27 28 29 30 2Introductory paragraph: 31 Assessing the impact of variation in chloroplast and mitochondrial DNA (collectively termed the 32 plasmotype) on plant phenotypes is challenging due to the difficulty in separating their effect from 33 nuclear derived variation (the nucleotype). Haploid inducer lines can be used as efficient plasmotype 34 donors to generate new plasmotype-nucleotype combinations (cybrids) (Ravi et al., 2014). We generated 35 a panel comprising all possible cybrids of seven Arabidopsis thaliana accessions and extensively 36 phenotyped these lines for 1859 phenotypes under stable and fluctuating conditions. We show that 37 natural variation in the plasmotype results in additive as well as epistatic effects across all phenotypic 38 categories. Plasmotypes which induce more additive phenotypic changes also cause more significant 39 epistatic effects, suggesting a possible common basis for both additive and epistatic effects. On average 40 epistatic interactions explained twice as much of the variance in phenotypes as additive plasmotype 41 effects. The impact of plasmotypic variation was also more pronounced under fluctuating and stressful 42 environmental conditions. Thus, the phenotypic impact of variation in plasmotypes is the outcome of 43 multilevel Nucleotype X Plasmotype X Environment interactions and, as such, the plasmotype is likely 44 to serve as a reservoir of variation which is only exposed under certain conditions. The production of 45 cybrids using haploid inducers is a quick and precise method for assessing the phenotypic effects of 46 natural variation in organellar genomes. It will facilitate efficient screening of unique nucleotype-47 plasmotype combinations to both improve our understanding of natural variation in nucleotype 48 plasmotype interactions and identify favourable combinations to improve plant performance.
49Chloroplasts and mitochondria play essential roles in metabolism, cellular homeostasis and 50 environmental sensing (Petrillo et al., 2014; Chan et al., 2016). Their genomes contain only a limited set 51 of genes whose functioning requires tight coordination with the nucleus through signaling pathways that 52 modulate nuclear and organellar gene expression (Petrillo et al., 2014; Kleine and Leister, 2016). 53 Plasmotype variation can be strongly additive, such as in the case of chloroplast encoded herbicide 54 tolerance (Flood et al., 2016), or can manifest in complex cytonuclear interactions as non-additive, non-55 linear effects (epistasis), such as found for secondary metabolites (Joseph et al., 2013). The phenotypic 56 consequences of epistasis can be detected when a plasmotype causes phenotypic effects in 57 combination with some, but not all nuclear backgrounds. Recent studies suggest that cytonuclear 58 epistasis is the main route through which variation in the plasmotype is expressed (Zeyl et al., 2005; 59 3 Montooth et al. , 2010; Joseph et al., 2013; Joseph et al., 2013; Tang et al., 2014; Roux et al., 2016; 60 Mossman et al., 2019) an...