3.3 GB-RFLP marker analysis for the assignment of the great lake species A. citrinellus and A. labiatus
Lastly, we designed markers for the two great lake species. As genetic differentiation between these species is very low (0.015–0.02) compared to the crater lake or allopatric species pairs (0.07–0.5), we used SNPs that we have identified based on genome-wide association mapping for the species–defining trait: lip size (A. labiatus has thick, hypertrophic lips that A. citrinellus lacks) (Kautt et al., 2020; Machado-Schiaffino et al., 2017). As the phenotype is largely driven by a single locus, the variants were on the same chromosome (distance: 113kb). For the two species-specific markers that we designed, we predicted an accuracy of 92 and 99%, respectively. When tested, the GB-RFLP markers obtained similar accuracies of 87 and 81%, respectively (Fig. S4a, b and Fig. 3d–f). Interestingly, even though both loci are in proximity on the same chromosome, combination of both markers lead to an accuracy of 100%.