W. Busch

Localization of the B-hordein locus on barley chromosomes using fluorescence in situ hybridization

The B-hordein gene family consists of at least 13 genes that appear to be clustered in two regions on the barley chromosome 1H (5). Using fluorescence in situ hybridization techniques, we have localized the B-hordein locus (Hor2) to the distal end of the short arm of chromosome 1H (5), corresponding to the genetically determined position, 91% distal on the short arm. This result is based on appropriate barley lines (i.e. the two cultivars Igri and Betzes, the telotrisomic and ditelotetrasomic lines 1HS and the translocation line T21), on two experimental approaches and on a signal frequency of between 20% and 60% on metaphase chromosomes.

Efficient preparation of plant chromosomes for high-resolution scanning electron microscopy

A highly reproducible technique to prepare plant chromosomes for high-resolution field emission scanning electron microscopy is presented. The procedure allows the production of relatively high numbers of chromosome spreads that can be viewed at high resolution, showing structural details below 10 nm. This preparation technique is not restricted to metaphase chromosomes, but also allows the observation of plant chromosomes during all stages of the cell cycle.

Sensitivity enhancement of fluorescencein situ hybridization on plant chromosomes

An improvedin situ hybridization procedure is presented, based on synchronization of root meristems of barley and wheat, enzymatic digestion, a protoplast drop technique, and the use of the fluorescent dye Cy3. The combination of these approaches resulted in a significant increase of well-spread metaphases suitable forin situ hybridization as compared to squash preparations, and to a significantly enhanced number and intensity of hybridization signals as demonstrated for a B-hordein-specific lowcopy probe of barley. In the case of Cy3 all metaphases displayed a signal, more than 60% of them on both chromatids of each gene-bearing chromosome.

Structural rearrangement in chromosome 2M of Aegilops comosa has prevented the utilization of the Compair and related wheat-Ae. comosa translocations in wheat improvement

Abstract The genetic constitutions of chromosome 2M of Aegilops comosa and the derived wheat-Ae. comosa translocations were analyzed by molecular cytogenetic techniques. Hybridization of 15 RFLP markers covering the entire length of the group-2 chromosomes revealed that chromosome 2M was structurally rearranged compared to the homoeologous chromosomes of wheat by either a pericentric inversion or a terminal intrachromosomal translocation. The breakpoint of the rearrangement was located in a region between the loci Xpsr131 and Xcdo405, resulting in the translocation of 47% of 2MS to 2ML. This aberrant structure of 2M allowed homoeologous recombination between 2M and its wheat counterpart only in the translocated segment on 2ML. C-banding and genomic in situ hybridization analyses confirmed that all translocation chromosomes consisted of the complete 2MS arm, a large part of 2ML, and very small distal segments derived from 2AS or 2DS, as expected from the aberrant structure of chromosome 2M. Thus, the translocation in the line 2A-2M?4/2 can be described as T2AS-2M?1L ⋅ 2M?1S and the translocations in the lines Compair and 2D-2M?3/8 as T2DS-2M?1L ⋅ 2M?1S. RFLP analysis determined the breakpoints in these translocation chromosomes to be within the telomeric 16% of the wheat chromosome arms. The breakpoint of the 2A/2M translocation was between Xbcd348 and Xcdo783, and that of the 2D/2M translocation was between Xcdo783 and Xpsr666. Because the translocation chromosomes retain the structural aberration found in chromosome 2M, further exploitation of the wheat-Ae. comosa translocations for cultivar improvement is questionable.

Changes in chromosomal ultrastructure during the cell cycle.

The surface structure of mitotic barley and rye chromosomes was studied by high-resolution scanning electron microscopy. Chromosomes with various degrees of chromatin condensation were prepared from untreated meristematic tissue of root tips. At lower magnifications the highly condensed chromosomes in metaphase and anaphase showed a compact structure with a smooth surface. The condensation starts from the centromeric region and the chromatics are often discernible in the still uncondensed telomeric region. Decondensation begins at the telomeric region during telophase. Parallel arrangement of fibres is a characteristic feature predominately seen in prophase and telophase chromosomes. Chromatin structures that resemble tiles on a roof or braided strands were often observed. Prophase and telophase chromosomes are particularly suitable for further studies of chromatin arrangement and organization in plant chromosomes.

Efficient preparation of plant metaphase spreads

A simple and efficient method for the preparation of a high number of plant metaphase spreads from a broad range of plant species suitable for light microscopy and high-resolution electron microscopy is described.

Repeated DNA sequences isolated by microdissection. II. Comparative analysis in Hordeum vulgare and Triticum aestivum

The genomic organization of two satellite DNA sequences, pHvMWG2314 and pHvMWG2315, of barley (Hordeum vulgare, 2n=14, HH) was studied by comparative in situ hybridization (ISH) and PCR analysis. Both sequences are members of different RsaI families. The sequence pHvMWG2314 is a new satellite element with a monomer unit of 73 bp which is moderately amplified in different grasses and occurs in interstitial clusters on D-genome chromosomes of hexaploid wheat (Triticum aestivum, 2n=42, AABBDD). The 331-bp monomer pHvMWG2315 belongs to a tandemly amplified repetitive sequence family that is present in the Poaceae and preferentially amplified in Aegilops squarrosa (2n=14, DD), H. vulgare and Agropyron elongatum. (2n=14, EE). The first described representative of this family was pAs 1 from Ae. squarrosa. Different sequences of one satellite DNA family were amplified from Ae. squarrosa, A. elongatum and H. vulgare using PCR. Characteristic differences between members of the D and H genome occurred in a variable region which is flanked by two conserved segments. The heterogeneity within this element was exploited for the cytogenetic analysis of Triticeae genomes and chromosomes. Comparative ISH with pHvMWG2315 identified individual wheat and barley chromosomes under low (75%) and high (85%) hybridization stringency in homologous and heterologous systems. We propose the designation Tas330 for the Triticeae amplified sequence (Tas) satellite family with a 330 bp average monomer length.

Chromosome Microdissection and Megabase Technology in Plant Genome Analysis; Plant Chromosomes and Genes at High Resolution

The analysis of the complex eukaryotic genomes with their huge amounts of DNA and compartmentalized genomes has made substantial progress during the past two decades (e. g., Bernatzky and Tanskley, 1989; Tanskley et al., 1989; Jordan, 1988; Herrmann et al., 1992). The development began with the analysis of the subgenomes in the organelles, continued with expression cloning, which allowed the serological selection of cDNAs for nuclear genes from which products were known, and culminated in the eighthies with various approaches to manage and elucidate the complexity of the nucleo/cytosolic compartment with its estimated 50.000 to 100.000 genes. Milestones represent the development of a molecular marker system based on restriction fragment length polymorphisms (RFLPs), of procedures for tagging individual genes (e. g., Gierl and Saedler, 1992), and of the PCR (Saiki et al., 1985) as well as the megabase technologies (Jordan, 1988). The latter have filled a long lasting gap between genetic mapping procedures (linkage analysis) and conventional plasmid- or lambda-based cloning techniques. Basically three techniques, namely pulsed field gel electrophoresis, cloning into yeast artificial chromosomes (YACs), and chromosome jumping, allow to handle and/or physically map long coherent DNA segments.