Dissecting Cold Tolerance in Drosophila ananassae: A Multi-Phenotypic and Bulk Segregant Analysis

As a major element of the environment, temperature impacts the geographical ranges of insects. Therefore, the worldwide distribution of insects follows adaptation to wide ranges of temperature. A population of Drosophila ananassae from the ancestral region showed variation in cold tolerance levels. The ancestral Bangkok population also differed from the derived Kathmandu population regarding cold tolerance. However, the phenotypic variation was only measured by chill coma assays, and no further characterization of the cold tolerance phenotype was performed. Here, we aimed to further characterize the iso-female lines of Bangkok and Kathmandu populations and the recombinant inbred lines generated from the ancestral population using lethal time and cold shock mortality in addition to chill coma recovery time. We showed that cold tolerance phenotypes differ between sexes, the additional phenotypes do not correlate significantly to chill coma recovery time, and some recombinant inbred lines have extreme phenotypes with higher tolerance than the tolerant founder or lower tolerance than the sensitive founder. We performed bulk segregant analysis using the recombinant inbred lines that exhibited extreme chill coma recovery time to identify genomic regions responsible for the phenotype. We identified 16 regions with significant association with the phenotype and showed that the genes in the putative regions were enriched in muscle development, metabolic processes, and cytoskeletal protein binding. Our results provide further evidence for the need for multiple cold tolerance phenotypes and shed light on the genetic architecture of adaptive phenotypic changes in natural populations of Drosophila.