Doležel Jaroslav, Kubaláková Marie, Cíhalíková Jarmila, Suchánková Pavla, Simková Hana
Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Olomouc, Czech Republic.
Methods Mol Biol. 2011;701:221-38. doi: 10.1007/978-1-61737-957-4_12.
Chromosome analysis and sorting using flow cytometry (flow cytogenetics) is an attractive tool for fractionating plant genomes to small parts. The reduction of complexity greatly simplifies genetics and genomics in plant species with large genomes. However, as flow cytometry requires liquid suspensions of particles, the lack of suitable protocols for preparation of solutions of intact chromosomes delayed the application of flow cytogenetics in plants. This chapter outlines a high-yielding procedure for preparation of solutions of intact mitotic chromosomes from root tips of young seedlings and for their analysis using flow cytometry and sorting. Root tips accumulated at metaphase are mildly fixed with formaldehyde, and solutions of intact chromosomes are prepared by mechanical homogenization. The advantages of the present approach include the use of seedlings, which are easy to handle, and the karyological stability of root meristems, which can be induced to high degree of metaphase synchrony. Chromosomes isolated according to this protocol have well-preserved morphology, withstand shearing forces during sorting, and their DNA is intact and suitable for a range of applications.
使用流式细胞术进行染色体分析和分选（流式细胞遗传学）是将植物基因组分割成小部分的一种有吸引力的工具。复杂性的降低极大地简化了大基因组植物物种的遗传学和基因组学研究。然而，由于流式细胞术需要颗粒的液体悬浮液，缺乏制备完整染色体溶液的合适方案延迟了流式细胞遗传学在植物中的应用。本章概述了一种从幼苗根尖制备完整有丝分裂染色体溶液并使用流式细胞术进行分析和分选的高产方法。处于中期的根尖用甲醛轻度固定，通过机械匀浆制备完整染色体溶液。本方法的优点包括使用易于处理的幼苗，以及根分生组织的核型稳定性，可诱导其达到高度的中期同步。按照此方案分离的染色体具有保存良好的形态，在分选过程中能承受剪切力，其DNA完整且适用于一系列应用。
