4.1 Non-random association and interactions between drivers of clock (DOC) loci and chloroplastic RpoC1
Since its establishment, the Barley1K infrastructure has been an instrumental research tool to explore landscape genomics (Hubneret al. , 2009; Hübner et al. , 2013; Chang et al. , 2022), plant biotic and abiotic interactions (Sade et al ., 2012; Dakhiya et al. 2017; Alegria Terrazas et al. 2020; Dakhiya & Green 2023), and more recently as reference for the whole barley Pan genome (Jayakodi et al. 2020). Here, we extended the use of the B1K by 1) developing a more dedicated genotyping platform for the wild barley to allow GWAS and 2) start exploring the chloroplast variation and its possible relationship with fitness traits. At first glance, the results obtained in this study could not directly be translated to crop improvement mainly since adaptation strategies in the wild and agricultural set up might be different. However, the B1K resource bear genetic attributes that could assist in targeting specific genes and gene complexes mainly owing to the non-randomness and long years of recombination within and between nuclear and chloroplastic genomes, and the way natural selection works on these intra and inter-loci combinations. For the nuclear genome, it is clear that the linkage disequilibrium decay is enormously shorter than that one could find in interspecific populations (Morrell et al. 2005). DOC3.2may be a good example for this “gene discovery” use of the B1K panel for zooming-in on a causal diversity that otherwise would be delimited to 7.7 Mbp in using the interspecific population (Prusty et al.,2021). Here, a closer look on at the allelic diversity indicates the QTL for amplitude under HT, which was identified in the previous study, most probably corresponds to the signal identified at chr3H_67267835. This marker is flanked by the non-significant markers chr3H_66,838,086 and chr3H_68,838,244 (Tables S5 and S6), therefore limiting the source of phenotypic variation to approximately 2 Mbp for this QTL.
For the chloroplast genome per se, and for the allelic combinations with the nuclear genome, this study clearly shows the rich diversity repertoire found in this organelle within the wild (Table S7 ) and its relevance to plant adaptation. Moreover, we could identify both linkage disequilibrium and CNI between DOC and the RpoC1alleles in the B1K panel, where in an extreme case of the DOC3.2this locus showed segregation only among carriers of theRpoC1G1713T (Figure 4c ). Non-random association of alleles in the nucleus and cytoplasmic organelles, or cyto-nuclear linkage disequilibrium, is both an important component of a number of evolutionary processes and a statistical indicator of others (Fields et al., 2014). We also followed up on the GWAS to examine inheritance of these interactions, and, beyond validating the environment-specific effects of the DOCs , we could also get indication for preferred homogeneity of the CNI. For example, in the case of CNI for the DOC5.1 combination of plasmotype and nuclear alleles from same origin (B1K-05-07 or B1K-50-04, the two parental lines of the F2) was associated with higher amplitude. In case of cereal evolution, it was hypothesized and tested in silico that in ancient hybridization, such as the one between the A and D-genomes of modern wheat there has been biased maintenance of maternal A-genome ancestry in nuclear genes encoding cytonuclear enzyme complexes (CECs) (Li et al., 2019). It is tempting to hypothesize that we observe such scenario in the case of the DOC and RpoC1 loci in the B1K. However, one way of testing the possible cause behind these linkage disequilibrium and CNI will require cloning of the underlying genes in the DOC loci and figuring out how they may interact withRpoC1 gene product and what makes one allele different than other. Another way to test the implications of these CNI for breeding would require importing these combinations into a rather cultivated genetic background. This approach is now followed in a new interspecific cytonuclear multi-parent population (CMPP) that we are currently testing for clock and for agricultural performance (manuscript in preparation).