Abstract
Cryptochrome (CRY) is a conserved protein associated with the circadian clock in a broad range of organisms, including plants, insects, and mammals. In Drosophila, cry is a pleiotropic gene that encodes a blue light-dedicated circadian photoreceptor, as well as an electromagnetic field sensor and a geotaxis behavior regulator. We have generated a panel of nearly-isogenic strains that originated from various wild populations and which carry different natural alleles of cry. Sequencing of these alleles revealed substantial polymorphism, the functional role of which was elusive. To link this natural molecular diversity to gene function, we relied on association mapping. Such analysis revealed two major haplogroups consisting of six linked nucleotides associated with circadian phase (haplotypes All1/All2). We also generated a maximum-likelihood gene-tree that uncovered an additional pair of haplogroups (B1/B2). Behavioral analysis of the different haplotypes indicated significant effect on circadian phase and period, as well on the amount of activity and sleep. The data also suggested substantial epistasis between the All and B haplogroups. Intriguingly, circadian photosensitivity, assessed by light-pulse experiments, did not differ between the genotypes. Using CRISPR-mediated transgenic flies, we verified the effect of B1/B2 polymorphism on circadian phase. The transgenic flies also exhibited substantially different levels of cry transcription. We, moreover, analyzed the geographical distribution of the B1/B2 haplotypes, focusing on a 12 bp insertion/deletion polymorphism that differentiates the two haplotypes. Analysis of cry sequences in wild populations across Europe revealed a geographical cline of B1/B2 indel frequency, which correlated with seasonal bioclimatic variables. This spatial distribution of cry polymorphism reinforces the functional importance of these haplotypes in the circadian system and local adaptation.
Highlights
The circadian clock system is one of the best examples of a molecular circuit that can generate an organized output without any external cues, yet which is amenable to environmental modulators, such as temperature and light
We further identified circadian phenotypes associated with the L232H single-nucleotide polymorphism (SNP), namely, those in which the phase of locomotor activity and adult eclosion were affected
We generated a panel of 33 nearisogenic lines (NIL) carrying cry alleles that were introgressed from various wild population strains. These lines were generated by backcrossing wild strains to deficiency line for eight generations and theoretically share 99.79% of their genomes, excluding the cry locus. We used these lines for fine association mapping of polymorphic sites in the gene with circadian clock properties
Summary
The circadian clock system is one of the best examples of a molecular circuit that can generate an organized output without any external cues, yet which is amenable to environmental modulators, such as temperature and light. The phenotype of JETLAG mutants, which are rhythmic in continuous light, is expressed only in strains carrying the ls-tim allele and not in those carrying the s-tim allele (Peschel et al, 2006) These discoveries demonstrate how understanding natural genetic variation in clock genes can help explain the evolution and molecular mechanisms that drive the clock. These lines were generated by backcrossing wild strains to deficiency line for eight generations and theoretically share 99.79% of their genomes, excluding the cry locus ( the possibility remains that residual genetic variability in other loci contributes to the phenotypes of interest) We used these lines for fine association mapping of polymorphic sites in the gene with circadian clock properties
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
