The mitochondrial and nuclear genomes contain genes encoding the protein subunits comprising mitochondrial electron transport chain complexes IV (Blanchard and Lynch 2000). This dynamic has established a co-evolutionary relationship between the two genomes: when a deleterious mutation occurs in one genome, a compensatory mutation in the other genome might restore efficient oxidative phosphorylation (Blier et al. 2001). The epistatic interactions between different co-evolved mito-nuclear alleles can then be disrupted when mating between two genetically diverse populations occurs (Rand et al. 2004). Such hybridization introduces non-compensatory nuclear alleles that can again disrupt efficient electron transport (Burton et al. 2013). The nematode Caenorhabditis briggsae exhibits such evidence of mito-nuclear epistasis, where experimental recombination of the nuclear genome from the wild isolate AF16 with the mitochondrial genome of wild isolate HK104 (and vice versa) resulted in hybrid dysfunction (Chang et al. 2016). Further, AF16-HK104 advanced-intercross recombinant inbred lines (AI-RIL) show strong epistatic interactions between the mitochondrial and nuclear genomes, particularly with the X chromosome (Haddad et al. 2018). AF16 and HK104 are representatives of the genetically divergent tropical and temperate clades of C. briggsae (Cutter et al. 2006). Because of this phylogeographic population structure and knowing that mitochondrial genetic variation can play important roles in environmental adaptation (Das 2006, Camus et al. 2017, Lamb et al. 2018), we tested the hypothesis that mitochondrial-nuclear epistatic interactions in C. briggsae are temperature-dependent. We hybridized AF16 with a different temperate population, HK105. We created ten (10) replicate AF16-HK105 AI-RIL for both (2) cross directions at both (2) 20C and 25C. We then genotyped these forty (10x2x2) RIL at a single locus on the X chromosome to measure segregation distortion and its dependence on cross direction (and thus on cytotype and mitotype).Samples of the wild isolate C. briggsae populations AF16 and HK105 were obtained from the Caenorhabditis Genetics Center. Nematodes were cultured on NGM plates with Escherichia coli strain OP50 as a food source according to (Steirnagle 2006). AF16-HK105 advanced-intercross recombinant inbred lines (AI-RIL) were produced as previously described (Ross et al. 2011) by crossing males from one population with a self sperm-depleted hermaphrodite from the other population to generate the F1 generation. Seven subsequent generations of sibling-mating and then ten generations of self-fertilization by single hermaphrodites were used to produce each AI-RIL. Ten biological replicate lines were established at the F1 generation for reciprocal crosses AF16xHK105 and HK105xAF16 (male population listed first). This cross design was performed both at 20C and 25C to produce lines FV225-234 (AF16xHK105 at 20C), FV235-244 (AF16xHK105 at 25C), FV245-254 (HK105xAF16 at 20C) and FV255-264 (HK105xAF16 at 25C). Genomic DNA was extracted from each line by phenol-chloroform extraction and isopropanol precipitation in preparation for PCR genotyping. Genomic DNA was diluted to 5 ng/L in 10mM TRIS buffer pH 8.0. Amplification and agarose gel electrophoresis was performed according to (Chang et al. 2016). The cb-
m127 insertion-deletion (indel)-amplifying primer sequences were obtained from (Koboldt et al. 2010), and the primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA). The cb-
m127 indel is X-linked (Haddad et al. 2018).