Cnops G, den Boer B, Gerats A, Van Montagu M, Van Lijsebettens M
Department of Genetics, Flanders Interuniversity Institute for Biotechnology, Universiteit Gent, Belgium.
Mol Gen Genet. 1996 Nov 27;253(1-2):32-41. doi: 10.1007/s004380050293.
The Arabidopsis tornado1 (trn1) mutation causes severe dwarfism combined with twisted growth of all organs. We present a chromosome landing strategy, using amplified restriction fragment length polymorphism (AFLP) marker technology, for the isolation of the TRN1 gene. The recessive trn1 mutation was identified in a C24 transgenic line and is located 5 cM from a T-DNA insertion. We mapped the TRN1 locus to the bottom half of chromosome 5 relative to visible and restriction fragment length polymorphism (RFLP) markers. Recombinant classes within a 3-cM region around TRN1 were used to build a high-resolution map in this region, using the AFLP technique. Approximately 300 primer combinations have been used to test about 26,000 fragments for polymorphisms. Seventeen of these AFLP markers were identified in the 3-cM region around TRN1. These markers were mapped within this region using individual recombinants. Four of these AFLP markers co-segregate with TRN1 whereas one maps at one recombinant below TRN1. We isolated and cloned three of these AFLP markers. These markers identified two yeast artificial chromosome (YAC) clones, containing the RFLP marker above and the AFLP marker below TRN1, demonstrating that these YACs span the TRN1 locus and that chromosome landing has been achieved, using an AFLP-based strategy.
拟南芥tornado1（trn1）突变导致严重矮化，并伴有所有器官的扭曲生长。我们提出了一种染色体着陆策略，利用扩增限制性片段长度多态性（AFLP）标记技术来分离TRN1基因。隐性trn1突变在一个C24转基因系中被鉴定出来，它位于距一个T-DNA插入位点5厘摩处。相对于可见标记和限制性片段长度多态性（RFLP）标记，我们将TRN1基因座定位到了第5号染色体的下半部分。利用AFLP技术，在TRN1周围3厘摩区域内的重组类型被用于构建该区域的高分辨率图谱。大约300种引物组合被用于检测约26000个片段的多态性。在TRN1周围3厘摩区域内鉴定出了17个这样的AFLP标记。利用单个重组体将这些标记定位在该区域内。其中4个AFLP标记与TRN1共分离，而有一个标记位于TRN1下方一个重组体处。我们分离并克隆了其中3个AFLP标记。这些标记鉴定出了两个酵母人工染色体（YAC）克隆，其中一个包含TRN1上方的RFLP标记，另一个包含TRN1下方的AFLP标记，这表明这些YAC跨越了TRN1基因座，并且利用基于AFLP的策略实现了染色体着陆。
