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Leafy spurge (Euphorbia esulaL.) is an invasive perennial weed infesting range and recreational lands of North America. Previous research and omics projects withE. esulahave helped develop it as a model for studying many aspects of perennial plant development and response to abiotic stress. However, the lack of an assembled genome forE. esulahas limited the power of previous transcriptomics studies to identify functional promoter elements and transcription factor binding sites. An assembled genome forE. esulawould enhance our understanding of signaling processes controlling plant development and responses to environmental stress and provide a better understanding of genetic factors impacting weediness traits, evolution, and herbicide resistance. A comprehensive transcriptome database would also assist in analyzing future RNA-seq studies and is needed to annotate and assess genomic sequence assemblies. Here, we assembled and annotated 56,234 unigenes from an assembly of 589,235 RNA-seq-derived contigs and a previously published Sanger-sequenced expressed sequence tag collection. The resulting data indicate that we now have sequence for >90% of the expressedE. esulaprotein-coding genes. We also assembled the gene space ofE. esulaby using a limited coverage (18X) genomic sequence database. In this study, the programs Velvet and Trinity produced the best gene-space assemblies based on representation of expressed and conserved eukaryotic genes. The results indicate thatE. esulacontains as much as 23% repetitive sequences, of which 11% are unique. Our sequence data were also sufficient for assembling a full chloroplast and partial mitochondrial genome. Further, marker analysis identified more than 150,000 high-quality variants in ourE. esulaL-RNA–scaffolded, whole-genome, Trinity-assembled genome. Based on these results,E. esulaappears to have limited heterozygosity. This study provides a blueprint for low-cost genomic assemblies in weed species and new resources for identifying conserved and novel promoter regions among coordinately expressed genes ofE. esula.

The variety and quantity of data being produced by biological research has grown dramatically in recent years, resulting in an expansion of our understanding of biological systems. However, this abundance of data has brought new challenges, especially in curation. The role of biocurators is in part to filter research outcomes as they are generated, not only so that information is formatted and consolidated into locations that can provide long-term data sustainability, but also to ensure that the relevant data that was captured is reliable, reusable, and accessible. In many ways, biocuration lies somewhere between an art and a science. At GrainGenes ( https://wheat.pw.usda.gov;https://graingenes.org ), a long-time, stably-funded centralized repository for data about wheat, barley, rye, oat, and other small grains, curators have implemented a workflow for locating, parsing, and uploading new data so that the most important, peer-reviewed, high-quality research is available to users as quickly as possible with rich links to past research outcomes. In this report, we illustrate the principles and practical considerations of curation that we follow at GrainGenes with three case studies for curating a gene, a quantitative trait locus (QTL), and genomic elements. These examples demonstrate how our work allows users, i.e., small grains geneticists and breeders, to harness high-quality small grains data at GrainGenes to help them develop plants with enhanced agronomic traits.

In March 2019, 45 scientists and software engineers from around the world converged at the University of California, Santa Cruz for the first pangenomics codeathon. The purpose of the meeting was to propose technical specifications and standards for a usable human pangenome as well as to build relevant tools for genome graph infrastructures. During the meeting, the group held several intense and productive discussions covering a diverse set of topics, including advantages of graph genomes over a linear reference representation, design of new methods that can leverage graph-based data structures, and novel visualization and annotation approaches for pangenomes. Additionally, the participants self-organized themselves into teams that worked intensely over a three-day period to build a set of pipelines and tools for specific pangenomic applications. A summary of the questions raised and the tools developed are reported in this manuscript.

GrainGenes (https://wheat.pw.usda.gov or https://graingenes.org) is an international centralized repository for curated, peer-reviewed datasets useful to researchers working on wheat, barley, rye and oat. GrainGenes manages genomic, genetic, germplasm and phenotypic datasets through a dynamically generated web interface for facilitated data discovery. Since 1992, GrainGenes has served geneticists and breeders in both the public and private sectors on six continents. Recently, several new datasets were curated into the database along with new tools for analysis. The GrainGenes homepage was enhanced by making it more visually intuitive and by adding links to commonly used pages. Several genome assemblies and genomic tracks are displayed through the genome browsers at GrainGenes, including the Triticum aestivum (bread wheat) cv. ‘Chinese Spring’ IWGSC RefSeq v1.0 genome assembly, the Aegilops tauschii (D genome progenitor) Aet v4.0 genome assembly, the Triticum turgidum ssp. dicoccoides (wild emmer wheat) cv. ‘Zavitan’ WEWSeq v.1.0 genome assembly, a T. aestivum (bread wheat) pangenome, the Hordeum vulgare (barley) cv. ‘Morex’ IBSC genome assembly, the Secale cereale (rye) select ‘Lo7’ assembly, a partial hexaploid Avena sativa (oat) assembly and the Triticum durum cv. ‘Svevo’ (durum wheat) RefSeq Release 1.0 assembly. New genetic maps and markers were added and can be displayed through CMAP. Quantitative trait loci, genetic maps and genes from the Wheat Gene Catalogue are indexed and linked through the Wheat Information System (WheatIS) portal. Training videos were created to help users query and reach the data they need. GSP (Genome Specific Primers) and PIECE2 (Plant Intron Exon Comparison and Evolution) tools were implemented and are available to use. As more small grains reference sequences become available, GrainGenes will play an increasingly vital role in helping researchers improve crops.

© The Author(s) 2018. Published by Oxford University Press. The future of agricultural research depends on data. The sheer volume of agricultural biological data being produced today makes excellent data management essential. Governmental agencies, publishers and science funders require data management plans for publicly funded research. Furthermore, the value of data increases exponentially when they are properly stored, described, integrated and shared, so that they can be easily utilized in future analyses. AgBioData (https://www.agbiodata.org) is a consortium of people working at agricultural biological databases, data archives and knowledgbases who strive to identify common issues in database development, curation and management, with the goal of creating database products that are more Findable, Accessible, Interoperable and Reusable. We strive to promote authentic, detailed, accurate and explicit communication between all parties involved in scientific data. As a step toward this goal, we present the current state of biocuration, ontologies, metadata and persistence, database platforms, programmatic (machine) access to data, communication and sustainability with regard to data curation. Each section describes challenges and opportunities for these topics, along with recommendations and best practices.

Plant height and spike length and angle are important agronomic traits in the production of barley ( Hordeum vulgare L.) due to strong correlations with lodging and disease. The objective of this study was to use QTL analysis to identify genetic regions associated with each trait in a recombinant inbred line (RIL) mapping population derived from a cross of Falcon by Azhul. Falcon is a spring six-row hulless feed barley with long spikes displaying obtuse angles, while Azhul is a spring dwarf, six-row hulless food barley with short spikes displaying acute angles. The population was genotyped using SNP, DArT and SSR markers and quantitative trait loci (QTL) were detected on chromosomes 2H (102.8 cM, spike length), 3H (89.2 cM, plant height and 38.2, spike angle and length), 4H (19.0 cM, spike length), and 5H (106.7 cM, spike angle). In conclusion, we developed a barley genetic map, which incorporated SNP, DArT and SSR markers, for detection of height and spike length and angle QTL. Three spike angle, one spike length and one plant height QTL were novel and by using comparative genomics we identified possible candidate genes involved in gibberellic acid signaling and auxin- and ethylene-responsive pathways. This knowledge can be used to generate suitable markers for barley breeding improvement.

Gene families often show degrees of differences in terms of exon–intron structures depending on their distinct evolutionary histories. Comparative analysis of gene structures is important for understanding their evolutionary and functional relationships within plant species. Here, we present a comparative genomics database named PIECE (http://wheat.pw.usda.gov/piece) for Plant Intron and Exon Comparison and Evolution studies. The database contains all the annotated genes extracted from 25 sequenced plant genomes. These genes were classified based on Pfam motifs. Phylogenetic trees were pre-constructed for each gene category. PIECE provides a user-friendly interface for different types of searches and a graphical viewer for displaying a gene structure pattern diagram linked to the resulting bootstrapped dendrogram for each gene family. The gene structure evolution of orthologous gene groups was determined using the GLOOME, Exalign and GECA software programs that can be accessed within the database. PIECE also provides a web server version of the software, GSDraw, for drawing schematic diagrams of gene structures. PIECE is a powerful tool for comparing gene sequences and provides valuable insights into the evolution of gene structure in plant genomes.

Natural rubber biosynthesis in guayule (Parthenium argentatum Gray) is associated with moderately cold night temperatures. To begin to dissect the molecular events triggered by cold temperatures that govern rubber synthesis induction in guayule, the transcriptome of bark tissue, where rubber is produced, was investigated. A total of 11,748 quality expressed sequence tags (ESTs) were obtained. The vast majority of ESTs encoded proteins that are similar to stress-related proteins, whereas those encoding rubber biosynthesis-related proteins comprised just over one percent of the ESTs. Sequence information derived from the ESTs was used to design primers for quantitative analysis of the expression of genes that encode selected enzymes and proteins with potential impact on rubber biosynthesis in field-grown guayule plants, including 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, small rubber particle protein, allene oxide synthase, and cis-prenyl transferase. Gene expression was studied for field-grown plants during the normal course of seasonal variation in temperature (monthly average maximum 41.7 °C to minimum 0 °C, from November 2005 through March 2007) and rubber transferase enzymatic activity was also evaluated. Levels of gene expression did not correlate with air temperatures nor with rubber transferase activity. Interestingly, a sudden increase in night temperature 10 days before harvest took place in advance of the highest CPT gene expression level.

Allotetraploid (2n = 4x = 28) Leymus triticoides and Leymus cinereus are divergent perennial grasses, which form fertile hybrids. Genetic maps with n = 14 linkage groups (LG) comprised with 1,583 AFLP and 67 heterologous anchor markers were previously used for mapping quantitative trait loci (QTLs) in these hybrids, and chromosomes of other Leymus wildryes have been transferred to wheat. However, identifications of the x = 7 homoeologous groups were tenuous and genetic research has been encumbered by a lack of functional, conserved gene marker sequences. Herein, we mapped 350 simple sequence repeats and 26 putative lignin biosynthesis genes from a new Leymus EST library and constructed one integrated consensus map with 799 markers, including 375 AFLPs and 48 heterologous markers, spanning 2,381 centiMorgans. LG1b and LG6b were reassigned as LG6b* and LG1b*, respectively, and LG4Ns and LG4Xm were inverted so that all 14 linkage groups are aligned to the x = 7 Triticeae chromosomes based on EST alignments to barley and other reference genomes. Amplification of 146 mapped Leymus ESTs representing six of the seven homoeologous groups was shown for 17 wheat-Leymus chromosome introgression lines. Reciprocal translocations between 4L and 5L in both Leymus and Triticum monococcum were aligned to the same regions of Brachypodium chromosome 1. A caffeic acid O-methyltransferase locus aligned to fiber QTL peaks on Leymus LG7a and brown midrib mutations of maize and sorghum. Glaucousness genes on Leymus and wheat chromosome 2 were aligned to the same region of Brachypodium chromosome 5. Markers linked to the S self-incompatibility gene on Leymus LG1a cosegregated with markers on LG2b, possibly cross-linked by gametophytic selection. Homoeologous chromosomes 1 and 2 harbor the S and Z gametophytic self-incompatibility genes of Phalaris, Secale, and Lolium, but the Leymus chromosome-2 self-incompatibility gene aligns to a different region on Brachypodium chromosome 5. Nevertheless, cosegregation of self-incompatibility genes on Leymus presents a powerful system for mapping these loci.

Single-nucleotide polymorphism was used in the construction of an expressed sequence tag map of Aegilops tauschii , the diploid source of the wheat D genome. Comparisons of the map with the rice and sorghum genome sequences revealed 50 inversions and translocations; 2, 8, and 40 were assigned respectively to the rice, sorghum, and Ae. tauschii lineages, showing greatly accelerated genome evolution in the large Triticeae genomes. The reduction of the basic chromosome number from 12 to 7 in the Triticeae has taken place by a process during which an entire chromosome is inserted by its telomeres into a break in the centromeric region of another chromosome. The original centromere–telomere polarity of the chromosome arms is maintained in the new chromosome. An intrachromosomal telomere–telomere fusion resulting in a pericentric translocation of a chromosome segment or an entire arm accompanied or preceded the chromosome insertion in some instances. Insertional dysploidy has been recorded in three grass subfamilies and appears to be the dominant mechanism of basic chromosome number reduction in grasses. A total of 64% and 66% of Ae. tauschii genes were syntenic with sorghum and rice genes, respectively. Synteny was reduced in the vicinity of the termini of modern Ae. tauschii chromosomes but not in the vicinity of the ancient termini embedded in the Ae. tauschii chromosomes, suggesting that the dependence of synteny erosion on gene location along the centromere–telomere axis either evolved recently in the Triticeae phylogenetic lineage or its evolution was recently accelerated.

A survey and analysis is made of all available ω-gliadin DNA sequences including ω-gliadin genes within a large genomic clone, previously reported gene sequences, and ESTs identified from the large wheat EST collection. A contiguous portion of the Gli-B3 locus is shown to contain two apparently active ω-gliadin genes, two pseudogenes, and four fragments of the 3′ portion of ω-gliadin sequences. Comparison of ω-gliadin sequences allows a phylogenetic picture of their relationships and genomes of origin. Results show three groupings of ω-gliadin active gene sequences assigned to each of the three hexaploid wheat genomes, and a fourth group thus far consisting of pseudogenes assigned to the A-genome. Analysis of ω-gliadin ESTs allows reconstruction of two full-length model sequences encoding the AREL- and ARQL-type proteins from the Gli-A3 and Gli-D3 loci, respectively. There is no DNA evidence of multiple active genes from these two loci. In contrast, ESTs allow identification of at least three to four distinct active genes at the Gli-B3 locus of some cultivars. Additional results include more information on the position of cysteines in some ω-gliadin genes and discussion of problems in studying the ω-gliadin gene family. Electronic supplementary material The online version of this article (doi:10.1007/s10142-009-0122-2) contains supplementary material, which is available to authorized users.

Brachypodium distachyon (Brachypodium) has been recently recognized as an emerging model system for both comparative and functional genomics in grass species. In this study, 55,221 repeat masked Brachypodium BAC end sequences (BES) were used for comparative analysis against the 12 rice pseudomolecules. The analysis revealed that approximately 26.4% of BES have significant matches with the rice genome and 82.4% of the matches were homologous to known genes. Further analysis of paired-end BES and approximately 1.0 Mb sequences from nine selected BACs proved to be useful in revealing conserved regions and regions that have undergone considerable genomic changes. Differential gene amplification, insertions/deletions and inversions appeared to be the common evolutionary events that caused variations of microcolinearity at different orthologous genomic regions. It was found that approximately 17% of genes in the two genomes are not colinear in the orthologous regions. Analysis of BAC sequences also revealed higher gene density (approximately 9 kb/gene) and lower repeat DNA content (approximately 13.1%) in Brachypodium when compared to the orthologous rice regions, consistent with the smaller size of the Brachypodium genome. The 119 annotated Brachypodium genes were BLASTN compared against the wheat EST database and deletion bin mapped wheat ESTs. About 77% of the genes retrieved significant matches in the EST database, while 9.2% matched to the bin mapped ESTs. In some cases, genes in single Brachypodium BACs matched to multiple ESTs that were mapped to the same deletion bins, suggesting that the Brachypodium genome will be useful for ordering wheat ESTs within the deletion bins and developing specific markers at targeted regions in the wheat genome.

The expression of 7,835 genes in developing wheat caryopses was analyzed using cDNA arrays. Using a mixed model analysis of variance (ANOVA) method, 29% (2,237) of the genes on the array were identified to be differentially expressed at the 6 different time-points examined, which covers the developmental stages from coenocytic endosperm to physiological maturity. Comparison of genes differentially expressed between two time-points revealed a dynamic transcript accumulation profile with major re-programming events that occur at 3-7, 7-14 and 21-28 DPA. A k-means clustering algorithm grouped the differentially expressed genes into 10 clusters, revealing co-expression of genes involved in the same pathway such as carbohydrate and protein synthesis or preparation for desiccation. Functional annotation of genes that show peak expression at specific time-points correlated with the developmental events associated with the respective stages. Results provide information on the temporal expression during caryopsis development for a significant number of differentially expressed genes with unknown function.

International audience; Generating a contiguous, ordered reference sequence of a complex genome such as hexaploid wheat (2n = 6x = 42; approximately 17 GB) is a challenging task due to its large, highly repetitive, and allopolyploid genome. In wheat, ordering of whole-genome or hierarchical shotgun sequencing contigs is primarily based on recombination and comparative genomics-based approaches. However, comparative genomics approaches are limited to syntenic inference and recombination is suppressed within the pericentromeric regions of wheat chromosomes, thus, precise ordering of physical maps and sequenced contigs across the whole-genome using these approaches is nearly impossible. We developed a whole-genome radiation hybrid (WGRH) resource and tested it by genotyping a set of 115 randomly selected lines on a high-density single nucleotide polymorphism (SNP) array. At the whole-genome level, 26 299 SNP markers were mapped on the RH panel and provided an average mapping resolution of approximately 248 Kb/cR1500 with a total map length of 6866 cR1500 . The 7296 unique mapping bins provided a five- to eight-fold higher resolution than genetic maps used in similar studies. Most strikingly, the RH map had uniform bin resolution across the entire chromosome(s), including pericentromeric regions. Our research provides a valuable and low-cost resource for anchoring and ordering sequenced BAC and next generation sequencing (NGS) contigs. The WGRH developed for reference wheat line Chinese Spring (CS-WGRH), will be useful for anchoring and ordering sequenced BAC and NGS based contigs for assembling a high-quality, reference sequence of hexaploid wheat. Additionally, this study provides an excellent model for developing similar resources for other polyploid species.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1852-2) contains supplementary material, which is available to authorized users.

GrainGenes (http://wheat.pw.usda.gov) is an international database for genetic and genomic information about Triticeae species (wheat [Triticum aestivum], barley [Hordeum vulgare], rye [Secale cereale], and their wild relatives) and oat (Avena sativa) and its wild relatives. A major strength of the GrainGenes project is the interaction of the curators with database users in the research community, placing GrainGenes as both a data repository and information hub. The primary intensively curated data classes are genetic and physical maps, probes used for mapping, classical genes, quantitative trait loci, and contact information for Triticeae and oat scientists. Curation of these classes involves important contributions from the GrainGenes community, both as primary data sources and reviewers of published data. Other partially automated data classes include literature references, sequences, and links to other databases. Beyond the GrainGenes database per se, the Web site incorporates other more specific databases, informational topics, and downloadable files. For example, unique BLAST datasets of sequences applicable to Triticeae research include mapped wheat expressed sequence tags, expressed sequence tag-derived simple sequence repeats, and repetitive sequences. In 2004, the GrainGenes project migrated from the AceDB database and separate Web site to an integrated relational database and Internet resource, a major step forward in database delivery. The process of this migration and its impacts on database curation and maintenance are described, and a perspective on how a genomic database can expedite research and crop improvement is provided.

This report describes the rationale, approaches, organization, and resource development leading to a large-scale deletion bin map of the hexaploid (2n = 6x = 42) wheat genome (Triticum aestivum L.). Accompanying reports in this issue detail results from chromosome bin-mapping of expressed sequence tags (ESTs) representing genes onto the seven homoeologous chromosome groups and a global analysis of the entire mapped wheat EST data set. Among the resources developed were the first extensive public wheat EST collection (113,220 ESTs). Described are protocols for sequencing, sequence processing, EST nomenclature, and the assembly of ESTs into contigs. These contigs plus singletons (unassembled ESTs) were used for selection of distinct sequence motif unigenes. Selected ESTs were rearrayed, validated by 5′ and 3′ sequencing, and amplified for probing a series of wheat aneuploid and deletion stocks. Images and data for all Southern hybridizations were deposited in databases and were used by the coordinators for each of the seven homoeologous chromosome groups to validate the mapping results. Results from this project have established the foundation for future developments in wheat genomics.

Because of the huge size of the common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) genome of 17,300 Mb, sequencing and mapping of the expressed portion is a logical first step for gene discovery. Here we report mapping of 7104 expressed sequence tag (EST) unigenes by Southern hybridization into a chromosome bin map using a set of wheat aneuploids and deletion stocks. Each EST detected a mean of 4.8 restriction fragments and 2.8 loci. More loci were mapped in the B genome (5774) than in the A (5173) or D (5146) genomes. The EST density was significantly higher for the D genome than for the A or B. In general, EST density increased relative to the physical distance from the centromere. The majority of EST-dense regions are in the distal parts of chromosomes. Most of the agronomically important genes are located in EST-dense regions. The chromosome bin map of ESTs is a unique resource for SNP analysis, comparative mapping, structural and functional analysis, and polyploid evolution, as well as providing a framework for constructing a sequence-ready, BAC-contig map of the wheat genome.

A total of 1918 loci, detected by the hybridization of 938 expressed sequence tag unigenes (ESTs) from 26 Triticeae cDNA libraries, were mapped to wheat (Triticum aestivum L.) homoeologous group 4 chromosomes using a set of deletion, ditelosomic, and nulli-tetrasomic lines. The 1918 EST loci were not distributed uniformly among the three group 4 chromosomes; 41, 28, and 31% mapped to chromosomes 4A, 4B, and 4D, respectively. This pattern is in contrast to the cumulative results of EST mapping in all homoeologous groups, as reported elsewhere, that found the highest proportion of loci mapped to the B genome. Sixty-five percent of these 1918 loci mapped to the long arms of homoeologous group 4 chromosomes, while 35% mapped to the short arms. The distal regions of chromosome arms showed higher numbers of loci than the proximal regions, with the exception of 4DL. This study confirmed the complex structure of chromosome 4A that contains two reciprocal translocations and two inversions, previously identified. An additional inversion in the centromeric region of 4A was revealed. A consensus map for homoeologous group 4 was developed from 119 ESTs unique to group 4. Forty-nine percent of these ESTs were found to be homoologous to sequences on rice chromosome 3, 12% had matches with sequences on other rice chromosomes, and 39% had no matches with rice sequences at all. Limited homology (only 26 of the 119 consensus ESTs) was found between wheat ESTs on homoeologous group 4 and the Arabidopsis genome. Forty-two percent of the homoeologous group 4 ESTs could be classified into functional categories on the basis of blastX searches against all protein databases.

The focus of this study was to analyze the content, distribution, and comparative genome relationships of 996 chromosome bin-mapped expressed sequence tags (ESTs) accounting for 2266 restriction fragments (loci) on the homoeologous group 3 chromosomes of hexaploid wheat (Triticum aestivum L.). Of these loci, 634, 884, and 748 were mapped on chromosomes 3A, 3B, and 3D, respectively. The individual chromosome bin maps revealed bins with a high density of mapped ESTs in the distal region and bins of low density in the proximal region of the chromosome arms, with the exception of 3DS and 3DL. These distributions were more localized on the higher-resolution group 3 consensus map with intermediate regions of high-mapped-EST density on both chromosome arms. Gene ontology (GO) classification of mapped ESTs was not significantly different for homoeologous group 3 chromosomes compared to the other groups. A combined analysis of the individual bin maps using 537 of the mapped ESTs revealed rearrangements between the group 3 chromosomes. Approximately 232 (44%) of the consensus mapped ESTs matched sequences on rice chromosome 1 and revealed large- and small-scale differences in gene order. Of the group 3 mapped EST unigenes ∼21 and 32% matched the Arabidopsis coding regions and proteins, respectively, but no chromosome-level gene order conservation was detected.

To localize wheat (Triticum aestivum L.) ESTs on chromosomes, 882 homoeologous group 6-specific ESTs were identified by physically mapping 7965 singletons from 37 cDNA libraries on 146 chromosome, arm, and sub-arm aneuploid and deletion stocks. The 882 ESTs were physically mapped to 25 regions (bins) flanked by 23 deletion breakpoints. Of the 5154 restriction fragments detected by 882 ESTs, 2043 (loci) were localized to group 6 chromosomes and 806 were mapped on other chromosome groups. The number of loci mapped was greatest on chromosome 6B and least on 6D. The 264 ESTs that detected orthologous loci on all three homoeologs using one restriction enzyme were used to construct a consensus physical map. The physical distribution of ESTs was uneven on chromosomes with a tendency toward higher densities in the distal halves of chromosome arms. About 43% of the wheat group 6 ESTs identified rice homologs upon comparisons of genome sequences. Fifty-eight percent of these ESTs were present on rice chromosome 2 and the remaining were on other rice chromosomes. Even within the group 6 bins, rice chromosomal blocks identified by 1–6 wheat ESTs were homologous to up to 11 rice chromosomes. These rice-block contigs were used to resolve the order of wheat ESTs within each bin.

D hordein, a prolamin storage protein of barley endosperms, is highly homologous to the high molecular weight (HWM) glutenin subunits, which are the major determinants of bread-making quality in wheat flour. In hexaploid wheat (AABBDD), each genome contains two paralogous copies of HMW-glutenin genes that encode the x- and y-type HMW-glutenin subunits. Previously, we reported the sequence analysis of a 102-kb genomic region that contains the HMW-glutenin locus of the D genome from Aegilops tauschii, the donor of the D genome of hexaploid wheat. Here, we present the sequence analysis of a 120-kb D-hordein region of the barley genome, a more distantly related member of the Triticeae grass tribe. Comparative sequence analysis revealed that gene content and order are generally conserved. Genes included in both of these orthologous regions are arranged in the following order: a Xa21-like receptor kinase, an endosperm globulin, an HMW prolamin, and a serine (threonine) protein kinase. However, in the wheat D genome, a region containing both the globulin and HMW-glutenin gene was duplicated, indicating that this duplication event occurred after the separation of the wheat and barley genomes. The intergenic regions are divergent with regard to the sequence and structural organization. It was found that different types of retroelements are responsible for the intergenic structure divergence in the wheat and barley genomes. In the barley region, we identified 16 long terminal repeat (LTR) retrotransposons in three distinct nested clusters. These retroelements account for 63% of the contig sequence. In addition, barley D hordein was compared with wheat HMW glutenins in terms of cysteine residue conservation and repeat domain organization.Key words: HMW glutenin, evolution, retrotransposon, comparative genomics.

The frequency of Ty1-copia-type and Ty3-gypsy-type retrotransposons in the International Triticeae EST Consortium (ITEC) database (61,942 sequences: 82% wheat, 10% barley, 8% rye) and the DuPont EST database (86,628 wheat sequences) was estimated using BLASTN searches. These ESTs were obtained from 94 cDNA libraries from different tissues (leaves, roots, spikes, flowers and seeds) and different growing conditions, excluding subtracted and normalized cDNA libraries. Triticeae EST databases were screened using four different Ty1--copia-type, 12 reverse transcriptase sequences, and three Ty3-gypsy-type Triticeae retrotransposon sequences. Using a selection threshold of BLASTN scores higher than 100 or E values smaller than e–20, 0.145% of the ESTs were found to be significantly similar to at least one of the retrotransposons used in the search (0.064% Ty1-copia, 0.081% Ty3-gypsy). This percentage increased to 0.176% when the BLASTN threshold was changed to E

Recognizing a need in cultivated hexaploid oat (Avena sativa L.) for a reliable set of reference single nucleotide polymorphisms (SNPs), we have developed a 6000 (6K) BeadChip design containing 257 Infinium I and 5486 Infinium II designs correspond ing to 5743 SNPs. Of those, 4975 SNPs yielded successful assays after array manufacturing. These SNPs were discovered based on a variety of bioinformatics pipelines in complementary DNA (cDNA) and genomic DNA originating from 20 or more diverse oat cultivars. The array was validated in 1100 samples from six recombinant inbred line (RIL) mapping populations and sets of diverse oat cultivars and breeding lines, and provided approximately 3500 discernible Mendelian polymorphisms. Here, we present an annotation of these SNPs, including methods of discovery, gene identification and orthology, population-genetic characteristics, and tentative positions on an oat consensus map. We also evaluate a new cluster-based method of calling SNPs. The SNP design sequences are made publicly available, and the full SNP genotyping platform is available for commercial purchase from an independent third party. O at is an important cereal crop grown throughout most of the world. In cultivated hexaploid oat, as in most other crop species, there has been considerable interest in developing efficient SNP genotyping methods to facilitate genomic discovery and molecular breeding. Discovery and application of SNPs in a hexaploid species is challenging due to the presence of homoeologous gene copies. Many putative SNPs are later found to be interlocus variants (homeo-SNPs), and true intralocus SNPs may be difficult to assay due to the confounding presence of homoeologous loci. Nevertheless, successful SNP assays in oat can be developed (Oliver et al., 2011) and a first-generation SNP array has been used to develop a physically anchored consensus map in oat (Oliver et al., 2013). The objectives of the present communication are to report the expansion of this SNP array, to evaluate its performance, and to formally announce its public availability. In addition, we report an annotation of the SNPs and the loci that they represent, we share lessons learned during the assay development, and we evaluate a new clusterbased method for calling SNP genotypes in a polyploid.

Background: Triticum monococcum (2n) is a close ancestor of T. urartu, the A-genome progenitor of cultivated hexaploid wheat, and is therefore a useful model for the study of components regulating photomorphogenesis in diploid wheat. In order to develop genetic and genomic resources for such a study, we constructed genome-wide transcriptomes of two Triticum monococcum subspecies, the wild winter wheat T. monococcum ssp. aegilopoides (accession G3116) and the domesticated spring wheat T. monococcum ssp. monococcum (accession DV92) by generating de novo assemblies of RNASeq data derived from both etiolated and green seedlings. Principal Findings: The de novo transcriptome assemblies of DV92 and G3116 represent 120,911 and 117,969 transcripts, respectively. We successfully mapped ,90% of these transcripts from each accession to barley and ,95% of the transcripts to T. urartu genomes. However, only ,77% transcripts mapped to the annotated barley genes and ,85% transcripts mapped to the annotated T. urartu genes. Differential gene expression analyses revealed 22% more light up-regulated and 35% more light down-regulated transcripts in the G3116 transcriptome compared to DV92. The DV92 and G3116 mRNA sequence reads aligned against the reference barley genome led to the identification of ,500,000 single nucleotide polymorphism (SNP) and ,22,000 simple sequence repeat (SSR) sites. Conclusions: De novo transcriptome assemblies of two accessions of the diploid wheat T. monococcum provide new empirical transcriptome references for improving Triticeae genome annotations, and insights into transcriptional programming during photomorphogenesis. The SNP and SSR sites identified in our analysis provide additional resources for the development of molecular markers.

Cotton (Gossypium spp) is the world's leading natural fiber crop. Genetic manipulation continues to play a key role in the improvement of fiber quality properties. By use of DNA-based molecular markers and a polymorphic mapping population derived from an inter specific cross between TM-1 (G. hirsutum) and 3-79 (G. barbadense), thirteen quantitative trait loci (QTLs) controlling fiber quality properties were identified in 3-79, an extra long staple (ELS) cotton. Four QTLs influenced bundle fiber strength, three influenced fiber length, and six influenced fiber fineness. These QTLs were located on different chromosomes or linkage groups and collectively explained 30% to 60%of the total phenotypic variance for each fiber quality property in the F2 population. The effects and modes of action for the individual QTLs were characterized with 3-79 alleles in TM-1 genetic background. The results indicated more recessive than dominant, with much less additive effect in the gene mode. Transgressive segregation was observed for fiber fineness that could be beneficial to improvement of this trait. Molecular markers linked to fiber quality QTLs would be most effective in marker-assisted selection (MAS) of these recessive alleles in cotton breeding programs.

Summary: High-throughput content fingerprinting techniques employing capillary electrophoresis place new demands on the editing of fingerprint files for the downstream contig assembly program, FPC. A cross-platform software application, GenoProfiler, was developed for automated editing of sized fingerprinting profiles generated by the ABI Genetic Analyzers. The batch-processing module extracts the sized fragment information directly from the ABI raw trace files, or from data files exported from GeneMapper or other size calling software, removes the background noise and undesired fragments, and generates fragment size files compatible with the FPC software.Availability: Contact: oandersn@pw.usda.gov

Tocochromanols are recognized for nutritional content, plant stress response, and seed longevity. Here we present a systems biological approach to characterize and develop predictive assays for genes affecting tocochromanol variation in barley. Major QTL, detected in three regions of a SNP linkage map, affected multiple tocochromanol forms. Candidate genes were identified through barley/rice orthology and sequenced in genotypes with disparate tocochromanol profiles. Gene-specific markers, designed based on observed polymorphism, mapped to the originating QTL, increasing R2 values at the respective loci. Polymorphism within promoter regions corresponded to motifs known to influence gene expression. Quantitative PCR analysis revealed a trend of increased expression in tissues grown at cold temperatures. These results demonstrate utility of a novel method for rapid gene identification and characterization, and provide a resource for efficient development of barley lines with improved tocochromanol profiles.

Starch granule surface-associated proteins were separated by HPLC and identified by direct protein sequencing. Among the proteins identified was one that consisted of two polypeptide chains of 11 and 19 kDa linked by disulfide bonds. Sequencing of tryptic peptides from each of the polypeptides revealed similarities between some of the peptides and avenin-like b proteins encoded by partial cDNAs in NCBI. To identify a contiguous sequence that matched all of the peptides, contigs encoding three avenin-like b proteins were constructed from ESTs of the cultivar Butte 86. All peptide sequences were found in a protein encoded by one of these contigs that had not been identified previously. Protein and DNA sequences indicated that the two polypeptide chains were derived from a parent protein that had been cleaved at the C-terminal position of an asparagine residue. The name farinin is suggested for this protein and other avenin-like b proteins. Evolutionary relationships of the protein are discussed and a simple computer molecular model was constructed. On the basis of its sequence, the new protein was likely to be allergenic but unlikely to be active in celiac disease.

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.

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.