Genomic and chromosomal distribution patterns of various repeated DNA sequences in wheat revealed by a fluorescence in situ hybridization procedure
Genomic and chromosomal distribution patterns of various repeated DNA sequences in wheat revealed by a fluorescence in situ hybridization procedure
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DOI：
10.1139/gen-2013-0003
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发表时间：
2013-03-01
期刊：
影响因子：
3.1
通讯作者：
Kato, Akio
中科院分区：
文献类型：
--
作者：
Komuro, Shirabe;Endo, Ryota;Kato, Akio
小麦（Triticum aestivum L.）是异源六倍体，其中三个基因组中的每一个都具有高的1C含量。这表明存在多个串联重复的序列，这应该是使用原位杂交检测。有些重复已经描述过了，但其他的还有待确认。为了发现其他的，在荧光原位杂交和显微镜信号观察之后，检测了2000个质粒小麦克隆的信号存在。其中，47个克隆在小麦染色体上产生了较强的离散信号。两个新鉴定的克隆（pTa-535和pTa-713）被确定具有用于染色体鉴定的特别有价值的序列。结合pTa-86（pSc 119同源序列），这些探针能够明确区分所有小麦染色体，包括方向。四个新鉴定的序列（pTa-465、pTa-k566、pTa-s120和pTa-s126）是有用的，因为它们在各种小麦染色体臂上产生离散信号。另外两个克隆（pTa-k288和pTa-k229）产生了类似GISH（基因组原位杂交）的信号，因为它们允许同时区分A、B和D基因组。此外，还鉴定了着丝粒、着丝粒相关和核糖体DNA克隆。还描述了载玻片制备和重新探测程序的改进。为了增强离散信号检测，采用了一种新的直接荧光标记方法，即Vent（R）（exo-）末端延伸方法。
Wheat (Triticum aestivum L.) is an allohexaploid, in which each of the three genomes has a high 1C content. This indicates the presence of multiple tandemly repeated sequences, which should be detectable using in situ hybridization. Some repeats have already been described, but others remain to be recognized. To discover others, 2000 plasmid wheat clones were examined for signal presence after fluorescence in situ hybridization and microscopic signal observation. Among them, 47 clones produced strong discrete signals on wheat chromosomes. Two of the newly identified clones (pTa-535 and pTa-713) were determined to have especially valuable sequences for chromosome identification. In combination with pTa-86 (the pSc119 homologous sequence), these probes enable unambiguous discrimination of all wheat chromosomes including orientation. Four newly identified sequences (pTa-465, pTa-k566, pTa-s120, and pTa-s126) were useful in that they produced discrete signals on various wheat chromosome arms. Two other clones (pTa-k288 and pTa-k229) produced GISH-like (genomic in situ hybridization) signals because they allowed the A, B, and D genomes to be distinguished simultaneously. In addition, centromere, centromere-related, and ribosomal DNA clones were identified. Also described are improvements on slide preparation and reprobing procedures. To enhance discrete signal detection, a new direct fluorescent-labeling procedure, namely the Vent(R) (exo-) terminal extension method, was employed.