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.

The CC-NB-LRR protein BSR1 from Brachypodium confers resistance to Barley stripe mosaic virus in gramineous plants by recognising TGB1 movement protein.

Although some nucleotide binding, leucine-rich repeat immune receptor (NLR) proteins conferring resistance to specific viruses have been identified in dicot plants, NLR proteins involved in viral resistance have not been described in monocots. We have used map-based cloning to isolate the CC-NB-LRR (CNL) Barley stripe mosaic virus (BSMV) resistance gene barley stripe resistance 1 (BSR1) from Brachypodium distachyon Bd3-1 inbred line. Stable BSR1 transgenic Brachypodium line Bd21-3, barley (Golden Promise) and wheat (Kenong 199) plants developed resistance against BSMV ND18 strain. Allelic variation analyses indicated that BSR1 is present in several Brachypodium accessions collected from countries in the Middle East. Protein domain swaps revealed that the intact LRR domain and the C-terminus of BSR1 are required for resistance. BSR1 interacts with the BSMV ND18 TGB1 protein in planta and shows temperature-sensitive antiviral resistance. The R390 and T392 residues of TGB1ND (ND18 strain) and the G196 and K197 residues within the BSR1 P-loop motif are key amino acids required for immune activation. BSR1 is the first cloned virus resistance gene encoding a typical CNL protein in monocots, highlighting the utility of the Brachypodium model for isolation and analysis of agronomically important genes for crop improvement.

Characterization of Pm68, a new powdery mildew resistance gene on chromosome 2BS of Greek durum wheat TRI 1796.

KEY MESSAGE: New powdery mildew resistance gene Pm68 was found in the terminal region of chromosome 2BS of Greek durum wheat TRI 1796. The co-segregated molecular markers could be used for MAS. Durum wheat (Triticum turgidum L. var. durum Desf.) is not only an important cereal crop for pasta making, but also a genetic resource for common wheat improvement. In the present study, a Greek durum wheat TRI 1796 was found to confer high resistance to all 22 tested isolates of Blumeria graminis f. sp. tritici (Bgt). Inheritance study on the F1 plants and the F2 population derived from the cross TRI 1796/PI 584832 revealed that the resistance in TRI 1796 was controlled by a single dominant gene, herein designated Pm68. Using the bulked segregant RNA-Seq (BSR-Seq) analysis combined with molecular analysis, Pm68 was mapped to the terminal part of the short arm of chromosome 2B and flanked by markers Xdw04 and Xdw12/Xdw13 with genetic distances of 0.22 cM each. According to the reference genome of durum wheat cv. Svevo, the corresponding physical region spanned the Pm68 locus was about 1.78-Mb, in which a number of disease resistance-related genes were annotated. This study reports the new powdery mildew resistance gene Pm68 that would be a valuable resource for improvement of both common wheat and durum wheat. The co-segregated markers (Xdw05-Xdw11) developed here would be useful tools for marker-assisted selection (MAS) in breeding.

Exploiting the reference genome sequence of hexaploid wheat: a proteomic study of flour proteins from the cultivar Chinese Spring.

Although the economic value of wheat flour is determined by the complement of gluten proteins, these proteins have been challenging to study because of the complexity of the major protein groups and the tremendous sequence diversity among wheat cultivars. The completion of a high-quality wheat genome sequence from the reference wheat Chinese Spring recently facilitated the assembly and annotation of a complete set of gluten protein genes from a single cultivar, making it possible to link individual proteins in the flour to specific gene sequences. In a proteomic analysis of total wheat flour protein from Chinese Spring using quantitative two-dimensional gel electrophoresis combined with tandem mass spectrometry, gliadins or low-molecular-weight glutenin subunits were identified as the predominant proteins in 72 protein spots. Individual spots were associated with 40 of 56 Chinese Spring gene sequences, including 16 of 26 alpha gliadins, 10 of 11 gamma gliadins, six of seven omega gliadins, one of two delta gliadins, and nine of ten LMW-GS. Most genes that were not associated with protein spots were either expressed at low levels in endosperm or encoded proteins with high similarity to other proteins. A wide range of protein accumulation levels were observed and discrepancies between transcript levels and protein levels were noted. This work together with similar studies using other commercial cultivars should provide new insight into the molecular basis of wheat flour quality and allergenic potential.

A rare single nucleotide variant in Pm5e confers powdery mildew resistance in common wheat.

Powdery mildew poses severe threats to wheat production. The most sustainable way to control this disease is through planting resistant cultivars. We report the map-based cloning of the powdery mildew resistance allele Pm5e from a Chinese wheat landrace. We applied a two-step bulked segregant RNA sequencing (BSR-Seq) approach in developing tightly linked or co-segregating markers to Pm5e. The first BSR-Seq used phenotypically contrasting bulks of recombinant inbred lines (RILs) to identify Pm5e-linked markers. The second BSR-Seq utilized bulks of genetic recombinants screened from a fine-mapping population to precisely quantify the associated genomic variation in the mapping interval, and identified the Pm5e candidate genes. The function of Pm5e was validated by transgenic assay, loss-of-function mutants and haplotype association analysis. Pm5e encodes a nucleotide-binding domain leucine-rich-repeat-containing (NLR) protein. A rare nonsynonymous single nucleotide variant (SNV) within the C-terminal leucine rich repeat (LRR) domain is responsible for the gain of powdery mildew resistance function of Pm5e, an allele endemic to wheat landraces of Shaanxi province of China. Results from this study demonstrate the value of landraces in discovering useful genes for modern wheat breeding. The key SNV associated with powdery mildew resistance will be useful for marker-assisted selection of Pm5e in wheat breeding programs.

Analysis of the Gli-D2 locus identifies a genetic target for simultaneously improving the breadmaking and health-related traits of common wheat.

Gliadins are a major component of wheat seed proteins. However, the complex homoeologous Gli-2 loci (Gli-A2, -B2 and -D2) that encode the α-gliadins in commercial wheat are still poorly understood. Here we analyzed the Gli-D2 locus of Xiaoyan 81 (Xy81), a winter wheat cultivar. A total of 421.091 kb of the Gli-D2 sequence was assembled from sequencing multiple bacterial artificial clones, and 10 α-gliadin genes were annotated. Comparative genomic analysis showed that Xy81 carried only eight of the α-gliadin genes of the D genome donor Aegilops tauschii, with two of them each experiencing a tandem duplication. A mutant line lacking Gli-D2 (DLGliD2) consistently exhibited better breadmaking quality and dough functionalities than its progenitor Xy81, but without penalties in other agronomic traits. It also had an elevated lysine content in the grains. Transcriptome analysis verified the lack of Gli-D2 α-gliadin gene expression in DLGliD2. Furthermore, the transcript and protein levels of protein disulfide isomerase were both upregulated in DLGliD2 grains. Consistent with this finding, DLGliD2 had increased disulfide content in the flour. Our work sheds light on the structure and function of Gli-D2 in commercial wheat, and suggests that the removal of Gli-D2 and the gliadins specified by it is likely to be useful for simultaneously enhancing the end-use and health-related traits of common wheat. Because gliadins and homologous proteins are widely present in grass species, the strategy and information reported here may be broadly useful for improving the quality traits of diverse cereal crops.

Rapid evolution of α-gliadin gene family revealed by analyzing Gli-2 locus regions of wild emmer wheat.

α-Gliadins are a major group of gluten proteins in wheat flour that contribute to the end-use properties for food processing and contain major immunogenic epitopes that can cause serious health-related issues including celiac disease (CD). α-Gliadins are also the youngest group of gluten proteins and are encoded by a large gene family. The majority of the gene family members evolved independently in the A, B, and D genomes of different wheat species after their separation from a common ancestral species. To gain insights into the origin and evolution of these complex genes, the genomic regions of the Gli-2 loci encoding α-gliadins were characterized from the tetraploid wild emmer, a progenitor of hexaploid bread wheat that contributed the AABB genomes. Genomic sequences of Gli-2 locus regions for the wild emmer A and B genomes were first reconstructed using the genome sequence scaffolds along with optical genome maps. A total of 24 and 16 α-gliadin genes were identified for the A and B genome regions, respectively. α-Gliadin pseudogene frequencies of 86% for the A genome and 69% for the B genome were primarily caused by C to T substitutions in the highly abundant glutamine codons, resulting in the generation of premature stop codons. Comparison with the homologous regions from the hexaploid wheat cv. Chinese Spring indicated considerable sequence divergence of the two A genomes at the genomic level. In comparison, conserved regions between the two B genomes were identified that included α-gliadin pseudogenes containing shared nested TE insertions. Analyses of the genomic organization and phylogenetic tree reconstruction indicate that although orthologous gene pairs derived from speciation were present, large portions of α-gliadin genes were likely derived from differential gene duplications or deletions after the separation of the homologous wheat genomes ~ 0.5 MYA. The higher number of full-length intact α-gliadin genes in hexaploid wheat than that in wild emmer suggests that human selection through domestication might have an impact on α-gliadin evolution. Our study provides insights into the rapid and dynamic evolution of genomic regions harboring the α-gliadin genes in wheat.

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.

Modular assembly of transposable element arrays by microsatellite targeting in the guayule and rice genomes.

BACKGROUND: Guayule (Parthenium argentatum A. Gray) is a rubber-producing desert shrub native to Mexico and the United States. Guayule represents an alternative to Hevea brasiliensis as a source for commercial natural rubber. The efficient application of modern molecular/genetic tools to guayule improvement requires characterization of its genome. RESULTS: The 1.6 Gb guayule genome was sequenced, assembled and annotated. The final 1.5 Gb assembly, while fragmented (N50 = 22 kb), maps > 95% of the shotgun reads and is essentially complete. Approximately 40,000 transcribed, protein encoding genes were annotated on the assembly. Further characterization of this genome revealed 15 families of small, microsatellite-associated, transposable elements (TEs) with unexpected chromosomal distribution profiles. These SaTar (Satellite Targeted) elements, which are non-autonomous Mu-like elements (MULEs), were frequently observed in multimeric linear arrays of unrelated individual elements within which no individual element is interrupted by another. This uniformly non-nested TE multimer architecture has not been previously described in either eukaryotic or prokaryotic genomes. Five families of similarly distributed non-autonomous MULEs (microsatellite associated, modularly assembled) were characterized in the rice genome. Families of TEs with similar structures and distribution profiles were identified in sorghum and citrus. CONCLUSION: The sequencing and assembly of the guayule genome provides a foundation for application of current crop improvement technologies to this plant. In addition, characterization of this genome revealed SaTar elements with distribution profiles unique among TEs. Satar targeting appears based on an alternative MULE recombination mechanism with the potential to impact gene evolution.

Towards reducing the immunogenic potential of wheat flour: omega gliadins encoded by the D genome of hexaploid wheat may also harbor epitopes for the serious food allergy WDEIA.

BACKGROUND: Omega-5 gliadins are a group of highly repetitive gluten proteins in wheat flour encoded on the 1B chromosome of hexaploid wheat. These proteins are the major sensitizing allergens in a severe form of food allergy called wheat-dependent exercise-induced anaphylaxis (WDEIA). The elimination of omega-5 gliadins from wheat flour through biotechnology or breeding approaches could reduce the immunogenic potential and adverse health effects of the flour. RESULTS: A mutant line missing low-molecular weight glutenin subunits encoded at the Glu-B3 locus was selected previously from a doubled haploid population generated from two Korean wheat cultivars. Analysis of flour from the mutant line by 2-dimensional gel electrophoresis coupled with tandem mass spectrometry revealed that the omega-5 gliadins and several gamma gliadins encoded by the closely linked Gli-B1 locus were also missing as a result of a deletion of at least 5.8 Mb of chromosome 1B. Two-dimensional immunoblot analysis of flour proteins using sera from WDEIA patients showed reduced IgE reactivity in the mutant relative to the parental lines due to the absence of the major omega-5 gliadins. However, two minor proteins showed strong reactivity to patient sera in both the parental and the mutant lines and also reacted with a monoclonal antibody against omega-5 gliadin. Analysis of the two minor reactive proteins by mass spectrometry revealed that both proteins correspond to omega-5 gliadin genes encoded on chromosome 1D that were thought previously to be pseudogenes. CONCLUSIONS: While breeding approaches can be used to reduce the levels of the highly immunogenic omega-5 gliadins in wheat flour, these approaches are complicated by the genetic linkage of different classes of gluten protein genes and the finding that omega-5 gliadins may be encoded on more than one chromosome. The work illustrates the importance of detailed knowledge about the genomic regions harboring the major gluten protein genes in individual wheat cultivars for future efforts aimed at reducing the immunogenic potential of wheat flour.

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.

GSP: a web-based platform for designing genome-specific primers in polyploids.

MOTIVATION: The sequences among subgenomes in a polyploid species have high similarity, making it difficult to design genome-specific primers for sequence analysis. RESULTS: We present GSP, a web-based platform to design genome-specific primers that distinguish subgenome sequences in a polyploid genome. GSP uses BLAST to extract homeologous sequences of the subgenomes in existing databases, performs a multiple sequence alignment, and design primers based on sequence variants in the alignment. An interactive primers diagram, a sequence alignment viewer and a virtual electrophoresis are displayed as parts of the primer design result. GSP also designs specific primers from multiple sequences uploaded by users. AVAILABILITY AND IMPLEMENTATION: GSP is a user-friendly and efficient web platform freely accessible at http://probes.pw.usda.gov/GSP Source code and command-line application are available at https://github.com/bioinfogenome/GSP CONTACTS: yong.gu@ars.usda.gov or devin.coleman-derr@ars.usda.gov SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

Development of a D genome specific marker resource for diploid and hexaploid wheat.

BACKGROUND: Mapping and map-based cloning of genes that control agriculturally and economically important traits remain great challenges for plants with complex highly repetitive genomes such as those within the grass tribe, Triticeae. Mapping limitations in the Triticeae are primarily due to low frequencies of polymorphic gene markers and poor genetic recombination in certain genetic regions. Although the abundance of repetitive sequence may pose common problems in genome analysis and sequence assembly of large and complex genomes, they provide repeat junction markers with random and unbiased distribution throughout chromosomes. Hence, development of a high-throughput mapping technology that combine both gene-based and repeat junction-based markers is needed to generate maps that have better coverage of the entire genome. RESULTS: In this study, the available genomics resource of the diploid Aegilop tauschii, the D genome donor of bread wheat, were used to develop genome specific markers that can be applied for mapping in modern hexaploid wheat. A NimbleGen array containing both gene-based and repeat junction probe sequences derived from Ae. tauschii was developed and used to map the Chinese Spring nullisomic-tetrasomic lines and deletion bin lines of the D genome chromosomes. Based on these mapping data, we have now anchored 5,171 repeat junction probes and 10,892 gene probes, corresponding to 5,070 gene markers, to the delineated deletion bins of the D genome. The order of the gene-based markers within the deletion bins of the Chinese Spring can be inferred based on their positions on the Ae. tauschii genetic map. Analysis of the probe sequences against the Chinese Spring chromosome sequence assembly database facilitated mapping of the NimbleGen probes to the sequence contigs and allowed assignment or ordering of these sequence contigs within the deletion bins. The accumulated length of anchored sequence contigs is about 155 Mb, representing ~ 3.2 % of the D genome. A specific database was developed to allow user to search or BLAST against the probe sequence information and to directly download PCR primers for mapping specific genetic loci. CONCLUSIONS: In bread wheat, aneuploid stocks have been extensively used to assign markers linked with genes/traits to chromosomes, chromosome arms, and their specific bins. Through this study, we added thousands of markers to the existing wheat chromosome bin map, representing a significant step forward in providing a resource to navigate the wheat genome. The database website ( http://probes.pw.usda.gov/ATRJM/ ) provides easy access and efficient utilization of the data. The resources developed herein can aid map-based cloning of traits of interest and the sequencing of the D genome of hexaploid wheat.

Dynamic evolution of resistance gene analogs in the orthologous genomic regions of powdery mildew resistance gene MlIW170 in Triticum dicoccoides and Aegilops tauschii.

KEY MESSAGE: Rapid evolution of powdery mildew resistance gene MlIW170 orthologous genomic regions in wheat subgenomes. Wheat is one of the most important staple grain crops in the world and also an excellent model for plant ploidy evolution research with different ploidy levels from diploid to hexaploid. Powdery mildew disease caused by Blumeria graminis f.sp. tritici can result in significant loss in both grain yield and quality in wheat. In this study, the wheat powdery mildew resistance gene MlIW170 locus located at the Triticum dicoccoides chromosome 2B short arm was further characterized by constructing and sequencing a BAC-based physical map contig covering a 0.3 cM genetic distance region (880 kb) and developing additional markers to delineate the resistance gene within a 0.16 cM genetic interval (372 kb). Comparative analyses of the T. dicoccoides 2BS region with the orthologous Aegilops tauschii 2DS region showed great gene colinearity, including the structure organization of both types of RGA1/2-like and RPS2-like resistance genes. Comparative analyses with the orthologous regions from Brachypodium and rice genomes revealed considerable dynamic evolutionary changes that have re-shaped this MlIW170 region in the wheat genome, resulting in a high number of non-syntenic genes including resistance-related genes. This result might reflect the rapid evolution in R-gene regions. Phylogenetic analysis on these resistance-related gene sequences indicated the duplication of these genes in the MlIW170 region, occurred before the separation of the wheat B and D genomes.

Accelerated evolution of the mitochondrial genome in an alloplasmic line of durum wheat.

BACKGROUND: Wheat is an excellent plant species for nuclear mitochondrial interaction studies due to availability of large collection of alloplasmic lines. These lines exhibit different vegetative and physiological properties than their parents. To investigate the level of sequence changes introduced into the mitochondrial genome under the alloplasmic condition, three mitochondrial genomes of the Triticum-Aegilops species were sequenced: 1) durum alloplasmic line with the Ae. longissima cytoplasm that carries the T. turgidum nucleus designated as (lo) durum, 2) the cytoplasmic donor line, and 3) the nuclear donor line. RESULTS: The mitochondrial genome of the T. turgidum was 451,678 bp in length with high structural and nucleotide identity to the previously characterized T. aestivum genome. The assembled mitochondrial genome of the (lo) durum and the Ae. longissima were 431,959 bp and 399,005 bp in size, respectively. The high sequence coverage for all three genomes allowed analysis of heteroplasmy within each genome. The mitochondrial genome structure in the alloplasmic line was genetically distant from both maternal and paternal genomes. The alloplasmic durum and the Ae. longissima carry the same versions of atp6, nad6, rps19-p, cob and cox2 exon 2 which are different from the T. turgidum parent. Evidence of paternal leakage was also observed by analyzing nad9 and orf359 among all three lines. Nucleotide search identified a number of open reading frames, of which 27 were specific to the (lo) durum line. CONCLUSIONS: Several heteroplasmic regions were observed within genes and intergenic regions of the mitochondrial genomes of all three lines. The number of rearrangements and nucleotide changes in the mitochondrial genome of the alloplasmic line that have occurred in less than half a century was significant considering the high sequence conservation between the T. turgidum and the T. aestivum that diverged from each other 10,000 years ago. We showed that the changes in genes were not limited to paternal leakage but were sufficiently significant to suggest that other mechanisms, such as recombination and mutation, were responsible. The newly formed ORFs, differences in gene sequences and copy numbers, heteroplasmy, and substoichiometric changes show the potential of the alloplasmic condition to accelerate evolution towards forming new mitochondrial genomes.

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.

Complete Genome of Agrobacterium fabrum Strain 1D1416.

This work reports the draft genome of Agrobacterium fabrum strain 1D1416. The assembled genome is composed of a 2,837,379-bp circular chromosome, a 2,043,296-bp linear chromosome, a 519,735-bp AT1 plasmid, a 188,396-bp AT2 plasmid, and a 196,706-bp Ti virulence plasmid. The nondisarmed strain produces gall-like structures in citrus tissue.

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.