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，在基因组中含有相当多的DNA。这使得利用适用于拟南芥等小基因组植物的方法分离基因变得困难。本研究试图用激光显微切割技术构建小麦染色体、臂和区域特异DNA文库。小麦(Triticum aestivum cv.)双染色体系中的端粒染色体和黑麦细胞质替代系中的小染色体。中国春)作为目标，在显微镜下观察，用调整到显微操作器上的细小玻璃针将其刮掉并拾起。然后将显微切割的染色体放入聚合酶链式反应管中，用简并寡核苷酸引物聚合酶链式反应(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，我们尝试只从小麦的一条染色体上扩增DNA，并与两个、四个和六个显微切割片段的结果进行了比较。在几乎所有的情况下，甚至可以从一条染色体上观察到DNA扩增。测序分析表明，在PCR片段中涉及较高比例的低拷贝序列。这表明目前的显微切割程序在创建绘制探针和产生区域特异性DNA标记方面是有效的。染色体专一性在小染色体的例子中得到了证实，因为它起源于黑麦。然而，在小麦染色体的显微切割中，它并没有被清楚地揭示出来。这可能是由于被用作探针的扩增DNA中含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.