Arabidopsis crossover hotspots and pollen-typing

We have greatly improved conventional pollen-typing for crossover measurements. New pollen-typing methods are simple and highly reproducible, that generate the finest -scale measurements of the landscape of CO distribution from 100 to 1,000 crossover molecules within single crossover hotspots. This approach allows to check if a mutant displays for changes to different CO rate and distribution at known hotspots, such as 3a and 3b (Choi et al, 2013). This assay requires two rounds of allele-specific PCR and needs two different Arabidopsis accessions, such as Col-0 and Ler-0. Thus CRISPR/Cas9 and RNAi will be applied to trans-acting genes in the required heterozygote stated by using a meiotic specific promoter. These CRISPR and RNAi approaches will also be introduced to different fluorescent reporter lines to determine if the mutation of candidate genes can lead to recombination changes. To study the relationship between hotspots on a chromosome, we will identify additional crossover hotspots along chromosomes, based on meiotic DSB maps and polymorphism information.

Diagram illustrating pollen typing methods for crossover hotspot analysis. (A) Purification of pollen grains. (B) Genomic DNA extraction from pollen. (C) 1st round of allele- specific PCR using oligonucleotides specific for Ler (red) or Col (blue) templates. ASO configurations for crossover or parental molecule amplifications are indicated. Polymorphic sites are represented by colored circles. (E) 2nd allele-specific PCR as for (C), but using a nested set of ASOs for (E) Crossover and parental molecules are quantified using titration (Subheading 3.6). (F) Single crossover molecule amplification products are Sanger sequenced to identify internal recombination sites. (G) Quantitative PCR analysis of cross- over frequency, following the first round of allele-specific PCR amplification. (H) Mass amplification and high-throughput sequencing of crossover molecules. (I) Identification of crossovers using paired-end sequencing reads