Li L, Lu S, O'Halloran D M, Garvin D F, Vrebalov J
USDA-ARS, Plant, Soil and Nutrition Laboratory, Cornell University, Ithaca, NY 14853, USA.
Mol Genet Genomics. 2003 Nov;270(2):132-8. doi: 10.1007/s00438-003-0904-5. Epub 2003 Aug 8.
Mutation in the cauliflower gene Or causes high levels of beta-carotene to accumulate in various tissues of the plant that are normally devoid of carotenoids. To decipher the molecular basis by which Or regulates carotenoid accumulation, we have undertaken the isolation of Or by a map-based cloning strategy. Two previously isolated, locus-specific, sequence-characterized amplified region (SCAR) markers that flank Or were employed for the analysis of a large segregating population consisting of 1632 F(2) individuals, and a high-resolution genetic linkage map of the Or locus region was developed. To facilitate positional cloning, we constructed a cauliflower genomic library in a bacterial artificial chromosome (BAC) vector, using high molecular weight DNA from Or homozygotes. The BAC library comprises 60,288 clones with an average insert size of 110 kb, and represents an estimated 10-fold coverage of the genome. A BAC contig encompassing the Or locus was established by screening the library with a marker that is closely linked to Or and by identifying overlapping BAC clones by chromosome walking. Physical mapping delimited the Or locus to a 50-kb DNA fragment within a single BAC clone, which corresponds to a genetic interval of 0.3 cM.
花椰菜基因Or发生突变会导致高水平的β-胡萝卜素在该植物通常不含类胡萝卜素的各种组织中积累。为了解析Or调控类胡萝卜素积累的分子基础，我们采用基于图谱的克隆策略对Or进行分离。利用两个先前分离的、位点特异性的、序列特征扩增区域（SCAR）标记（它们位于Or两侧）对由1632个F2个体组成的大型分离群体进行分析，并构建了Or位点区域的高分辨率遗传连锁图谱。为便于进行定位克隆，我们使用来自Or纯合子的高分子量DNA，构建了一个以细菌人工染色体（BAC）为载体的花椰菜基因组文库。该BAC文库包含60288个克隆，平均插入片段大小为110 kb，估计覆盖基因组10倍。通过用与Or紧密连锁的标记筛选文库，并通过染色体步移鉴定重叠BAC克隆，建立了一个包含Or位点的BAC重叠群。物理图谱将Or位点定位到单个BAC克隆内的一个50 kb DNA片段上，该片段对应于0.3 cM的遗传区间。
