Potato miRNAs and their role in potato tuber formation

Potato is the third most important food crop, and it is cultivated worldwide for its underground storage stems (tubers), rich in starch and other nutrients. Potato is unique among the major world food crops in tuber formation and the processes by which tubers develop are not well understood. It is essential to know the basic biological processes which lead to tuber formation to be able to select better cultivars (with higher yield, specific shape, texture or skin colour) for the future.
In this proposal we aim to identify small regulatory RNAs which play a role in tuber formation. These RNA molecules are called 'micro' RNAs (miRNAs). This new, recently discovered, layer of gene regulation involves small RNAs (including miRNAs) as regulatory molecules for post-transcriptional gene regulation. This mechanism is widespread in animals and plants and the discovery of small RNAs has changed our basic knowledge about the regulation of genes. miRNAs play important roles in development and gene regulation upon biotic and abiotic stresses.
In our experiments we aim to explore the miRNA transcriptome of potato. Using a photoperiod inducible tuberization system we aim to find miRNAs involoved in this biological process. By generating high-throughput sequence data for small RNAs the availability of the potato genome enables us to identify the miRNA 'genes' using bioinformatics tools. We aim to identify conserved as well as potato specific miRNAs and investigate the role of candidate miRNAs in tuberization. We will characterize the candidate miRNAs and validate them in plants. These miRNAs will help to predict and validate the target molecules for these regulatory molecules. Such genes might play important roles in tuber development or other biological processes connected to tuber formation. The miRNAs and the target genes will help to influence and modify the pathways which lead to tuber formation and we might be able to influence or modify this process, ultimately leading to the ability to breed improved cultivars of this important crop. This knowledge will ultimately help us to produce an important food source with higher yields and improved quality.

A class of small RNAs known as microRNAs (miRNAs) play important and diverse roles in gene regulation at the post-transcriptional level in eukaryotes. Knowledge of the potato miRNA transcriptome is poor. The aim of this proposal is to find miRNAs involved in one of the most important biological process of potato, tuberization. A next generation sequencing (NGS) approach will be used to identify and explore the potato miRNA transcriptome.

We will use a photoperiod inducible tuberization system to analyze a time course series of plant samples and identify miRNAs by use of NGS. We will validate samples putatively selected for NGS by Northern blotting for miRNAs and quantitative or semi-quantitative RT-PCRs for genes involved in tuberization. Bioinformatics analysis of NGS data will be performed using established pipelines for small RNA analysis of plant derived RNAs developed by the Dalmay group at UEA.

After identification of candidate miRNAs we will predict their target genes using the potato genome and perform genome-wide analyses of miRNA cutting sites of mRNAs (PARE analyses). Candidate miRNA targets will be validated by 5' RACE. A transgenic approach will be used to over-express candidate miRNAs for tuberization or alternatively using constructs which can reduce the activity of a specific miRNA in planta (artificial miRNA target mimics). Levels of miRNAs and related transcripts (including targets) will be monitored. The transgenic plants will be assessed for tuberization traits (e.g. yield, tuberization time).

This work could ultimately increases the knowledge of miRNAs in crop plants and will provide new data about the miRNAs for further studies in this important crop. Additionally, we might find new genes having a role in tuber formation.

Who will benefit from this research?
The proposal is directly relevant to the BBSRC priority area in Crop Science/Food Security and Living with Environmental Change. It builds on their investment in potato genome sequencing by using the genome to identify potato miRNAs. It will also add value to previous funding of miRNA research in model plants, providing evidence that these molecuales play important roles in crops.
First of all the RAs and the investigators will benefit from this inter-institutional research because the project requires intensive collaboration between JHI and UEA. As this project is focusing on potato the smaller potato community will benefit from this research having the first view on miRNA transcriptome. Additionally, the project will be of interest for scientists working on other species (e.g. tomato, pepper) because many miRNAs might be shared between these closely related species. This pioneering work will deliver new knowledge about miRNAs in crop plants further facilitating the research of small regulatory RNAs in crops.

Downstream there will be benefits for different sectors of the potato industry. If, as seems likely, miRNAs are found to influence tuber development traits breeders will ultimately be able to select better cultivars which may lead to increased yields, better control of tuber development parameters (shape, size, size distribution etc).

For biotechnologists (public or private) the research will facilitate development of transgenic strategies for improved tuber traits; however progress to consumers will depend on public opinion and is likely to take up to 10 years for this to become commercial reality. In the medium to long term, strategies for improvement should be advanced thereby contributing to the societal objectives of improved availability of nutritious food. In the wider context of a globalised world economy, improved human health anywhere in the world will improve overall global security.

How will they benefit from this research?
The research scientists will benefit from the continuous communication and will be able to initiate new collaborations. The project as a basic research on the miRNAs in potato will contribute to the expanding field of miRNAs in crops and will add new knowledge to it. Scientists working with other species could translate the findings to their research which can initiate new research directions. All of these will be possible through publications, conference attendances and personal communications, meetings.
This project will supply breeders with new data about how miRNAs can influence and regulate important crop traits and the identified miRNAs might be molecular markers for the future to be able to select better cultivars. The target genes might represent new candidates for transgenic research and facilitate the development of the crop depending on public opinion. The project will also further develop laboratory and bioinformatic techniques for handling miRNAs and associated datasets.

Development of staff skills
The PI, Co-Is and researchers will develop their scientific and communication skills. The project will provide multi-disciplinary training activities for the RAs employed on the project, delivering scientists with a high skills base and applied perspective. The RAs will develop their communication skills through the interaction between and within JHI and UEA. C. Hornyik (Researcher Co-I) will benefit from managing the RA and gain more experience in project management. JHI and UEA also benefit from the cooperation having a better understanding of how science is carried out at the two sites with the possibility to build future interactions. The data generated will be presented at national and international conferences further developing communication skills.