Cytology and Microscopy: Immunolocalization of Covalently Modified Histone Marks on Barley Mitotic Chromosomes

• Published in: Bio-protocol 2016-06, Vol.6 (12)
• Main Authors: Colas, Isabelle, Baker, Katie, Flavell, Andrew
• Format: Article
• Language: English
• ISSN: 2331-8325; 2331-8325
• DOI: 10.21769/BioProtoc.1841

Barley is a diploid inbreeding crop with a genome of 5 GB organized into seven chromosomes. The relatively low chromosome number and their large size make barley an excellent model for chromosome cytogenetic studies of large genome cereal crops. Chromatin can be defined as euchromatin or heterochromatin. Euchromatin is gene-rich, less condensed, and transcriptionally active while the heterochromatin is gene-poor, remains highly condensed and has low transcriptional activity (Bartova et al., 2008; Sharakhov and Sharakhova, 2015). However, the mapping of nine Histone modifications has shown that this simple description is not accurate in barley. Instead, it has been shown that combinations of histones carrying different covalent modifications reveal 10 chromatin states partitioning barley chromosomes into three global environments (Baker et al., 2015). Briefly, in this protocol, barley roots (cv Morex) were collected, fixed in paraformaldehyde and squashed onto slides. Chromosome spreads were immunostained using antibodies against specific histone modifications, in particular H3K27me3, K3K27me1 and H3K9me2. We used confocal imaging to acquire stacked images and confirm the locations of these histone modifications on barley chromosomes.