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

Abstract

Crossovers (COs) shuffle genetic information and allow balanced segregation of homologous chromosomes during the first division of meiosis. 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 fluorescent probes with the high resolution of electron microscopy to localize and characterize COs 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 and II 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.

Keywords: MLH1; MUS81; genetic interference; recombination nodule; synaptonemal complex.

Fig. 1.

Identifying MLH1-positive and MLH1-negative RNs using consecutive light and electron microscopy of the same SC spread (SI Appendix, Fig. S1). (A) LM view of a complete set of 12 tomato SCs labeled with antibodies to MLH1 (green) and SMC1, an SC component (magenta). (B) Area boxed in A, enlarged. (C) Fluorescent LM image from B superimposed over the corresponding portion of the EM image. Two complete SCs, each with a kinetochore (k), are shown along with a part of another SC. Numbered arrowheads point to RNs that are visible by EM. Four RNs (numbers 2–5) correspond to sites of MLH1 foci (MLH1-positive RNs), whereas two RNs (numbers 1 and 6) do not correspond to MLH1 foci (MLH1-negative RNs). (D) Enlarged view of RNs without the LM overlay. MLH1-positive RNs (numbers 2–5) are larger than MLH1-negative RNs (numbers 1 and 6). (Scale bars: 4 µm for A–C and 250 nm for D.)

Fig. 2.

Distributions of MLH1-positive RNs (blue bars) and MLH1-negative RNs (red bars) for all tomato chromosomes and chromosome groups. Histogram bars are in 2% SC length intervals (X-axis) and show the number of RNs observed per interval (left y axis). The red histogram has been superimposed on the blue histogram for each chromosome. To highlight differences between the individual histograms, lines showing the cumulative frequency (right y axis) of all RNs (black), of MLH1-positive RNs (blue), and of MLH1-negative RNs (red) have been added. The length of each chromosome has been scaled to represent its appropriate length relative to the other chromosomes. The short arm of chromosome 2 is entirely heterochromatic, and the distal segment is usually asynapsed and/or broken off (and often lost) in SC spreads, explaining the lack of any RNs in that region. Only MLH1-negative RNs were observed on the retained short arm of chromosome 2. Except for the near-metacentric chromosomes 5/12 and 11, all chromosomes have a higher fraction of MLH1-negative RNs on the short arm and in the pericentric heterochromatin compared with MLH1-positive RNs (SI Appendix, Table S6). Black dots on the X-axis represent centromere positions.

Fig. 3.

Distribution of distances (in µm SC) between different types of RNs, pooled over all chromosomes. (A) Distribution of distances between MLH1-positive RNs (class I COs) and MLH1-negative RNs (class II COs). (B) Distribution of distances (in µm SC) between pairs of MLH1-positive RNs (class I COs). Histograms represent the experimental data. Vertical bars represent 95% confidence intervals for each bin of the histogram. The expected distribution in the absence of any interaction between class I and class II COs is plotted as a solid line with the associated 95% confidence intervals (dashed lines along the solid line). Interference between class I and class II COs (A) and between class I COs (B) is indicated by the lower than expected frequency of events at short distances. Similar figures for individual chromosomes are shown in SI Appendix, Figs. S10 and S12.