Evolution of Gene Regulation through small RNA-mediated neofunctionalisation.

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The genetic makeup of every plant or animal comprises many thousands of genes, raising the question of where genes come from. A major source is the process of "neofunctionalization", whereby a gene becomes duplicated and one of the copies acquires a new function. This process is critical for the evolution of biological diversity and provides variation important for breeding or genetic engineering. Two types of neofunctionalisation have been traditionally described: changes in regions coding for proteins (C-NF), and changes in regions regulating where and when a gene is switched on (R-NF). Recent discoveries have raised the possibility of a third type, which we call small RNA-mediated neofunctionalisation (S-NF). S-NF involves mirror-image duplications of a gene which leads to the production of regulatory molecules, small RNAs, which inhibit another gene's activity. In this way a gene may adopt a new regulatory role. We have recently obtained evidence for S-NF contributing to the evolution of flower colour differences between species of snapdragon, Antirrhinum. In the longer term, some S-NF inverted duplications may become shorter and produce fewer small RNAs, giving rise to microRNA genes. The aim of this proposal is to test the idea that S-NF provides a natural mechanism of broad significance, complementing R-NF and C-NF, for creating new genes and refining gene activity during evolution.We will integrate several approaches to test this idea. First, we will use advances in DNA sequencing, allowing us to define genome sequence variation across a range of different plant species. Based on these findings, we will be able to map the origin, evolution and diversification of S-NF duplications and clarify their relation to microRNAs. Second, we will use genetic analysis, whereby individuals are crossed and progeny analysed, to separate the contribution of particular S-NF genes. Third, we will investigate how S-NF genes exert their effects by measuring their activity, and that of their targets, in different tissues and regions. Fourth, we will use phylogenetics, whereby evolutionary trees for genes and genomes may be constructed, to allow likely paths evolution has taken to be defined. Fifth, we will use population genomics, whereby the frequencies of genetic variants can be measured in natural populations, to infer which genes are likely under selection. Sixth, we will use transgenic approaches, introducing S-NF into species by genetic engineering, to investigate and evaluate how it can be used to influence and refine gene activity patterns. We will apply these approaches to snapdragon species and their relatives. S-NF has already been established in this system in relation to flower colour, which provides a convenient and sensitive way to detect variation in patterns of gene activity. Moreover, the ability to inter-cross snapdragon species that differ widely in colour or other traits, allows the genetic contribution of genes to be readily followed. Engineering and breeding flower colours is also a test bed for evaluating the contributions of S-NF. Taken together our studies should provide new insights into how new gene activities arise and the contribution of S-NF to evolution and breeding.