Monna L, Miyao A, Zhong H S, Sasaki T, Minobe Y
Rice Genome Research Program, National Institute of Agrobiological Resources, Tsukuba, Japan.
DNA Res. 1995 Jun 30;2(3):101-6. doi: 10.1093/dnares/2.3.101.
Bulked segregant analysis was used to determine randomly amplified polymorphic DNA (RAPD) markers in a specific interval in the middle of chromosome 6 of rice for tagging the photoperiod sensitivity gene. Two pools of F2 individuals (japonica cv. Nipponbare and indica cv. Kasalath) were constructed according to the genotypes of three restriction fragment length polymorphism (RFLP) markers located at both ends and the middle of the targeted interval. Then another pair of pools were constructed based on the "graphical genotype," which was made with our high density linkage map. RAPD analysis was performed using these DNA pools as templates, and polymorphic fragments were detected and mapped. Using 80 primers, either singly or pairwise, we tested 2,404 primer pairs and established 14 markers tightly linked to the photoperiod sensitivity gene. The obtained RAPD markers were converted into sequence-tagged sites by cloning and sequencing of the polymorphic fragments and they can be used directly for construction of physical maps. This bulked segregant method can be applied for any species and any region of interest in which detailed linkage maps or physical maps are needed.
利用混合分离群体分析法来确定水稻第6号染色体中部特定区间的随机扩增多态性DNA（RAPD）标记，以对光周期敏感基因进行标记。根据位于目标区间两端和中间的三个限制性片段长度多态性（RFLP）标记的基因型，构建了两个F2个体池（粳稻品种日本晴和籼稻品种Kasalath）。然后基于用我们的高密度连锁图谱构建的“图形基因型”构建了另一对池。以这些DNA池为模板进行RAPD分析，检测并定位多态性片段。使用80个引物，单独或成对使用，我们测试了2404对引物，并建立了14个与光周期敏感基因紧密连锁的标记。通过对多态性片段进行克隆和测序，将获得的RAPD标记转化为序列标签位点，它们可直接用于构建物理图谱。这种混合分离群体方法可应用于任何需要详细连锁图谱或物理图谱的物种和任何感兴趣的区域。
