TY - JOUR T1 - Direct estimation of the spontaneous mutation rate by short-term mutation accumulation lines in <em>Chironomus riparius</em> JF - bioRxiv DO - 10.1101/086207 SP - 086207 AU - Ann-Marie Oppold AU - Markus Pfenninger Y1 - 2016/01/01 UR - http://biorxiv.org/content/early/2016/11/07/086207.abstract N2 - Mutations are the ultimate basis of evolution, yet their occurrence rate is known only for few species. We directly estimated the spontaneous mutation rate and the mutational spectrum in the non-biting midge C. riparius with a new approach. Individuals from ten mutation accumulation lines over five generations were deep genome sequenced to count de novo mutations (DNMs) that were not present in a pool of F1 individuals, representing parental genotypes. We identified 51 new single site mutations of which 25 were insertions or deletions and 26 single point mutations. We estimated a mutation rate of 4.2 × 10−9 (95% confidence interval: 2.8 × 10−9 – 6.1 × 10−9) which is in the range of recent estimates for other insects and supports the drift barrier hypothesis. We show that accurate mutation rate estimation from a high number of observed mutations is feasible with moderate effort even for non-model species.Being the ultimate source of genetic variation for evolution to act upon, mutation is certainly among the most important genetic processes. The per generation rate at which spontaneous mutations occur in the genome is the central parameter to estimate the effective population size on recent time scales1 or in the course of population history2, equilibrium of genomic base composition3 and divergence times4. Yet, the spontaneous mutation rate (µ) is so difficult to measure directly that it has been rarely estimated up to now. Consequently, only very few eukaryotic direct µ estimates are currently available5-11, scarcely representing biodiversity. More estimates, in particular of non-model species would be highly desirable because they would shed light on the evolution of µ, its associated ecological and evolutionary circumstances12 as for example the drift-barrier hypothesis13 and Lewontin’s paradox14.Currently, two approaches are applied to directly estimate µ: mutation-accumulation (MA) lines experiments and parent-offspring trios6,⇓,8,⇓,⇓. In the MA line approach, inbred lines are established and bred over many generations15. Due to the almost absent effect of selection, all except of the most deleterious mutations become eventually fixed and are thus readily identified and confirmed5. However, establishing inbred lines is not possible for all organisms, often require complex logistics to transfer generations, intensive care over long time spans and recessively deleterious mutations will be lost with their respective MA-line. In addition, mutator alleles may become fixed, altering the estimated mutation rate16. In the trio approach, parents together with their offspring are full genome sequenced17,18. This has the advantage that the observed mutational spectrum includes also recessively deleterious mutations as they appear heterozygously in the offspring. Limitations arise from the large number of offspring needed to be screened for an appreciable number of mutations and the requirement to know the parents6, which is difficult in some species.In our estimation of µ in the non-biting midge Chironomus riparius, we drew on the advantages of both approaches. We established ten MA-lines over five generations and deep sequenced the genomes of a single individual per MA-line. Because individual parenthood is difficult to determine in the swarm breeding C. riparius, we compared these individuals with the pooled full-sibling offspring from the single egg-clutch the MA-lines were established from. This yielded an appreciable number of mutations, allowing an accurate estimation of µ. ER -