Hobza Roman, Vyskot Boris
Laboratory of Plant Developmental Genetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, CZ-612 65 Brno, Czech Republic.
Methods Cell Biol. 2007;82:433-53. doi: 10.1016/S0091-679X(06)82015-7.
A recent progress in plant molecular biology has led to enormous available data of DNA sequences, including complete nuclear genomes of Arabidopsis, rice, and poplar. On the other hand, in plant species with more complex genomes, containing widespread repetitive sequences, it is important to establish genomic resources that help us to focus on particular part of genomes. Laser technology enables to handle with specific subcellular structures or even individual chromosomes. Here we present a comprehensive protocol to isolate and characterize DNA sequences derived from the sex chromosomes of white campion (Silene latifolia). This dioecious plant has become the most favorite model to study the structure, function, and evolution of plant sex chromosomes due to a large and distinguishable size of both the X and Y chromosomes. The protocol includes a versatile technique to prepare metaphase chromosomes from either germinating seeds or in vitro cultured hairy roots. Such slides can be used for laser chromosome microdissection, fluorescence in situ-hybridization mapping, and immunostaining. Here we also demonstrate some applications of the laser-dissected chromosome template, especially a modified FAST-FISH technique to paint individual chromosomes, and construction and screening of chromosome-specific DNA libraries.
植物分子生物学的最新进展产生了大量可用的DNA序列数据，包括拟南芥、水稻和杨树的完整核基因组。另一方面，在基因组更为复杂、含有广泛重复序列的植物物种中，建立有助于我们聚焦于基因组特定部分的基因组资源很重要。激光技术能够处理特定的亚细胞结构甚至单个染色体。在此，我们展示了一个全面的方案，用于分离和鉴定源自白麦瓶草（Silene latifolia）性染色体的DNA序列。这种雌雄异株植物已成为研究植物性染色体结构、功能和进化的最热门模式植物，因为其X和Y染色体都很大且易于区分。该方案包括一种通用技术，可从萌发种子或体外培养的毛状根制备中期染色体。这样的载玻片可用于激光染色体显微切割、荧光原位杂交图谱绘制和免疫染色。在此，我们还展示了激光切割染色体模板的一些应用，特别是一种改良的快速荧光原位杂交技术来描绘单个染色体，以及构建和筛选染色体特异性DNA文库。
