Construction of a 10,000-marker ultradense genetic recombination map of potato: providing a framework for accelerated gene isolation and a genomewide physical map.

An ultradense genetic linkage map with >10,000 AFLP loci was constructed from a heterozygous diploid potato population. To our knowledge, this is the densest meiotic recombination map ever constructed. A fast marker-ordering algorithm was used, based on the minimization of the total number of recombination events within a given marker order in combination with genotyping error-detection software. This resulted in "skeleton bin maps," which can be viewed as the most parsimonious marker order. The unit of distance is not expressed in centimorgans but in "bins." A bin is a position on the genetic map with a unique segregation pattern that is separated from adjacent bins by a single recombination event. Putative centromeres were identified by a strong clustering of markers, probably due to cold spots for recombination. Conversely, recombination hot spots resulted in large intervals of up to 15 cM without markers. The current level of marker saturation suggests that marker density is proportional to physical distance and independent of recombination frequency. Most chromatids (92%) recombined once or never, suggesting strong chiasma interference. Absolute chiasma interference within a chromosome arm could not be demonstrated. Two examples of contig construction and map-based cloning have demonstrated that the marker spacing was in accordance with the expected physical distance: approximately one marker per BAC length. Currently, the markers are used for genetic anchoring of a physical map of potato to deliver a sequence-ready minimal tiling path of BAC contigs of specific chromosomal regions for the potato genome sequencing consortium (http://www.potatogenome.net).

Specific patterns of changes in wheat gene expression after treatment with three antifungal compounds.

The two fungicides azoxystrobin and fenpropimorph are used against powdery mildew and rust diseases in wheat (Triticum aestivumL). Azoxystrobin, a strobilurin, inhibits fungal mitochondrial respiration and fenpropimorph, a morpholin, represses biosynthesis of ergosterol, the major sterol of fungal membranes. Although the fungitoxic activity of these compounds is well understood, their effects on plant metabolism remain unclear. In contrast to the fungicides which directly affect pathogen metabolism, benzo(1,2,3) thiadiazole-7-carbothioic acid S-methylester (BTH) induces resistance against wheat pathogens by the activation of systemic acquired resistance in the host plant. In this study, we monitored gene expression in spring wheat after treatment with each of these agrochemicals in a greenhouse trial using a microarray containing 600 barley cDNA clones. Defence-related genes were strongly induced after treatment with BTH, confirming the activation of a similar set of genes as in dicot plants following salicylic acid treatment. A similar gene expression pattern was observed after treatment with fenpropimorph and some defence-related genes were induced by azoxystrobin, demonstrating that these fungicides also activate a defence reaction. However, less intense responses were triggered than with BTH. The same experiments performed under field conditions gave dramatically different results. No gene showed differential expression after treatment and defence genes were already expressed at a high level before application of the agrochemicals. These differences in the expression patterns between the two environments demonstrate the importance of plant growth conditions for testing the impact of agrochemicals on plant metabolism.

Ancient haplotypes resulting from extensive molecular rearrangements in the wheat A genome have been maintained in species of three different ploidy levels.

Plant genomes, in particular grass genomes, evolve very rapidly. The closely related A genomes of diploid, tetraploid, and hexaploid wheat are derived from a common ancestor that lived <3 million years ago and represent a good model to study molecular mechanisms involved in such rapid evolution. We have sequenced and compared physical contigs at the Lr10 locus on chromosome 1AS from diploid (211 kb), tetraploid (187 kb), and hexaploid wheat (154 kb). A maximum of 33% of the sequences were conserved between two species. The sequences from diploid and tetraploid wheat shared all of the genes, including Lr10 and RGA2 and define a first haplotype (H1). The 130-kb intergenic region between Lr10 and RGA2 was conserved in size despite its activity as a hot spot for transposon insertion, which resulted in >70% of sequence divergence. The hexaploid wheat sequence lacks both Lr10 and RGA2 genes and defines a second haplotype, H2, which originated from ancient and extensive rearrangements. These rearrangements included insertions of retroelements and transposons deletions, as well as unequal recombination within elements. Gene disruption in haplotype H2 was caused by a deletion and subsequent large inversion. Gene conservation between H1 haplotypes, as well as conservation of rearrangements at the origin of the H2 haplotype at three different ploidy levels indicate that the two haplotypes are ancient and had a stable gene content during evolution, whereas the intergenic regions evolved rapidly. Polyploidization during wheat evolution had no detectable consequences on the structure and evolution of the two haplotypes.

Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region.

To isolate genes of interest in plants, it is essential to construct bacterial artificial chromosome (BAC) libraries from specific genotypes. Construction and organisation of BAC libraries is laborious and costly, especially from organisms with large and complex genomes. In the present study, we developed the pooled BAC library strategy that allows rapid and low cost generation and screening of genomic libraries from any genotype of interest. The BAC library is constructed, directly organised into a few pools and screened for BAC clones of interest using PCR and hybridisation steps, without requiring organization into individual clones. As a proof of concept, a pooled BAC library of approximately 177,000 recombinant clones has been constructed from the barley cultivar Cebada Capa that carries the Rph7 leaf rust resistance gene. The library has an average insert size of 140 kb, a coverage of six barley genome equivalents and is organised in 138 pools of about 1,300 clones each. We rapidly established a single contig of six BAC clones spanning 230 kb at the Rph7 locus on chromosome 3HS. The described low-cost cloning strategy is fast and will greatly facilitate direct targeting of genes and large-scale intra- and inter-species comparative genome analysis.

Large intraspecific haplotype variability at the Rph7 locus results from rapid and recent divergence in the barley genome.

To study genome evolution and diversity in barley (Hordeum vulgare), we have sequenced and compared more than 300 kb of sequence spanning the Rph7 leaf rust disease resistance gene in two barley cultivars. Colinearity was restricted to five genic and two intergenic regions representing <35% of the two sequences. In each interval separating the seven conserved regions, the number and type of repetitive elements were completely different between the two homologous sequences, and a single gene was absent in one cultivar. In both cultivars, the nonconserved regions consisted of approximately 53% repetitive sequences mainly represented by long-terminal repeat retrotransposons that have inserted <1 million years ago. PCR-based analysis of intergenic regions at the Rph7 locus and at three other independent loci in 41 H. vulgare lines indicated large haplotype variability in the cultivated barley gene pool. Together, our data indicate rapid and recent divergence at homologous loci in the genome of H. vulgare, possibly providing the molecular mechanism for the generation of high diversity in the barley gene pool. Finally, comparative analysis of the gene composition in barley, wheat (Triticum aestivum), rice (Oryza sativa), and sorghum (Sorghum bicolor) suggested massive gene movements at the Rph7 locus in the Triticeae lineage.

Analysis of two alleles of the urease gene from potato: polymorphisms, expression, and extensive alternative splicing of the corresponding mRNA.

Globally, urea is the most widely used nitrogen fertilizer and is made accessible to plants via the urease reaction. However, sequence information for the plant enzyme is scarce. A cDNA encoding urease from soybean (Glycine max) has been cloned and sequence information has been obtained for two alleles (11 and 19 kbp, respectively) of the potato (Solanum tuberosum, cv. Desiree) urease gene and the corresponding cDNAs. It was found that urease is encoded by a single copy gene in several solanaceous species, and maps to chromosome V in potato. By contrast, the presence of two urease genes was reported for soybean. Comparative analysis of 11 kbp overlapping allelic DNA allowed the quantification of single nucleotide polymorphisms and revealed the presence of a truncated Ty1-copia retrotransposon in one of the alleles. Both alleles contained a copy of a terminal-repeat retrotransposon in miniature (TRIM). 25-50% of urease pre-mRNAs from both alleles were alternatively spliced in a variety of different ways. The retrotransposons had no effect on splicing. While urease is expressed in all tissues tested, its mRNA and protein are of low abundance. The TATA-less urease promoter appears to drive low-level housekeeping transcription. An in silico analysis showed that eukaryotic urease protein sequences are very similar to sequences from prokaryotic enzymes, conserving all residues of known functional importance. It is therefore likely that all known ureases are structurally similar, employing the same catalytic mechanism.

Toward a marker-dense meiotic map of the potato genome: lessons from linkage group I.

Segregation data were obtained for 1260 potato linkage group I-specific AFLP loci from a heterozygous diploid potato population. Analytical tools that identified potential typing errors and/or inconsistencies in the data and that assembled cosegregating markers into bins were applied. Bins contain multiple-marker data sets with an identical segregation pattern, which is defined as the bin signature. The bin signatures were used to construct a skeleton bin map that was based solely on observed recombination events. Markers that did not match any of the bin signatures exactly (and that were excluded from the calculation of the skeleton bin map) were placed on the map by maximum likelihood. The resulting maternal and paternal maps consisted of 95 and 101 bins, respectively. Markers derived from EcoRI/MseI, PstI/MseI, and SacI/MseI primer combinations showed different genetic distributions. Approximately three-fourths of the markers placed into a bin were considered to fit well on the basis of an estimated residual "error rate" of 0-3%. However, twice as many PstI-based markers fit badly, suggesting that parental PstI-site methylation patterns had changed in the population. Recombination frequencies were highly variable across the map. Inert, presumably centromeric, regions caused extensive marker clustering while recombination hotspots (or regions identical by descent) resulted in empty bins, despite the level of marker saturation.