Meiotic Crossover Analysis in Plants

Young spikes were collected from 7 to 10-week-old plants and carefully dissected to isolate anthers. For each dissected floret, one of the three developmentally equivalent anthers was squashed in aceto-carmine staining solution and meiocytes visualised using a ZEISS Optima microscope. When meiocytes at metaphase I were identified (for chiasma frequency analysis) or other defined stages (RNA analysis), the two remaining anthers were either fixed in 100% ethanol/acetic acid 3:1 (v/v) for 48 h and then subsequently transferred to 70% ethanol or snap frozen in liquid N₂ for later RNA-based analyses. Fixed anthers can eventually be stored at 4°C for a few months. For cytological analysis of meiocytes at metaphase I, pollen mother cells (PMCs) were released from the anther by crushing it on a slide in a drop of aceto-carmine staining solution. Anther debris was carefully removed, and a coverslip placed on the slide. The slides were then heated until separation of the chromosomes and aceto-carmine solution replaced by acetic acid 45%. Coverslips were then vertically pressed to spread out the chromosomes. Chromosome configurations of ~50 PMC per anther were analysed under a ZEISS Axio Observer Z1 inverted microscope. For each cell, the number of univalents, rod bivalents (pair of chromosomes linked by a unique chiasma), ring bivalents (pair of chromosomes linked by two chiasmata), trivalents (three chromosomes linked by two chiasmata) and quadrivalents (four chromosomes linked by three or four chiasmata) were counted. Frequency of chiasmata (the cytological manifestation of meiotic crossovers) was then calculated. Significant differences between mutant and corresponding wild-type control chiasma frequencies were assessed using Mann–Whitney tests adjusted for multiple comparisons.