Evolution of Gene Regulation through sRNA-mediated Neofunctionalisation

Lead Research Organisation: University of East Anglia
Department Name: Biological Sciences


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.

Technical Summary

Through a combination of genomic, genetic, transcriptomic, phylogenetic, population and transgenic approaches, we will test the hypothesis that small RNA-mediated neofunctionalisation (S-NF) provides a natural mechanism for refining gene expression patterns during evolution. Through bioinformatics analysis of genomes of Antirrhinum species with diverse morphologies, we will identify candidate S-NF inverted duplications and analyse their function through genetic and expression analysis of segregating populations, and clarify their relationship to microRNAs. Through phylogenetic analysis we will determine when and how a particular case of S-NF, the SULF locus which restricts yellow flower colour in Antirrhinum, arose in relation to the duplication and diversification of its homologues, and whether it was a one-off event, specific to the Antirrhinum lineage or whether similar events have occurred in other clades. By analysing genetic and molecular interactions between SULF and promoter changes in its target, Am4'CGT (Antirrhinum majus chalcone 4'-O-glucosyltransferase) we will determine how S-NF combines with regulatory neofunctionalisation (R-NF) to control gene expression. The role of these interactions in a natural context will be evaluated by measuring selective sweeps in natural populations and cline steepness at a hybrid zone. Mechanisms of interaction and potential for engineering gene expression control will be evaluated by recreating the SULF system transgenically in Torenia. We will also analyse the role of S-NF and R-NF in the evolution of flower colour changes in other species (Linaria) to determine whether evolution follows the same or different routes. Taken together these approaches should provide deeper insights into how S-NF evolves and contributes to variation within and between species, providing potential for refining gene expression through evolution and breeding.

Planned Impact

This project will benefit non-academic beneficiaries, in the following ways:

1. Breeders will benefit from knowledge of how S-NF may contribute to genetic variation. Neofunctionalisation provides a major source of genetic variation exploited by breeders, and understanding the origins and properties of all its forms is critical for the long-term goal of predictive breeding. For example, candidate S-NF loci are involved in oil content of sunflower and olives, and microRNA loci (which likely evolve from S-NF loci) influence barley cleistogamy and wheat spike formation. By applying the knowledge, methods and pipelines developed in this project to crop genomes, it should be possible to identify S-NF candidates and evaluate the roles they play in control of expression patterns. This knowledge may then inform breeding programmes which target particular traits. The expected time frame for this beneficial impact will be 10 years after the start of the project.

2. Biotech industries will benefit from our work through improved ability to control gene expression patterns. A typical initial goal of genetic modification is to introduce a new gene activity. In the longer term, gene activities need to be refined and restricted to particular tissues or regions. One approach is to engineer promoters to target gene expression. However, promoters may still be leaky and by exploiting the principles of S-NF it may be possible to enhance specificity by expressing inverted duplications in complementary domains. This may be a natural mechanism that has been employed during the evolution of flower colour. By showing how this system operates in both natural and engineered systems, the project will underpin such approaches. Genetic modification is still some way from gaining public acceptance, but having the tools for controlling expression patterns more precisely will be invaluable should attitudes change. The time frame for this type of impact is expected to be 10-20 years.

3. The general public and school children will benefit directly from this project through the proposed hands-on events and through dissemination of latest research findings in an accessible way via media routes like youtube videos and press articles. Through these events they will learn how genetic variation underlies evolution and breeding; how genetic engineers may employ methods that have already been explored naturally through the course of evolution; and how genes and environment interact to modify organisms. The public will also benefit in the longer term because of the contribution that this project will make to maintaining and developing forward-looking scientific research that provides the foundations of a modern healthy and growing economy. These activities will have immediate impact on audiences (throughout the 3 years of the project) as well as longer term impact on career choices and society (10-20 years).

4. BBSRC will benefit because the project is directly relevant to the research priorities in food security, synthetic biology, new strategic approaches to industrial biotechnology, data-driven biology and interdisciplinary research. The project also meets the BBSRC objective of building partnerships, through the involvement of an interdisciplinary team that involves a BBSRC institute, university and researchers in Japan, Israel and Spain. The time frame for this type of impact is 3-20 years.


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Description Lab/project website 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact We are currently in the process of publishing a lab website which will showcase publications and software tools produced by the lab as part of this and other funding - this is still under development at the time of writing. We have also set up GitHub repositories for all software developed and published this under an open source license. My group members have agreed to write an article for the website describing their work in simple terms to the public. We will also aim to publish more technical articles on methods and best practices used in data analysis.
Year(s) Of Engagement Activity 2020
URL http://bioinformatics.info
Description Youth STEMM Showcase - JIC 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Co-supervised PhD student was involved in setting up and manning a stall at the event. Student communicated their research to visiting Year 9 / Year 10 children as well as explaining the breadth of scientific research that is carried out at the Norwich Research Park. Feedback from attendees was very good.
Year(s) Of Engagement Activity 2019
URL https://ysawards.co.uk/