Data from: Combined fluorescent and electron microscopic imaging unveils the specific properties of two classes of meiotic crossovers
Data files
Dec 20, 2023 version files 887.18 KB
Abstract
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, we combine the efficiency of light microscopy for revealing cellular functions using a fluorescent probe to MLH1 proteins to mark class I COs with the high resolution of electron microscopy to localize and characterize all COs (RNs) in the same sample of meiotic pachytene chromosomes from wild-type tomato. To our knowledge, for the first time, every CO along each chromosome can be identified by class to unveil specific characteristics of each pathway. We find that class I (MLH1-positive) and class II (MLH1-negative) COs have different recombination profiles along chromosomes. In particular, class II COs, which represent about 18% of all COs, exhibit no interference and are disproportionately represented in pericentric heterochromatin, a feature potentially exploitable in plant breeding. Finally, our results demonstrate that the two pathways are not independent because there is interference between class I and II COs.
README: Combined fluorescent and electron microscopic imaging unveils the specific properties of two classes of meiotic crossovers
https://doi.org/10.5061/dryad.0vt4b8h5m
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 MLH1-negative (code=blue). In addition, the positions of overlying fluorescent foci that do not correspond to RNs (background) are also noted (code=yellow). Other notes explaining the nuances of the data and the reference in which this data was used for publication are also presented in the Notes sheet.
Sheet 3 (“SC1”): Number, type and position of each RN on each tomato SC1.
Sheet 4 (“SC2”): Number, type and position of each RN on each tomato SC2.
Sheet 5 (“SC3”): Number, type and position of each RN on each tomato SC3.
Sheet 6 (“SC4”): Number, type and position of each RN on each tomato SC4.
Sheet 7 (“SC5/12”): Number, type and position of each RN on each tomato SC5/12.
Sheet 8 (“SC6”): Number, type and position of each RN on each tomato SC6.
Sheet 9 (“SC7/9”): Number, type and position of each RN on each tomato SC7/9.
Sheet 10 (“SC8”): Number, type and position of each RN on each tomato SC8.
Sheet 11 (“SC10”): Number, type and position of each RN on each tomato SC10.
Sheet 12 (“SC11”): Number, type and position of each RN on each tomato SC11.
Methods
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 individually using a cooled Hamamatsu monochrome 1344X1044 pixel camera and IP Lab software (ver 4). Because some MLH1 foci were quite small and dim, we used long exposure times to be sure that even dim foci would be imaged. Images for each spread were artificially colored using IPLab and merged using Photoshop CS2. After LM images were captured, the cover glass on each slide was removed carefully, and slides were stained with phosphotungstic acid (46). Plastic was lifted from each slide onto grids, and SC spreads previously imaged by fluorescence were photographed at a magnification of 3000X (generally requiring 3 – 6 images each) using a JEOL 2000 EM.
LM-EM image analysis: EM negatives were scanned at 800 dpi using an Epson Perfection V700 Photo scanner. A montage of each SC spread was assembled using Adobe Photoshop CS2, and RNs were identified (18). The corresponding fluorescent image was then layered over the EM montage, and each SC was analyzed individually by precisely aligning the red (SC) and green (MLH1) combined fluorescent image over the EM image of the same SC. Then, the MLH1 fluorescent signal at each previously identified RN position was assessed. Each “unlabeled” RN was then more carefully evaluated using only the green channel (instead of the red and green combined image), after additional temporary enhancement of the green signal. If no green signal was observed under these conditions, the RN was marked as an MLH1-negative RN. Only informative SC spreads in which each chromosome had at least one (obligate) MLH1 focus were selected for measurement using MicroMeasure 3.0 and subsequent analysis. In some cases, one or more SCs in a set had to be excluded due to a lack of distinct kinetochores or to the presence of stain precipitate at the EM level that partially obscured SCs. SCs from these groups were used for counting the number of RNs per SC set but were not used for mapping RN positions.