Development of chromosome-specific painting and its application in plants

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Chromosome painting is desired in plant cytogenetics but the reproducible method has not appeared in plant. This program is intended to develop chromosome painting technique for plant chromosomes. Microdissection of chromosomes, recover of dissected chromosome or chromosome segment, amplification of small amount of DNA of dissected chromosomes, chromosome painting method using DNA probe amplified from dissected chromosome were investigated and following results were obtained.1.The technique of microdisectiton using a micromanipu ; ater for dissection from the chromosome preparation prepared by enzymic maceration were established. The DNA contained in dissected chromosomes was amplified by DOP-PCR.2.In allopolyploid of Scilla scilloides the chromosomes belonging to genome A and B were painted with genomic in situ hybridizaton using total genome DNA of diploids with each gemone. This tequniqe is useful for detection of intergenomic translocation.3.Allium cepa has two sized 5S rDNAs and t … More wo loci on the short arm of the 7th chromosome. The chromosome segments containing a 5S rDNA locus were dissected and 5S rDNA were amplified from the dissected chromosome segments. The correspondence between size of 5S rDNA and chromosome locus was revieled.4.As the 6th chromosome with a small satellite is easily identified among the chromosome complement, 20 of this chromosomes were dissected and collected by mincrodisection and then the DNA was amplified by DOP-PCR.The amplified DNA was used as a chromosome-specific probe for chromosome painting to chromosomesof A.wakegi. This plant is hybrid between A.cepa and A.fistulosum. All eight chromosomes of A.cepa were signaled but the 6th chromosome was not specific.5.Y sex chromosomes were dissected in Rumex acetosa and their DNA was amplified by DOP-PCR.The Y chromosomes were specifically painted with the DOP-PCR product from Y chromosomes by FISH.In a case of Y chromosome in rumex acetosa the chromosomes painting is successful, but the DNA probe amplified from certain dissected chromosome haybridizaed homogeneously on all chromosomes and not specific for dissected chromosome. In plant genome a large amount of genome specific repetitive DNA distributed over whole chromosomes. Less