SummaryWithin the framework of the International Solanaceae Genome Project, the genome of tomato (Solanum lycopersicum) is currently being sequenced. We follow a 'BAC-by-BAC' approach that aims to deliver highquality sequences of the euchromatin part of the tomato genome. BACs are selected from various libraries of the tomato genome on the basis of markers from the F2.2000 linkage map. Prior to sequencing, we validated the precise physical location of the selected BACs on the chromosomes by five-colour high-resolution fluorescent in situ hybridization (FISH) mapping. This paper describes the strategies and results of cytogenetic mapping for chromosome 6 using 75 seed BACs for FISH on pachytene complements. The cytogenetic map obtained showed discrepancies between the actual chromosomal positions of these BACs and their markers on the linkage group. These discrepancies were most notable in the pericentromere heterochromatin, thus confirming previously described suppression of cross-over recombination in that region. In a so called pooled-BAC FISH, we hybridized all seed BACs simultaneously and found a few large gaps in the euchromatin parts of the long arm that are still devoid of seed BACs and are too large for coverage by expanding BAC contigs. Combining FISH with pooled BACs and newly recruited seed BACs will thus aid in efficient targeting of novel seed BACs into these areas. Finally, we established the occurrence of repetitive DNA in heterochromatin/ euchromatin borders by combining BAC FISH with hybridization of a labelled repetitive DNA fraction (Cot-100). This strategy provides an excellent means to establish the borders between euchromatin and heterochromatin in this chromosome.

Interspecific hybridization of Lilium longiflorum (L) with Asiatic (A) lily hybrids results in so-called LA-hybrids. Some of these hybrids produce 2n-pollen, which were used to perform crosses on Asiatic and Oriental (O) hybrids, resulting in ALA- and OLA-hybrids. Recombination between homoeologous chromosomes (introgression) and the mechanism of 2n-pollen formation in these hybrids were studied using genomic in situ hybridization (GISH). A clear differentiation between the chromosomes of L. longiflorum, Asiatic, and Oriental hybrids was observed in four ALA- and one OLA-hybrid using GISH. Two ALA-hybrids showed 3 and 5 recombinant chromosomes with a total of 5 and 10 crossover sites per hybrid, respectively. These occurred at random positions on the chromosomes. The number and the location of the rDNA-sites were determined using in situ hybridization and provided a tool, the FISH-marker, for identifying the NOR-bearing chromosomes in the lily hybrids. Evidence for the occurrence of the FDR-mechanism (first division restitution) of 2n-pollen formation in the LA-hybrids was obtained on the basis of absence of homologous chromosomes of L. longiflorum in the ALA- and OLA-hybrids.Key words: Lilium longiflorum, introgression, FDR, interspecific hybridization, FISH.

BackgroundThe chromosome preparation is a crucial step for obtaining satisfactory results in molecular cytogenetic researches. The preparation of plant chromosomes for molecular cytogenetic purposes remains a challenge for some species. In contrast to human chromosome preparation, the processes occurring during plant chromosome preparation and causing chromosome spreading are still poorly understood.ResultsWe studied the dynamics of plant chromosome spreading after dropping cell suspension on slides. We showed that steam stimulates cytoplasm hydrolysis and rapid chromosome spreading and that chromosomes stretch during this chromosome spreading. Based on these observations, we developed a novel method, named “SteamDrop”, for the preparation of well-spread mitotic and pachytene chromosomes and successfully used it for 28 plant species with large and small chromosomes. We applied cell suspensions in ethanol instead of the commonly used ethanol/acetic acid fixative. Mitotic and meiotic chromosomes prepared via “SteamDrop” were used in fluorescent in situ hybridization (FISH) experiments with repetitive and unique DNA probes. Long storage of cell suspensions in ethanol did not impair the quality of chromosome preparations.ConclusionThe SteamDrop procedure provides a robust and routine method for high quality plant chromosome preparations. The method can be applied for metaphase as well as pachytene chromosome preparation in wide range of species. The chromosomes prepared by SteamDrop are well suitable for repetitive and unique DNA visualization.

Downy mildew resistance originating from Allium roylei Stearn provides a complete resistance to onions and is based on one, dominant gene. Since A. roylei can successfully be hybridized with onion (A. cepa L.), a breeding scheme aimed at the introgression of this gene was initiated ca. 20 years ago. Several setbacks in this programme were encountered, firstly the identified molecular marker linked to the downy mildew resistance locus became increasingly difficult to use and finally lost its discriminating power and secondly the final step, making homozygous introgression lines (ILs), turned out to be more difficult then was hoped. GISH analysis showed that the chromosomal region harbouring the resistance locus was the only remaining piece of A. roylei in the nuclear background of onion and it also confirmed that this region was located on the distal end of chromosome 3. It was hypothesized that some factor present in the remaining A. roylei region was lethal when homozygously present in an onion genetic background. The identification of an individual with a smaller and more distally located introgression fragment and homozygous ILs in its progeny validated this hypothesis. With the help of these nearly isogenic lines four AFLP Ò markers closely linked to the resistance gene were identified, which can be used for marker-aided selection. The introduction of downy mildew resistance caused by Peronospora destructor into onion is a significant step forward in the development of environmentally-friendly onion cultivars.