Combined fluorescent and electron microscopic imaging unveils the specific properties of two classes of meiotic crossovers

Crossovers (COs) shuffle genetic information and allow balanced segregation of homologous chromosomes during the first division of meiosis. Recombination nodules (RNs) are closely correlated with crossing over, and, because they are observed by electron microscopy of synaptonemal complexes (SCs) in extended pachytene chromosomes, RNs provide the highest-resolution cytological marker currently available for defining the frequency and distribution of crossovers along the length of chromosomes. In several organisms, mutants demonstrate that two molecularly distinct pathways produce COs. One pathway produces class I COs that exhibit interference (lowered probability of nearby COs), and the other pathway produces class II COs with little or no interference. However, the relative contributions, genomic distributions, and interactions of these two pathways are essentially unknown in nonmutant organisms because marker segregation only indicates that a CO has occurred, not its class type. Here, ..., Additional details of experimental and statistical procedures are provided in Anderson et al. 2014 (see SI Appendix, Materials and Methods).
SC spreading & Immunolabeling: Primary microsporocytes in pachytene from the highly inbred cherry tomato line (LA4444) were used to prepare SC spreads on 0.6% Falcon plastic-coated slides as described in (45, 46). SC spreads were treated with DNase I, then immunolabeled with affinity-purified chicken anti-SlSMC1 diluted 1:50 and affinity-purified rabbit anti-SlMLH1 diluted 1:50 followed by goat anti-chicken Dylight 649 and goat anti-rabbit AlexaFluor 488, both from Jackson Labs and diluted 1:500 (19, 45-47). Fluorescence microscopy was performed using a 100X Plan-Apo objective with an adjustable iris and a Leica DM5000 microscope equipped for both phase contrast and fluorescence microscopy with narrow band-pass FITC and TRITC filter cubes and zero pixel shift.
Fluorescence LM and EM: Red and green signals for each spread were captured individua..., , # Combined fluorescent and electron microscopic imaging unveils the specific properties of two classes of meiotic crossovers

The data are presented in an Excel spreadsheet with 12 sheets that contain the following information:

Sheet 1 (“Karyotype”): Contains details about SC length and arm ratio that were used to identify each tomato SC.  Information about each tomato SC is presented in terms of relative length (% of the total SC set length), SC length (µm), arm ratio, short arm (SA) length (µm) and long arm (LA) length (µm). Additional details are included about SC2 (in which the SA containing the NOR is often broken) and SCs 7/9 and SCs 5/12 (each pair of which cannot be distinguished by relative length and arm ratio so RN data from each SC pair were pooled together).

 Sheet 2 (“Notes”) contains definitions of the headings used for the 10 sheets labeled SC1 through SC11 where RN positions on each SC have been mapped.  Each RN has been identified as MLH1-positive (code = green) or...