Ribosomal DNA variation in multi-locus systems

Recent technological advances have led to a dramatic drop in both the cost and the time taken to obtain the genome sequence of a chosen organism. As a consequence, the genomes of thousands of organisms are currently being sequenced around the world. Once these genome sequences have been obtained, researchers may then analyse them using a growing toolkit of software. Much of the effort analysing these sequences is naturally spent on examining the genes, which make proteins that are used in cells for growth and development.

Despite the quantity of genome sequences now publicly available, one part of them that has received scant attention is the ribosomal DNA (rDNA). The rDNA is essential for life, as it is involved in "reading" the sequence of a gene and from that sequence constructing a protein. The rDNA itself is a short sequence (of a few thousand "letters" long) that in many organisms is repeated over and over again, in tens or hundreds of copies, at one or more locations within a genome. Until recently, researchers believed that all the tens or hundreds of copies of the rDNA within a single organism were identical. However, recent studies have shown that there are indeed differences between rDNA copies, both in terms of the number of copies and their DNA sequences. Furthermore, the rDNA is now being shown to play a role in important biological processes such as ageing but we have yet to discover how these rDNA differences affect such processes.

Over the last decade, we have meticulously analysed the rDNA in species of yeast that package it within just a single location within their genome. We have shown that the differences between copies of the rDNA both within and between organisms encapsulate a rich source of evolutionary information. An important part of this work was developing two software tools, TURNIP and VariantLister, that enabled us to find those rDNA differences. Here, we will extend our knowledge of rDNA differences to include species that organise their rDNA across two or more genomic locations. We will do this by analysing special sequence datasets that comprise just a single chromosome within a genome - analogous to a chapter within a book - for the yeast species Candida glabrata (2 rDNA locations) and bread wheat (5 rDNA locations). Such an analysis is important as many species that humans depend upon, including farm animals and cereal crops, organise their rDNA across multiple locations and finding out how the rDNA differs between locations may help us to develop better breeds and varieties in the future. We will then test whether we could in fact have used DNA sequences from whole genomes to determine the same information, which will have broad implications for how we analyse organisms with multiple rDNA locations in the future. These tasks will require us to first improve the VariantLister software so that it can accurately find rDNA differences without the need for us to edit its results by eye.

We will then determine which of the rDNA differences that we have identified are actually used by yeast and wheat to construct proteins. In particular, we will discover if the rDNA differences they use depend on the genomic location at which they are found and the environmental conditions in which the organism is living (e.g. temperature, water availability). These results, which may indicate rDNA differences that change aspects of how an organism functions (i.e. its traits), will be communicated to relevant crop and yeast improvement projects that are aiming to develop new varieties and strains tailored to specific purposes (e.g. crops that grow best in certain environmental conditions).

Finally, we will make all project datasets and the VariantLister tool freely available on a dedicated project website, to the benefit of researchers around the world, so that others may carry out their own studies on rDNA variation, evolution and function.

The ribosomal DNA (rDNA) evolves under a balance of heterogeneity-inducing point mutations acting against homogeneity-inducing concerted evolutionary processes. This balance is now known to be imperfect, leading to intra- and inter-organism variation in rDNA copy number and unit sequence. This situation is made even more complex by the presence, in many organisms, of multiple rDNA loci that are believed to be homogenised by processes such as gene conversion. Evidence is growing rapidly that rDNA variation is tightly linked to phenotype. Furthermore, the rDNA has now been implicated in vital biological phenomena such as genome integrity and ageing. We therefore urgently need to discover how rDNA variation is organised within a genome, how it is expressed and how it underpins the functionality of an organism.

We have developed VariantLister, a software tool that enables us to systematically characterise rDNA variation in organisms with a single rDNA locus. However, to date we have been unable to attribute rDNA variants to a specific locus in organisms with multiple rDNA loci. Through analysis of single chromosome datasets and further VariantLister development, we will carry out such an analysis for the first time, in yeast and wheat. We will also carefully assess whether clustering of rDNA variants called from whole genome sequence datasets can be accurately ascribed to distinct loci for organisms with multiple rDNA loci. Finally, by analysis of transcriptome datasets, we will discover which of the identified rDNA variants are expressed and how a variant's expression fate depends on its locus and the environment.

The results of these tasks will provide vital new knowledge on the organisation and expression of rDNA variants in two key eukaryotes, which will underpin future investigations of rDNA function and ultimately species improvement programs. Finally we will disseminate all project software and data on a dedicated project website.

This project has considerable promise to impact on the UK society and economy, the general public and the project participants. While economic and societal impact will be derived in the long-term, benefits to the general public and project participants are expected to be realised both during and following the project.

1) The UK society and economy
Our society is currently facing significant challenges stemming from threats ranging from climate change to a growing and ageing population. We have an urgent need to secure and optimise future food production while also utilising food and agricultural waste in the replacement of petroleum as sustainable sources of key chemicals. This project will impact on both of these needs, to the benefit of our society and economy.

a) Crop breeders/Agri-food industry
Wheat is the UK's most important cereal crop, yielding 16.68 million tonnes in 2015, and a vital component of the UK diet. New knowledge of rDNA variation and expression in bread wheat will be communicated to crop breeders and the agri-food industry, to be used for the development of new varieties of wheat tailored to specific environmental conditions. In particular, analysis of RNA-Seq datasets will kick-start the identification of rDNA variants that are preferentially expressed under stress, including conditions of high temperature and low water. Dr Davey's position in the wheat community, including the BBSRC Wheat ISP, will be key to effective knowledge dissemination in the pursuit of continued food security.

b) Industrial biotechnology/Biopharma
The vast quantities of wheat straw left over from food production (e.g. 6.3 million tonnes in 2007), in particular in the East of England close to the project's location, is a key target substrate for secondary biorefining in the UK. Here, sugars released from the straw are fermented by yeast to produce a wide spectrum of platform chemicals and fuels. Harnessing the vast biodiversity of yeast is a fast emerging area of interest to a wide range of UK companies and NCYC has recently developed a new collaboration on yeast natural products with Croda, a FTSE 250 company. Yeast rDNA variants and expression profiles discovered in this project are expected to lead to the development of new strains that efficiently produce optimal quantities of a required chemical product. Consortia such as Sc2.0 and existing relationships with key companies such as Croda will ensure broad communication of our results.

2) The general public
There is a growing public appetite for scientific knowledge, with a wide recognition of the enormous impact that science has on our prosperity and continued well-being. The project team are highly committed to public scientific outreach, each tending to focus on a different part of this broad sector. Within this project, we will engage directly with members of the public, from schoolchildren to our society's most senior members, to educate them in its most important aspects. In particular, we will use our existing contacts within local organisations such as the SAW Trust and BBC Look East to introduce concepts such as genetic variation, synthetic biology and industrial biotechnology, to explain why we are carrying out this project and what benefits we anticipate it will bring to the local population and to the wider UK community.

3) The project participants
The three project investigators are all highly skilled in the training of new members of their field, and combined they have passed on a wide range of scientific and transferable skills to dozens of scientists in the UK and beyond, many of them now holding senior scientific positions of their own. Within this project, the post-doctoral research assistant will benefit from this expertise, gaining excellent inter-disciplinary training, with the project focus ensuring they possess the skill sets essential to the next generation of UK scientists.