Laser microdissection-based analysis of plant sex chromosomes

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.