Preparing Maize Synaptonemal Complex Spreads and Sequential Immunofluorescence and Fluorescence In Situ Hybridization.

Immunofluorescence and fluorescence in situ hybridization (FISH) can be used to locate specific proteins and DNA sequences, respectively, in chromosomes by light microscopy. Here we describe sequential use of these techniques on spreads of maize synaptonemal complexes (SCs) to determine whether crossing over can occur in knob heterochromatin. We used antibodies to AFD1, an SC protein, and MLH1, a class I (interference-sensitive) crossover protein found in most recombination nodules (RNs) to identify crossovers (COs) along SCs.

Meiotic Crossing Over in Maize Knob Heterochromatin.

There is ample evidence that crossing over is suppressed in heterochromatin associated with centromeres and nucleolus organizers (NORs). This characteristic has been attributed to all heterochromatin, but the generalization may not be justified. To investigate the relationship of crossing over to heterochromatin that is not associated with centromeres or NORs, we used a combination of fluorescence hybridization of the maize 180-bp knob repeat to show the locations of knob heterochromatin and fluorescent immunolocalization of MLH1 protein and AFD1 protein to show the locations of MLH1 foci on maize synaptonemal complexes (SCs, pachytene chromosomes).

Interspecific reproductive barriers between sympatric populations of wild tomato species (Solanum section Lycopersicon).

Premise Of The Study: Interspecific reproductive barriers (IRBs) often prevent hybridization between closely related species in sympatry. In the tomato clade (Solanum section Lycopersicon), interspecific interactions between natural sympatric populations have not been evaluated previously. In this study, we assessed IRBs between members of the tomato clade from nine sympatric sites in Peru.

Fluorescence in situ hybridization and optical mapping to correct scaffold arrangement in the tomato genome.

The order and orientation (arrangement) of all 91 sequenced scaffolds in the 12 pseudomolecules of the recently published tomato (Solanum lycopersicum, 2n = 2x = 24) genome sequence were positioned based on marker order in a high-density linkage map. Here, we report the arrangement of these scaffolds determined by two independent physical methods, bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and optical mapping. By localizing BACs at the ends of scaffolds to spreads of tomato synaptonemal complexes (pachytene chromosomes), we showed that 45 scaffolds, representing one-third of the tomato genome, were arranged differently than predicted by the linkage map.

Preparing SC spreads with RNs for EM analysis.

Recombination nodules (RNs) are associated with synaptonemal complexes (SCs) during early prophase I of meiosis. RNs are too small to be resolved by light microscopy and can be observed directly only by electron microscopy. The patterns of RNs on SCs can be analyzed using three-dimensional reconstructions of nuclei using serial thin sections, but this method is time consuming and technically difficult.

Interspecific reproductive barriers in the tomato clade: opportunities to decipher mechanisms of reproductive isolation.

The tomato clade within the genus Solanum has numerous advantages for mechanistic studies of reproductive isolation. Its thirteen closely related species, along with four closely allied Solanum species, provide a defined group with diverse mating systems that display complex interspecific reproductive barriers. Several kinds of pre- and postzygotic barriers have already been identified within this clade.

Two types of meiotic crossovers coexist in maize.

We apply modeling approaches to investigate the distribution of late recombination nodules in maize (Zea mays). Such nodules indicate crossover positions along the synaptonemal complex. High-quality nodule data were analyzed using two different interference models: the "statistical" gamma model and the "mechanical" beam film model.

Electron microscopic immunogold localization of recombination-related proteins in spreads of synaptonemal complexes from tomato microsporocytes.

Many of the structures involved in meiotic synapsis and recombination such as synaptonemal complexes (SCs) and recombination nodules (RNs) can be resolved only by electron microscopy. Therefore, electron microscopic (EM) immunolocalization using gold-conjugated antibodies is the best way to verify whether certain proteins are components of SCs or RNs. Here, we describe (1) preparing tomato primary microsporocyte protoplasts in leptotene, zygotene, and pachytene stages; (2) hypotonically bursting the protoplasts on glow-discharged glass and plastic-coated slides to make spreads of SCs; (3) immunolabeling proteins in SCs and RNs with colloidal gold; (4) staining SC spreads for EM; and (5) transferring SC spreads on plastic films to grids for EM.

A weak effect of background selection on trinucleotide microsatellites in maize.

Artificial selection during the domestication of maize is thought to have been predominantly positive and to have had little effect on the surrounding neutral diversity because linkage disequilibrium breaks down rapidly when physical distance increases. However, the degree to which indirect selection has shaped neutral diversity in the maize genome during domestication remains unclear. In this study, we investigate the relationship between local recombination rate and neutral polymorphism in maize and in teosinte using both sequence and microsatellite data.

Predicting and testing physical locations of genetically mapped loci on tomato pachytene chromosome 1.

Predicting the chromosomal location of mapped markers has been difficult because linkage maps do not reveal differences in crossover frequencies along the physical structure of chromosomes. Here we combine a physical crossover map based on the distribution of recombination nodules (RNs) on Solanum lycopersicum (tomato) synaptonemal complex 1 with a molecular genetic linkage map from the interspecific hybrid S. lycopersicum x S.

Uneven distribution of expressed sequence tag loci on maize pachytene chromosomes.

Examining the relationships among DNA sequence, meiotic recombination, and chromosome structure at a genome-wide scale has been difficult because only a few markers connect genetic linkage maps with physical maps. Here, we have positioned 1195 genetically mapped expressed sequence tag (EST) markers onto the 10 pachytene chromosomes of maize by using a newly developed resource, the RN-cM map. The RN-cM map charts the distribution of crossing over in the form of recombination nodules (RNs) along synaptonemal complexes (SCs, pachytene chromosomes) and allows genetic cM distances to be converted into physical micrometer distances on chromosomes.

Integrating genetic linkage maps with pachytene chromosome structure in maize.

Genetic linkage maps reveal the order of markers based on the frequency of recombination between markers during meiosis. Because the rate of recombination varies along chromosomes, it has been difficult to relate linkage maps to chromosome structure. Here we use cytological maps of crossing over based on recombination nodules (RNs) to predict the physical position of genetic markers on each of the 10 chromosomes of maize.

High-resolution crossover maps for each bivalent of Zea mays using recombination nodules.

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. Using the maize inbred line KYS, we prepared an SC karyotype in which each SC was identified by relative length and arm ratio and related to the proper linkage group using inversion heterozygotes. We mapped 4267 RNs on 2080 identified SCs to produce high-resolution maps of RN frequency and distribution on each bivalent.

Patterns of diversity and recombination along chromosome 1 of maize (Zea mays ssp. mays L.).

We investigate the interplay between genetic diversity and recombination in maize (Zea mays ssp. mays). Genetic diversity was measured in three types of markers: single-nucleotide polymorphisms, indels, and microsatellites.

Crossing over as assessed by late recombination nodules is related to the pattern of synapsis and the distribution of early recombination nodules in maize.

Recombination nodules (RNs) are multicomponent proteinaceous ellipsoids found in association with the synaptonemal complex (SC) during prophase I of meiosis. Numerous early RNs (ENs) are observed during zygotene, and they may be involved in homologous synapsis and early events in recombination. Fewer late RNs (LNs) are observed during pachytene, and they occur at crossover sites.