Analyzing Meiosis in Barley

This chapter contains a detailed description of the immunological and cytological techniques developed to determine factors involved in crossover control during meiosis in barley. The immunological technique involves digesting fresh anthers followed by chromosome spreading to remove cytoplasm that causes unwanted background, whilst protecting the fragile chromosome structure from being compromised. Specific antibodies raised against meiotic proteins are then incubated with nuclei, detected with secondary antibodies conjugated to fluorescent dyes, and visualized with either wide-field or confocal microscopes. In the cytological technique, barley inflorescences are fixed, followed by dissecting out the anthers, digesting the cell walls and then spreading the meiotic chromosomes. Both techniques can be used in conjunction with specific DNA probes for fluorescent in situ hybridization (FISH) to label telomeres, centromeres, and ribosomal DNA; to identify DNA modifications such as 5-methylcytosine; and to detect the incorporation of DNA base analogues such as 5-bromo-2′-deoxyuridine (BrdU) or 5-ethynyl-2′-deoxyuridine (EdU) to be used for a meiotic time-course or assaying newly synthesized DNA. Although these techniques have been specifically developed for barley, they should be directly transferable to other cereal crop species such as wheat and rice.