Pieter Vos

Evaluation of the DNA fingerprinting method AFLP as a new tool in bacterial taxonomy

We investigated the usefulness of a novel DNA fingerprinting technique, AFLP, which is based on the selective amplification of genomic restriction fragments by PCR, to differentiate bacterial strains at the subgeneric level. In totals, 147 bacterial strains were subjected to AFLP fingerprinting: 36 Xanthomonas strains, including 23 pathovars of Xanthomonas axonopodis and six pathovars of Xanthomonas vasicola, one strain of Stenotrophomonas, 90 genotypically characterized strains comprising all 14 hybridization groups currently described in the genus Aeromonas, and four strains of each of the genera Clostridium, Bacillus, Acinetobacter, Pseudomonas and Vibrio. Depending on the genus, total genomic DNA of each bacterium was digested with a particular combination of two restriction endonucleases and the resulting fragments were ligated to restriction halfsite-specific adaptors. These adaptors served as primer-binding sites allowing the fragments to be amplified by selective PCR primers that extend beyond the adaptor and restriction site sequences. Following electrophoretic separation on 5% (w/v) polyacrylamide/8.3 M urea, amplified products could be visualized by autoradiography because one of the selective primers was radioactively labelled. The resulting banding patterns, containing approximately 30-50 visualized PCR products in the size range 80-550 bp, were captured by a high-resolution densitoscanner and further processed for computer-assisted analysis to determine band-based similarity coefficients. This study reveals extensive evidence for the applicability of AFLP in bacterial taxonomy through comparison of the newly obtained data with results previously obtained by well-established genotypic and chemotaxonomic methods such as DNA-DNA hybridization and cellular fatty acid analysis. In addition, this study clearly demonstrates the superior discriminative power of AFLP towards the differentiation of highly related bacterial strains that belong to the same species or even biovar (i.e. to characterize strains at the infrasubspecific level), highlighting the potential of this novel fingerprinting method in epidemiological and evolutionary studies.

Combined mapping of AFLP and RFLP markers in barley

AFLP marker technology allows efficient DNA fingerprinting and the analysis of large numbers of polymorphic restriction fragments on polyacrylamide gels. Using the doubled haploids from the F1 of the cross Proctor × Nudinka, 118 AFLP markers were mapped onto a barley (Hordeum vulgare L.) RFLP map, also including five microsatellite and four protein marker loci. The AFLP markers mapped to all parts of the barley chromosomes and filled in the gaps on barley chromosomes 2L, 4L and 6 in which no RFLP loci had been mapped. Interestingly, the AFLP markers seldom interrupted RFLP clusters, but grouped next to them. The combined map covers 1873 cM, with a total of 282 markers. The merging of AFLP and RFLP markers increased the total map length; 402 cM were added to the map at the tips of chromosomes or in regions corresponding to earlier gaps. Another 375 cM resulted from mapping AFLP markers near to RFLP clusters or in between non-clustered RFLP markers.

Dissection of the fusarium I2 gene cluster in tomato reveals six homologs and one active gene copy.

The I2 locus in tomato confers resistance to race 2 of the soil-borne fungus Fusarium oxysporum f sp lycopersici. The selective restriction fragment amplification (AFLP) positional cloning strategy was used to identify I2 in the tomato genome. A yeast artificial chromosome (YAC) clone covering ~750 kb encompassing the I2 locus was isolated, and the AFLP technique was used to derive tightly linked AFLP markers from this YAC clone. Genetic complementation analysis in transgenic R1 plants using a set of overlapping cosmids covering the I2 locus revealed three cosmids giving full resistance to F. o. lycopersici race 2. These cosmids shared a 7-kb DNA fragment containing an open reading frame encoding a protein with similarity to the nucleotide binding site leucine-rich repeat family of resistance genes. At the I2 locus, we identified six additional homologs that included the recently identified I2C-1 and I2C-2 genes. However, cosmids containing the I2C-1 or I2C-2 gene could not confer resistance to plants, indicating that these members are not the functional resistance genes. Alignments between the various members of the I2 gene family revealed two significant variable regions within the leucine-rich repeat region. They consisted of deletions or duplications of one or more leucine-rich repeats. We propose that one or both of these leucine-rich repeats are involved in Fusarium wilt resistance with I2 specificity.

The tomato Mi-1 gene confers resistance to both root-knot nematodes and potato aphids.

Mi-1, a Lycopersicon peruvianum gene conferring resistance to the agricultural pests, root-knot nematodes, and introgressed into tomato, has been cloned using a selective restriction fragment amplification based strategy. Complementation analysis of a susceptible tomato line with a 100 kb cosmid array yielded a single cosmid clone capable of conferring resistance both to the root-knot nematode Meloidogyne incognita and to an unrelated pathogen, the potato aphid Macrosiphum euphorbiae. This resistance was stable. The Mi-1 gene encodes a protein sharing structural features with the nucleotide-binding site leucine-rich repeat–containing type of plant resistance genes.

Evidence for an ancient chromosomal duplication in Arabidopsis thaliana by sequencing and analyzing a 400-kb contig at the APETALA2 locus on chromosome 41

As part of the European Scientists Sequencing Arabidopsis program, a contiguous region (396 607 bp) located on chromosome 4 around the APETALA2 gene was sequenced. Analysis of the sequence and comparison to public databases predicts 103 genes in this area, which represents a gene density of one gene per 3.85 kb. Almost half of the genes show no significant homology to known database entries. In addition, the first 45 kb of the contig, which covers 11 genes, is similar to a region on chromosome 2, as far as coding sequences are concerned. This observation indicates that ancient duplications of large pieces of DNA have occurred in Arabidopsis.