Cytological Analysis of Meiotic Recombination

A minimum of 10 plants were characterized for each mutant. Chromosome spreads were stained with DAPI and centromere FISH, and immuno-localization experiments were carried out as described previously [79 (link)]. Rabbit polyclonal AtRAD51 and γ-H2AX antibodies were used at 1:200 fold dilutions and Alexa Fluor 488 Goat Anti-Rat IgG (H+L) secondary antibody (A-21428, Invitrogen, Carlsbad, CA, USA) was used at a 1:1000 fold dilution [80 (link)]. Chiasmata distribution statistics were performed following the protocol of Sanchez et al. [81 (link)]. BAC DNA extraction (F19K16) and probe labeling were described previously [43 (link)]. Images of chromosome spreads were obtained using an Axio Imager A2 microscope (Zeiss, Heidelberg, Germany) equipped with a digital camera (Canon, Tokyo, Japan), and processed using Photoshop CS (Adobe Systems, Mountain View, CA). Images were initially captured in black & white and, if necessary, globally false-colored post-capture for visual contrast. AtRAD51 and γ-H2AX foci in WT and mutant lines were counted and statistically analyzed using ImageTool version 3.0 software (University of Texas Health Science Center, San Antonio, USA).
In mutants that lacked synapsis, we distinguished zygotene from pachytene chromosomes by their relative condensation, with pachytene being more condensed than zygotene chromosomes.