Construction of chromosome -specific DNA libraries by laser-microdissection-Molecular analysis of homoeologous group 1 chromosomes in wheat.

小麦和大麦等重要谷物的基因组中含有大量的DNA。这使得难以通过使用适用于具有小基因组的植物如拟南芥的方法来分离基因。本研究利用激光显微切割技术构建了小麦染色体、臂和区域特异性DNA文库。小麦（Triticum aestivum cv.中国春）作为目标，刮下并在显微镜观察下用调整到显微操作器的细玻璃针拾取。然后将显微切割的染色体收集到PCR管中，并使用简并寡核苷酸引物聚合酶链反应（DOP-PCR）扩增其染色体DNA。为了评估我们的显微切割程序的效率，我们试图从小麦的一个单一的染色体上扩增DNA，并与两个，四个和六个显微切割片段的结果进行比较。在几乎所有情况下，即使是单个染色体也可以观察到DNA扩增。测序分析表明，一个相对较高比例的低拷贝序列参与的PCR片段。这表明本显微切割程序是有效的，在创建绘画探针和产生区域特异性DNA标记。染色体特异性在侏儒染色体的情况下得到证实，因为它起源于黑麦。但在小麦染色体的显微切割中，这一点并没有得到很好的揭示。这可能是由于在用作探针的扩增DNA中存在大量的逆转录酶样重复序列。

Important cereals such as wheat and barley have considerably large amounts of DNA in the genome. This makes it difficult to isolate the genes by using the methods, which are applicable to plants with small genome such as Arabidopsis. In this study, chromosome-, arm- and region-specific DNA libraries were attempted to be constructed from wheat by laser-microdissection. Telocentric chromosomes in ditelosomic lines and the midget chromosome in the rye-cytoplasm substitution line of wheat (Triticum aestivum cv. Chinese Spring) were chosen as targets, scratched off and picked up with fine glass needles adjusted to a micromanipulator under microscopic observation. The microdissected chromosomes were then harvested into a PCR tube, and their chromosomal DNA was amplified using degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). In order to evaluate the efficiency of our microdissection procedure, we attempted to amplify DNA from only one single chromosome of wheat, and compared with the results from two, four and six microdissected fragments. In almost all cases, DNA amplification could be observed even from a single chromosome. Sequencing analysis revealed that a relatively high proportion of low-copy sequences are involved in the PCR fragments. This suggests the present microdissection procedure is effective in creating painting probes and in generating region-specific DNA markers. Chromosome-specificity was confirmed in the case of the midget chromosome, because it has originated from rye. However, it was not clearly revealed in the microdissection of wheat chromosomes. It may be due to the large amount of retroposon-like repeat sequences in the amplified DNA used as probes.