Genome. 1995 Jun;38(3):458-66. doi: 10.1139/g95-060.
A targeted mapping strategy using representational difference analysis (RDA) was employed to isolate new restriction fragment length polymorphism probes for the long arm of chromosome 6 in rye (6RL), which carries a gene for resistance to Hessian fly larvae. Fragments from the 6RL arm were specifically isolated using a 'Chinese Spring' (CS) wheat - rye ditelosomic addition line (CSDT6RL) as tester, and CS and (or) CS4R as the driver for the genomic subtraction. Three RDA experiments were performed using BamHI amplicons, two of which were successful in producing low-copy clones. All low-copy clones were confirmed to have originated from 6RL, indicating substantial enrichment for target sequences. Two mapping populations, both of which are derived from a cross between two similar wheat-rye translocation lines, were used to map five RDA probes as well as five wheat probes. One of the populations was prescreened for recombinants by C-banding analysis. Fifteen loci, including seven new RDA markers, were placed on a map of the distal half of 6RL. The Hessian fly resistance gene was localized by mapping and C-banding analysis to approximately the terminal 1% of the arm. The utility of RDA as a method of targeted mapping in cereals and prospects for map-based cloning of the resistance gene are discussed.
采用代表性差异分析（RDA）的靶向作图策略，分离携带抗黑森瘿蚊幼虫基因的黑麦 6 号染色体长臂的新的限制性片段长度多态性探针。使用“中国春”（CS）小麦-黑麦双端体附加系（CSDT6RL）作为测试者，CS 和（或）CS4R 作为基因组消减的驱动子，从 6RL 臂特异性分离片段。使用 BamHI 扩增子进行了三次 RDA 实验，其中两次成功产生了低拷贝克隆。所有低拷贝克隆均确认源自 6RL，表明目标序列得到了实质性富集。两个源自两个类似的小麦-黑麦易位系杂交的作图群体，用于定位 5 个 RDA 探针和 5 个小麦探针。其中一个群体通过 C-带分析进行了重组体的预筛选。在 6RL 远侧半部分的图谱上定位了 15 个位点，包括 7 个新的 RDA 标记。通过作图和 C-带分析，将黑森瘿蚊抗性基因定位到臂的末端约 1%。讨论了 RDA 在谷物中作为靶向作图方法的实用性以及基于图谱克隆抗性基因的前景。
