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.

A technique to detect DNA sequences on extended DNA fibres (EDF) prepared from interphase nuclei from tomato (Lycopersicon esculentum) and Arabidopsis thaliana leaves by fluorescence in situ hybridization (FISH) is described. Three nuclear lysis procedures have been tested for their ability to decondense chromatin and to generate highly extended intact DNA fibres on microscopic slides. DNA probes of various sizes have been used in FISH experiments to EDFs to establish the resolution and sensitivity of the technique. The fluorescent signals of a 5S rDNA probe hybridized to tomato EDFs revealed continuous strings of about 200 microns, that corresponded to a molecular size of about 660 kb. In A. thaliana, a contig of three cosmids spanning a genomic region with a total length of about 89 kb was analysed. By means of multicolour hybridization the physical positions of the cosmids were visualized as red and green fluorescence strings with overlapping regions in yellow. Comparison of the length of the fluorescent signals with the molecular data revealed a stretching degree of the DNA fibres at 3.27 kb microns-1, which is close to the Watson-Crick DNA length estimate of 2.9 kb microns-1. Other experiments on small size molecular probes with both lambda clones (13.5-17 kb insert sizes) and plasmids (4.2 and 5 kb) in a contig of A. thaliana, and the 5S rDNA region in tomato showed close agreement with molecular data. The lower limit of the detection, which was established in a hybridization experiment with two DNA probes from the 45S ribosomal gene on extended fibres of tomato, was about 0.7 kb. Consistent patterns of alternating fluorescent red and green spots were obtained reflecting the tandemly repeated arrangement of the 18S and 25S ribosomal sequences. On the basis of the microscopic distance between these hybridization spots the size of the ribosomal unit was estimated at 8.2 kb. This implies a drastic improvement of high-resolution physical mapping of DNA sequences by FISH on plant DNA.

As part of a map-based cloning strategy designed to isolate the root-knot nematode resistance gene Mi, tomato F2 populations were analyzed in order to identify recombination points close to this economically important gene. A total of 21,089 F2 progeny plants were screened using morphological markers. An additional 1887 F2 were screened using PCR-based flanking markers. Fine-structure mapping of recombinants with newly developed AFLP markers, and RFLP markers derived from physically mapped cosmid subclones, localized Mi to a genomic region of about 550 kb. The low frequency of recombinants indicated that recombination was generally suppressed in these crosses and that crossovers were restricted to particular regions. To circumvent this problem, a population of Lycopersicon peruvianum, the species from which Mi was originally introgressed, that was segregating for resistance was developed. Screening of this population with PCR, RFLP and AFLP markers identified several plants with crossovers near Mi. Recombination frequency was approximately eight-fold higher in the Mi region of the L. peruvianum cross. However, even within the wild species cross, recombination sites were not uniformly distributed in the region. By combining data from the L. esculentum and L. peruvianum recombinant analyses, it was possible to localize Mi to a region of the genome spanning less than 65 kb.

A new DNA polymorphism assay was developed in 1990 that is based on the amplification by the polymerase chain reaction (PCR) of random DNA segments, using single primers of arbitrary nucleotide sequence. The amplified DNA fragments, referred to as RAPD markers, were shown to be highly useful in the construction of genetic maps ("RAPD mapping"). We have now adapted the RAPD assay to tomato. Using a set of 11 oligonucleotide decamer primers, each primer directed the amplification of a genome-specific "fingerprint" of DNA fragments. The potential of the original RAPD assay to generate polymorphic DNA markers with a given set of primers was further increased by combining two primers in a single PCR. By comparing "fingerprints" of L. esculentum, L. pennellii, and the L. esculentum chromosome 6 substitution line LA1641, which carries chromosome 6 from L. pennellii, three chromosome 6-specific RAPD markers could be directly identified among the set of amplified DNA fragments. Their chromosomal position on the classical genetic map of tomato was subsequently established by restriction fragment length polymorphism (RFLP) linkage analysis. One of the RAPD markers was found to be tightly linked to the nematode resistance gene Mi.

Variation in recombinant frequencies at two adjacent intervals on chromosome 6 of tomato (Lycopersicon esculentum Mill.) has been studied in seven lines that differ in the amount and origin of introgressed segments from wild species. These lines were all crossed to a genotype homozygous recessive for the markers tl, yv, and c, which define the centromere spanning region tl-yv and the long arm region yv-c. Recombinants were identified in large F2, populations consisting of over 30 000 plants in total. Application of molecular markers provided additional information on the distribution of crossover events within the centromere-containing interval tl-yv. A decrease in recombination at the marked intervals correlated with the presence of an alien segment. Suppression of recombination was up to sixfold in the centromere spanning interval tl-yv depending on the source and size of the introgression, and was restricted to the alien segments with no strong effect on the neighbouring intervals. Key words : recombinant frequency, Lycopersicon esculentum, morphological markers, introgressions, centromere.