Genome sequence of the progenitor of the wheat D genome Aegilops tauschii.

Aegilops tauschii is the diploid progenitor of the D genome of hexaploid wheat (Triticum aestivum, genomes AABBDD) and an important genetic resource for wheat. The large size and highly repetitive nature of the Ae. tauschii genome has until now precluded the development of a reference-quality genome sequence. Here we use an array of advanced technologies, including ordered-clone genome sequencing, whole-genome shotgun sequencing, and BioNano optical genome mapping, to generate a reference-quality genome sequence for Ae. tauschii ssp. strangulata accession AL8/78, which is closely related to the wheat D genome. We show that compared to other sequenced plant genomes, including a much larger conifer genome, the Ae. tauschii genome contains unprecedented amounts of very similar repeated sequences. Our genome comparisons reveal that the Ae. tauschii genome has a greater number of dispersed duplicated genes than other sequenced genomes and its chromosomes have been structurally evolving an order of magnitude faster than those of other grass genomes. The decay of colinearity with other grass genomes correlates with recombination rates along chromosomes. We propose that the vast amounts of very similar repeated sequences cause frequent errors in recombination and lead to gene duplications and structural chromosome changes that drive fast genome evolution.

New insights into structural organization and gene duplication in a 1.75-Mb genomic region harboring the α-gliadin gene family in Aegilops tauschii, the source of wheat D genome.

Among the wheat prolamins important for its end-use traits, α-gliadins are the most abundant, and are also a major cause of food-related allergies and intolerances. Previous studies of various wheat species estimated that between 25 and 150 α-gliadin genes reside in the Gli-2 locus regions. To better understand the evolution of this complex gene family, the DNA sequence of a 1.75-Mb genomic region spanning the Gli-2 locus was analyzed in the diploid grass, Aegilops tauschii, the ancestral source of D genome in hexaploid bread wheat. Comparison with orthologous regions from rice, sorghum, and Brachypodium revealed rapid and dynamic changes only occurring to the Ae. tauschii Gli-2 region, including insertions of high numbers of non-syntenic genes and a high rate of tandem gene duplications, the latter of which have given rise to 12 copies of α-gliadin genes clustered within a 550-kb region. Among them, five copies have undergone pseudogenization by various mutation events. Insights into the evolutionary relationship of the duplicated α-gliadin genes were obtained from their genomic organization, transcription patterns, transposable element insertions and phylogenetic analyses. An ancestral glutamate-like receptor (GLR) gene encoding putative amino acid sensor in all four grass species has duplicated only in Ae. tauschii and generated three more copies that are interspersed with the α-gliadin genes. Phylogenetic inference and different gene expression patterns support functional divergence of the Ae. tauschii GLR copies after duplication. Our results suggest that the duplicates of α-gliadin and GLR genes have likely taken different evolutionary paths; conservation for the former and neofunctionalization for the latter.

Analysis of the bread wheat genome using whole-genome shotgun sequencing.

Bread wheat (Triticum aestivum) is a globally important crop, accounting for 20 per cent of the calories consumed by humans. Major efforts are underway worldwide to increase wheat production by extending genetic diversity and analysing key traits, and genomic resources can accelerate progress. But so far the very large size and polyploid complexity of the bread wheat genome have been substantial barriers to genome analysis. Here we report the sequencing of its large, 17-gigabase-pair, hexaploid genome using 454 pyrosequencing, and comparison of this with the sequences of diploid ancestral and progenitor genomes. We identified between 94,000 and 96,000 genes, and assigned two-thirds to the three component genomes (A, B and D) of hexaploid wheat. High-resolution synteny maps identified many small disruptions to conserved gene order. We show that the hexaploid genome is highly dynamic, with significant loss of gene family members on polyploidization and domestication, and an abundance of gene fragments. Several classes of genes involved in energy harvesting, metabolism and growth are among expanded gene families that could be associated with crop productivity. Our analyses, coupled with the identification of extensive genetic variation, provide a resource for accelerating gene discovery and improving this major crop.

Rapid evolutionary dynamics in a 2.8-Mb chromosomal region containing multiple prolamin and resistance gene families in Aegilops tauschii.

Prolamin and resistance gene families are important in wheat food use and in defense against pathogen attacks, respectively. To better understand the evolution of these multi-gene families, the DNA sequence of a 2.8-Mb genomic region, representing an 8.8 cM genetic interval and harboring multiple prolamin and resistance-like gene families, was analyzed in the diploid grass Aegilops tauschii, the D-genome donor of bread wheat. Comparison with orthologous regions from rice, Brachypodium, and sorghum showed that the Ae. tauschii region has undergone dramatic changes; it has acquired more than 80 non-syntenic genes and only 13 ancestral genes are shared among these grass species. These non-syntenic genes, including prolamin and resistance-like genes, originated from various genomic regions and likely moved to their present locations via sequence evolution processes involving gene duplication and translocation. Local duplication of non-syntenic genes contributed significantly to the expansion of gene families. Our analysis indicates that the insertion of prolamin-related genes occurred prior to the separation of the Brachypodieae and Triticeae lineages. Unlike in Brachypodium, inserted prolamin genes have rapidly evolved and expanded to encode different classes of major seed storage proteins in Triticeae species. Phylogenetic analyses also showed that the multiple insertions of resistance-like genes and subsequent differential expansion of each R gene family. The high frequency of non-syntenic genes and rapid local gene evolution correlate with the high recombination rate in the 2.8-Mb region with nine-fold higher than the genome-wide average. Our results demonstrate complex evolutionary dynamics in this agronomically important region of Triticeae species.

A 4-gigabase physical map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the wheat D-genome progenitor.

The current limitations in genome sequencing technology require the construction of physical maps for high-quality draft sequences of large plant genomes, such as that of Aegilops tauschii, the wheat D-genome progenitor. To construct a physical map of the Ae. tauschii genome, we fingerprinted 461,706 bacterial artificial chromosome clones, assembled contigs, designed a 10K Ae. tauschii Infinium SNP array, constructed a 7,185-marker genetic map, and anchored on the map contigs totaling 4.03 Gb. Using whole genome shotgun reads, we extended the SNP marker sequences and found 17,093 genes and gene fragments. We showed that collinearity of the Ae. tauschii genes with Brachypodium distachyon, rice, and sorghum decreased with phylogenetic distance and that structural genome evolution rates have been high across all investigated lineages in subfamily Pooideae, including that of Brachypodieae. We obtained additional information about the evolution of the seven Triticeae chromosomes from 12 ancestral chromosomes and uncovered a pattern of centromere inactivation accompanying nested chromosome insertions in grasses. We showed that the density of noncollinear genes along the Ae. tauschii chromosomes positively correlates with recombination rates, suggested a cause, and showed that new genes, exemplified by disease resistance genes, are preferentially located in high-recombination chromosome regions.

The gene space in wheat: the complete γ-gliadin gene family from the wheat cultivar Chinese Spring.

The complete set of unique γ-gliadin genes is described for the wheat cultivar Chinese Spring using a combination of expressed sequence tag (EST) and Roche 454 DNA sequences. Assemblies of Chinese Spring ESTs yielded 11 different γ-gliadin gene sequences. Two of the sequences encode identical polypeptides and are assumed to be the result of a recent gene duplication. One gene has a 3' coding mutation that changes the reading frame in the final eight codons. A second assembly of Chinese Spring γ-gliadin sequences was generated using Roche 454 total genomic DNA sequences. The 454 assembly confirmed the same 11 active genes as the EST assembly plus two pseudogenes not represented by ESTs. These 13 γ-gliadin sequences represent the complete unique set of γ-gliadin genes for cv Chinese Spring, although not ruled out are additional genes that are exact duplications of these 13 genes. A comparison with the ESTs of two other hexaploid cultivars (Butte 86 and Recital) finds that the most active genes are present in all three cultivars, with exceptions likely due to too few ESTs for detection in Butte 86 and Recital. A comparison of the numbers of ESTs per gene indicates differential levels of expression within the γ-gliadin gene family. Genome assignments were made for 6 of the 13 Chinese Spring γ-gliadin genes, i.e., one assignment from a match to two γ-gliadin genes found within a tetraploid wheat A genome BAC and four genes that match four distinct γ-gliadin sequences assembled from Roche 454 sequences from Aegilops tauschii, the hexaploid wheat D-genome ancestor.

The B-hordein prolamin family of barley.

The spectrum of B-hordein prolamins and genes in the single barley cultivar Barke is described from an in silico analysis of 1452 B-hordein ESTs and available genomic DNA. Eleven unique B-hordein proteins are derived from EST contigs. Ten contigs encode apparent full-length B-hordeins and the eleventh contains a premature stop codon that will lead to a truncated B-hordein. The 11 sequences are placed within the two previously described classes, i.e., the B1- and B3-type B-hordeins. The number of ESTs assigned to each sequence is used as an estimate of relative gene transcription and expression. Three of the sequences account for 79% of the total ESTs, with one sequence comprises 32% of the total ESTs and has a variant C-terminus caused by an undefined sequence change history near the 3' coding terminus. The 70× difference in EST distribution among sequences points to the importance of understanding differential rates of expression within closely related gene families. Analysis of available genomic sequences confirms the EST assembly and reveals one full-length and two partial sequences of pseudogenes as evidenced by no matching ESTs for the sequences and premature stop codons and frame shifts.

Exploring the diploid wheat ancestral A genome through sequence comparison at the high-molecular-weight glutenin locus region.

The polyploid nature of hexaploid wheat (T. aestivum, AABBDD) often represents a great challenge in various aspects of research including genetic mapping, map-based cloning of important genes, and sequencing and accurately assembly of its genome. To explore the utility of ancestral diploid species of polyploid wheat, sequence variation of T. urartu (A(u)A(u)) was analyzed by comparing its 277-kb large genomic region carrying the important Glu-1 locus with the homologous regions from the A genomes of the diploid T. monococcum (A(m)A(m)), tetraploid T. turgidum (AABB), and hexaploid T. aestivum (AABBDD). Our results revealed that in addition to a high degree of the gene collinearity, nested retroelement structures were also considerably conserved among the A(u) genome and the A genomes in polyploid wheats, suggesting that the majority of the repetitive sequences in the A genomes of polyploid wheats originated from the diploid A(u) genome. The difference in the compared region between A(u) and A is mainly caused by four differential TE insertion and two deletion events between these genomes. The estimated divergence time of A genomes calculated on nucleotide substitution rate in both shared TEs and collinear genes further supports the closer evolutionary relationship of A to A(u) than to A(m). The structure conservation in the repetitive regions promoted us to develop repeat junction markers based on the A(u) sequence for mapping the A genome in hexaploid wheat. Eighty percent of these repeat junction markers were successfully mapped to the corresponding region in hexaploid wheat, suggesting that T. urartu could serve as a useful resource for developing molecular markers for genetic and breeding studies in hexaploid wheat.

RJPrimers: unique transposable element insertion junction discovery and PCR primer design for marker development.

Transposable elements (TE) exist in the genomes of nearly all eukaryotes. TE mobilization through 'cut-and-paste' or 'copy-and-paste' mechanisms causes their insertions into other repetitive sequences, gene loci and other DNA. An insertion of a TE commonly creates a unique TE junction in the genome. TE junctions are also randomly distributed along chromosomes and therefore useful for genome-wide marker development. Several TE-based marker systems have been developed and applied to genetic diversity assays, and to genetic and physical mapping. A software tool 'RJPrimers' reported here allows for accurate identification of unique repeat junctions using BLASTN against annotated repeat databases and a repeat junction finding algorithm, and then for fully automated high-throughput repeat junction-based primer design using Primer3 and BatchPrimer3. The software was tested using the rice genome and genomic sequences of Aegilops tauschii. Over 90% of repeat junction primers designed by RJPrimers were unique. At least one RJM marker per 10 Kb sequence of A. tauschii was expected with an estimate of over 0.45 million such markers in a genome of 4.02 Gb, providing an almost unlimited source of molecular markers for mapping large and complex genomes. A web-based server and a command line-based pipeline for RJPrimers are both available at http://wheat.pw.usda.gov/demos/RJPrimers/.

Genome-wide gene expression analysis supports a developmental model of low temperature tolerance gene regulation in wheat (Triticum aestivum L.).

BACKGROUND: To identify the genes involved in the development of low temperature (LT) tolerance in hexaploid wheat, we examined the global changes in expression in response to cold of the 55,052 potentially unique genes represented in the Affymetrix Wheat Genome microarray. We compared the expression of genes in winter-habit (winter Norstar and winter Manitou) and spring-habit (spring Manitou and spring Norstar)) cultivars, wherein the locus for the vernalization gene Vrn-A1 was swapped between the parental winter Norstar and spring Manitou in the derived near-isogenic lines winter Manitou and spring Norstar. Global expression of genes in the crowns of 3-leaf stage plants cold-acclimated at 6°C for 0, 2, 14, 21, 38, 42, 56 and 70 days was examined. RESULTS: Analysis of variance of gene expression separated the samples by genetic background and by the developmental stage before or after vernalization saturation was reached. Using gene-specific ANOVA we identified 12,901 genes (at p < 0.001) that change in expression with respect to both genotype and the duration of cold-treatment. We examined in more detail a subset of these genes (2,771) where expression was highly influenced by the interaction between these two main factors. Functional assignments using GO annotations showed that genes involved in transport, oxidation-reduction, and stress response were highly represented. Clustering based on the pattern of transcript accumulation identified genes that were up or down-regulated by cold-treatment. Our data indicate that the cold-sensitive lines can up-regulate known cold-responsive genes comparable to that of cold-hardy lines. The levels of expression of these genes were highly influenced by the initial rate and the duration of the gene's response to cold. We show that the Vrn-A1 locus controls the duration of gene expression but not its initial rate of response to cold treatment. Furthermore, we provide evidence that Ta.Vrn-A1 and Ta.Vrt1 originally hypothesized to encode for the same gene showed different patterns of expression and therefore are distinct. CONCLUSION: This study provides novel insight into the underlying mechanisms that regulate the expression of cold-responsive genes in wheat. The results support the developmental model of LT tolerance gene regulation and demonstrate the complex genotype by environment interactions that determine LT adaptation in winter annual cereals.

Annotation-based genome-wide SNP discovery in the large and complex Aegilops tauschii genome using next-generation sequencing without a reference genome sequence.

BACKGROUND: Many plants have large and complex genomes with an abundance of repeated sequences. Many plants are also polyploid. Both of these attributes typify the genome architecture in the tribe Triticeae, whose members include economically important wheat, rye and barley. Large genome sizes, an abundance of repeated sequences, and polyploidy present challenges to genome-wide SNP discovery using next-generation sequencing (NGS) of total genomic DNA by making alignment and clustering of short reads generated by the NGS platforms difficult, particularly in the absence of a reference genome sequence. RESULTS: An annotation-based, genome-wide SNP discovery pipeline is reported using NGS data for large and complex genomes without a reference genome sequence. Roche 454 shotgun reads with low genome coverage of one genotype are annotated in order to distinguish single-copy sequences and repeat junctions from repetitive sequences and sequences shared by paralogous genes. Multiple genome equivalents of shotgun reads of another genotype generated with SOLiD or Solexa are then mapped to the annotated Roche 454 reads to identify putative SNPs. A pipeline program package, AGSNP, was developed and used for genome-wide SNP discovery in Aegilops tauschii-the diploid source of the wheat D genome, and with a genome size of 4.02 Gb, of which 90% is repetitive sequences. Genomic DNA of Ae. tauschii accession AL8/78 was sequenced with the Roche 454 NGS platform. Genomic DNA and cDNA of Ae. tauschii accession AS75 was sequenced primarily with SOLiD, although some Solexa and Roche 454 genomic sequences were also generated. A total of 195,631 putative SNPs were discovered in gene sequences, 155,580 putative SNPs were discovered in uncharacterized single-copy regions, and another 145,907 putative SNPs were discovered in repeat junctions. These SNPs were dispersed across the entire Ae. tauschii genome. To assess the false positive SNP discovery rate, DNA containing putative SNPs was amplified by PCR from AL8/78 and AS75 and resequenced with the ABI 3730 xl. In a sample of 302 randomly selected putative SNPs, 84.0% in gene regions, 88.0% in repeat junctions, and 81.3% in uncharacterized regions were validated. CONCLUSION: An annotation-based genome-wide SNP discovery pipeline for NGS platforms was developed. The pipeline is suitable for SNP discovery in genomic libraries of complex genomes and does not require a reference genome sequence. The pipeline is applicable to all current NGS platforms, provided that at least one such platform generates relatively long reads. The pipeline package, AGSNP, and the discovered 497,118 Ae. tauschii SNPs can be accessed at (http://avena.pw.usda.gov/wheatD/agsnp.shtml).

A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening.

BACKGROUND: A five-dimensional (5-D) clone pooling strategy for screening of bacterial artificial chromosome (BAC) clones with molecular markers utilizing highly-parallel Illumina GoldenGate assays and PCR facilitates high-throughput BAC clone and BAC contig anchoring on a genetic map. However, this strategy occasionally needs manual PCR to deconvolute pools and identify truly positive clones. RESULTS: A new implementation is reported here for our previously reported clone pooling strategy. Row and column pools of BAC clones are divided into sub-pools with 1~ 2 x genome coverage. All BAC pools are screened with Illumina's GoldenGate assay and the BAC pools are deconvoluted to identify individual positive clones. Putative positive BAC clones are then further analyzed to find positive clones on the basis of them being neighbours in a contig. An exhaustive search or brute force algorithm was designed for this deconvolution and integrated into a newly developed software tool, FPCBrowser, for analyzing clone pooling data. This algorithm was used with empirical data for 55 Illumina GoldenGate SNP assays detecting SNP markers mapped on Aegilops tauschii chromosome 2D and Ae. tauschii contig maps. Clones in single contigs were successfully assigned to 48 (87%) specific SNP markers on the map with 91% precision. CONCLUSION: A new implementation of 5-D BAC clone pooling strategy employing both GoldenGate assay screening and assembled BAC contigs is shown here to be a high-throughput, low cost, rapid, and feasible approach to screening BAC libraries and anchoring BAC clones and contigs on genetic maps. The software FPCBrowser with the integrated clone deconvolution algorithm has been developed and is downloadable at http://avena.pw.usda.gov/wheatD/fpcbrowser.shtml.

Feasibility of physical map construction from fingerprinted bacterial artificial chromosome libraries of polyploid plant species.

BACKGROUND: The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy. RESULTS: The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries. CONCLUSIONS: The negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes.

Physical mapping of a large plant genome using global high-information-content-fingerprinting: the distal region of the wheat ancestor Aegilops tauschii chromosome 3DS.

BACKGROUND: Physical maps employing libraries of bacterial artificial chromosome (BAC) clones are essential for comparative genomics and sequencing of large and repetitive genomes such as those of the hexaploid bread wheat. The diploid ancestor of the D-genome of hexaploid wheat (Triticum aestivum), Aegilops tauschii, is used as a resource for wheat genomics. The barley diploid genome also provides a good model for the Triticeae and T. aestivum since it is only slightly larger than the ancestor wheat D genome. Gene co-linearity between the grasses can be exploited by extrapolating from rice and Brachypodium distachyon to Ae. tauschii or barley, and then to wheat. RESULTS: We report the use of Ae. tauschii for the construction of the physical map of a large distal region of chromosome arm 3DS. A physical map of 25.4 Mb was constructed by anchoring BAC clones of Ae. tauschii with 85 EST on the Ae. tauschii and barley genetic maps. The 24 contigs were aligned to the rice and B. distachyon genomic sequences and a high density SNP genetic map of barley. As expected, the mapped region is highly collinear to the orthologous chromosome 1 in rice, chromosome 2 in B. distachyon and chromosome 3H in barley. However, the chromosome scale of the comparative maps presented provides new insights into grass genome organization. The disruptions of the Ae. tauschii-rice and Ae. tauschii-Brachypodium syntenies were identical. We observed chromosomal rearrangements between Ae. tauschii and barley. The comparison of Ae. tauschii physical and genetic maps showed that the recombination rate across the region dropped from 2.19 cM/Mb in the distal region to 0.09 cM/Mb in the proximal region. The size of the gaps between contigs was evaluated by comparing the recombination rate along the map with the local recombination rates calculated on single contigs. CONCLUSIONS: The physical map reported here is the first physical map using fingerprinting of a complete Triticeae genome. This study demonstrates that global fingerprinting of the large plant genomes is a viable strategy for generating physical maps. Physical maps allow the description of the co-linearity between wheat and grass genomes and provide a powerful tool for positional cloning of new genes.

Nucleotide diversity maps reveal variation in diversity among wheat genomes and chromosomes.

BACKGROUND: A genome-wide assessment of nucleotide diversity in a polyploid species must minimize the inclusion of homoeologous sequences into diversity estimates and reliably allocate individual haplotypes into their respective genomes. The same requirements complicate the development and deployment of single nucleotide polymorphism (SNP) markers in polyploid species. We report here a strategy that satisfies these requirements and deploy it in the sequencing of genes in cultivated hexaploid wheat (Triticum aestivum, genomes AABBDD) and wild tetraploid wheat (Triticum turgidum ssp. dicoccoides, genomes AABB) from the putative site of wheat domestication in Turkey. Data are used to assess the distribution of diversity among and within wheat genomes and to develop a panel of SNP markers for polyploid wheat. RESULTS: Nucleotide diversity was estimated in 2114 wheat genes and was similar between the A and B genomes and reduced in the D genome. Within a genome, diversity was diminished on some chromosomes. Low diversity was always accompanied by an excess of rare alleles. A total of 5,471 SNPs was discovered in 1791 wheat genes. Totals of 1,271, 1,218, and 2,203 SNPs were discovered in 488, 463, and 641 genes of wheat putative diploid ancestors, T. urartu, Aegilops speltoides, and Ae. tauschii, respectively. A public database containing genome-specific primers, SNPs, and other information was constructed. A total of 987 genes with nucleotide diversity estimated in one or more of the wheat genomes was placed on an Ae. tauschii genetic map, and the map was superimposed on wheat deletion-bin maps. The agreement between the maps was assessed. CONCLUSIONS: In a young polyploid, exemplified by T. aestivum, ancestral species are the primary source of genetic diversity. Low effective recombination due to self-pollination and a genetic mechanism precluding homoeologous chromosome pairing during polyploid meiosis can lead to the loss of diversity from large chromosomal regions. The net effect of these factors in T. aestivum is large variation in diversity among genomes and chromosomes, which impacts the development of SNP markers and their practical utility. Accumulation of new mutations in older polyploid species, such as wild emmer, results in increased diversity and its more uniform distribution across the genome.

Conserved globulin gene across eight grass genomes identify fundamental units of the loci encoding seed storage proteins.

The wheat high molecular weight (HMW) glutenins are important seed storage proteins that determine bread-making quality in hexaploid wheat (Triticum aestivum). In this study, detailed comparative sequence analyses of large orthologous HMW glutenin genomic regions from eight grass species, representing a wide evolutionary history of grass genomes, reveal a number of lineage-specific sequence changes. These lineage-specific changes, which resulted in duplications, insertions, and deletions of genes, are the major forces disrupting gene colinearity among grass genomes. Our results indicate that the presence of the HMW glutenin gene in Triticeae genomes was caused by lineage-specific duplication of a globulin gene. This tandem duplication event is shared by Brachypodium and Triticeae genomes, but is absent in rice, maize, and sorghum, suggesting the duplication occurred after Brachypodium and Triticeae genomes diverged from the other grasses ~35 Ma ago. Aside from their physical location in tandem, the sequence similarity, expression pattern, and conserved cis-acting elements responsible for endosperm-specific expression further support the paralogous relationship between the HMW glutenin and globulin genes. While the duplicated copy in Brachypodium has apparently become nonfunctional, the duplicated copy in wheat has evolved to become the HMW glutenin gene by gaining a central prolamin repetitive domain.

Structural and transcriptional analysis of plant genes encoding the bifunctional lysine ketoglutarate reductase saccharopine dehydrogenase enzyme.

BACKGROUND: Among the dietary essential amino acids, the most severely limiting in the cereals is lysine. Since cereals make up half of the human diet, lysine limitation has quality/nutritional consequences. The breakdown of lysine is controlled mainly by the catabolic bifunctional enzyme lysine ketoglutarate reductase - saccharopine dehydrogenase (LKR/SDH). The LKR/SDH gene has been reported to produce transcripts for the bifunctional enzyme and separate monofunctional transcripts. In addition to lysine metabolism, this gene has been implicated in a number of metabolic and developmental pathways, which along with its production of multiple transcript types and complex exon/intron structure suggest an important node in plant metabolism. Understanding more about the LKR/SDH gene is thus interesting both from applied standpoint and for basic plant metabolism. RESULTS: The current report describes a wheat genomic fragment containing an LKR/SDH gene and adjacent genes. The wheat LKR/SDH genomic segment was found to originate from the A-genome of wheat, and EST analysis indicates all three LKR/SDH genes in hexaploid wheat are transcriptionally active. A comparison of a set of plant LKR/SDH genes suggests regions of greater sequence conservation likely related to critical enzymatic functions and metabolic controls. Although most plants contain only a single LKR/SDH gene per genome, poplar contains at least two functional bifunctional genes in addition to a monofunctional LKR gene. Analysis of ESTs finds evidence for monofunctional LKR transcripts in switchgrass, and monofunctional SDH transcripts in wheat, Brachypodium, and poplar. CONCLUSIONS: The analysis of a wheat LKR/SDH gene and comparative structural and functional analyses among available plant genes provides new information on this important gene. Both the structure of the LKR/SDH gene and the immediately adjacent genes show lineage-specific differences between monocots and dicots, and findings suggest variation in activity of LKR/SDH genes among plants. Although most plant genomes seem to contain a single conserved LKR/SDH gene per genome, poplar possesses multiple contiguous genes. A preponderance of SDH transcripts suggests the LKR region may be more rate-limiting. Only switchgrass has EST evidence for LKR monofunctional transcripts. Evidence for monofunctional SDH transcripts shows a novel intron in wheat, Brachypodium, and poplar.

Nonadditive expression of homoeologous genes is established upon polyploidization in hexaploid wheat.

Effects of polyploidy in allohexaploid wheat (Triticum aestivum L.) have primarily been ascribed to increases in coding sequence variation and potential to acquire new gene functions through mutation of redundant loci. However, regulatory variation that arises through new promoter and transcription factor combinations or epigenetic events may also contribute to the effects of polyploidization. In this study, gene expression was characterized in a synthetic T. aestivum line and the T. turgidum and Aegilops tauschii parents to establish a timeline for such regulatory changes and estimate the frequency of nonadditive expression of homoeologous transcripts in newly formed T. aestivum. Large-scale analysis of nonadditive gene expression was assayed by microarray expression experiments, where synthetic T. aestivum gene expression was compared to additive model values (mid-parent) calculated from parental T. turgidum and Ae. tauschii expression levels. Approximately 16% of genes were estimated to display nonadditive expression in synthetic T. aestivum. A certain fraction of the genes (2.9%) showed overdominance or underdominance. cDNA-single strand conformation polymorphism analysis was applied to measure expression of homoeologous transcripts and further verify microarray data. The results demonstrate that allopolyploidization, per se, results in rapid initiation of differential expression of homoeologous loci and nonadditive gene expression in T. aestivum.

ConservedPrimers 2.0: a high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery.

BACKGROUND: In some genomic applications it is necessary to design large numbers of PCR primers in exons flanking one or several introns on the basis of orthologous gene sequences in related species. The primer pairs designed by this target gene approach are called "intron-flanking primers" or because they are located in exonic sequences which are usually conserved between related species, "conserved primers". They are useful for large-scale single nucleotide polymorphism (SNP) discovery and marker development, especially in species, such as wheat, for which a large number of ESTs are available but for which genome sequences and intron/exon boundaries are not available. To date, no suitable high-throughput tool is available for this purpose. RESULTS: We have developed, the ConservedPrimers 2.0 pipeline, for designing intron-flanking primers for large-scale SNP discovery and marker development, and demonstrated its utility in wheat. This tool uses non-redundant wheat EST sequences, such as wheat contigs and singleton ESTs, and related genomic sequences, such as those of rice, as inputs. It aligns the ESTs to the genomic sequences to identify unique colinear exon blocks and predicts intron lengths. Intron-flanking primers are then designed based on the intron/exon information using the Primer3 core program or BatchPrimer3. Finally, a tab-delimited file containing intron-flanking primer pair sequences and their primer properties is generated for primer ordering and their PCR applications. Using this tool, 1,922 bin-mapped wheat ESTs (31.8% of the 6,045 in total) were found to have unique colinear exon blocks suitable for primer design and 1,821 primer pairs were designed from these single- or low-copy genes for PCR amplification and SNP discovery. With these primers and subsequently designed genome-specific primers, a total of 1,527 loci were found to contain one or more genome-specific SNPs. CONCLUSION: The ConservedPrimers 2.0 pipeline for designing intron-flanking primers was developed and its utility demonstrated. The tool can be used for SNP discovery, genetic variation assays and marker development for any target genome that has abundant ESTs and a related reference genome that has been fully sequenced. The ConservedPrimers 2.0 pipeline has been implemented as a command-line tool as well as a web application. Both versions are freely available at http://wheat.pw.usda.gov/demos/ConservedPrimers/.

A BAC-based physical map of Brachypodium distachyon and its comparative analysis with rice and wheat.

BACKGROUND: Brachypodium distachyon (Brachypodium) has been recognized as a new model species for comparative and functional genomics of cereal and bioenergy crops because it possesses many biological attributes desirable in a model, such as a small genome size, short stature, self-pollinating habit, and short generation cycle. To maximize the utility of Brachypodium as a model for basic and applied research it is necessary to develop genomic resources for it. A BAC-based physical map is one of them. A physical map will facilitate analysis of genome structure, comparative genomics, and assembly of the entire genome sequence. RESULTS: A total of 67,151 Brachypodium BAC clones were fingerprinted with the SNaPshot HICF fingerprinting method and a genome-wide physical map of the Brachypodium genome was constructed. The map consisted of 671 contigs and 2,161 clones remained as singletons. The contigs and singletons spanned 414 Mb. A total of 13,970 gene-related sequences were detected in the BAC end sequences (BES). These gene tags aligned 345 contigs with 336 Mb of rice genome sequence, showing that Brachypodium and rice genomes are generally highly colinear. Divergent regions were mainly in the rice centromeric regions. A dot-plot of Brachypodium contigs against the rice genome sequences revealed remnants of the whole-genome duplication caused by paleotetraploidy, which were previously found in rice and sorghum. Brachypodium contigs were anchored to the wheat deletion bin maps with the BES gene-tags, opening the door to Brachypodium-Triticeae comparative genomics. CONCLUSION: The construction of the Brachypodium physical map, and its comparison with the rice genome sequence demonstrated the utility of the SNaPshot-HICF method in the construction of BAC-based physical maps. The map represents an important genomic resource for the completion of Brachypodium genome sequence and grass comparative genomics. A draft of the physical map and its comparisons with rice and wheat are available at http://phymap.ucdavis.edu/brachypodium/.