Large-scale genomic correlations in Arabidopsis thaliana relate to chromosomal structure

Background

The genome of classical laboratory strains of mice is an artificial mosaic of genomes originated from several mouse subspecies with predominant representation (>90%) of the Mus m. domesticus component. Mice of another subspecies, East European/Asian Mus m. musculus, can interbreed with the classical laboratory strains to generate hybrids with unprecedented phenotypic and genotypic variations. To study these variations in depth we prepared the first genomic large insert BAC library from an inbred strain derived purely from the Mus m. musculus-subspecies. The library will be used to seek and characterize genomic sequences controlling specific monogenic and polygenic complex traits, including modifiers of dominant and recessive mutations.

Results

A representative mouse genomic BAC library was derived from a female mouse of the PWD/Ph inbred strain of Mus m. musculus subspecies. The library consists of 144 768 primary clones from which 97% contain an insert of 120 kb average size. The library represents an equivalent of 6.7 × mouse haploid genome, as estimated from the total number of clones carrying genomic DNA inserts and from the average insert size. The clones were arrayed in duplicates onto eight high-density membranes that were screened with seven single-copy gene probes. The individual probes identified four to eleven positive clones, corresponding to 6.9-fold coverage of the mouse genome. Eighty-seven BAC-ends of PWD/Ph clones were sequenced, edited, and aligned with mouse C57BL/6J (B6) genome. Seventy-three BAC-ends displayed unique hits on B6 genome and their alignment revealed 0.92 single nucleotide polymorphisms (SNPs) per 100 bp. Insertions and deletions represented 0.3% of the BAC end sequences.

Conclusion

Analysis of the novel genomic library for the PWD/Ph inbred strain demonstrated coverage of almost seven mouse genome equivalents and a capability to recover clones for specific regions of PWD/Ph genome. The single nucleotide polymorphism between the strains PWD/Ph and C57BL/6J was 0.92/100 bp, a value significantly higher than between classical laboratory strains. The library will serve as a resource for dissecting the phenotypic and genotypic variations between mice of the Mus m. musculus subspecies and classical laboratory mouse strains.     

Recombination and gene conversion in a 170-kb genomic region of Arabidopsis thaliana

Arabidopsis thaliana is a highly selfing plant that nevertheless appears to undergo substantial recombination. To reconcile its selfing habit with the observations of recombination, we have sampled the genetic diversity of A. thaliana at 14 loci of approximately 500 bp each, spread across 170 kb of genomic sequence centered on a QTL for resistance to herbivory. A total of 170 of the 6321 nucleotides surveyed were polymorphic, with 169 being biallelic. The mean silent genetic diversity (pi(s)) varied between 0.001 and 0.03. Pairwise linkage disequilibria between the polymorphisms were negatively correlated with distance, although this effect vanished when only pairs of polymorphisms with four haplotypes were included in the analysis. The absence of a consistent negative correlation between distance and linkage disequilibrium indicated that gene conversion might have played an important role in distributing genetic diversity throughout the region. We tested this by coalescent simulations and estimate that up to 90% of recombination is due to gene conversion.     

AtMap1: a DNA microarray for genomic deletion mapping in Arabidopsis thaliana

We have designed a novel tiling array, AtMap1, for genomic deletion mapping. AtMap1 is a 60-mer oligonucleotide microarray consisting of 42 497 data probes designed from the genomic sequence of Arabidopsis thaliana Col-0. The average probe interval is 2.8 kb. The performance of the AtMap1 array was assessed using the deletion mutants mag2-2, rot3-1 and zig-2. Eight of the probes showed threefold lower signals in mag2-2 than Col-0. Seven of these probes were located in one region on chromosome 3. We considered these adjacent probes to represent one deletion. This deletion was consistent with a reported deleted region. The other probe was located near the end of chromosome 4. A newly identified deletion around the probe was confirmed by PCR. We also detected the responsible deletions for rot3-1 and zig-2. Thus we concluded that the AtMap1 array was sufficiently sensitive to identify a deletion without any a priori knowledge of the deletion. An analysis of the result of hybridization of Ler and previously reported polymorphism data revealed that the signal decrease tended to depend on the overlap size of sequence polymorphisms. Mutation mapping is time-consuming, laborious and costly. The AtMap1 array removes these limitations.     

Computational identification and analysis of immune-associated nucleotide gene family in Arabidopsis thaliana

GTP-binding proteins represent a ubiquitous regulatory mechanism in controlling growth and development in eukaryotes under normal and stress conditions. The IAN/GIMAP proteins belong to a novel family of functionally uncharacterized GTP-binding proteins expressed in both plant and vertebrate cells during anti-pathogenic responses. To gain novel insights into their roles in plants, we did genome-wide analysis of the IAN/GIMAP gene family. We identified 13 Arabidopsis IAN/GIMAP genes, which share similar gene structures and mostly reside in a tandem cluster on chromosomes. Sequence comparison reveals that these genes encode 26–52 kDa proteins with one GTP-binding domain and a conserved box unique to the family. Phylogenetic analysis suggests that the IAN/GIMAP genes of angiosperms and vertebrates may have evolved by independent gene duplication events. GENEVESTIGATOR sources were mined for comprehensive and comparative Arabidopsis IAN/GIMAP gene family expression analysis. These data reveal that IAN/GIMAPs exhibit diverse expression patterns during development and in response to external stimuli, indicating that these paralogous genes are likely involved in complex biological processes in Arabidopsis. Our present findings provide a basis for elucidating the novel GTPase family protein-mediated regulatory mechanisms in the future.     

The evolutionary origin and genomic organization of SINEs in Arabidopsis thaliana.

We have characterized the two families of SINE retroposons present in Arabidopsis thaliana. The origin, distribution, organization, and evolutionary history of RAthE1 and RAthE2 elements were studied and compared to the well-characterized SINE S1 element from Brassica. Our studies show that RAthE1, RAthE2, and S1 retroposons were generated independently from three different tRNAs. The RAthE1 and RAthE2 families are older than the S1 family and are present in all tested Cruciferae species. The evolutionary history of the RAthE1 family is unusual for SINEs. The 144 RAthE1 elements of the Arabidopsis genome cannot be classified in distinct subfamilies of different evolutionary ages as is the case for S1, RAthE2, and mammalian SINEs. Instead, most RAthE1 elements were probably derived steadily from a single source gene that was maintained intact and active for at least 12-20 Myr, a result suggesting that the RAthE1 source gene was under selection. The distribution of RAthE1 and RAthE2 elements on the Arabidopsis physical map was studied. We observed that, in contrast to other Arabidopsis transposable elements, SINEs are not concentrated in the heterochromatic regions. Instead, SINEs are grouped in the euchromatic chromosome territories several hundred kilobase pairs long. In these territories, SINE elements are closely associated with genes. A retroposition partnership between Arabidopsis SINEs and LINEs is proposed.     

Evaluation of gene prediction software using a genomic data set: application to Arabidopsis thaliana sequences

MOTIVATION: The annotation of the Arabidopsis thaliana genome remains a problem in terms of time and quality. To improve the annotation process, we want to choose the most appropriate tools to use inside a computer-assisted annotation platform. We therefore need evaluation of prediction programs with Arabidopsis sequences containing multiple genes. RESULTS: We have developed AraSet, a data set of contigs of validated genes, enabling the evaluation of multi-gene models for the Arabidopsis genome. Besides conventional metrics to evaluate gene prediction at the site and the exon levels, new measures were introduced for the prediction at the protein sequence level as well as for the evaluation of gene models. This evaluation method is of general interest and could apply to any new gene prediction software and to any eukaryotic genome. The GeneMark.hmm program appears to be the most accurate software at all three levels for the Arabidopsis genomic sequences. Gene modeling could be further improved by combination of prediction software. AVAILABILITY: The AraSet sequence set, the Perl programs and complementary results and notes are available at http://sphinx.rug.ac.be:8080/biocomp/napav/. CONTACT: Pierre.Rouze@gengenp.rug.ac.be.     

Prediction of glycosylphosphatidylinositol-anchored proteins in Arabidopsis. A genomic analysis

Glycosylphosphatidylinositol (GPI) anchoring of proteins provides a potential mechanism for targeting to the plant plasma membrane and cell wall. However, relatively few such proteins have been identified. Here, we develop a procedure for database analysis to identify GPI-anchored proteins (GAP) based on their possession of common features. In a comprehensive search of the annotated Arabidopsis genome, we identified 167 novel putative GAP in addition to the 43 previously described candidates. Many of these 210 proteins show similarity to characterized cell surface proteins. The predicted GAP include homologs of beta-1,3-glucanases (16), metallo- and aspartyl proteases (13), glycerophosphodiesterases (6), phytocyanins (25), multi-copper oxidases (2), extensins (6), plasma membrane receptors (19), and lipid-transfer-proteins (18). Classical arabinogalactan (AG) proteins (13), AG peptides (9), fasciclin-like proteins (20), COBRA and 10 homologs, and novel potential signaling peptides that we name GAPEPs (8) were also identified. A further 34 proteins of unknown function were predicted to be GPI anchored. A surprising finding was that over 40% of the proteins identified here have probable AG glycosylation modules, suggesting that AG glycosylation of cell surface proteins is widespread. This analysis shows that GPI anchoring is likely to be a major modification in plants that is used to target a specific subset of proteins to the cell surface for extracellular matrix remodeling and signaling.     

Non-random association of transposable elements with duplicated genomic blocks in Arabidopsis thaliana

The genome of Arabidopsis thaliana is known to contain numerous open reading frames apparently encoding transposases. In order to test the hypothesis that transposable elements have played a role in segmental duplication in this species, we compared the distribution of transposable elements with that of genomic windows that shared gene families to a greater extent than expected by chance. Phylogenetic analyses indicated that duplication of these segments occurred after the monocot-dicot divergence and probably after the eurosid I-eurosid II divergence. Known transposable elements were found to occur in putatively duplicated segments to a far greater extent than expected on the basis of their genome-wide distribution, suggesting that transposition may have played a role in segmental duplication in this species.     

Direct gene transfer to protoplasts of Arabidopsis thaliana

We performed a series of direct gene transfer experiments with protoplasts of Arabidopsis thaliana ecotype Zürich. An average of more than 100 transformants were selected per 10⁶6 treated protoplasts. Stable transformation was confirmed by integration of the marker gene into high molecular weight DNA and by its genetic transmission to subsequent offspring generations.Abbreviations ATF absolute transformation frequency - PEG polyethyleneglycol - hpt hygromycin phosphotransferase gene - CTAB N-Cetyl-N,N,N-trimethyl-ammonium bromide - MES 2-(N-morpholino)ethanesulfonic acid     

Human intrinsic factor expressed in the plant Arabidopsis thaliana.

Intrinsic factor (IF) is the gastric protein that promotes the intestinal uptake of vitamin B12. Gastric IF from animal sources is used in diagnostic tests and in vitamin pills. However, administration of animal IF to humans becomes disadvantageous because of possible pathogenic transmission and contamination by other B12 binders. We tested the use of recombinant plants for large-scale production of pathogen-free human recombinant IF. Human IF was successfully expressed in the recombinant plant Arabidopsis thaliana. Extract from fresh plants possessed high B12-binding capacity corresponding to 70 mg IF per 1 kg wet weight. The dried plants still retained 60% of the IF activity. The purified IF preparation consisted of a 50-kDa glycosylated protein with the N-terminal sequence of mature IF. Approximately one-third of the protein was cleaved at the internal site em leader PSNP downward arrow GPGP. The key properties of the preparation obtained were identical to those of native IF: the binding curves of vitamin B12 to recombinant IF and gastric IF were the same, as were those for a B12 analogue cobinamide, which binds to IF with low affinity. The absorbance spectra of the vitamin bound to recombinant IF and gastric IF were alike, as was the interaction of recombinant and native IF with the specific receptor cubilin. The data presented show that recombinant plants have a great potential as a large-scale source of human IF for analytical and therapeutic purposes.