This proposal is to prove the concept and develop a high throughput methodology for screening virus infections and immunities in wild plant and insect communities. We propose to obtain small RNA profiles of the plant and insect communities from the Wytham Wood, Oxfordshire, by using Solexa high throughput sequencing. The anti-virus small interfering (avsi)RNAs that are produced by the host gene silencing systems against the virus RNAs will be screened for viral origins. We anticipate the detection of the avsiRNAs against the known prevalent viruses at the site and will use these viruses as positive controls to optimize conditions of sample preparation, sequencing, and bioinformatics. We also expect to discover the prevalence of previously unconfirmed and unknown viruses, and we plan to validate these newly detected infections by using the conventional methods (e.g., RTPCR, cloning and sequencing, Northern Blotting, etc.) to determine the sensitivity and accuracy of the high throughput methodology. To enable the conventional method assessment, we plan to label samples for each sampled species by using sequence tags. The sampling regime is designed for achieving a sensitivity of shotgun detection of 5% infection rate for plant populations. The tagged samples will be pooled together for high throughput sequencing runs to achieve cost effectiveness. The resulting sequences will be sorted back to their original sample identities and analyzed. Results will be validated by using the conventional methods with the sorted specific samples. Mass post-sequencing analyses will also be performed without sorting the samples to their original identities. Results from the specific analyses and mass analyses will be compared. The mass analyses without the requirement of sorting samples are designed for testing a capacity of genetic random sampling from an ecosystem without restriction of sampling regimes. The technology will offer a broad range of applications from large scale random sampling in natural conditions during the environment change, to defined survey in agricultural and the other managed conditions.

Rice can be considered the most important worldwide crop for human nutrition, currently providing ~20% of worldwide daily dietary energy. Due to a growing worldwide population, and the effects of climate change, research into improvements in rice yield, resilience to drought and resistance to pathogens is urgently needed. Such research must be underpinned by public databases storing high-quality information about the rice genome, genetic variants carried by varieties with desirable traits, and information about the function of each gene/protein. This project is a collaboration between research teams based at the University of Liverpool, the Beijing Institute of Genomics and the BGI Education Centre. Our teams have considerable track record in the development of methods for studying the abundance of genes (transcriptomics) and proteins (proteomics) on a large scale for rice and other species, as well as computational approaches for interpreting and integrating data from these different techniques. At present, the public databases storing the rice genome and information known about gene/protein function are disconnected from experimental data (transcriptomics/proteomics) being collected in laboratories all over the world. These experimental data can be used directly to improve the annotation of the genome, by showing how strongly particular genes or proteins are expressed under particular growth conditions or for a given rice variety (which gives clues as to functional importance). These data also show how genes or proteins differ in a given variety from the "reference" genome contained in the database. Our groups are developing software tools for integrating and analysing these data in new ways, so that when laboratories submit their data to a public repository, it can be directly integrated and viewed alongside the genome - which at present is not possible. We are also going to generate and analyse new data sets for several important rice varieties, so we can study how these gene and protein sequences differ from the reference genome. Our results will help to improve the sequences and annotation of rice genes and proteins, and will be made easily available to all other rice researchers through the most widely accessed international public databases.

Understanding the impacts of environmental change and changing land use on biodiversity and how ecosystems work require comprehensive knowledge of communities and their ecology. Molecular biodiversity identification is emerging as a high throughput and cost effective alternative to traditional approaches and in particular, the analysis of environmental DNA (eDNA) provides an opportunity to measure biodiversity in space and time at unprecedented scales. Unlike DNA obtained through direct analysis of communities, eDNA refers to shed cells or free-DNA from organisms as they pass through an environment, or die and decay. eDNA is being applied for various uses such as identification and monitoring of endangered/invasive species and analysis of biodiversity. It is very clear that researchers can detect eDNA from a variety of natural environments and in particular, freshwater environments. However, understanding how those sources of eDNA relate to living biodiversity and associated ecological function in ecologically and socio-economically important river ecosystems is at the heart of the eDNA:LOFRESH proposal. Focusing on a range of exemplar experimental semi-natural and natural freshwater catchment systems from local to national scales, we will (a.) improve understanding of the movement, and persistence of lotic eDNA, (b.) quantify the relationship between lotic eDNA and the in situ community using different combinations of genetic and genomic approaches, (c.) improve methodological approaches for eDNA data acquisition and interpreting eDNA data using novel ecological and phylogenetic algorithms, (e.) develop and test new models relating lotic eDNA to stream biodiversity and ecosystem function and their variation in response to land use pressures. Over a 4 year period, five work packages (WPs) will be delivered by the Universities of Bangor, Birmingham, Cardiff and the Centre for Ecology and Hydrology. In WP1, we will use artificial stream channels in a series of experiments to assess the effects of a range of physical and chemical drivers on the loss of lotic eDNA and to compare and contrast genetic and genomic approaches for assessing known sources of lotic eDNA. In WP2, we will test our experimental findings from WP1 by tracking natural lentic (i.e. lake) and experimentally introduced control lotic eDNA through the natural stream network of the intensely studied Conwy River research catchment in north Wales. WP2 will also assess relationships between observed lotic eDNA and the in situ community in selected tributaries of the Conwy displaying a range of physicochemical characteristics and experiencing different land use pressures. WP3 will sample lotic eDNA in coordination with an on-going national survey in Wales to up-scale the experimental and catchment-scale findings of WP1 and WP2 to the Welsh landscape and national scales. WP4 will provide informatics support, but specifically, develop workflows to identify species level diversity in eDNA datasets. Finally, in WP5 we will further test our model findings, by manipulating the experimental stream systems with emulated land use pressures, quantify the ecosystem functions of decomposition and food web structure and test linkages with eDNA signals. Effective engagement with a broad range of stakeholder groups (government, end-users, environmental agencies) and project partners (research institutions and academic partners specialising in eDNA, sequencing and informatics) will optimise impact and research synergies of potentially transformative science throughout the consortium network.