SNIP-SNP Analysis of Recombination Patterns in C. elegans: The Role of PCH-2 and Sexual Dimorphism in Meiotic Recombination

Kealani Holland and Needhi Bhalla

Molecular, Cell, and Developmental Biology

University of California Santa Cruz

Introduction

Figure 1. Meiosis schematic. Cartoon depiction of the two rounds of cell division in meiosis, resulting in the generation of haploid gametes.

Meiosis

Meiosis is a fundamental process of cell division that ensures the production of haploid gametes with the correct number of chromosomes. Meiosis halves the chromosome number in the resulting gametes, ensuring that when a sperm and egg fuse during fertilization, the resulting zygote has the correct diploid number of chromosomes. Errors in this process can result in aneuploidy, leading to infertility, miscarriages, or cancer.

Background

Figure 2. Crossover formation. Crossover event between parental homologous chromosomes that depicts possible gamete types.

What are Crossovers (CO)?

Meiotic crossover recombination (CO) is the reciprocal exchange of genetic material between homologous chromosomes. COs are essential for ensuring accurate chromosome segregation, generating new haplotypes, and avoiding aneuploidy. The number and distribution of COs is a tightly regulated process to avoid aneuploidy and other consequences.

C. elegans gametogenesis

Gametogenesis is the production of gametes like egg and sperm. In C. elegans, spermatogenesis (the production of sperm) is significantly faster than oogenesis (the production of eggs). We are specifically interested in this sexual dimorphism regarding timing. As mentioned, COs are tightly regulated, and the timing of COs and meiosis can have significant effects on meiotic fidelity.

Figure 3. Immunofluorescence images of C. elegans hermaphrodite and male germlines stained for PCH-2. Top figure shows hermaphrodite, bottom shows male. DNA is in magenta and PCH-2 is in green.

Figure 4. PCH-2 distributes its regulation of meiotic prophase events by remodeling different meiotic HORMADs. Cartoon depicting proposed model for PCH-2’s regulation of meiotic prophase events. PCH-2 regulates the progression of pairing and by acting through the HORMADS HTP-3 and HIM-3.

Why PCH-2?

PCH-2, a highly conserved AAA+-ATPase, coordinates homolog pairing, synapsis, and recombination and is necessary for regulating both the number and distribution of COs. PCH-2 achieves this by antagonizing CO formation. PCH-2 and its orthologs structurally remodel a family of proteins with HORMA domains (HORMADs) to control their function. During meiosis, specific forms of meiotic HORMADs are essential for pairing, synapsis, and recombination between homologous chromosomes.

Previously in the Bhalla Lab

Previous research has identified PCH-2’s essential role in proper CO distribution in C. elegans oogenesis. The proposed mechanism is that PCH-2 restrains meiotic prophase to coordinate its events, reach specific CO conditions, and ensure fidelity. Mutants with a pch-2 knockout show defects in CO regulation and meiotic checkpoints, with varied consequences depending on the meiotic stage. Mutants experience a shift of CO towards the pairing center of the chromosome and away from the gene-rich centers. PCH-2’s function was thus essential in ensuring a wider distribution of CO across chromosomes.

Figure 5. PCH-2 controls the number and distribution of crossovers similarly on multiple chromosomes. Genetic analysis of meiotic recombination in wildtype and pch-2 mutants. DCO indicates double crossovers. Physical and genetic maps of Chromosome I, III, IV and the X chromosome. Genetic distance is shown in centimorgans (cM).

Hypothesis

My project focuses on spermatogenesis and expands on previous work in the Bhalla Lab. It challenges the assumption that meiotic regulators like PCH-2 function identically across sexes. My hypothesis is that the faster timing of spermatogenesis will result in a different recombination pattern compared to oogenesis and that pch-2 mutants will disrupt that recombination distribution.

Methods

Figure 6. Simplified strain construction for chromosome 4. Cartoon of chromosome 4 during various crosses. Parental (P) and recombinant (R) genotypes simplified.

Strain Construction

I am studying spermatogenesis and want to track recombination in males. We mated Bristol (N2) and Hawaiian (HI) worms together, generating the F1 progeny. All of these worms should be heterozygous, and from these, we selected males. These hybrid males were then mated with HI hermaphrodites, resulting in our F2 progeny. Now, we know that the progeny will always have a full HI chromosome from the hermaphrodite, and we can track recombination from the male in the other chromosome.

SNIP-SNP analysis

Single Nucleotide Polymorphism (SNP)
- Single nucleotide changes in the DNA sequence
- We use two well-characterized strains with SNPs throughout their genomes: Bristol (N2) and Hawaiian (HI)
SNIP-SNP
- SNPs at specific sites recognized by restriction enzymes
- SNIP-SNPs produce different length DNA fragments after digestion
- We are “cutting SNPs”

Figure 7. A single nucleotide polymorphism (SNP) between the N2 and HI genomes affects DraI's ability to digest DNA. PCR amplicons for N2 and HI genomes are given. A SNP in the HI genome (T to G) prevents recognition of the cut site by the restriction enzyme DraI (highlighted region). Band patterns following gel resolution will reflect this difference.

Figure 8. Workflow of SNIP-SNP Analysis. Schematic illustrates the SNIP-SNP genotyping process.

SNIP-SNP analysis workflow

Lysates: the DNA from one worm in the F2 generation
PCR: amplify target sequences surrounding each SNP
Restriction Digestion: digest PCR products with enzymes specific to each SNP to identify allelic variants in the F2 generation
Gel Electrophoresis: visualize DNA fragments and interpret recombination
Data Interpretation: analyze band patterns following restriction digestions

Example gel

Following restriction digestion, samples were analyzed by agarose gel electrophoresis. Banding patterns corresponding to N2 and HI SNPs were identified based on known fragment sizes. All worms have HI SNPs due to strain construction, so only the presence of N2 SNPs was recorded for each worm. Recombination events were scored by identifying points of inflection, locations where the pattern shifted from N2 to HI or vice versa, across SNP intervals. Recombination frequency was calculated as the number of crossover (CO) events in a given interval divided by the total number of animals analyzed.

Figure 9. SNP 4E HI/R; bcIs39/pch-2 gel electrophoresis. Agarose gel electrophoresis run following PCR and restriction enzyme digestion. N2 and HI band fragment sizes are given.

Results and Future Directions

Preliminary results

Chromosome 1: wildtype
- There is a difference in recombination frequencies between oogenesis and spermatogenesis
- Seeing more COs at the ends of chromosomes and less in the middle in spermatogenesis
Chromosome 1: pch-2 mutants
- In contrast to oogenesis (26.9 cM), we don’t see as many COs near the pairing center end (8.7cM) in spermatogenesis
- There is a difference in the number of DCOs in oogenesis (0 DCOs) and spermatogenesis (3 DCOs)

Figure 10. Preliminary results of spermatogenesis recombination landscape. (A) Physical and genetic map of Chromosome I is depicted to scale. (B) Genetic analysis of meiotic recombination in wildtype animals during oogenesis and spermatogenesis. (C) Genetic analysis of meiotic recombination in pch-2 mutants during spermatogenesis. DCO indicates double crossovers. Genetic distance is shown in centimorgans (cM).

Future Directions

Complete chromosome 4 in both backgrounds
Complete chromosome 3 in both backgrounds
Generate and test additional strains

Page updated

Report abuse