DOI: 10.5061/dryad.0vt4b8h5m
title: Data from: Combined fluorescent and electron microscopic imaging unveils the specific properties of two classes of meiotic crossovers

description: 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.
description: Additional details of experimental and statistical procedures are
 provided in Anderson et al. 2014 (see SI Appendix, Materials and
 Methods). <strong>SC spreading &
 Immunolabeling:</strong> 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.
 <strong>Fluorescence LM and EM:</strong> 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.
 <strong>LM-EM image analysis:</strong> 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. 
description: # 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
 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.