We sequenced the Steinernema hermaphroditum genome.

We analyzed S. hermaphroditum chromosomes. We assembled long, short, and Hi-C reads into five chromosomes of 16.7 to 18.4 Mb, totalling 87.9 Mb; we used conserved synteny (Nigon groups, shown here) to identify four autosomes and one X chromosome; and we predicted 19,628 genes encoding 43,884 proteins with a BUSCO completeness of 89.0%.

We developed molecular genetics. We demonstrated classical mutagenesis and genetics to define loci by mapping and complementation. We used the genome sequence to find the gene mutated in multiple alleles of a locus controlling Steinernema body size. We developed CRISPR-Cas9 gene knockout and used it to confirm this identification.

We have three high quality X. griffiniae bacterial genomes. Our comparative sequence analyses are yielding high resolution information on X. griffiniae relatedness and genomic variation. The X. griffiniae symbiont of S. hermaphroditum is genetically tractable; X. griffiniae can accept exogenous DNA by conjugation and it can be genetically modified by homologous recombination and transposon-based methods, used to create X. griffiniae:

• strains with stable in-genome, neutral site insertions of genes encoding fluorescent proteins. Fluorescent X. griffiniae associated with S. hermaphroditum can be visualized by microscopy throughout the lifecycle.
• strains with allelic exchange deletion mutations in three genes predicted to encode colonization and/or virulence factors. We currently are complementing these mutants, and testing their host association phenotypes.
• RbTnseq mutant library. Quality control testing of this library revealed that insertions were non-random and enriched around the origin of replication. Modified protocols are being developed to increase randomness of insertion and chromosome coverage. However, the library has yielded X. griffiniae mutants defective in essential metabolic biosynthetic pathways.