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.E ukaryotic sexual reproduction involves meiosis, a specialized cell division in which DNA duplication in a diploid cell is followed by two cell divisions to produce four haploid cells. The first division, Meiosis I, involves crossing over and chiasmata formation between each pair of homologous chromosomes, thereby ensuring separation of the homologs and formation of two haploid cells, each with one complete set of replicated chromosomes. The second division, Meiosis II, is a mitosis-like division in which the two sister chromatids separate to yield four haploid cells that directly or indirectly form gametes. Because these four products are genetically unique due to crossing over and independent segregation of homologous chromosomes during Meiosis I, meiosis plays an important role in creating genetic diversity in sexually reproducing organisms.Crossing over during meiosis is tightly controlled so each pair of homologs has at least one "obligate" crossover (CO) that ensures balanced reductional segregation, but the presence of a CO reduces the likelihood of another CO in its vicinity, a phenomenon referred to as CO interference (1, 2). Significant progress has been made recently in illuminating the molecular events of meiotic recombination and the control of crossing over (3)(4)(5)(6)(7)(8). The initiating event of meiotic recombination in most organisms is formation of numerous DNA double-strand breaks (DSBs). Homolog-dependent repair of a DSB may follow any one of at least three pathways: (i) non-CO that may result in a short gene conversion; (ii) CO ...

Meiotic Crossing Over in Maize Knob Heterochromatin

“…Together, these results indicate that the reported frequencies of MLH1 foci in and around knobs are probably minimal estimates of crossing over, and that the actual amount of crossing over in these locations may be substantially higher. Furthermore, in tomato, type I and type II crossovers have different distributions, with type II crossovers being observed more often than expected in the short arms of acrocentric chromosomes and in pericentric heterochromatin compared to type I crossovers (Anderson et al 2014). If the same pattern applies to maize, it could explain why only a few MLH1 foci were observed at the ends of the terminal knob-carrying short arms of SC 1 and SC 9, with most of the many RNs observed at those positions presumably representing type II crossovers (Figure 1) (Anderson et al 2003).…”

“…Most SCs have one or two MLH1 foci, and we found an average of 13.9 MLH1 foci per SC set (n = 18) ( Figure 5). Individual MLH1 foci varied in brightness, possibly depending on the amount of the MLH1 protein present in individual RNs (Anderson et al 2014). Using the 180-bp knob repeat for FISH on these previously immunolabeled SC spreads allowed us to compare the location of knobs and MLH1 fluorescent foci simultaneously on the same SCs ( Figure 5).…”

“…While we could not see knobs on spreads of maize SCs because the chromatin was dispersed, we knew where knobs were supposed to be based on the work of others (Chen et al 2000). Because RNs occur at sites of crossing over (Carpenter 1975;Sherman and Stack 1995;Anderson et al 2003Anderson et al , 2014Marcon and Moens 2003), this result indicated that crossing over occurs along the SC in knob heterochromatin at rates similar to adjacent euchromatin. However, in deference to the assumption that crossing over is suppressed in heterochromatin, we suggested a model for the structure of homozygous knobs in which the knob on each homolog consists of a few long loops of condensed chromatin attached to a short segment of SC compared to the dimensions of the knob.…”

“…This was accomplished by fluorescence in situ hybridization (FISH) of the 180-bp repeat found in maize knobs (Peacock et al 1981;Ananiev et al 1998a,b) and immunofluorescent labeling of maize SC lateral element protein AFD1 (Golubovskaya et al 2006) and RN protein MLH1 to show crossover sites on SCs (Baker et al 1996;Anderson et al 1999Anderson et al , 2014. We found that long loops of knob heterochromatin attach to SCs along the entire length of knobs, not just at tiny sites, and that MLH1 foci occur on SCs within knob heterochromatin at frequencies similar to SCs in adjacent euchromatin.…”

The Chromosomes of Birds during Meiosis

“…2 c, d). MLH1 is required for wild-type levels of crossing over during meiosis and is a component of RNs [Marcon and Moens, 2003;Anderson et al, 2014]. Current evidence in yeast and other organisms indicates that MLH1 foci tag the CO events showing chiasmatic interference, while a second type of COs (non-interfering) follows a molecular pathway lacking MLH1 [reviewed in Mezard et al, 2015].…”