Microarrays for global expression constructed with a low redundancy set of 27,500 sequenced cDNAs representing an array of developmental stages and physiological conditions of the soybean plant

BACKGROUND: Microarrays are an important tool with which to examine coordinated gene expression. Soybean (Glycine max) is one of the most economically valuable crop species in the world food supply. In order to accelerate both gene discovery as well as hypothesis-driven research in soybean, global expression resources needed to be developed. The applications of microarray for determining patterns of expression in different tissues or during conditional treatments by dual labeling of the mRNAs are unlimited. In addition, discovery of the molecular basis of traits through examination of naturally occurring variation in hundreds of mutant lines could be enhanced by the construction and use of soybean cDNA microarrays. RESULTS: We report the construction and analysis of a low redundancy 'unigene' set of 27,513 clones that represent a variety of soybean cDNA libraries made from a wide array of source tissue and organ systems, developmental stages, and stress or pathogen-challenged plants. The set was assembled from the 5' sequence data of the cDNA clones using cluster analysis programs. The selected clones were then physically reracked and sequenced at the 3' end. In order to increase gene discovery from immature cotyledon libraries that contain abundant mRNAs representing storage protein gene families, we utilized a high density filter normalization approach to preferentially select more weakly expressed cDNAs. All 27,513 cDNA inserts were amplified by polymerase chain reaction. The amplified products, along with some repetitively spotted control or 'choice' clones, were used to produce three 9,728-element microarrays that have been used to examine tissue specific gene expression and global expression in mutant isolines. CONCLUSIONS: Global expression studies will be greatly aided by the availability of the sequence-validated and low redundancy cDNA sets described in this report. These cDNAs and ESTs represent a wide array of developmental stages and physiological conditions of the soybean plant. We also demonstrate that the quality of the data from the soybean cDNA microarrays is sufficiently reliable to examine isogenic lines that differ with respect to a mutant phenotype and thereby to define a small list of candidate genes potentially encoding or modulated by the mutant phenotype

New data about the suspensor of succulent angiosperms : ultrastructure and cytochemical study of the embryo-suspensor of Sempervivum arachnoideum L. and Jovibarba sobolifera (Sims) Opiz

The development of the suspensor in two species - Sempervivum arachnoideum and Jovibarba sobolifera - was investigated using cytochemical methods, light and electron microscopy. Cytological processes of differentiation in the embryo-suspensor were compared with the development of embryo-proper. The mature differentiated suspensor consists of a large basal cell and three to four chalazal cells. The basal cell produces haustorial branched invading ovular tissues. The walls of the haustorium and the micropylar part of the basal cell form the wall ingrowths typical for a transfer cells. The ingrowths also partially cover the lateral wall and the chalazal wall separating the basal cell from the other embryo cells. The dense cytoplasm filling the basal cell is rich in: numerous polysomes lying free or covering rough endoplasmic reticulum (RER), active dictyosomes, microtubules, bundles of microfilaments, microbodies, mitochondria, plastids and lipid droplets. Cytochemical tests (including proteins, insoluble polysaccharides and lipids are distributed in the suspensor during different stages of embryo development) showed the presence of high amounts of macromolecules in the suspensor cells, particularly during the globular and heart-shaped phases of embryo development. The protein bodies and lipid droplets are the main storage products in the cells of the embryo-proper. The results of Auramine 0 indicate that a cuticular material is present only on the surface walls of the embryo-proper, but is absent from the suspensor cell wall. The ultrastructural features and cytochemical tests indicate that in the two species - S. arachnoideum and J. sobolifera - the embryo-suspensor is mainly involved in the absorption and transport of metabolites from the ovular tissues to the developing embryo-proper

A BAC pooling strategy combined with PCR-based screenings in a large, highly repetitive genome enables integration of the maize genetic and physical maps

BACKGROUND: Molecular markers serve three important functions in physical map assembly. First, they provide anchor points to genetic maps facilitating functional genomic studies. Second, they reduce the overlap required for BAC contig assembly from 80 to 50 percent. Finally, they validate assemblies based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy in combination with a high-throughput PCR-based screening method to anchor the maize genetic and physical maps. RESULTS: A total of 110,592 maize BAC clones (~ 6x haploid genome equivalents) were pooled into six different matrices, each containing 48 pools of BAC DNA. The quality of the BAC DNA pools and their utility for identifying BACs containing target genomic sequences was tested using 254 PCR-based STS markers. Five types of PCR-based STS markers were screened to assess potential uses for the BAC pools. An average of 4.68 BAC clones were identified per marker analyzed. These results were integrated with BAC fingerprint data generated by the Arizona Genomics Institute (AGI) and the Arizona Genomics Computational Laboratory (AGCoL) to assemble the BAC contigs using the FingerPrinted Contigs (FPC) software and contribute to the construction and anchoring of the physical map. A total of 234 markers (92.5%) anchored BAC contigs to their genetic map positions. The results can be viewed on the integrated map of maize [1,2]. CONCLUSION: This BAC pooling strategy is a rapid, cost effective method for genome assembly and anchoring. The requirement for six replicate positive amplifications makes this a robust method for use in large genomes with high amounts of repetitive DNA such as maize. This strategy can be used to physically map duplicate loci, provide order information for loci in a small genetic interval or with no genetic recombination, and loci with conflicting hybridization-based information

Crowdsourcing biocuration: The Community Assessment of Community Annotation with Ontologies (CACAO).

Experimental data about gene functions curated from the primary literature have enormous value for research scientists in understanding biology. Using the Gene Ontology (GO), manual curation by experts has provided an important resource for studying gene function, especially within model organisms. Unprecedented expansion of the scientific literature and validation of the predicted proteins have increased both data value and the challenges of keeping pace. Capturing literature-based functional annotations is limited by the ability of biocurators to handle the massive and rapidly growing scientific literature. Within the community-oriented wiki framework for GO annotation called the Gene Ontology Normal Usage Tracking System (GONUTS), we describe an approach to expand biocuration through crowdsourcing with undergraduates. This multiplies the number of high-quality annotations in international databases, enriches our coverage of the literature on normal gene function, and pushes the field in new directions. From an intercollegiate competition judged by experienced biocurators, Community Assessment of Community Annotation with Ontologies (CACAO), we have contributed nearly 5,000 literature-based annotations. Many of those annotations are to organisms not currently well-represented within GO. Over a 10-year history, our community contributors have spurred changes to the ontology not traditionally covered by professional biocurators. The CACAO principle of relying on community members to participate in and shape the future of biocuration in GO is a powerful and scalable model used to promote the scientific enterprise. It also provides undergraduate students with a unique and enriching introduction to critical reading of primary literature and acquisition of marketable skills

Mu Transposon Insertion Sites and Meiotic Recombination Events Co-Localize with Epigenetic Marks for Open Chromatin across the Maize Genome

The Mu transposon system of maize is highly active, with each of the ∼50–100 copies transposing on average once each generation. The approximately one dozen distinct Mu transposons contain highly similar ∼215 bp terminal inverted repeats (TIRs) and generate 9-bp target site duplications (TSDs) upon insertion. Using a novel genome walking strategy that uses these conserved TIRs as primer binding sites, Mu insertion sites were amplified from Mu stocks and sequenced via 454 technology. 94% of ∼965,000 reads carried Mu TIRs, demonstrating the specificity of this strategy. Among these TIRs, 21 novel Mu TIRs were discovered, revealing additional complexity of the Mu transposon system. The distribution of >40,000 non-redundant Mu insertion sites was strikingly non-uniform, such that rates increased in proportion to distance from the centromere. An identified putative Mu transposase binding consensus site does not explain this non-uniformity. An integrated genetic map containing more than 10,000 genetic markers was constructed and aligned to the sequence of the maize reference genome. Recombination rates (cM/Mb) are also strikingly non-uniform, with rates increasing in proportion to distance from the centromere. Mu insertion site frequencies are strongly correlated with recombination rates. Gene density does not fully explain the chromosomal distribution of Mu insertion and recombination sites, because pronounced preferences for the distal portion of chromosome are still observed even after accounting for gene density. The similarity of the distributions of Mu insertions and meiotic recombination sites suggests that common features, such as chromatin structure, are involved in site selection for both Mu insertion and meiotic recombination. The finding that Mu insertions and meiotic recombination sites both concentrate in genomic regions marked with epigenetic marks of open chromatin provides support for the hypothesis that open chromatin enhances rates of both Mu insertion and meiotic recombination

Evolution of Genome Size and Complexity in Pinus

BACKGROUND: Genome evolution in the gymnosperm lineage of seed plants has given rise to many of the most complex and largest plant genomes, however the elements involved are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Gymny is a previously undescribed retrotransposon family in Pinus that is related to Athila elements in Arabidopsis. Gymny elements are dispersed throughout the modern Pinus genome and occupy a physical space at least the size of the Arabidopsis thaliana genome. In contrast to previously described retroelements in Pinus, the Gymny family was amplified or introduced after the divergence of pine and spruce (Picea). If retrotransposon expansions are responsible for genome size differences within the Pinaceae, as they are in angiosperms, then they have yet to be identified. In contrast, molecular divergence of Gymny retrotransposons together with other families of retrotransposons can account for the large genome complexity of pines along with protein-coding genic DNA, as revealed by massively parallel DNA sequence analysis of Cot fractionated genomic DNA. CONCLUSIONS/SIGNIFICANCE: Most of the enormous genome complexity of pines can be explained by divergence of retrotransposons, however the elements responsible for genome size variation are yet to be identified. Genomic resources for Pinus including those reported here should assist in further defining whether and how the roles of retrotransposons differ in the evolution of angiosperm and gymnosperm genomes