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

重要的谷物，如小麦和大麦，在基因组中有相当多的DNA。这使得用这种方法分离基因变得困难，这种方法适用于像拟南芥这样基因组小的植物。本研究尝试用激光显微解剖技术从小麦中构建染色体特异性、臂特异性和区域特异性DNA文库。小麦二染色体系的远心染色体和黑麦细胞质代换系的侏儒染色体。选用中国春)作为靶材，用调整到显微操作器上的细玻璃针刮去并拾取，在显微镜下观察。然后将微解剖的染色体收集到PCR管中，使用退化寡核苷酸引物聚合酶链反应（dopp -PCR）扩增染色体DNA。为了评估我们的微解剖程序的效率，我们试图扩增小麦的一条染色体的DNA，并与两个，四个和六个微解剖片段的结果进行比较。在几乎所有的情况下，DNA扩增甚至可以从单个染色体上观察到。测序分析表明，PCR片段中有较高比例的低拷贝序列参与。这表明目前的显微解剖程序在创建绘画探针和产生区域特异性DNA标记方面是有效的。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。然而，在小麦染色体的显微解剖中并没有明显的发现。这可能是由于用作探针的扩增DNA中有大量的反转录子样重复序列。

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.