An integrated approach to understanding spontaneous mutation and natural selection in the Chlamydomonas genome

Evolution by natural selection is fueled by genetic variation among individuals that ultimately originates from new mutations. Thus, the rate of appearance of new spontaneous mutations, whether they are beneficial or harmful and the magnitudes of their effects have profound consequences for the mode and speed of evolution. For example, if the rate of appearance of new beneficial mutations is very low, adaptation will frequently be limited by the supply of these mutations, whereas if the rate of beneficial mutations is high, adaptation will be limited by other processes, such as the speed with which natural selection can alter gene frequencies. Unfortunately, estimating mutational parameters has proved difficult, and, in spite of the importance of new mutations for evolution, there are few reliable estimates. We shall combine two approaches that aim to provide a fuller picture of the mutational process in our model system, the single-celled alga Chlamydomonas reinhardtii. Part of the difficulty in examining the supply of mutations is that natural selection operates in natural populations and removes deleterious mutations and increases the frequency of beneficial ones. Thus, the genetic differences that we observe within populations and between species are filtered and biased subsets of those mutations that arose in the first place. Our first approach will be to maintain replicate lines of C. reinhardtii for several hundred generations in conditions in which the effects of natural selection are minimised. This will allow us to examine the effects of the full spectrum of new mutations, not just those that pass through the filter of selection. We will then use state-of-the-art genome sequencing technologies to examine the complete genome sequences of a subset of these lines, which will allow us to measure directly the number and kinds of mutational changes at the DNA level. Taken together this information will provide us with a detailed picture of the fuel available to natural selection. Our second approach will also harness the power of whole genome sequencing to obtain a detailed picture of variation in the genome of C. reinhardtii sampled from a natural population. By comparing the amount and distribution of variation in different parts of the genome, we will be able to quantify how selection in natural conditions has acted on the mutations that originally generated the variation we observe. By comparing the genetic differences between different regions of the genome we will also be able to dissect the functional organisation of the genome, and determine which regions are constrained and so have remained relatively unchanged and which areas have more freedom to vary. We also plan to obtain the genome sequence of the closest relative of C. reinhardtii, C. incerta. By comparing the sequences of the C. reinhardtii and C. incerta genomes we shall be able to infer the principal adaptive changes in the genome between the two species. Answers to these questions will provide important insights in many areas of evolutionary biology, from helping us to understand why most eukaryotic species reproduce sexually to allowing us to predict whether the future responses of populations to environmental change is likely to be based on existing or new genetic variation.

A lack of knowledge of the nature of variation from new mutations limits progress in population, quantitative and evolutionary genetics. We propose to use two integrated approaches that will provide detailed information on the rates, molecular properties and fitness effects of new mutations in our model system, the microalga, Chlamydomonas reinhardtii. In our first approach, we will carry out long-term mutation accumulation (MA) experiments to quantify the rate, distribution of effects, and interactions between new mutations affecting fitness traits. By sequencing the genomes of a sample of MA lines, we shall obtain detailed information on the molecular properties of new mutations and estimate the mutation rate per nucleotide site in the nuclear, chloroplast and mitochondrial genomes. We will integrate these findings with our second approach in which we will infer the distribution of fitness effects of new mutations by analysing polymorphism data obtained by sequencing the genomes of a sample of natural isolates of C. reinhardtii. We will use these data to infer the fraction of selectively constrained sites in the genome. Combined with our estimate of the mutation rate per nucleotide site from our MA lines, this will allow us to estimate the genomic deleterious mutation rate, U, which is a crucial parameter in many evolutionary models. We will also combine our estimate of the mutation rate with the level of nucleotide diversity in C. reinhardtii to infer the recent effective population size, a parameter that determines the effectiveness of natural selection. We propose to obtain the genome sequence of the closest known relative of C. reinhardtii (C. incerta), and use it to leverage information on the prevalence of adaptive evolution in the genome in a joint analysing with our genome-wide polymorphism data. Our findings will provide new and detailed information on the processes of mutation and natural selection in the genome of a model plant species.

Whilst the research we propose is pure rather than applied, we envisage several areas where it will have impact outside the academic community. The work we propose is of direct relevance to questions of interest to the general public (for example, extinction and adaptation in the face of global change, emergence of herbicide and pesticide resistance in weeds and crop pests), so we predict positive impact on both the public interest and the public understanding of science. We see the public understanding of science as critical in maintaining public support for scientists (and their funding) and a scientifically literate public. Engaging young minds with science is critical if the UK is to continue producing the top quality science graduates required to remain competitive in and reap the benefits of scientific discovery. We will maximize these potential impacts by engaging with the public directly through talks at science festivals, cafe scientifique events (http://www.cafescientifique.org/) and also through the media. Both the PI and Co-PI have a proven track record in presenting their science to the public directly and through the media. Systems based on our study organism are currently being developed in several industrial contexts, including the production of biofuels and drugs and also as potential bioremediation agents. Whilst less direct, the research we propose to carry out into the basic biology of the organism will potentially impact on any or all of these applied aspects of its biology. Ultimately this may lead to improved carbon efficiencies, lower cost drug production and reclamation of contaminated land, which will have UK-wide and global benefits, although realistically these potential benefits may take many years to be realised. By exploring the potential for collaborative projects, we plan to develop links in these areas so as to ensure that any potential impacts in these areas are realised. The genomics resources we produce, in the form of polymorphism data for C. reinhardtii and the genome sequence of C. incerta, may be of interest for such research. The wealth of genetic information that we plan to make available potentially represents a considerable resource for the biotechnology industry. Similarly, the software that we propose to develop or maintain as part of this research may also be of interest outside the academic community, and we will ensure that our programs are available via our websites. PDK has already demonstrated successful collaboration with industrial partners in his previous research and software developed in his previous projects are used outside of the academic community.