Palindromic sequences and A+T-rich DNA elements promote illegitimate recombination in Nicotiana tabacum.

Illegitimate recombination is the prevailing molecular mechanism for the integration of recombinant DNA into the genome of most eukaryotic systems and the generation of deletions by intrachromosomal recombination. We developed a ?selectable marker system to screen for intrachromosomal illegitimate recombination events in order to assess the sequence and structure-specific requirements for illegitimate recombination in tobacco. In 12 illegitimate recombination products analysed, we found that all deletion termini localise to sites of palindromic structures or to A+T-rich DNA elements. All deletion termini showed microhomologies of two to six nucleotides. In three plants, the recombination products contained filler-DNA or an inversion of an endogenous segment. Our data strongly suggest that illegitimate recombination in plants is mediated by a DNA synthesis-dependent process, and that this mechanism is promoted by DNA regions that can form palindromic structures or facilitate DNA unwinding.

Visualization of the Brassica self-incompatibility S-locus on identified oilseed rape chromosomes.

Seventy years after Karpechenko [15] first reported the accurate chromosome number of oilseed rape (Brassica napus L., 2n=38), we have developed a quantitative chromosome map of rape using computer imaging technology. The capacity to identify individual rape chromosomes will facilitate a wide range of genetic studies. Here we demonstrate the use of imaging methods in combination with fluorescence in situ hybridization to localize, on identified chromosomes, the single copy S-locus glycoprotein and S-locus-related genes involved in the self-incompatibility system of Brassica. These techniques have a broader application in plant genome research involving the mapping of single-copy genes and markers, irrespective of the plant species.

Laser isolation of plant sex chromosomes: studies on the DNA composition of the X and Y sex chromosomes of Silene latifolia.

X and Y sex chromosomes from the dioecious plant Silene latifolia (white campion) were isolated from mitotic metaphase chromosome preparations on polyester membranes. Autosomes were ablated using an argon ion laser microbeam and isolated sex chromosomes were then recovered on excised fragments of polyester membrane. Sex chromosome associated DNA sequences were amplified using the degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and pools of DOP-PCR products were used to investigate the genomic organization of the S. latifolia sex chromosomes. The chromosomal locations of cloned sex chromosome repeat sequences were analysed by fluorescence in situ hybridization and data complementary to laser ablation studies were obtained by genomic in situ hybridization. In combination, these studies demonstrate that the X and Y sex chromosomes of S. latifolia are of very similar DNA composition and also that they share a significant repetitive DNA content with the autosomes. The evolution of sex chromosomes in Silene is discussed and compared with that in another dioecious species, Rumex acetosa.

Mapping of C-banded Crepis chromosomes by imaging methods.

Crepis capillaris is a highly suitable model plant for a computer-aided analysis of chromosomes because of its small number of chromosomes and well-defined bands. The identification of both large and small bands by means of profiles [grey value-function (position)] is frequently not possible with simple discrimination. In this case, dynamic curve fitting gives exact results by sliding discrimination. The application of low-pass filters, with a filter matrix size of 75 x 75 pixels, to the original image generates a reference image that can be used for sliding discrimination. The relevant bands are visible by the subtraction of the filtered image from the original image. The computerized bands correspond exactly to band regions recognized visually. Human vision is another approach to improve the quantification in cytogenetic results. The connection between the visual inspection and the imaging methods will reduce the subjective interpretation of individual researchers. The knowledge-based analysis may be a significant factor in the near future to answer unsolved questions.

Physical mapping of the 5S ribosomal RNA genes on rice chromosome 11.

One 5S ribosomal RNA gene (5S rDNA) locus was localized on chromosome 11 of japonica rice by in situ hybridization. The biotinylated DNA probe used was prepared by direct cloning and direct labeling methods, and the locus was localized to the proximal region of the short arm of chromosome 11 (11p1.1) by imaging methods. The distance between the signal site and the centromere is 4.0 arbitrary units, where the total length of the short arm is 43.3 units. The 5SrDNA locus physically identified and mapped in rice was designated as 5SRrn. The position of the 5S rDNA locus reported here differs from that in indica rice; possible reasons for this difference are discussed. DNA sequences of 5S rDNA are also reported.

Physical mapping of 5S rDNA loci by direct-cloned biotinylated probes in barley chromosomes.

5S rDNA loci have been mapped on barley chromosomes by in situ hybridization using five reciprocal translocation lines. Two kinds of DNA probes covering either the 5S rDNA coding region or the 5S rDNA coding and flanking noncoding regions were used. They were prepared by direct cloning from interphase nuclei and simultaneous direct labeling in PCR. Four 5S rDNA loci were detected in a haploid genome by the 5S rDNA coding region, whereas in addition, the four or six 5S rDNA related sites, depending on the variety used, were revealed by the probe covering the flanking region. The four 5S rDNA loci revealed and mapped on the barley chromosomes: 2 (2I), 3 (3I), 1 (7I), and 4 (4I) were designated 5SRrn-I1, 5SRrn-I2, 5SRrn-I3 and 5SRrn-I4, respectively, in descending order of copy number of 5S rRNA genes.

Computer-aided automatic identification of rice chromosomes by image parameters.

An automatic, computer-aided method for identification of rice chromosomes was developed based on image parameters obtained by image analysis. Numerical data of the 360 condensation patterns (CPs) or the density profiles of rice chromosomes obtained from 30 chromosomal spreads of haploid rice were subjected to three different discrimination methods for identifying the chromosome: (1) discrimination flow chart, (2) linear discrimination functions, and (3) minimum distance classifier based on standardized Euclidean distance. Discrimination based on the minimum distance classifier resulted in the correct identification of more than 92% of the rice chromosomes. The first and second methods gave a correct identification of 91% and 84%, respectively. It is concluded that the 12 rice chromosomes in most spreads could be identified by a computer.

Recovery of dissected C-band regions in Crepis chromosomes.

Chromosome samples were prepared on a plastic coverslip covered with a polyester membrane and were subjected to the C-banding treatment. The C-band pattern was obtained after Giemsa staining. The C-band positive regions of the Crepis chromosomes were identified, dissected out by irradiation with a micro-laser beam and recovered in Eppendorf tubes.

Microdissection of plant chromosomes by argon-ion laser beam.

Rice and barley chromosomal samples were prepared both on a polyester membrane and on an ordinary glass slide and subjected to microdissection by an argon-ion laser. The intensity and the position of the laser beam were controlled by a microcomputer. The most suitable intensity to obtain chromosomal fragments was determined experimentally. As a result, specific regions of the centromere, satellite, short arm, or long arm, of the barley and rice chromosomes were dissected out from the chromosomal spreads. Chromosomal fragments were also successfully transferred from the sample into an Eppendorf tube.

Automatic karyotyping of plant chromosomes by imaging techniques.

Chromosomes of Crepis capillaris (L.) Wallr. were analyzed using a newly developed chromosome image analyzing system, CHIAS-mini. The CHIAS-mini is a desktop system which consists of an ordinary 16-bit personal computer and an image processor as the main frames. Data acquisition, karyotyping and idiogramming of the plant chromosomes were automatically carried out with some manual interaction. The data obtained were comparable to those obtained manually and by a standard image analyzer already developed.