Chromosome painting and synteny analysis in cereals by multicolor FISH using bacterial artificial chromosomes

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1.Chromosome painting was carried out on chromosomes of cereals using the probes in which rice or Aegilops squarrosa bacterial artificial chromosome(BAC) clones were pooled for each chromosome group. We succeeded in developing a probe labeling-detection system which discriminate each probe with different color by combining three fluorochromes. The seven pooled probe sets were visualized simultaneously in a single FISH experiment. Using this technique, genome donor species to polyploid species were identified in useful plants such as, finger millet, peanut and coffee.2.We have selected 500 clones (including agronomically important genes) from an Ae squarrosa BAC library and applied multicolor FISH to mitotic metaphase chromosomes of Ae. squarrosa and common wheat. Most genes were mapped on the distal ends of chromosomes. The synteny analysis was conducted by comparing the order of FISH signals. Synteny of some genes was conserved beyond plant family.3.We have achieved the direct visualization of gene organization on extended nuclear DNA fibers using FISH in agronomically important genes of cereals. We demonstrated the repetitive nature of the secalin-1 gene cluster of rye, which consists of up to 15 genes arranged in tandem repeats. We also developed high-resolution FISH on BAC DNAs by molecular combing. The fiber FISH technique contributes to construction of BAC contig.4.We have isolated 20 clones containing centromeric repeated sequences from a BAC library of Ae. squarrosa and characterized these clones. We found that several kinds of sequences have high homology to the sequences reported previously in rice, sorghum, barley and corn. FISH analysis has revealed that these sequences are mostly conserved in the centromeric regions of cereals. Fine structure of centromeres was visualized by fiber FISH.