Rye chromosome-specific polymerase chain reaction products developed by primers designed from the EcoO109I recognition site.
Rye chromosome-specific polymerase chain reaction products developed by primers designed from the EcoO109I recognition site.
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DOI：
10.1139/g2012-024
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发表时间：
2012-05
期刊：
影响因子：
3.1
通讯作者：
M. Tomita;Akiyoshi Seno
中科院分区：
文献类型：
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作者：
M. Tomita;Akiyoshi Seno
根据我们对黑麦基因组中重复序列的分析，已经预测了EcoO 109 I（5 '-PuGGNCCP y-3'）在整个基因组中存在多个限制性位点。首先，通过在聚合酶链反应（PCR）中使用设计成含有EcoO 109 I位点的引物，有效地获得了多态性DNA标记。以黑麦自交可育系IR 27和普通小麦“中国春”（CS）基因组DNA为模板，设计了43种含有EcoO 109 I位点的10-mer引物，进行PCR扩增。利用22个引物对小麦和黑麦染色体间的多态性进行了检测，并以一系列小麦-帝国黑麦异附加系为模板进行了PCR扩增。从凝胶上收集了5条染色体上的9个染色体特异性扩增片段，并与来自小麦-黑麦染色体附加系的尼龙膜转移PCR产物杂交。仅在收集的片段之间观察到凝胶印迹;因此，确认这些片段具有染色体特异性。对这些片段进行测序并转化为序列标记位点（STS）引物。因此，我们介绍了一种新的方法来构建染色体特异性DNA标记：（i）多个多态性片段可以从EcoO 109 I引物和（ii）添加三个核苷酸的EcoO 109 I位点限制扩增区域，以产生染色体特异性片段。
From our analysis of repeat sequences in the rye genome, the presence of multiple restriction sites of EcoO109I (5'-PuGGNCCPy-3') across the genome has been predicted. By first using primers designed to contain EcoO109I sites in polymerase chain reaction (PCR), polymorphic DNA markers were effectively obtained. A total of 43 types of 10-mer primers containing EcoO109I sites were applied for PCR by using genomic DNA of Secale cereale self-fertile line IR27 and Triticum aestivum 'Chinese Spring' (CS) as the template. Twenty two primers detected polymorphisms between wheat and rye, and they were applied for PCR using a series of CS wheat--'Imperial' rye chromosome addition lines as templates. Nine chromosome-specific amplification fragments identified on five chromosomes were collected from gels and hybridized with nylon membrane-transferred PCR products from the wheat-rye chromosome addition lines. The gel blot was only observed between the collected fragments; therefore, these fragments were confirmed to be chromosome-specific. These fragments were sequenced and converted to sequence-tagged site (STS) primers. We therefore introduce a new method for building chromosome-specific DNA markers: (i) multiple polymorphic fragments can be obtained from EcoO109I primers and (ii) the addition of three nucleotides to the EcoO109I site restricts the amplification region to generate chromosome-specific fragments.