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

1.用水稻或山羊草细菌人工染色体(BAC)克隆为每组染色体的探针，对谷类作物染色体进行染色体染色。我们成功地开发了一种探针标记检测系统，通过结合三种荧光染料来区分不同颜色的每个探针。在一次鱼类实验中，七个集合的探针组被同时可视化。利用这一技术，从谷子、花生和咖啡等有用植物中鉴定了多倍体物种的基因组供体物种。2.我们从Ae squarrosa BAC文库中筛选了500个克隆(包括农学上重要的基因)，并将多色FISH应用于Ae.大麦和普通小麦。大多数基因定位在染色体的远端。通过比较FISH信号的顺序进行共时分析。一些基因的共性在植物家族之外是保守的。3.我们利用FISH技术实现了谷物重要农艺基因在延伸的核DNA纤维上的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，它由多达15个基因组成，以串联重复的形式排列。我们还通过分子梳理的方法在BAC DNA上开发了高分辨率的FISH。纤维FISH技术有助于构建BAC重叠序列。4.我们从Ae的BAC文库中分离到20个含有着丝粒重复序列的克隆。Squarrosa，并对这些克隆进行了表征。我们发现有几种序列与以前在水稻、高粱、大麦和玉米中报道的序列有很高的同源性。FISH分析表明，这些序列大多在谷物的着丝粒区域保守。纤维FISH显示着丝粒的精细结构。

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.