Towards Interoperability in Genome Databases: The MAtDB (MIPS Arabidopsis Thaliana Database) Experience

Increasing numbers of whole-genome sequences are available, but to interpret them fully requires more than listing all genes. Genome databases are faced with the challenges of integrating heterogenous data and enabling data mining. In comparison to a data warehousing approach, where integration is achieved through replication of all relevant data in a unified schema, distributed approaches provide greater flexibility and maintainability. These are important in a field where new data is generated rapidly and our understanding of the data changes. Interoperability between distributed data sources allows data maintenance to be separated from integration and analysis. Simple ways to access the data can facilitate the development of new data mining tools and the transition from model genome analysis to comparative genomics. With the MIPS Arabidopsis thaliana genome database (MAtDB, http://mips.gsf.de/proj/thal/db) our aim is to go beyond a data repository towards creating an integrated knowledge resource. To this end, the Arabidopsis genome has been a backbone against which to structure and integrate heterogenous data. The challenges to be met are continuous updating of data, the design of flexible data models that can evolve with new data, the integration of heterogenous data, e.g. through the use of ontologies, comprehensive views and visualization of complex information, simple interfaces for application access locally or via the Internet, and knowledge transfer across species

Softening-up mannan-rich cell walls

The softening and degradation of the cell wall (CW), often mannan enriched, is involved in several processes during development of higher plants, such as meristematic growth, fruit ripening, programmed cell death, and endosperm rupture upon germination. Mannans are also the predominant hemicellulosic CW polymers in many genera of green algae. The endosperm CWs of dry seeds often contain mannan polymers, sometimes in the form of galactomannans (Gal-mannans). The endo-beta-mannanases (MANs) that catalyse the random hydrolysis of the beta-linkage in the mannan backbone are one of the main hydrolytic enzymes involved in the loosening and remodelling of CWs. In germinating seeds, the softening of the endosperm seed CWs facilitates the emergence of the elongating radicle. Hydrolysis and mobilization of endosperm Gal-mannans by MANs also provides a source of nutrients for early seedling growth, since Gal-mannan, besides its structural role, serves as a storage polysaccharide. Therefore, the role of mannans and of their hydrolytic enzymes is decisive in the life cycle of seeds. This review updates and discusses the significance of mannans and MANs in seeds and explores the increasing biotechnological potential of MAN enzymes

Computational Tools for Brassica–Arabidopsis Comparative Genomics

Recent advances, such as the availability of extensive genome survey sequence (GSS) data and draft physical maps, are radically transforming the means by which we can dissect Brassica genome structure and systematically relate it to the Arabidopsis model. Hitherto, our view of the co-linearities between these closely related genomes had been largely inferred from comparative RFLP data, necessitating substantial interpolation and expert interpretation. Sequencing of the Brassica rapa genome by the Multinational Brassica Genome Project will, however, enable an entirely computational approach to this problem. Meanwhile we have been developing databases and bioinformatics tools to support our work in Brassica comparative genomics, including a recently completed draft physical map of B. rapa integrated with anchor probes derived from the Arabidopsis genome sequence. We are also exploring new ways to display the emerging Brassica–Arabidopsis sequence homology data. We have mapped all publicly available Brassica sequences in silico to the Arabidopsis TIGR v5 genome sequence and published this in the ATIDB database that uses Generic Genome Browser (GBrowse). This in silico approach potentially identifies all paralogous sequences and so we colour-code the significance of the mappings and offer an integrated, real-time multiple alignment tool to partition them into paralogous groups. The MySQL database driving GBrowse can also be directly interrogated, using the powerful API offered by the Perl Bio∷DB∷GFF methods, facilitating a wide range of data-mining possibilities

Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB-LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations

RenSeq is a NB-LRR (nucleotide binding-site leucine-rich repeat) gene-targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB-LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB-LRRs and can be accessed through a genome browser that we provide. We compared these NB-LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB-LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co-segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi-ber2) and S. ruiz-ceballosii (Rpi-rzc1), we were able to apply RenSeq successfully to identify markers that co-segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy-to-adapt Galaxy pipelines

Large-Scale Trends in the Evolution of Gene Structures within 11 Animal Genomes

We have used the annotations of six animal genomes (Homo sapiens, Mus musculus, Ciona intestinalis, Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans) together with the sequences of five unannotated Drosophila genomes to survey changes in protein sequence and gene structure over a variety of timescales—from the less than 5 million years since the divergence of D. simulans and D. melanogaster to the more than 500 million years that have elapsed since the Cambrian explosion. To do so, we have developed a new open-source software library called CGL (for “Comparative Genomics Library”). Our results demonstrate that change in intron–exon structure is gradual, clock-like, and largely independent of coding-sequence evolution. This means that genome annotations can be used in new ways to inform, corroborate, and test conclusions drawn from comparative genomics analyses that are based upon protein and nucleotide sequence similarities

The roles of the cation transporters CHX21 and CHX23 in the development of Arabidopsis thaliana

The Arabidopsis thaliana genome encodes a family of 28 proteins whose members have been associated with the transport of monovalent cations across membranes. Experiments have been performed to elucidate the biochemical function and the role in plant development of two closely related members of this CHX family. A genotype carrying a knockout of the AtCHX23 gene (At1g05580) showed no phenotype when grown in glasshouse conditions. In particular, it did not exhibit the reduced root growth phenotype observed for a knockout of its homologue AtCHX21 when exposed to elevated sodium concentration. However, it was not possible to produce plants that were homozygous knockout for both AtCHX21 and AtCHX23. Reverse transcription-PCR (RT-PCR) experiments revealed that both genes are highly expressed in flower buds, flowers, and pollen. However, examination of pollen grain viability and pollen tube growth through excised styles did not reveal a phenotypic difference between the chx21–chx23– condition and other haplotypes. Crosses between selected mutants and wild-type plants in which the chx21–chx23– haplotype was produced by either the male or female parent demonstrated unequivocally that the chx21–chx23– haplotype could not pass through the female line. This suggests that the genes share a critical function in the development and/or function of the female gametophyte and that this function cannot be provided by other members of the AtCHX gene family. Experiments were carried out using the heterologous expression of AtCHX23 in Saccharomyces cerevisiae genotypes carrying combinations of deletions of genes involved in the transport of sodium or potassium across membranes. The results show that CHX23 would only complement the poor colony growth phenotype associated with the deletion of the yeast gene kha1. The conclusion is that both AtCHX21 and AtCHX23 act in potassium homeostasis within the female gametophyte and this is discussed in terms of the diversification of gene sequence and function within the CHX gene family

The Biofuel Feedstock Genomics Resource: a web-based portal and database to enable functional genomics of plant biofuel feedstock species

Major feedstock sources for future biofuel production are likely to be high biomass producing plant species such as poplar, pine, switchgrass, sorghum and maize. One active area of research in these species is genome-enabled improvement of lignocellulosic biofuel feedstock quality and yield. To facilitate genomic-based investigations in these species, we developed the Biofuel Feedstock Genomic Resource (BFGR), a database and web-portal that provides high-quality, uniform and integrated functional annotation of gene and transcript assembly sequences from species of interest to lignocellulosic biofuel feedstock researchers. The BFGR includes sequence data from 54 species and permits researchers to view, analyze and obtain annotation at the gene, transcript, protein and genome level. Annotation of biochemical pathways permits the identification of key genes and transcripts central to the improvement of lignocellulosic properties in these species. The integrated nature of the BFGR in terms of annotation methods, orthologous/paralogous relationships and linkage to seven species with complete genome sequences allows comparative analyses for biofuel feedstock species with limited sequence resources

Characterization of genes in the ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES (AS2/LOB) family in Arabidopsis thaliana, and functional and molecular comparisons between AS2 and other family members

The ASYMMETRIC LEAVES2 (AS2) gene is required for the generation of the flat and symmetrical shape of the leaf lamina in Arabidopsis. AS2 encodes a plant-specific protein with an AS2/LATERAL ORGAN BOUNDARIES (AS2/LOB) domain that includes a cysteine repeat, a conserved single glycine residue and a leucine-zipper-like sequence in its amino-terminal half. The Arabidopsis genome contains 42 genes, including AS2, that encode proteins with an AS2/LOB domain in their amino-terminal halves, and these genes constitute the AS2/LOB gene family. In the present study, we cloned and characterized cDNAs that covered the putative coding regions of all members of this family, and investigated patterns of transcription systematically in Arabidopsis plants. Comparisons among amino acid sequences that had been deduced from the cloned cDNAs revealed eight groups of genes, with two or three members each, and high degrees of identity among entire amino acid sequences, suggesting that some members of the AS2/LOB family might have redundant function(s). Moreover, no member of the family exhibited significant similarity, in terms of the deduced amino acid sequence of the carboxy-terminal half, to AS2. Results of domain swapping between AS2 and other members of the family showed that the AS2/LOB domain of AS2 cannot be functionally replaced by those of other members of the family, and that only a few dissimilarities among respective amino acid residues of the AS2/LOB domain of AS2 and those of other members are important for the specific functions of AS2

REGIA, An EU Project on Functional Genomics of Transcription Factors From Arabidopsis Thaliana

Transcription factors (TFs) are regulatory proteins that have played a pivotal role in the evolution of eukaryotes and that also have great biotechnological potential. REGIA (REgulatory Gene Initiative in Arabidopsis) is an EU-funded project involving 29 European laboratories with the objective of determining the function of virtually all transcription factors from the model plant, Arabidopsis thaliana. REGIA involves: 1. the definition of TF gene expression patterns in Arabidopsis; 2. the identification of mutations at TF loci; 3. the ectopic expression of TFs (or derivatives) in Arabidopsis and in crop plants; 4. phenotypic analysis of the mutants and mis-expression lines, including both RNA and metabolic profiling; 5. the systematic analysis of interactions between TFs; and 6. the generation of a bioinformatics infrastructure to access and integrate all this information. We expect that this programme will establish the full biotechnological potential of plant TFs, and provide insights into hierarchies, redundancies, and interdependencies, and their evolution. The project involves the preparation of both a TF gene array for expression analysis and a normalised full length open reading frame (ORF) library of TFs in a yeast two hybrid vector; the applications of these resources should extend beyond the scope of this programme