Identification of genetic basis of Hybrid Incompatibility (HI) in hybrids between closely related species leads to a comprehensive understanding of speciation. Although model organism C. elegans is well-established in laboratory, it has performed little contribution to this research area, because C. elegans failed to mate with other sister species and produce viable progeny. As a sister species of C. briggsae, which is close to C. elegans, newly discovered C. nigoni made it possible to identify the genetic basis of HI in nematode species. In this study, a new species pair including C. nigoni and C. briggsae was used to study the genetic and molecular bases of HI. 96 GFP markers were randomly integrated into the genome of C. briggsae by biolistic bombardment. Next-Generation Sequencing (NGS) combined with single worm PCR were performed to identify the location of GFP markers. By tracking those markers, the genomic fragments of C. briggsae linked to GFP were backcrossed into C. nigoni. Such process was repeated for at least 15 generations and total 111 strains carrying independent introgressions were generated. Widespread HI loci were identified on a genome-wide scale for the first time in nematode species, which also supported Haldane's Rule and large X-effect theory between the two species. In this study, C. nigoni genome "cn1" was de novo assembled by using a hybrid approach, which combined Illumina synthetic long-read technology and massive parallel sequencing of Fosmid mate-pair library. Two lines of hybrid sterile males each carrying an independent introgression fragment from C. briggsae X chromosome in an otherwise C. nigoni background, demonstrate similar defects in spermatogenesis. A similar pattern of downregulated genes that are specific for spermatogenesis between the two hybrids and wild type control was observed. Importantly, the downregulated genes caused by the X chromosome introgressions are significantly enriched on autosomes, suggesting an epistatic interaction between the X chromosome and autosomes. By measuring small RNAs, the results shows that a subset of 22G RNAs specifically targeting the downregulated spermatogenesis genes are significantly upregulated in hybrids, indicating that perturbation of small RNA-mediated regulation may contribute to the X-autosome interaction.

The human opportunistic pathogen, Pseudomonas aeruginosa PAO1, has been shown to kill the nematode Caenorhabditis elegans. C. elegans has been a valuable model for the study of bacterial pathogenesis, and has reinforced the notion that common virulence and host defense mechanisms exist. Recently, the pyrimidine pathway was shown to regulate virulence levels. Therefore, mutations in the pyrimidine pathway of PAO1 showed decrease virulence in the nematode. When starving the nematode, bacterial resistance was also shown to increase. It was hypothesized that starvation induced the DAF pathway, which regulates the transcription of genes involved with the antibacterial defense mechanism. Further research will be conducted to test this theory by performing RNAi experiments for the genes functioning in the antibacterial defense mechanism.

Hybrid incompatibility (HI) is frequently manifested as lethality or sterility in hybrid progeny between related species, and plays a key role in speciation. The genetic basis of HI has been intensively studied in model organisms such as yeast and fruit fly over decades, and "Two rules of speciation" have been observed across species. C. elegans as a nematode model organism contributes little to speciation research mainly due to lack of a close relative with which it can mate and produce viable progeny. Such limitation has recently been alleviated by identification of C. nigoni, a close relative, also termed as sister species, of C. briggsae. The two can make and produce a handful of viable hybrids. Both species are members of Elegans supergroup. Hybrid cross between the two species uncovered asymmetric hybrid incompatibilities, i.e. crossing direction-dependent hybrid male sterility and inviability. Asymmetry was also observed in F1 hybrids from reciprocal crosses exclusively in male but not female (Woodruff, Eke, Baird, Félix, & Haag, 2010). Asymmetry was also observed in backcrosses between the F1 female hybrids and the parental species. For example, F2 progeny fathered by C. briggsae suffered almost 100% embryonic lethality for both males and females, whereas those fathered by C. nigoni were partially viable and fertile. Further study of HI between these two species was initiated by investigating how C. briggsae chromosomal fragments in an otherwise pure C. nigoni genome affect fitness of hybrid worms. The hybrid worms were generated by repeatedly backcrossing C. briggsae genomic fragments each bearing a visible chromosomal-integrated marker to C. nigoni to produce introgression lines. Characterization of the introgression lines provided a detailed HI landscape of between the two species. Multiple intervals on the C. briggsae X chromosome were responsible for hybrid male inviability or sterility while most of the C. briggsae autosomes were not involved in these male phenotypes (Bi et al., 2015). RNA sequencing was performed in sterile male worms bearing independent introgressions, revealing a down-regulated gene expression pattern (Li et al., 2016). To uncover the HI mechanism underlying the asymmetric HI phenotypes exhibited in hybrids in F1 generation, I performed a genome-wide screening to identify HI loci that are responsible for the hybrid male inviability and sterility in F1 as well as hybrid breakdown in F2. By crossing between C. briggsae and C. nigoni introgression lines bearing a known C. briggsae fragment, I was able to construct hybrid animals homozygous or heterozygous for C. briggsae alleles on the introgression while those on counterpart of C. nigoni were absent. Contrasting the HI phenotypes here and those between two wild-type parents allows mapping of the loci responsible for the hybrid asymmetric phenotypes. The aggregated introgressions cover 94.6% of the C. briggsae genome, including 100% of the X chromosome. Surprisingly, I identified another two C. briggsae genomic intervals on chromosomes II and IV that can rescue the hybrid male inviability but not the male sterility in F1 fathered by C. nigoni, suggesting the involvement of differential epistatic interactions in the asymmetric hybrid male fertility and inviability. What's more, I observed that two independent C. briggsae X fragments that produce male sterility in C. nigoni as an introgression rescued hybrid male sterility in F1 fathered by C. briggsae. Backcrossing of the rescued sterile F1 male to its parental species showed that they can alleviate the F2 hybrid breakdown by a handful of viable F2 mothered by C. briggsae. Subsequent backcrossing of the rescued sterile males with C. nigoni led to the isolation of a 1.1-Mb genomic interval that specifically interacts with an X-linked introgression, which is essential for hybrid male fertility. In addition, I further identified three C. briggsae genomic intervals on chromosome I, II, and IV that produced inviability in all F1 progeny, dependent on or independent of the parent-of-origin. Taken together, I identified multiple independent interacting loci that are responsible for asymmetric HI phenotypes especially hybrid male sterility and inviability, which lays a foundation for their molecular characterization.

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