Development of chromosome-specific painting and its application in plants

染色体涂染是植物细胞遗传学研究的一个热点，但在植物中还没有可重复的方法。本程序旨在开发植物染色体的染色技术。本文对染色体的显微切割、切割后染色体或染色体片段的回收、切割后染色体的少量DNA扩增、切割后染色体的DNA探针涂染法等进行了研究，取得了以下结果：1.建立了酶解制备染色体标本的显微切割技术。2.以异源多倍体Scilla scilloides为材料，利用二倍体的全基因组DNA对A、B染色体进行了基因组原位杂交。3.洋葱具有两个大小不等的5S rDNAs，且其5S rDNAs的大小与洋葱的5S rDNAs的大小一致，这为洋葱的基因组间易位检测提供了一种新的方法。 ...更多信息 第7号染色体短臂上的两个位点。将含有5SrDNA位点的染色体片段切开，并从切开的染色体片段扩增5SrDNA。4.由于第6条染色体具有小随体，在染色体组中比较容易识别，因此，本研究将其中的20条染色体切取收集，用DOP-PCR方法扩增，并将扩增的DNA作为染色体特异性探针，用于尖吻蝮蛇染色体的涂染。这种植物是洋葱和洋葱的杂交种。5.解剖了酸模Y染色体，采用DOP-PCR方法扩增了Y染色体DNA，用荧光原位杂交（FISH）技术将Y染色体的DOP-PCR产物特异性地涂染到Y染色体上，并成功地对1例酸模Y染色体进行了涂染，但从某些解剖染色体上扩增的DNA探针在所有染色体上均表现为杂交，对解剖染色体无特异性。在植物基因组中，大量的基因组特异性重复DNA分布在整条染色体上.少

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