Unified display of Arabidopsis thaliana physical maps from AtDB, the A.thaliana database.

In the past several years, there has been a tremendous effort to construct physical maps and to sequence the genome of Arabidopsis thaliana. As a result, four of the five chromosomes are completely covered by overlapping clones except at the centromeric and nucleolus organizer regions (NOR). In addition, over 30% of the genome has been sequenced and completion is anticipated by the end of the year 2000. Despite these accomplishments, the physical maps are provided in many formats on laboratories' Web sites. These data are thus difficult to obtain in a coherent manner for researchers. To alleviate this problem, AtDB (Arabidopsis thaliana DataBase, URL: http://genome-www.stanford.edu/Arabidopsis/) has constructed a unified display of the physical maps where all publicly available physical-map data for all chromosomes are presented through the Web in a clickable, 'on-the-fly' graphic, created by CGI programs that directly consult our relational database.     

Poorly known relatives of Arabidopsis thaliana

Non-model Arabidopsis species have been widely used as outgroup taxa in studies of molecular evolution. In Arabidopsis lyrata, Arabidopsis halleri and Arabidopsis arenosa, traits pertaining to self-incompatibility, heavy metal tolerance and inter-specific hybridization have been subjected to detailed genetic analysis. However, the full potential for exploring the causes and consequences of natural variation in complex traits within the genus Arabidopsis has not been widely appreciated or realized. Here, we draw on broadly dispersed information to characterize the basic biology, ecology, population genetics and molecular evolution for these three non-model Arabidopsis species. We illustrate how the wealth of functional and genomic tools pioneered in A. thaliana can be applied to gain insights into adaptive evolution of ecologically important traits and genome-wide processes, such as polyploidy, speciation and reticulate evolution, within and among Arabidopsis species.     

DAtA: database of Arabidopsis thaliana annotation

The Database of Arabidopsis thaliana Annotation (D At A) was created to enable easy access to and analysis of all the Arabidopsis genome project annotation. The database was constructed using the completed A.thaliana genomic sequence data currently in GenBank. An automated annotation process was used to predict coding sequences for GenBank records that do not include annotation. D At A also contains protein motifs and protein similarities derived from searches of the proteins in D At A with motif databases and the non-redundant protein database. The database is routinely updated to include new GenBank submissions for Arabidopsis genomic sequences and new Blast and protein motif search results. A web interface to D At A allows coding sequences to be searched by name, comment, blast similarity or motif field. In addition, browse options present lists of either all the protein names or identified motifs present in the sequenced A.thaliana genome. The database can be accessed at http://baggage. stanford.edu/group/arabprotein/     

Birth, death and subfunctionalization in the Arabidopsis genome

Arabidopsis thaliana is now a model system, not just for plant biology but also for comparative genomics. The completion of the sequences of two closely related species, Arabidopsis lyrata and Brassica rapa, is complemented by genomic comparisons among A. thaliana accessions and mutation accumulation lines. Together these genomic data document the birth of new genes via gene duplication, transposon exaptation and de novo formation of new genes from noncoding sequence. Most novel loci exhibit low expression, and are undergoing pseudogenization or subfunctionalization. Comparatively, A. thaliana has lost large amounts of sequence through deletion, particularly of transposable elements. Intraspecific genomic variation indicates high rates of deletion mutations and deletion polymorphisms across accessions, shedding light on the history of Arabidopsis genome architecture.     

GARNet, the Genomic Arabidopsis Resource Network

GARNet, the Genomic Arabidopsis Resource Network, was created to establish UK-based facilities for functional genomic research on Arabidopsis thaliana. In addition, GARNet provides a platform for international Arabidopsis research and for research on other plant species. To use the GARNet facilities apply via the website: http://garnet.arabidopsis.org.uk. All GARNet services and resources are publicly available, and data created using the GARNet resources will be freely distributed via databases held at the Nottingham Arabidopsis Stock Centre and the John Innes Centre.     

Genome evolution among cruciferous plants: a lecture from the comparison of the genetic maps of three diploid species--Capsella rubella, Arabidopsis lyrata subsp. petraea, and A. thaliana

Comparative mapping in cruciferous plants is ongoing, and recently two additional genetic maps of diploid Capsella and Arabidopsis lyrata subsp. petraea have been presented. We compared both maps with each other using the sequence map and genomic data resources from Arabidopsis thaliana as a reference. The ancestors of the species pair Capsella-Arabidopsis diverged from one another approximately 10-14 million years ago (mya), whereas Arabidopsis thaliana and Arabidopsis lyrata have been separated since roughly 5-6 mya. Our analysis indicated that among diploid Capsella and Arabidopsis lyrata all eight genetic linkage groups are totally colinear to each other, with only two inversions significantly differentiating these two species.By minimizing the number of chromosomal rearrangements during genome evolution, we presented a model of chromosome evolution involving all three species. From this scenario, it is obvious that Arabidopsis thaliana underwent a dramatic genome reconstruction, with a base chromosome number reduction from five to eight and with approximately 1.3 chromosomal rearrangements per million years. In contrast, the terminal lineage leading to Capsella has only undergone less than 0.09 rearrangements per million years. This is the same rate as calculated for Arabidopsis lyrata since its separation from the Capsella lineage 10-14 mya. These results are in strong contrast to all overestimated rates calculated from comparisons of the systems Arabidopsis thaliana and Brassica, and our data demonstrate the problematic nature of both model systems.     

The Arabidopsis Information Resource (TAIR): a comprehensive database and web-based information retrieval, analysis, and visualization system for a model plant

Arabidopsis thaliana, a small annual plant belonging to the mustard family, is the subject of study by an estimated 7000 researchers around the world. In addition to the large body of genetic, physiological and biochemical data gathered for this plant, it will be the first higher plant genome to be completely sequenced, with completion expected at the end of the year 2000. The sequencing effort has been coordinated by an international collaboration, the Arabidopsis Genome Initiative (AGI). The rationale for intensive investigation of Arabidopsis is that it is an excellent model for higher plants. In order to maximize use of the knowledge gained about this plant, there is a need for a comprehensive database and information retrieval and analysis system that will provide user-friendly access to Arabidopsis information. This paper describes the initial steps we have taken toward realizing these goals in a project called The Arabidopsis Information Resource (TAIR) (www.arabidopsis.org).     

Natural genetic variation in Arabidopsis: tools, traits and prospects for evolutionary ecology

BACKGROUND: The model plant Arabidopsis thaliana (Arabidopsis) shows a wide range of genetic and trait variation among wild accessions. Because of its unparalleled biological and genomic resources, the potential of Arabidopsis for molecular genetic analysis of this natural variation has increased dramatically in recent years. SCOPE: Advanced genomics has accelerated molecular phylogenetic analysis and gene identification by quantitative trait loci (QTL) mapping and/or association mapping in Arabidopsis. In particular, QTL mapping utilizing natural accessions is now becoming a major strategy of gene isolation, offering an alternative to artificial mutant lines. Furthermore, the genomic information is used by researchers to uncover the signature of natural selection acting on the genes that contribute to phenotypic variation. The evolutionary significance of such genes has been evaluated in traits such as disease resistance and flowering time. However, although molecular hallmarks of selection have been found for the genes in question, a corresponding ecological scenario of adaptive evolution has been difficult to prove. Ecological strategies, including reciprocal transplant experiments and competition experiments, and utilizing near-isogenic lines of alleles of interest will be a powerful tool to measure the relative fitness of phenotypic and/or allelic variants. CONCLUSIONS: As the plant model organism, Arabidopsis provides a wealth of molecular background information for evolutionary genetics. Because genetic diversity between and within Arabidopsis populations is much higher than anticipated, combining this background information with ecological approaches might well establish Arabidopsis as a model organism for plant evolutionary ecology.     

Mapping salinity tolerance during Arabidopsis thaliana germination and seedling growth

To characterize and dissect genetic variation for salinity tolerance, we assessed variation in salinity tolerance during germination and seedling growth for a worldwide sample of Arabidopsis thaliana accessions. By combining QTL mapping, association mapping and expression data, we identified genomic regions involved in salinity response. Among the worldwide sample, we found germination ability within a moderately saline environment (150 mM NaCl) varied considerable, from >90% among the most tolerant lines to complete inability to germinate among the most susceptible. Our results also demonstrated wide variation in salinity tolerance within A. thaliana RIL populations and identified multiple genomic regions that contribute to this variation. These regions contain known candidate genes, but at least four of the regions contain loci not yet associated with salinity tolerance response phenotypes. Our observations suggest A. thaliana natural variation may be an underutilized resource for investigating salinity stress response.     

Comparative physical mapping reveals features of microsynteny between Glycine max, Medicago truncatula, and Arabidopsis thaliana.

To gain insight into genomic relationships between soybean (Glycine max) and Medicago truncatula, eight groups of bacterial artificial chromosome (BAC) contigs, together spanning 2.60 million base pairs (Mb) in G. max and 1.56 Mb in M. truncatula, were compared through high-resolution physical mapping combined with sequence and hybridization analysis of low-copy BAC ends. Cross-hybridization among G. max and M. truncatula contigs uncovered microsynteny in six of the contig groups and extensive microsynteny in three. Between G. max homoeologous (within genome duplicate) contigs, 85% of coding and 75% of noncoding sequences were conserved at the level of cross-hybridization. By contrast, only 29% of sequences were conserved between G. max and M. truncatula, and some kilobase-scale rearrangements were also observed. Detailed restriction maps were constructed for 11 contigs from the three highly microsyntenic groups, and these maps suggested that sequence order was highly conserved between G. max duplicates and generally conserved between G. max and M. truncatula. One instance of homoeologous BAC contigs in M. truncatula was also observed and examined in detail. A sequence similarity search against the Arabidopsis thaliana genome sequence identified up to three microsyntenic regions in A. thaliana for each of two of the legume BAC contig groups. Together, these results confirm previous predictions of one recent genome-wide duplication in G. max and suggest that M. truncatula also experienced ancient large-scale genome duplications.     

Linkage maps for Arabidopsis lyrata subsp. lyrata and Arabidopsis lyrata subsp. petraea combining anonymous and Arabidopsis thaliana-derived markers.

Arabidopsis lyrata, a close relative of the model plant Arabidopsis thaliana, is 1 of a few plant species for which the genome is to be entirely sequenced, which promises to yield important insights into genome evolution. Only 2 sparse linkage maps have been published, and these were based solely on markers derived from the A. thaliana genome. Because the genome of A. lyrata is practically twice as large as that of A. thaliana, the extent of map coverage of the A. lyrata genome remains uncertain. In this study, a 2-way pseudo-testcross strategy was used to construct genetic linkage maps of A. lyrata subsp. petraea and A. lyrata subsp. lyrata, using simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) markers from the A. thaliana genome, and anonymous amplified fragment length polymorphism (AFLP) markers that could potentially uncover regions unique to the A. lyrata genome. The SSR and CAPS markers largely confirmed the relationships between linkage groups in A. lyrata and A. thaliana. AFLP markers slightly increased the coverage of the A. lyrata maps, but mostly increased marker density on the linkage groups. We noted a much lower level of polymorphism and a greater segregation distortion in A. lyrata subsp. lyrata markers. The implications of these findings for the sequencing of the A. lyrata genome are discussed.     

Analysis of Arabidopsis genome sequence reveals a large new gene family in plants

A detailed analysis of the currently available Arabidopsis thaliana genomic sequence has revealed the presence of a large number of open reading frames with homology to the stigmatic self-incompatibility (S) genes of Papaver rhoeas. The products of these potential genes are all predicted to be relatively small, basic, secreted proteins with similar predicted secondary structures. We have named these potential genes SPH (S-protein homologues). Their presence appears to have been largely missed by the prediction methods currently used on the genomic sequence. Equivalent homologues could not be detected in the human, microbial, Drosophila or C. elegans genomic databases, suggesting a function specific to plants. Preliminary RT-PCR analysis indicates that at least two members of the family (SPH1, SPH8) are expressed, with expression being greatest in floral tissues. The gene family may total more than 100 members, and its discovery not only illustrates the importance of the genome sequencing efforts, but also indicates the extent of information which remains hidden after the initial trawl for potential genes.     

Partial shotgun sequencing of the Boechera stricta genome reveals extensive microsynteny and promoter conservation with Arabidopsis

Comparative genomics provides insight into the evolutionary dynamics that shape discrete sequences as well as whole genomes. To advance comparative genomics within the Brassicaceae, we have end sequenced 23,136 medium-sized insert clones from Boechera stricta, a wild relative of Arabidopsis (Arabidopsis thaliana). A significant proportion of these sequences, 18,797, are nonredundant and display highly significant similarity (BLASTn e-value < or = 10(-30)) to low copy number Arabidopsis genomic regions, including more than 9,000 annotated coding sequences. We have used this dataset to identify orthologous gene pairs in the two species and to perform a global comparison of DNA regions 5' to annotated coding regions. On average, the 500 nucleotides upstream to coding sequences display 71.4% identity between the two species. In a similar analysis, 61.4% identity was observed between 5' noncoding sequences of Brassica oleracea and Arabidopsis, indicating that regulatory regions are not as diverged among these lineages as previously anticipated. By mapping the B. stricta end sequences onto the Arabidopsis genome, we have identified nearly 2,000 conserved blocks of microsynteny (bracketing 26% of the Arabidopsis genome). A comparison of fully sequenced B. stricta inserts to their homologous Arabidopsis genomic regions indicates that indel polymorphisms >5 kb contribute substantially to the genome size difference observed between the two species. Further, we demonstrate that microsynteny inferred from end-sequence data can be applied to the rapid identification and cloning of genomic regions of interest from nonmodel species. These results suggest that among diploid relatives of Arabidopsis, small- to medium-scale shotgun sequencing approaches can provide rapid and cost-effective benefits to evolutionary and/or functional comparative genomic frameworks.     

PFGE-resolved RFLP analysis and long range restriction mapping of the DNA of Arabidopsis thaliana using whole YAC clones as probes.

The cleavage patterns of 23 rare-cutting restriction endonucleases (rcREs) on high molecular weight DNA, isolated from leaves of Arabidopsis thaliana (Arabidopsis), have been analysed using pulsed field gel electrophoresis (PFGE). The DNA digested with rcREs can be used for restriction fragment length polymorphism (RFLP) analysis. We show that RFLPs are more readily identified in restriction fragments that require resolution by PFGE than in smaller restriction fragments. Taking advantage of the low dispersed repetitive DNA content of the Arabidopsis genome, whole yeast artificial chromosomes (YACs) were used as probes to PFGE resolved genomic DNA. This enabled whole YAC clones to be used as RFLP markers and long range restriction maps to be constructed. These techniques should enhance the analysis of regions of the genome of Arabidopsis (and other organisms with low levels of dispersed repetitive DNA) that are the subject of chromosome walking strategies to isolate particular loci.     

Characterization of minisatellites in Arabidopsis thaliana with sequence similarity to the human minisatellite core sequence.

A strategy based on random PCR amplification was used to isolate new repetitive elements of Arabidopsis thaliana. One of the random PCR product analyzed by this approach contained a tandem repetitive minisatellite sequence composed of 33 bp repeated units. The genomic locus corresponding to this PCR product was isolated by screening a lambda genomic library. New related loci were also isolated from the genomic library by screening with a 14 mer oligonucleotide representing a region conserved among the different repeated units. Alignment of the consensus sequence for each minisatellite locus allowed the definition of an Arabidopsis thaliana core sequence that shows strong sequence similarities with the human core sequence and with the generalized recombination signal Chi of Escherichia coli. The minisatellites were tested for their ability to detect polymorphism, and their chromosomal position was established.     

A multiplex GC-MS/MS technique for the sensitive and quantitative single-run analysis of acidic phytohormones and related compounds,and its application to Arabidopsis thaliana

A highly sensitive and accurate multiplex gas chromatography-tandem mass spectrometry (GC-MS/MS) technique is reported for indole-3-acetic acid, abscisic acid, jasmonic acid, 12-oxo-phytodienoic acid and salicylic acid. The optimized setup allows the routine processing and analysis of up to 60 plant samples of between 20 and 200 mg of fresh weight per day. The protocol was designed and the equipment used was chosen to facilitate implementation of the method into other laboratories and to provide access to state-of-the-art analytical tools for the acidic phytohormones and related signalling molecules. Whole-plant organ-distribution maps for indole-3-acetic acid, abscisic acid, jasmonic acid, 12-oxo-phytodienoic acid and salicylic acid were generated for Arabidopsis thaliana (L.) Heynh. For leaves of A. thaliana, a spatial resolution of hormone quantitation down to approximately 2 mm(2) was achieved.     

Design, implementation and maintenance of a model organism database for Arabidopsis thaliana

The Arabidopsis Information Resource (TAIR) is a web-based community database for the model plant Arabidopsis thaliana. It provides an integrated view of genes, sequences, proteins, germplasms, clones, metabolic pathways, gene expression, ecotypes, polymorphisms, publications, maps and community information. TAIR is developed and maintained by collaboration between software developers and biologists. Biologists provide specification and use cases for the system, acquire, analyse and curate data, interact with users and test the software. Software developers design, implement and test the database and software. In this review, we briefly describe how TAIR was built and is being maintained.