Frequency and Distribution of Crossovers in Caenorhabditis elegans Meiosis by SNP Genotyping using Real-time PCR

Caenorhabditis elegans is an excellent model organism for studying meiosis. In addition to general advantages, such as a short reproductive cycle, many progenies, and a transparent body for imaging, there are six pairs of homologous holocentric chromosomes. There is strong crossover interference and regulation of crossover distribution. To measure the crossover frequency, visible marker mapping, snip-SNP mapping, and quantification of cytological markers as precursors of crossover formation have been developed. Here, we introduce a modified SNP genotyping method to measure the crossover frequency and distribution in C. elegans oogenesis. This method can omit the laborious steps of restriction digestion and gel electrophoresis and avoid the ambiguous judgment of uncut DNA bands. Single-nucleotide polymorphism (SNP) genotyping is performed using the Bristol/Hawaiian hybrid strain. Crossing hermaphrodites with myo-3p::GFP-expressing males enables us to focus on oogenesis. A single worm lysate produces sufficient DNA template for all six chromosomes. The 5' exonuclease-based TaqMan chemistry and SNP-specific minor groove binder (MGB) probes allow the precise detection of SNP at a genotyping rate of approximately 97.7%. We utilize the accurate detection of SNPs to measure crossover frequency in C. elegans. This method can also be applied to determine the crossover frequency in male or crossover-defective mutants, as well as for more specific chromosomal intervals.