Isolation, characterization, and analysis of Leymus-specific DNA sequences
Isolation, characterization, and analysis of Leymus-specific DNA sequences
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DOI：
10.1139/g03-029
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发表时间：
2003-08-01
期刊：
影响因子：
3.1
通讯作者：
Anamthawat-Jónsson, K
中科院分区：
文献类型：
--
作者：
Bödvarsdóttir, SK;Anamthawat-Jónsson, K
使用标记的羊草总基因组 DNA 作为探针进行基因组 Southern 杂交，在来自五种羊草和四种黑穗草的所有限制性酶消化的 DNA 上显示出强烈的杂交信号。使用相同的毛球菌探针但使用未标记的来自刚毛竹的封闭 DNA 的实验表明根本没有杂交。这两个属显然具有相同的基因组内容。无需封闭的 Southern 杂交可鉴定出羊草和刚玉中丰富的 DNA 片段。克隆了可能对羊草具有特异性的片段。对来自 L. mollis 和 L. arenarius 的 5 个重复 DNA 克隆进行了表征：pLmIs1、pLmIs44、pLmIs51、pLmIs53 和 pLaIs56。这些克隆在 Southern 印迹上与羊草属和刚穗属杂交——没有克隆只与这些属之一杂交。 Southern印迹和荧光原位杂交（FISH）实验均表明所有克隆均含有分散的重复序列。他们均匀地绘制了除着丝粒、端粒和核仁组织区域外的所有染色体。这些克隆中的三个，即 pLmIs1、pLmIs44 和 pLmls53，基本上对羊草属和刚穗属具有特异性，在其他属（如小麦属、大麦属、堇菜属或披碱草属）中很少或没有检测到杂交。序列分析进一步表明，这些克隆是逆转录元件的一部分。特别是，克隆 pLmls44 产生了适合于分析物种间遗传相关性的杂交图谱。本研究表明，羊草属和刚毛属具有相同的基本基因组 N-s，因此为这两个属的结合提供了强有力的证据。
Genomic Southern hybridization using labeled total genomic DNA of Leymus mollis as probe showed intense hybridization signals on all restriction enzyme digested DNA from five species of Leymus Hochst., and four species of Psathyrostachys Nevski. Experiments using the same L. mollis probe, but with unlabeled blocking DNA from Psathyrostachys, showed no hybridization at all. These two genera evidently had the same genomic content. Southern hybridization without blocking allowed identification of DNA fragments abundant in Leymus and Psathyrostachys. Fragments potentially specific to Leymus were cloned. Five repetitive DNA clones from L. mollis and L. arenarius were characterized: pLmIs1, pLmIs44, pLmIs51, pLmIs53, and pLaIs56. These clones hybridized to both Leymus and Psathyrostachys on Southern blots - no clone hybridized to only one of these genera. Both Southern blot and fluorescence in situ hybridization (FISH) experiments showed that all the clones contained dispersed repetitive sequences. They painted all and whole chromosomes uniformly except at centromeres, telomeres, and nucleolar organiser regions. Three of these clones, i.e., pLmIs1, pLmIs44, and pLmls53, were essentially specific to Leymus and Psathyrostachys little or no hybridization was detected in other genera such as Triticum, Hordeum, Thinopyrum, or Elymus. Sequence analysis further revealed that the clones were part of retroelements. In particular, the clone pLmls44 produced hybridization profiles suitable for analysis of genetic relatedness among species. The present study shows that Leymus and Psathyrostachys share the same basic genome, N-s, and therefore provides strong evidence for combining these two genera.