Molecular and cytogenetic mapping of plant genomes

Three principal approaches are used in our laboratory to analyze plant genomes: (i) The construction of high density molecular maps: We have established a refined microdissection procedure to construct chromosomal and subchromosomal libraries. Synchronized meristematic root tip tissue from which metaphase spreads can be prepared with a novel dropspread technique in combination with nl-scale PCR allowed the cloning of DNA of 10 or less chromosomes or chromosome segments. The construction of high-resolution maps from discrete genome regions can greatly facilitate genetic fingerprinting, gene isolation and QTL studies. (ii) Synteny analysis: The Aegilops-based deletion mapping system in wheat with an array of deleted chromosome parts and chromosome-specific RFLP markers has lead to the construction of a high density physical consensus map of wheat. The integration of wheat, barley and oat RFLP markers proves the colinearity between the wheat A-, B- and D-genomes, the H- genome of barley and the E-genome of Agropyron. (iii) Gene mapping in situ and chromosomes at high resolution: For the sensitivity enhancement of fluorescence in situ hybridization (FISH), the efficient preparation of plant chromosomes for high resolution scanning electron microscopy, mapping of low-copy sequences, and for comparative in situ hybridization a modified drop technique for plant protoplasts was developed. A tandemly amplified repetitive sequence element from microdissected barley chromosomes has enabled the karyotyping of single Gramineae genomes in a single step. These sequences are also useful for simultaneous double or triple hybridization experiments in an attempt to localize new sequence on specific chromosomes or chromosome segments. The physical mapping of the Sec-1 locus has been refined on the satellite of chromosome 1R of rye, and the syntenic locus on barley chromosome 1H identified. A method was developed for in situ hybrization and signal detection at high resolution using a field emission scanning electron microscope and a backscattered electron detector. Colloidal gold particles were localized on chromosome structures resembling the 30 mm fibre. Comparatively, an rDNA probe was located in the secondary constriction and highly compact adjacent regions of barley chromosomes.