Teraishi M, Hirochika H, Okumoto Y, Horibata A, Yamagata H, Tanisaka T
Laboratory of Plant Breeding, Graduate School of Agriculture, Kyoto University, Japan.
Genome. 2001 Feb;44(1):1-6. doi: 10.1139/gen-44-1-1.
A mutable slender glume gene slg, which often reverts to the wild-type state, was induced by gamma-ray irradiation of seeds of the japonica rice cultivar 'Gimbozu'. The final goal was to understand whether the slender glume mutation was associated with the insertion of a transposable element, utilizing map-based cloning techniques. The RFLP (restriction fragment length polymorphism) analysis revealed that the slg locus was located between two RFLP loci, XNpb33 and R1440, on chromosome 7 with recombination values of 3.1% and 1.0%, respectively. Using these two RFLP loci as probes, five YAC (yeast artificial chromosome) clones containing either of these two loci were selected from a YAC library. Subsequently, both end fragments of these YAC clones, amplified by the inverse PCR (IPCR) method, were used to select new YAC clones more closely located to the slg locus. After repeating such a procedure, we successfully constructed a 6-cM YAC contig, and identified four overlapping YAC clones, Y1774, Y3356, Y5124, and Y5762, covering the slg locus. The chromosomal location of the slg was narrowed down to the region with a physical distance of less than 280 kb between the right-end fragments of Y1774 and Y3356.
通过对粳稻品种‘Gimbozu’种子进行γ射线辐照，诱导出了一个易发生回复突变的细长颖壳基因slg，该基因常回复为野生型状态。最终目标是利用基于图谱的克隆技术，了解细长颖壳突变是否与转座元件的插入有关。限制性片段长度多态性（RFLP）分析表明，slg基因座位于第7号染色体上的两个RFLP基因座XNpb33和R1440之间，重组值分别为3.1%和1.0%。以这两个RFLP基因座为探针，从酵母人工染色体（YAC）文库中筛选出了包含这两个基因座之一的5个YAC克隆。随后，通过反向PCR（IPCR）方法扩增这些YAC克隆的两端片段，用于筛选更靠近slg基因座的新YAC克隆。重复这一过程后，我们成功构建了一个6厘摩的YAC重叠群，并鉴定出4个重叠的YAC克隆Y1774、Y3356、Y5124和Y5762，它们覆盖了slg基因座。slg基因在染色体上的定位被缩小到Y1774和Y3356右端片段之间物理距离小于280 kb的区域。
