Characterization of Meiotic Non-crossover Molecules from Arabidopsis thaliana Pollen

Abstract

Meiotic recombination is essential for proper segregation of homologous chromosomes and thus for formation of viable gametes. Recombination generates either crossovers (COs), which are reciprocal exchanges between chromosome segments, or gene conversion not associated with crossovers (NCOs). Both kinds of events occur in narrow regions (less than 10 kb) called hotspots, which are distributed along chromosomes. While NCOs may represent a large fraction of meiotic recombination events in plants, as in many other higher eukaryotes, they have been poorly characterized due to the technical difficulty of detecting them. Here, we present a powerful approach, based on allele-specific PCR amplification of single molecules from pollen genomic DNA, allowing detection, quantification and characterization of NCO events arising at low frequencies at recombination hotspots.

Contrasted Patterns of Crossover and Non-crossover at Arabidopsis thaliana Meiotic Recombination Hotspots

“…In plants, very few meiotic NCOs have been characterized because of the difficulty in detecting molecular events unless they are linked to a phenotypic change. We characterized NCO events at both the 14a1 and 130× hotspots, with different molecular approaches adapted to the polymorphisms available at each hotspot ( Figure 1 ; see Material and Methods; [30] ).…”

“…For 14a1, the polymorphisms at the center of the hotspot were not suitable for a pollen typing strategy. Thus we used a cloning strategy based on a method described in [31] ( Figure 1 ; see Material and Methods, [30] ): after two rounds of allele-specific PCR performed on pollen DNA, the fragment corresponding to the 14a1 hotspot region was cloned. 3000 clones were individually genotyped at three SNPs: two (#35 and #37) located on opposite sides of the center of the hotspot and one (#33) on the left border ( Figure 5 ).…”

“…At 130×, NCO molecules were characterized using a PCR-based “pollen-typing” strategy (see Materials and Methods ; Figure 1 ; [30] ). Allele-specific PCR was performed using either Col specific or L er specific primers on 96 samples, each containing 4,145 F1 pollen genomes or 4,800 F1 leaf genomes.…”

“…Allele-specific PCR was performed using either Col specific or L er specific primers on 96 samples, each containing 4,145 F1 pollen genomes or 4,800 F1 leaf genomes. Then, to specifically detect NCO molecules, allele-specific PCR was carried out at three different SNPs ( Figure 1 ; see Material and Methods; [30] ): the three polymorphic sites #21, #44 and #52 were 2339 and 2052 bp away, respectively (see green dots in Figure 3B ). SNP#44 is next to the center of the hotspot where the CO frequency is maximal, #21 is located in the left section of 130× where the CO rate is low and #52 is to the right where CO rates were average.…”

Sequencing of Single Pollen Nuclei Reveals Meiotic Recombination Events at Megabase Resolution and Circumvents Segregation Distortion Caused by Postmeiotic Processes

“…Future attempts to overcome these restrictions will require efficient methods to assay such effects. There are numerous methods to measure meiotic recombination in plants, including molecular markers (Salome et al, 2012 ), cytological visualization of crossovers (Sybenga, 1966 ; Anderson et al, 2003 ; Phillips et al, 2013 ), tetrad analysis (Copenhaver et al, 2000 ), fluorescent protein-tagged loci expressed in pollen (Yelina et al, 2013 ), and several pollen genotyping approaches (Drouaud and Mezard, 2011 ; Khademian et al, 2013 ; Dreissig et al, 2015 ). Although these methods have been successfully used to characterize recombination patterns and improve our understanding of meiosis, each of them has its specific advantages and disadvantages.…”