~800-kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone-by-clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC-contig physical map and validate sequence assembly on a chromosome-arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome-by-chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules.

Reticulate evolution of the rye genome.

Rye (Secale cereale) is closely related to wheat (Triticum aestivum) and barley (Hordeum vulgare). Due to its large genome (~8 Gb) and its regional importance, genome analysis of rye has lagged behind other cereals. Here, we established a virtual linear gene order model (genome zipper) comprising 22,426 or 72% of the detected set of 31,008 rye genes. This was achieved by high-throughput transcript mapping, chromosome survey sequencing, and integration of conserved synteny information of three sequenced model grass genomes (Brachypodium distachyon, rice [Oryza sativa], and sorghum [Sorghum bicolor]). This enabled a genome-wide high-density comparative analysis of rye/barley/model grass genome synteny. Seventeen conserved syntenic linkage blocks making up the rye and barley genomes were defined in comparison to model grass genomes. Six major translocations shaped the modern rye genome in comparison to a putative Triticeae ancestral genome. Strikingly dissimilar conserved syntenic gene content, gene sequence diversity signatures, and phylogenetic networks were found for individual rye syntenic blocks. This indicates that introgressive hybridizations (diploid or polyploidy hybrid speciation) and/or a series of whole-genome or chromosome duplications played a role in rye speciation and genome evolution.

Chromosomal genomics facilitates fine mapping of a Russian wheat aphid resistance gene.

KEY MESSAGE: Making use of wheat chromosomal resources, we developed 11 gene-associated markers for the region of interest, which allowed reducing gene interval and spanning it by four BAC clones. Positional gene cloning and targeted marker development in bread wheat are hampered by high complexity and polyploidy of its nuclear genome. Aiming to clone a Russian wheat aphid resistance gene Dn2401 located on wheat chromosome arm 7DS, we have developed a strategy overcoming problems due to polyploidy and enabling efficient development of gene-associated markers from the region of interest. We employed information gathered by GenomeZipper, a synteny-based tool combining sequence data of rice, Brachypodium, sorghum and barley, and took advantage of a high-density linkage map of Aegilops tauschii. To ensure genome- and locus-specificity of markers, we made use of survey sequence assemblies of isolated wheat chromosomes 7A, 7B and 7D. Despite the low level of polymorphism of the wheat D subgenome, our approach allowed us to add in an efficient and cost-effective manner 11 new gene-associated markers in the Dn2401 region and narrow down the target interval to 0.83 cM. Screening 7DS-specific BAC library with the flanking markers revealed a contig of four BAC clones that span the Dn2401 region in wheat cultivar 'Chinese Spring'. With the availability of sequence assemblies and GenomeZippers for each of the wheat chromosome arms, the proposed strategy can be applied for focused marker development in any region of the wheat genome.

Flow sorting of C-genome chromosomes from wild relatives of wheat Aegilops markgrafii, Ae. triuncialis and Ae. cylindrica, and their molecular organization.

BACKGROUND AND AIMS: Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (U(t)U(t)C(t)C(t)) and Ae. cylindrica (D(c)D(c)C(c)C(c)) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting. METHODS: The flow karyotypes obtained after the analysis of 4',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA. KEY RESULTS: FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7C(t), T6U(t)S.6U(t)L-5C(t)L, 1C(c) and 5D(c) could be sorted with purities ranging from 66 to 91 %, while the remaining chromosomes could be sorted in groups of 2-5. This identified a partial wheat-C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C-2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected. CONCLUSIONS: The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat.

Selfish supernumerary chromosome reveals its origin as a mosaic of host genome and organellar sequences.

Supernumerary B chromosomes are optional additions to the basic set of A chromosomes, and occur in all eukaryotic groups. They differ from the basic complement in morphology, pairing behavior, and inheritance and are not required for normal growth and development. The current view is that B chromosomes are parasitic elements comparable to selfish DNA, like transposons. In contrast to transposons, they are autonomously inherited independent of the host genome and have their own mechanisms of mitotic or meiotic drive. Although B chromosomes were first described a century ago, little is known about their origin and molecular makeup. The widely accepted view is that they are derived from fragments of A chromosomes and/or generated in response to interspecific hybridization. Through next-generation sequencing of sorted A and B chromosomes, we show that B chromosomes of rye are rich in gene-derived sequences, allowing us to trace their origin to fragments of A chromosomes, with the largest parts corresponding to rye chromosomes 3R and 7R. Compared with A chromosomes, B chromosomes were also found to accumulate large amounts of specific repeats and insertions of organellar DNA. The origin of rye B chromosomes occurred an estimated ∼1.1-1.3 Mya, overlapping in time with the onset of the genus Secale (1.7 Mya). We propose a comprehensive model of B chromosome evolution, including its origin by recombination of several A chromosomes followed by capturing of additional A-derived and organellar sequences and amplification of B-specific repeats.

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

BACKGROUND: The ~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. RESULTS: We 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. CONCLUSIONS: The 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 ACCESSIONS: EBI European Nucleotide Archive, Study no. ERP002330.

SNP Discovery for mapping alien introgressions in wheat.

BACKGROUND: Monitoring alien introgressions in crop plants is difficult due to the lack of genetic and molecular mapping information on the wild crop relatives. The tertiary gene pool of wheat is a very important source of genetic variability for wheat improvement against biotic and abiotic stresses. By exploring the 5Mg short arm (5MgS) of Aegilops geniculata, we can apply chromosome genomics for the discovery of SNP markers and their use for monitoring alien introgressions in wheat (Triticum aestivum L). RESULTS: The short arm of chromosome 5Mg of Ae. geniculata Roth (syn. Ae. ovata L.; 2n = 4x = 28, UgUgMgMg) was flow-sorted from a wheat line in which it is maintained as a telocentric chromosome. DNA of the sorted arm was amplified and sequenced using an Illumina Hiseq 2000 with ~45x coverage. The sequence data was used for SNP discovery against wheat homoeologous group-5 assemblies. A total of 2,178 unique, 5MgS-specific SNPs were discovered. Randomly selected samples of 59 5MgS-specific SNPs were tested (44 by KASPar assay and 15 by Sanger sequencing) and 84% were validated. Of the selected SNPs, 97% mapped to a chromosome 5Mg addition to wheat (the source of t5MgS), and 94% to 5Mg introgressed from a different accession of Ae. geniculata substituting for chromosome 5D of wheat. The validated SNPs also identified chromosome segments of 5MgS origin in a set of T5D-5Mg translocation lines; eight SNPs (25%) mapped to TA5601 [T5DL · 5DS-5MgS(0.75)] and three (8%) to TA5602 [T5DL · 5DS-5MgS (0.95)]. SNPs (gsnp_5ms83 and gsnp_5ms94), tagging chromosome T5DL · 5DS-5MgS(0.95) with the smallest introgression carrying resistance to leaf rust (Lr57) and stripe rust (Yr40), were validated in two released germplasm lines with Lr57 and Yr40 genes. CONCLUSION: This approach should be widely applicable for the identification of species/genome-specific SNPs. The development of a large number of SNP markers will facilitate the precise introgression and monitoring of alien segments in crop breeding programs and further enable mapping and cloning novel genes from the wild relatives of crop plants.

Major haplotype divergence including multiple germin-like protein genes, at the wheat Sr2 adult plant stem rust resistance locus.

BACKGROUND: The adult plant stem rust resistance gene Sr2 was introgressed into hexaploid wheat cultivar (cv) Marquis from tetraploid emmer wheat cv Yaroslav, to generate stem rust resistant cv Hope in the 1920s. Subsequently, Sr2 has been widely deployed and has provided durable partial resistance to all known races of Puccinia graminis f. sp. tritici. This report describes the physical map of the Sr2-carrying region on the short arm of chromosome 3B of cv Hope and compares the Hope haplotype with non-Sr2 wheat cv Chinese Spring. RESULTS: Sr2 was located to a region of 867 kb on chromosome 3B in Hope, which corresponded to a region of 567 kb in Chinese Spring. The Hope Sr2 region carried 34 putative genes but only 17 were annotated in the comparable region of Chinese Spring. The two haplotypes differed by extensive DNA sequence polymorphisms between flanking markers as well as by a major insertion/deletion event including ten Germin-Like Protein (GLP) genes in Hope that were absent in Chinese Spring. Haplotype analysis of a limited number of wheat genotypes of interest showed that all wheat genotypes carrying Sr2 possessed the GLP cluster; while, of those lacking Sr2, some, including Marquis, possessed the cluster, while some lacked it. Thus, this region represents a common presence-absence polymorphism in wheat, with presence of the cluster not correlated with presence of Sr2. Comparison of Hope and Marquis GLP genes on 3BS found no polymorphisms in the coding regions of the ten genes but several SNPs in the shared promoter of one divergently transcribed GLP gene pair and a single SNP downstream of the transcribed region of a second GLP. CONCLUSION: Physical mapping and sequence comparison showed major haplotype divergence at the Sr2 locus between Hope and Chinese Spring. Candidate genes within the Sr2 region of Hope are being evaluated for the ability to confer stem rust resistance. Based on the detailed mapping and sequencing of the locus, we predict that Sr2 does not belong to the NB-LRR gene family and is not related to previously cloned, race non-specific rust resistance genes Lr34 and Yr36.

Flow sorting and sequencing meadow fescue chromosome 4F.

The analysis of large genomes is hampered by a high proportion of repetitive DNA, which makes the assembly of short sequence reads difficult. This is also the case in meadow fescue (Festuca pratensis), which is known for good abiotic stress resistance and has been used in intergeneric hybridization with ryegrasses (Lolium spp.) to produce Festulolium cultivars. In this work, we describe a new approach to analyze the large genome of meadow fescue, which involves the reduction of sample complexity without compromising information content. This is achieved by dissecting the genome to smaller parts: individual chromosomes and groups of chromosomes. As the first step, we flow sorted chromosome 4F and sequenced it by Illumina with approximately 50× coverage. This provided, to our knowledge, the first insight into the composition of the fescue genome, enabled the construction of the virtual gene order of the chromosome, and facilitated detailed comparative analysis with the sequenced genomes of rice (Oryza sativa), Brachypodium distachyon, sorghum (Sorghum bicolor), and barley (Hordeum vulgare). Using GenomeZipper, we were able to confirm the collinearity of chromosome 4F with barley chromosome 4H and the long arm of chromosome 5H. Several new tandem repeats were identified and physically mapped using fluorescence in situ hybridization. They were found as robust cytogenetic markers for karyotyping of meadow fescue and ryegrass species and their hybrids. The ability to purify chromosome 4F opens the way for more efficient analysis of genomic loci on this chromosome underlying important traits, including freezing tolerance. Our results confirm that next-generation sequencing of flow-sorted chromosomes enables an overview of chromosome structure and evolution at a resolution never achieved before.

Frequent gene movement and pseudogene evolution is common to the large and complex genomes of wheat, barley, and their relatives.

All six arms of the group 1 chromosomes of hexaploid wheat (Triticum aestivum) were sequenced with Roche/454 to 1.3- to 2.2-fold coverage and compared with similar data sets from the homoeologous chromosome 1H of barley (Hordeum vulgare). Six to ten thousand gene sequences were sampled per chromosome. These were classified into genes that have their closest homologs in the Triticeae group 1 syntenic region in Brachypodium, rice (Oryza sativa), and/or sorghum (Sorghum bicolor) and genes that have their homologs elsewhere in these model grass genomes. Although the number of syntenic genes was similar between the homologous groups, the amount of nonsyntenic genes was found to be extremely diverse between wheat and barley and even between wheat subgenomes. Besides a small core group of genes that are nonsyntenic in other grasses but conserved among Triticeae, we found thousands of genic sequences that are specific to chromosomes of one single species or subgenome. By examining in detail 50 genes from chromosome 1H for which BAC sequences were available, we found that many represent pseudogenes that resulted from transposable element activity and double-strand break repair. Thus, Triticeae seem to accumulate nonsyntenic genes frequently. Since many of them are likely to be pseudogenes, total gene numbers in Triticeae are prone to pronounced overestimates.

Unlocking the barley genome by chromosomal and comparative genomics.

We used a novel approach that incorporated chromosome sorting, next-generation sequencing, array hybridization, and systematic exploitation of conserved synteny with model grasses to assign ~86% of the estimated ~32,000 barley (Hordeum vulgare) genes to individual chromosome arms. Using a series of bioinformatically constructed genome zippers that integrate gene indices of rice (Oryza sativa), sorghum (Sorghum bicolor), and Brachypodium distachyon in a conserved synteny model, we were able to assemble 21,766 barley genes in a putative linear order. We show that the barley (H) genome displays a mosaic of structural similarity to hexaploid bread wheat (Triticum aestivum) A, B, and D subgenomes and that orthologous genes in different grasses exhibit signatures of positive selection in different lineages. We present an ordered, information-rich scaffold of the barley genome that provides a valuable and robust framework for the development of novel strategies in cereal breeding.

Flow cytometric chromosome sorting from diploid progenitors of bread wheat, T. urartu, Ae. speltoides and Ae. tauschii.

KEY MESSAGE: Chromosomes 5A (u) , 5S and 5D can be isolated from wild progenitors, providing a chromosome-based approach to develop tools for breeding and to study the genome evolution of wheat. The three subgenomes of hexaploid bread wheat originated from Triticum urartu (A(u)A(u)), from a species similar to Aegilops speltoides (SS) (progenitor of the B genome), and from Ae. tauschii (DD). Earlier studies indicated the potential of chromosome genomics to assist gene transfer from wild relatives of wheat and discover novel genes for wheat improvement. This study evaluates the potential of flow cytometric chromosome sorting in the diploid progenitors of bread wheat. Flow karyotypes obtained by analysing DAPI-stained chromosomes were characterized and the contents of the chromosome peaks were determined. FISH analysis with repetitive DNA probes proved that chromosomes 5A(u), 5S and 5D could be sorted with purities of 78-90 %, while the remaining chromosomes could be sorted in groups of three. Twenty-five conserved orthologous set (COS) markers covering wheat homoeologous chromosome groups 1-7 were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA. These assays validated the cytomolecular results as follows: peak I on flow karyotypes contained chromosome groups 1, 4 and 6, peak II represented homoeologous group 5, while peak III consisted of groups 2, 3 and 7. The isolation of individual chromosomes of wild progenitors provides an attractive opportunity to investigate the structure and evolution of the polyploid genome and to deliver tools for wheat improvement.

Dispersion and domestication shaped the genome of bread wheat.

Despite the international significance of wheat, its large and complex genome hinders genome sequencing efforts. To assess the impact of selection on this genome, we have assembled genomic regions representing genes for chromosomes 7A, 7B and 7D. We demonstrate that the dispersion of wheat to new environments has shaped the modern wheat genome. Most genes are conserved between the three homoeologous chromosomes. We found differential gene loss that supports current theories on the evolution of wheat, with greater loss observed in the A and B genomes compared with the D. Analysis of intervarietal polymorphisms identified fewer polymorphisms in the D genome, supporting the hypothesis of early gene flow between the tetraploid and hexaploid. The enrichment for genes on the D genome that confer environmental adaptation may be associated with dispersion following wheat domestication. Our results demonstrate the value of applying next-generation sequencing technologies to assemble gene-rich regions of complex genomes and investigate polyploid genome evolution. We anticipate the genome-wide application of this reduced-complexity syntenic assembly approach will accelerate crop improvement efforts not only in wheat, but also in other polyploid crops of significance.

Flow cytometric chromosome sorting in plants: the next generation.

Genome analysis in many plant species is hampered by large genome size and by sequence redundancy due to the presence of repetitive DNA and polyploidy. One solution is to reduce the sample complexity by dissecting the genomes to single chromosomes. This can be realized by flow cytometric sorting, which enables purification of chromosomes in large numbers. Coupling the chromosome sorting technology with next generation sequencing provides a targeted and cost effective way to tackle complex genomes. The methods outlined in this article describe a procedure for preparation of chromosomal DNA suitable for next-generation sequencing.

Chromosomes in the flow to simplify genome analysis.

Nuclear genomes of human, animals, and plants are organized into subunits called chromosomes. When isolated into aqueous suspension, mitotic chromosomes can be classified using flow cytometry according to light scatter and fluorescence parameters. Chromosomes of interest can be purified by flow sorting if they can be resolved from other chromosomes in a karyotype. The analysis and sorting are carried out at rates of 10(2)-10(4) chromosomes per second, and for complex genomes such as wheat the flow sorting technology has been ground-breaking in reducing genome complexity for genome sequencing. The high sample rate provides an attractive approach for karyotype analysis (flow karyotyping) and the purification of chromosomes in large numbers. In characterizing the chromosome complement of an organism, the high number that can be studied using flow cytometry allows for a statistically accurate analysis. Chromosome sorting plays a particularly important role in the analysis of nuclear genome structure and the analysis of particular and aberrant chromosomes. Other attractive but not well-explored features include the analysis of chromosomal proteins, chromosome ultrastructure, and high-resolution mapping using FISH. Recent results demonstrate that chromosome flow sorting can be coupled seamlessly with DNA array and next-generation sequencing technologies for high-throughput analyses. The main advantages are targeting the analysis to a genome region of interest and a significant reduction in sample complexity. As flow sorters can also sort single copies of chromosomes, shotgun sequencing DNA amplified from them enables the production of haplotype-resolved genome sequences. This review explains the principles of flow cytometric chromosome analysis and sorting (flow cytogenetics), discusses the major uses of this technology in genome analysis, and outlines future directions.