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

使用细菌人工染色体通过多色 FISH 对谷物进行染色体涂色和同线性分析

基本信息

批准号：
09490024
负责人：
MUKAI Yasuhiko
金额：
$ 6.66万
依托单位：
Osaka Kyoiku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

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.

1.使用探针对谷物的染色体进行染色体涂色，其中每个染色体组汇集了水稻或糙山羊草细菌人工染色体（BAC）克隆。我们成功开发了一种探针标记检测系统，该系统通过组合三种荧光染料来区分具有不同颜色的每种探针。七个混合探针组在单个 FISH 实验中同时可视化。利用该技术，在有用植物（如小米、花生和咖啡）中鉴定了多倍体物种的基因组供体物种。 2.我们从糙麦草 BAC 文库中选择了 500 个克隆（包括农艺学上重要的基因），并应用多色 FISH 进行有丝分裂Ae 的中期染色体。 squarrosa 和普通小麦。大多数基因被定位在染色体的远端。通过比较 FISH 信号的顺序进行同线性分析。一些基因的同线性在植物科之外是保守的。3.我们利用FISH技术在谷物的农艺重要基因中实现了延伸核DNA纤维上基因组织的直接可视化。我们证明了黑麦 secalin-1 基因簇的重复性质，该基因簇由多达 15 个串联重复排列的基因组成。我们还通过分子梳对 BAC DNA 开发了高分辨率 FISH。纤维FISH技术有助于BAC重叠群的构建。4.我们从Ae的BAC文库中分离出20个含有着丝粒重复序列的克隆。 squarrosa 并表征了这些克隆。我们发现几种序列与之前报道的水稻、高粱、大麦和玉米的序列具有较高的同源性。 FISH 分析表明，这些序列大多在谷物的着丝粒区域中保守。通过纤维 FISH 观察着丝粒的精细结构。