Mapping, molecular cloning and functional characterization of an autosome-linked locus that leads to 100% lethality of the hybrid embryos between two closely related nematodes

Consecutive loss of three or more pregnancies is referred to as recurrent miscarriage (RM), and it affects approximately 1% of couples. Approximately half of RM cases cannot be explained by any existing knowledge, suggesting the presence of some cryptic genetic changes in either or both of the parent genomes, which make them genetically incompatible, a phenomenon called hybrid incompatibility (HI). HI generally refers to the compromised fitness of hybrid progeny, including reduced viability, lethality and sterility. It can be found frequently between two closely related species and less frequently between two populations of the same species. HI is vital for maintaining the relative genetic stability of a species, by limiting genetic exchanges with other species. The identification of HI loci and the underlying genes is crucial for understanding not only the genetic mechanism for maintaining biodiversity but also the mechanism underlying RM.

The identification of HI genes is particularly challenging because these genes usually are not under selection pressure, leading to their little or no conservation between species. This makes conventional mapping techniques unsuitable for mapping and cloning of these genes. Although HI genes are more frequently seen between closely related species than between different populations of the same species, the mapping of HI loci is more challenging between different species than between populations. This is because homologous chromosomes between different species are often resistant to recombination due to their highly divergent sequences and chromosomal rearrangements. We previously mapped more than 30 HI loci between two nematode species, Caenorhabditis briggsae and C. nigoni. In particular, we found a single locus tightly linked to chromosome IV that led to the complete death of their hybrid F1 embryos. However, we encountered major difficulties in identifying the underlying genes, because most regions of the C. briggsae genome cannot recombine with their syntenic regions in the C. nigoni genome, presumably due to the high degree of sequence divergence. Fortunately, recent attempts at forced recombination have been successful, which is achieved through the artificial introduction of double-stranded DNA breaks within the targeted region using the CRISPR/Cas9 system. Adoption of this method allowed us to reduce the chromosome IV-linked interval from approximately 6 Mbp to approximately 1 Mbp. In this study, we propose to further map the interval to about 50 kbp and establish the molecular identity of the HI locus that produces complete embryonic lethality. The identification of such a gene/element will not only provide mechanistic insights into how a cryptic genetic change leads to 100% embryonic lethality in the hybrid embryos, but also will shed light on the genetic mechanism underlying RM.