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

Our research focuses on the regulation of meiotic recombination in Caenorhabditis elegans. The question we are trying to answer is how the frequency and distribution of crossover are regulated at the molecular level during meiosis. SNP genotyping is one with the primary methods for measuring crossover frequency in C.elegans.

It is commonly referred to as snip-SNP, including PCR, restriction digestion, and gel electrophoresis. This kind of multi-step approach to SNP genotyping is labor-intensive and sometimes inaccurate. Therefore, our current experimental challenge is to modify it into simple and accurate method.

Our method is a qPCR-based approach for detecting multi-crossover combination. It can be widely applied in genetic mutants, different sexes, and various growth constraints as well. To begin, obtain the required hermaphrodites for the procedure.

Place three L4 stage hermaphrodites from the Bristol N2 strain or from mutants on a Bristol background, along with nine young adult Hawaiian CB4856 males, onto a mating plate. Incubate the mating plate for 1.5 days at 20 degrees Celsius to allow mating between the two strains. After incubation, transfer each hermaphrodite to a new individual nematode growth medium plate seeded with OP50 bacteria.

For the second cross, pick six L4 stage crossed hermaphrodites that are Bristol-Hawaiian hybrids and transfer them to a new mating plate along with 12 to 15 TTS24 Bristol males carrying the ccIs4251 transgene array. Incubate the plate for 1.5 days at 20 degrees Celsius to enable mating. After incubation, transfer each hermaphrodite to an individual nematode growth medium plate containing OP50.

Incubate the new plate at 20 degrees Celsius for one day to allow the hermaphrodites to lay fertilized eggs. After one day, transfer each hermaphrodite again to a new individual plate with OP50. Check the genotypes of the parental worms as Bristol or Hawaiian hybrids using PCR in a region of chromosome five.

Separate the amplified bands by performing electrophoresis on a 2.5%agarose gel. After checking GFP expression, pick a single GFP-positive worm and place it in a PCR tube preloaded with three microliters of worm lysis buffer. Then, place the tube in a minus 80 degrees Celsius freezer four or five minutes before lysis.

For lysis, set the PCR tube in the thermal cycler and run the program at 60 degrees Celsius for one hour, followed by 95 degrees Celsius for 15 minutes. After the thermal cycling, add 100 microliters of nuclease-free water to each tube and mix thoroughly. Now, prepare the mixture containing the buffer, probes, and the primers.

Mix the solution thoroughly by pipetting several times. Using a 12-channel micropipette, dispense four microliters of the mixture into each well of a 96-well PCR plate placed on the support base. Next, add one microliter of lysate containing genomic DNA to each well and pipette the content several times to mix thoroughly.

Seal the plate with an optical adhesive film. Use an applicator to press the film down firmly, especially around the rims of each well. Centrifuge the sealed plate briefly to collect the liquid at the bottom and eliminate any air bubbles.

Now, load the plate onto the realtime PCR instrument. On the software screen, click on the create new experiment button. After adjusting the settings, click on the run button to initiate the protocol.

Review the allelic discrimination plot generated from the data to visualize genotype groupings. Identify the genotypes based on their position for Bristol homozygotes, Bristol-Hawaiian hybrids, Hawaiian homozygotes, and negative controls. Note the applied color codes of red for Bristol, blue for Hawaiian, green for heterozygous, and black for negative control.

Finally, examine the SNP patterns to identify the crossovers. For instance, Bristol-Hawaiian heterozygote, Bristol homozygote, Bristol homozygote, Bristol homozygote indicates a single crossover between positions one and two. Chromosome two was divided into three regions, the left arm, center, and right arm, using four selected single nucleotide polymorphism markers.

The crossover frequency in the left arm of chromosome two was significantly higher at 23.90 centimorgans compared to the center region at 5.80 centimorgans. The crossover frequency in the right arm was also significantly higher at 19.80 centimorgans compared to the center region. No significant difference was observed between the crossover frequencies of the left and right arms.