Rex T. Nelson

RNA-Seq Atlas of Glycine max : A guide to the soybean transcriptome

BackgroundNext generation sequencing is transforming our understanding of transcriptomes. It can determine the expression level of transcripts with a dynamic range of over six orders of magnitude from multiple tissues, developmental stages or conditions. Patterns of gene expression provide insight into functions of genes with unknown annotation.ResultsThe RNA Seq-Atlas presented here provides a record of high-resolution gene expression in a set of fourteen diverse tissues. Hierarchical clustering of transcriptional profiles for these tissues suggests three clades with similar profiles: aerial, underground and seed tissues. We also investigate the relationship between gene structure and gene expression and find a correlation between gene length and expression. Additionally, we find dramatic tissue-specific gene expression of both the most highly-expressed genes and the genes specific to legumes in seed development and nodule tissues. Analysis of the gene expression profiles of over 2,000 genes with preferential gene expression in seed suggests there are more than 177 genes with functional roles that are involved in the economically important seed filling process. Finally, the Seq-atlas also provides a means of evaluating existing gene model annotations for the Glycine max genome.ConclusionsThis RNA-Seq atlas extends the analyses of previous gene expression atlases performed using Affymetrix GeneChip technology and provides an example of new methods to accommodate the increase in transcriptome data obtained from next generation sequencing. Data contained within this RNA-Seq atlas of Glycine max can be explored at http://www.soybase.org/soyseq.

SoyBase, the USDA-ARS soybean genetics and genomics database

SoyBase, the USDA-ARS soybean genetic database, is a comprehensive repository for professionally curated genetics, genomics and related data resources for soybean. SoyBase contains the most current genetic, physical and genomic sequence maps integrated with qualitative and quantitative traits. The quantitative trait loci (QTL) represent more than 18 years of QTL mapping of more than 90 unique traits. SoyBase also contains the well-annotated ‘Williams 82’ genomic sequence and associated data mining tools. The genetic and sequence views of the soybean chromosomes and the extensive data on traits and phenotypes are extensively interlinked. This allows entry to the database using almost any kind of available information, such as genetic map symbols, soybean gene names or phenotypic traits. SoyBase is the repository for controlled vocabularies for soybean growth, development and trait terms, which are also linked to the more general plant ontologies. SoyBase can be accessed at http://soybase.org.

Phenotypic and genomic analyses of a fast neutron mutant population resource in soybean

Mutagenized populations have become indispensable resources for introducing variation and studying gene function in plant genomics research. In this study, fast neutron (FN) radiation was used to induce deletion mutations in the soybean (Glycine max) genome. Approximately 120,000 soybean seeds were exposed to FN radiation doses of up to 32 Gray units to develop over 23,000 independent M2 lines. Here, we demonstrate the utility of this population for phenotypic screening and associated genomic characterization of striking and agronomically important traits. Plant variation was cataloged for seed composition, maturity, morphology, pigmentation, and nodulation traits. Mutants that showed significant increases or decreases in seed protein and oil content across multiple generations and environments were identified. The application of comparative genomic hybridization (CGH) to lesion-induced mutants for deletion mapping was validated on a midoleate x-ray mutant, M23, with a known FAD2-1A (for fatty acid desaturase) gene deletion. Using CGH, a subset of mutants was characterized, revealing deletion regions and candidate genes associated with phenotypes of interest. Exome resequencing and sequencing of PCR products confirmed FN-induced deletions detected by CGH. Beyond characterization of soybean FN mutants, this study demonstrates the utility of CGH, exome sequence capture, and next-generation sequencing approaches for analyses of mutant plant genomes. We present this FN mutant soybean population as a valuable public resource for future genetic screens and functional genomics research.

Distinct Biphasic mRNA Changes in Response to Asian Soybean Rust Infection

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is now established in all major soybean-producing countries. Currently, there is little information about the molecular basis of ASR-soybean interactions, which will be needed to assist future efforts to develop effective resistance. Toward this end, abundance changes of soybean mRNAs were measured over a 7-day ASR infection time course in mock-inoculated and infected leaves of a soybean accession (PI230970) carrying the Rpp2 resistance gene and a susceptible genotype (Embrapa-48). The expression profiles of differentially expressed genes (ASR-infected compared with the mock-inoculated control) revealed a biphasic response to ASR in each genotype. Within the first 12 h after inoculation (hai), which corresponds to fungal germination and penetration of the epidermal cells, differential gene expression changes were evident in both genotypes. mRNA expression of these genes mostly returned to levels found in mock-inoculated plants by 24 hai. In the susceptible genotype, gene expression remained unaffected by rust infection until 96 hai, a time period when rapid fungal growth began. In contrast, gene expression in the resistant genotype diverged from the mock-inoculated control earlier, at 72 h, demonstrating that Rpp2-mediated defenses were initiated prior to this time. These data suggest that ASR initially induces a nonspecific response that is transient or is suppressed when early steps in colonization are completed in both soybean genotypes. The race-specific resistance phenotype of Rpp2 is manifested in massive gene expression changes after the initial response prior to the onset of rapid fungal growth that occurs in the susceptible genotype.

SoyTEdb: a comprehensive database of transposable elements in the soybean genome

BackgroundTransposable elements are the most abundant components of all characterized genomes of higher eukaryotes. It has been documented that these elements not only contribute to the shaping and reshaping of their host genomes, but also play significant roles in regulating gene expression, altering gene function, and creating new genes. Thus, complete identification of transposable elements in sequenced genomes and construction of comprehensive transposable element databases are essential for accurate annotation of genes and other genomic components, for investigation of potential functional interaction between transposable elements and genes, and for study of genome evolution. The recent availability of the soybean genome sequence has provided an unprecedented opportunity for discovery, and structural and functional characterization of transposable elements in this economically important legume crop.DescriptionUsing a combination of structure-based and homology-based approaches, a total of 32,552 retrotransposons (Class I) and 6,029 DNA transposons (Class II) with clear boundaries and insertion sites were structurally annotated and clearly categorized, and a soybean transposable element database, SoyTEdb, was established. These transposable elements have been anchored in and integrated with the soybean physical map and genetic map, and are browsable and visualizable at any scale along the 20 soybean chromosomes, along with predicted genes and other sequence annotations. BLAST search and other infrastracture tools were implemented to facilitate annotation of transposable elements or fragments from soybean and other related legume species. The majority (> 95%) of these elements (particularly a few hundred low-copy-number families) are first described in this study.ConclusionSoyTEdb provides resources and information related to transposable elements in the soybean genome, representing the most comprehensive and the largest manually curated transposable element database for any individual plant genome completely sequenced to date. Transposable elements previously identified in legumes, the third largest family of flowering plants, are relatively scarce. Thus this database will facilitate structural, evolutionary, functional, and epigenetic analyses of transposable elements in soybean and other legume species.

Identification and analysis of gene families from the duplicated genome of soybean using EST sequences

BackgroundLarge scale gene analysis of most organisms is hampered by incomplete genomic sequences. In many organisms, such as soybean, the best source of sequence information is the existence of expressed sequence tag (EST) libraries. Soybean has a large (1115 Mbp) genome that has yet to be fully sequenced. However it does have the 6th largest EST collection comprised of ESTs from a variety of soybean genotypes. Many EST libraries were constructed from RNA extracted from various genetic backgrounds, thus gene identification from these sources is complicated by the existence of both gene and allele sequence differences. We used the ESTminer suite of programs to identify potential soybean gene transcripts from a single genetic background allowing us to observe functional classifications between gene families as well as structural differences between genes and gene paralogs within families. The identification of potential gene sequences (pHaps) from soybean allows us to begin to get a picture of the genomic history of the organism as well as begin to observe the evolutionary fates of gene copies in this highly duplicated genome.ResultsWe identified approximately 45,000 potential gene sequences (pHaps) from EST sequences of Williams/Williams82, an inbred genotype of soybean (Glycine max L. Merr.) using a redundancy criterion to identify reproducible sequence differences between related genes within gene families. Analysis of these sequences revealed single base substitutions and single base indels are the most frequently observed form of sequence variation between genes within families in the dataset. Genomic sequencing of selected loci indicate that intron-like intervening sequences are numerous and are approximately 220 bp in length. Functional annotation of gene sequences indicate functional classifications are not randomly distributed among gene families containing few or many genes.ConclusionThe predominance of single nucleotide insertion/deletions and substitution events between genes within families (individual genes and gene paralogs) is consistent with a model of gene amplification followed by single base random mutational events expected under the classical model of duplicated gene evolution. Molecular functions of small and large gene families appear to be non-randomly distributed possibly indicating a difference in retention of duplicates or local expansion.

Biphasic Gene Expression Changes Elicited by Phakopsora pachyrhizi in Soybean Correlate with Fungal Penetration and Haustoria Formation

Inoculation of soybean (Glycine max) plants with Phakopsora pachyrhizi, the causal organism of Asian soybean rust, elicits a biphasic response characterized by a burst of differential gene expression in the first 12 h. A quiescent period occurs from 24 to 48 h after inoculation, in which P. pachyrhizi continues to develop but does not elicit strong host responses, followed by a second phase of intense gene expression. To correlate soybean responses with P. pachyrhizi growth and development, we inoculated the soybean cultivar Ankur (accession PI462312), which carries the Rpp3 resistance gene, with avirulent and virulent isolates of P. pachyrhizi. The avirulent isolate Hawaii 94-1 elicits hypersensitive cell death that limits fungal growth on Ankur and results in an incompatible response, while the virulent isolate Taiwan 80-2 grows extensively, sporulates profusely, and produces a compatible reaction. Inoculated leaves were collected over a 288-h time course for microarray analysis of soybean gene expression and microscopic analysis of P. pachyrhizi growth and development. The first burst in gene expression correlated with appressorium formation and penetration of epidermal cells, while the second burst of gene expression changes followed the onset of haustoria formation in both compatible and incompatible interactions. The proliferation of haustoria coincided with the inhibition of P. pachyrhizi growth in the incompatible interaction or the beginning of accelerated growth in the compatible interaction. The temporal relationships between P. pachyrhizi growth and host responses provide an important context in which to view interacting gene networks that mediate the outcomes of their interactions.

An ontology approach to comparative phenomics in plants

BackgroundPlant phenotype datasets include many different types of data, formats, and terms from specialized vocabularies. Because these datasets were designed for different audiences, they frequently contain language and details tailored to investigators with different research objectives and backgrounds. Although phenotype comparisons across datasets have long been possible on a small scale, comprehensive queries and analyses that span a broad set of reference species, research disciplines, and knowledge domains continue to be severely limited by the absence of a common semantic framework.ResultsWe developed a workflow to curate and standardize existing phenotype datasets for six plant species, encompassing both model species and crop plants with established genetic resources. Our effort focused on mutant phenotypes associated with genes of known sequence in Arabidopsis thaliana (L.) Heynh. (Arabidopsis), Zea mays L. subsp. mays (maize), Medicago truncatula Gaertn. (barrel medic or Medicago), Oryza sativa L. (rice), Glycine max (L.) Merr. (soybean), and Solanum lycopersicum L. (tomato). We applied the same ontologies, annotation standards, formats, and best practices across all six species, thereby ensuring that the shared dataset could be used for cross-species querying and semantic similarity analyses. Curated phenotypes were first converted into a common format using taxonomically broad ontologies such as the Plant Ontology, Gene Ontology, and Phenotype and Trait Ontology. We then compared ontology-based phenotypic descriptions with an existing classification system for plant phenotypes and evaluated our semantic similarity dataset for its ability to enhance predictions of gene families, protein functions, and shared metabolic pathways that underlie informative plant phenotypes.ConclusionsThe use of ontologies, annotation standards, shared formats, and best practices for cross-taxon phenotype data analyses represents a novel approach to plant phenomics that enhances the utility of model genetic organisms and can be readily applied to species with fewer genetic resources and less well-characterized genomes. In addition, these tools should enhance future efforts to explore the relationships among phenotypic similarity, gene function, and sequence similarity in plants, and to make genotype-to-phenotype predictions relevant to plant biology, crop improvement, and potentially even human health.

SoyBase: A Comprehensive Database for Soybean Genetic and Genomic Data

SoyBase, the USDA-ARS soybean genetics and genomics database, provides a comprehensive collection of data, analysis tools, and links to external resources of interest to soybean researchers. The SoyBase home page (https://soybase.org) contains the SoyBase Toolbox which provides quick access to a search of the SoyBase database or the SoyCyc metabolic pathways database, access to the data download page, a genome sequence BLAST tool, and direct links to the genetic and sequence maps. An extensive navigation menu and site description provides facile access to all sections of SoyBase. Comprehensive information for a number of data types is available at SoyBase including the current genetic maps, the soybean reference genome sequence with tracks covering genetic markers, genome organization, gene annotation and expression, and gene knockout mutants. SoyBase includes an extensive RNA-Seq gene atlas and innovative tools for identifying fast neutron-induced mutants. SoyBase is an actively curated database, with new data regularly being incorporated, including additions to the controlled vocabularies (ontologies) for soybean growth, development and phenotypic traits, soybean genes, and quantitative trait loci. New “omics” tools enable sophisticated queries on lists of genes. These features are all accessed using intuitive interfaces and are linked together wherever possible.