Monogram Early Career Excellence Award 2014

We seek to recognize outstanding young scientists and researchers in the field of small grain cereal and grass research in the UK. The excellence award will consider research contributions in both basic and more applied disciplines, as well as contributions of excellence to scientific outreach efforts.

Applicants Short List

Sacha

Sacha Allen: Development and validation of a flexible genotyping platform for wheat

The development of a high-throughput genotyping platform for hexaploid wheat was a project that I was involved in from conception and throughout its development. The basis for this work was next-generation sequencing data from the BBSRC funded project ‘Mining the allohexaploid wheat genome for useful sequence polymorphisms’, which culminated in the release of the 5X Chinese Spring sequence data and a publication in Nature (Brenchley et al., 2012). To establish a suitable genotyping platform to validate markers identified in this study I initially tested several different strategies and assessed each for their accuracy, reproducibility and affordability. This led to a close collaboration with the UK bioscience company KBiosciences (now LGC Genomics) and I was responsible for interacting with the research and development team to create an initial panel of 1114 SNP assays (Allen et al., 2011). I set- up and performed the lab-based screening of this panel against a set of UK varieties at Bristol and also travelled to the KBiosciences laboratories to perform high-throughput screening of a UK mapping population. This work led to the first ever publication of a wheat genetic map based on several hundred SNP markers (Allen et al., 2011); a key publication in the wheat community that has been cited 50 times.

The response to these initial findings from both the academic and commercial breeding communities was very positive and led to the funding of the BBSRC CIRC award ‘Development and validation of a flexible genotyping platform for wheat’. This award was a collaboration between Bristol University, JIC and LGC Genomics and it focussed upon developing the KASP platform further for wheat genotyping. My specific roles in this project were to manage the selection and screening of assays, analyse the resulting data, create genetic maps, co-ordinate activities between Bristol, JIC and LGC Genomics and to report the progress of the project to the steering committee at six-monthly meetings. KASP was proven to be a cost-effective, flexible platform for wheat genotyping, allowing academics and breeders to perform screening in their own labs, using simple microplate technology.

By implementing high-throughput KASP technology I was able to increase the number of validated markers to over 7000, with over 4000 assigned a genetic map position. Additional outcomes of this work that benefitted the wheat community are an improvement in the efficiency of the SNP validation rate to 96%, and an increased number of D-genome and co-dominant markers (Allen et al., 2013). The co-dominant markers have been particularly useful in the WISP project where populations created from diverse crosses need to be screened by markers capable of detecting heterozygotes. In order to enable wheat researchers worldwide to make immediate use of the new information resulting from this research an important feature of this project was to make the genotyping and associated data rapidly available to the public. I was responsible for preparing the data for release on a public web-based database, often to tight deadlines. The website (http://www.cerealsdb.uk.net/) has been highly accessed, with an average of ~6000 unique hits per month from global addresses including USA, Canada, India, China and Japan indicating the global impact of this data. In addition I have also been involved in developing the Illumina 90K iSelect array (Wang et al., in press) and more recently the Affymetrix 820K Axiom array.

To build awareness of the huge progress made as a result of the work described here I have presented the data and publicised the database at several international conferences.

Chris

Tina Blackmore

Diversity and population structure in European ecotypes and varieties in Lolium perenne Grassland ecosystems account for approximately 40% of the terrestrial land mass of our planet and are of critical importance to carbon sequestration, the bio-geochemistry of soils and the maintenance of biodiversity. Perennial ryegrass (Lolium perenne L.) is a dominant species of temperate grassland ecosystems providing an important resource to study adaptations to various environmental and ecological factors. In addition to which, in temperate regions it is the primary forage grass species and thus the foundation of grassland based agricultural systems. Phenotypic diversity within the species has also allowed its selection and utilisation as a major component of seed mixtures used for sports, amenity and landscape purposes. Therefore, L. perenne has a significant commercial and environmental importance. However, little was known on the true extent of genetic diversity in naturally occurring populations (ecotypes) and the comparison to commercial varieties available.

A large collection of L. perenne ecotypes have been gathered within IBERS, Aberystwyth University. The remit of my position as Post Doctoral Research Assistant within the Translational Genomics group, lead by Prof Wayne Powell and Dr Matthew Hegarty, was to use a custom Illumina Infinium Array to genotype ~1000 different L. perenne individuals across 3425 markers (manuscript submitted). This has involved the generation of a cluster file, data management and analysis for this project.

The initial questions to be addressed focused on determining the amount of genetic diversity across 716 European L. perenne ecotypes using this substantial increase in genome wide markers. Principal component analysis revealed a genetic distribution that mirrored the geographic location of the individuals, with a significant East-West and North-South correlation to PC1 and PC2, respectively (fig. 1). The generation of a linkage map using 2 different mapping populations, has also facilitated the application of genome wide association studies (GWAS) in this collection of ecotypes. A number of phenotypic measures have been collected on the individual plants, including ge(Ao) graphic and climatic measures from original sample site, to growth characteristics in plots in the UK to disease response. GWAS provides a method for identifying novel sources of variation, desirable traits and offers insight in the biological pathways and their control in respect to different challenges. Candidate genes for a number of traits have been identified and are already being investigated further. In parallel to elucidating the natural variation in L. perenne, a range of commercial varieties have been genotyped using the same custom Infinium array. This has allowed the comparison of the genetic diversity currently contained in varieties and the extent to which this utilises the range of diversity seen in ecotypes (manuscript in preparation). The combination of these datasets has allowed the exploration of the effect of incorporating ecotypes into a breeding programme and the subsequent recurrent selection, as provided by the commercial Aber high sugar grasses. This particular population has already started to provide further information on the genetic control (via candidate genes) and the biological pathways involved in the production of water soluble carbohydrate. This trait is commercially important and has been correlated to a reduction in the methane production in ruminants, thus a potentially powerful tool in the reduction of greenhouse gas emission and mitigating climate change.

Phil

Philippa Borrill - Personal Statement

Wheat grain nutrient content and yield are influenced by an array of developmental and environmental processes. We are dissecting this complex network using the transcription factor Grain Protein Content (Gpc-B1) which increases grain protein content, alters grain micronutrient content and affects senescence but does not adversely affect yield. During my PhD I have identified direct target genes of Gpc-B1 which may allow more precise regulation of grain nutrient content and senescence for use in improving varieties in the field.

Gpc-B1 is a NAC transcription factor which binds and regulates other genes to bring about effects on grain nutrient content and senescence. We wish to further understand the pathways by which Gpc-B1 acts to enable more precise manipulation of downstream phenotypes such as altering individual micronutrients. I developed transgenic wheat lines expressing Gpc-B1 tagged with a FLAG peptide which has a commercially available antibody. I then carried out chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) to identify the genes directly bound by Gpc-B1. This study, to our knowledge, is the first example of the use of ChIP- seq in wheat. We have produced a list of candidate target genes which are both directly bound by Gpc-B1 and whose expression level is altered when Gpc-B1 homologue expression is reduced, as determined by RNA-seq. I am now working on identifying TILLING mutants for these candidate genes to further understand the control of grain nutrient composition, yield and senescence at a molecular level.

In addition to identifying target genes of Gpc-B1 I have worked on characterising the interaction between senescence and yield. I have used transgenic plants with reduced expression of Gpc-B1 homologues (RNAi lines) which have a dramatically delayed senescence of ~3 weeks, yet which do not show an increase in yield. This is surprising because a paradigm in wheat breeding has been that delaying senescence will increase yield due to an extended grain filling period. To investigate what is happening during this stay-green period I measured the rate of photosynthesis after anthesis in RNAi and control lines using an infra-red gas analyser and explored grain development via moisture content and grain weight. I found that during the extended stay-green period the RNAi plants continue to photosynthesise, however by this stage the grain have already reached physiological maturity, i.e. maximum dry weight, so the extra photosynthate is not imported into the grain. Instead approximately 25% of the photosynthate produced during the extended photosynthetic period in RNAi lines accumulates in stem tissues as fructan and sucrose. This work shows that to improve yield by delaying senescence the sink capacity must also be enhanced.

Alongside my work on Gpc-B1, throughout my PhD I have carried out a great deal of scientific outreach as a STEM ambassador, particularly focussing on the importance of crop science. For example I have made oral presentations about Gpc-B1 and wheat research to members of the general public, a Norfolk Young Farmers Club and school children. I have contributed to the training of younger scientists by supervising several PhD, Masters’ and Undergraduate students, organising transferrable and scientific skills training for the International Undergraduate Summer School at the John Innes Centre and mentoring sixth-form students to develop an eco-friendly business plan. The opportunity to communicate my research to a range of audiences has not only allowed me to share my passion for science but also given me the chance to reflect on the wider value of my research. My commitment to outreach was recognised by the award of the 2012 John Innes Foundation Prize for Excellence in Science Communication.

My activities in science communication have complemented my research which has led to a greater understanding of source-sink relations in the context of delayed senescence and identified promising candidate genes to understand and manipulate wheat grain nutrient content and senescence at the molecular level.


Goddard

Rachel Goddard - Avoiding trade-off when enhancing Fusarium head blight resistance of barley

Fusarium head blight (FHB) is a major disease of barley caused by mycotoxin-producing Fusarium species. FHB causes yield loss and poses a potential health risk to organisms which consume contaminated grain. Like wheat, no single barley cultivar currently used in breeding programs demonstrates consistent resistance.

Resistance to FHB in both barley and wheat has been linked to height, with taller varieties thought to be more resistant. However increased plant height can lead to lodging, creating a trade- off between disease resistance and agronomic traits. Rht gibberellic acid (GA) insensitive wheat alleles give a favourable semi-dwarf phenotype, but increase susceptibility to FHB (Srinivisachary et al., 2009). They also confer a resistance trade-off between fungi of differing trophic lifestyles, increasing susceptibility to biotrophs but increasing resistance to necrotrophs (Saville et al., 2012). It is therefore important to identify semi-dwarfing genes which do not affect resistance to FHB or other diseases. I investigated the response of barley semi-dwarf near isogenic lines (NILs), which have impaired signalling in the brassinosteroid (BR) pathway due to a mutation in the BRI1 (BR- insensitive 1) BR receptor gene, to FHB and a range of other fungal pathogens. Semi-dwarf bri1 lines show an increase in resistance to necrotrophic and hemibiotrophic pathogens including Magnaporthe oryzae, Oculimacula acuformis, O. yallundae and Gaeumannomyces graminis. They do not however, show altered susceptibility to either FHB or the biotroph Blumeria graminis compared to tall BRI1 lines (manuscript submitted to Plant Physiology). My results demonstrate that alteration of signalling through the BR pathway reduced height without increasing susceptibility to FHB, and also gives increased resistance to necrotrophic pathogens without increasing susceptibility to biotrophs. Application of this research may help improve disease resistance without compromising disease resistance in the favourable shorter stem phenotype.

Chevallier, a heritage malting barley shows significant resistance to FHB, yet also possesses the tall height trait. A bi-parental cross (CxT) between Chevallier and Tipple, a shorter modern malting cultivar, was created to determine if the FHB resistance of Chevallier is associated with height through linkage or pleiotropy. I phenotyped 200 CxT F5 lines, generated a genetic map and identified QTLs for several traits (FHB, height, heading date, tillering, growth habit, spike density) within the population. I identified a single large-effect FHB resistance QTL on 6H which explained 29.2% of the variance. I also identified two height QTL that mapped to chromosome 3H. Thus the FHB QTL does not co-locate with any of the height QTLs, suggesting that FHB resistance within Chevallier is not due to the pleiotropic effects of height genes. I have developed a separate CxT F7 population to fine map the FHB QTL. I will identify SNP markers to enable breeders to introgress the FHB resistance of Chevallier into other varieties without a trade-off with other desirable traits such as shorter height. I will also undertake QTL analysis of malting and yeast fermentability traits to determine whether important quality traits are inherited independently of FHB resistance. This will increase knowledge on the safe production of barley grain products without a trade-off between FHB resistance, agronomic or malting quality traits.


Matt

Industry Applicant: Dr. Matt Kerton, Wheat Breeder, DSV UK Ltd

My scientific career began at the University of Birmingham where I completed a PhD in Crop Physiology. I was charged with investigating the causes of tip burn in Coriander and enjoyed the applied nature of my research, gaining an understanding in plant physiology and molecular genetics as well as experience in a wide range of techniques such as radioisotope mapping, electron microscopy, EDAX, thermal imaging, ion chromatography, liquid scintillation counting and bioinformatics. Tip burn in coriander was found to be due to a lack of calcium at the leaf edge due to an uncoupling of calcium and water transport (Kerton et al. 2009).

After graduating, I took up the position of wheat breeder at DSV UK Ltd. Prior to my employment, DSV UK had only screened wheat material arising from our German breeding programme. I was instructed to set up the wheat breeding programme in the UK, develop breeding lines and manage the programme on a daily basis.

Within one year of my appointment, I had made the first DSV UK crosses in a purpose-built glasshouse with elite varieties and UK-type lines from our German programme. Today these crosses are in F5 trials within our pedigree breeding programme. Beyond this stage, DSV UK continues to screen German lines for UK suitability. From these lines and since my appointment, we have produced the recommended variety Chilton and three other nationally listed varieties, Skylark, Sherwood and Dexter. I have developed a double haploid programme to compliment the pedigree lines and routinely make use of our service laboratory in Germany to employ MAS and to screen my breeding lines for traits relevant to UK agriculture.

Within 4 years of employment, it has been my responsibility to design crosses, carry out these crosses and see the breeding lines through to official trials application. I work closely with my trials manager to grow our trials and nursery to a high standard. Phenotyping is carried out across the UK and four other EU countries before the data is analysed, selections made and applications put forward.

One of the enjoyable aspects of my job is the day-to-day variation. I spend a lot of time in the field or greenhouse and enjoy driving the plot combine allowing me to see the data as it is generated. I also involve myself with academic research by attending conferences and steering groups. The integration and harmony between academic research and our industry is vital for the long-term strategy of plant breeding and will underpin the success in the future.

Keyes

Samuel Keyes: Visualisation and nutrient uptake simulations of undisturbed, soil-grown root hairs in 3D

Root hairs are micro-scale, filamentous extensions of root epidermal cells. Their dominant function is thought to be the exploration of rhizosphere soil, enhancing plant uptake of sparingly soluble nutrients, especially phosphate (P). However, virtually all understanding of hair morphology has been derived from sterile and structurally homogenous gel assays, meaning significant trophomorphogenetic responses are not expressed. To better understand the role of hairs in rhizosphere-scale nutrient transport processes, there exists a pressing need for novel, in-situ visualisation methodologies.

We have developed high-resolution synchrotron X-ray CT (SRXCT) imaging techniques to image the rhizosphere of intact roots for the first time, uncovering undisturbed hair, soil and fluid interactions in unprecedented detail (Figure 1). By developing an assay allowing single roots of rice and wheat to be grown in small soil volumes (~4.5 mm diameter), a sophisticated suite of synchrotron-based methods can now be applied to the intact rhizosphere zone. Plants at a range of developmental stages have been investigated using the X02DA beamline of the Swiss Light Source. This facility uses a high- brilliance X-ray source to acquire full 3D structure at spatial resolutions of ~0.6-1.5 μm. These data permit the measurement of rhizosphere physical parameters such as hair density, porosity and fluid volume with extremely high accuracy as compared to existing methods.

Furthermore, for the first time, real undisturbed rhizosphere geometry can now be used to parameterise explicit numerical models of nutrient desorption, transport and uptake1. Figure 2 shows post-processed simulation data for a mechanistic P uptake model, showing both the spatial heterogeneity of uptake and the paths of ion flow from sorption sites to uptake membranes. By developing a complementary suite of novel in-vivo imaging methods and modelling approaches that explicitly consider complex soil, fluid, hair and root interactions, we are now in a position to explore in silico the influence different parameter combinations on nutrient uptake. By uncovering the significance of hair patterning traits (such as length, density and morphology) to uptake, knowledge is already being generated that will aid targeted breeding of new cereal varieties for enhanced nutrient acquisition in the world’s increasingly infertile soils.

  • 1 - Keyes SD, Daly K, Gostling NJ, Jones DL, Talboys P, Pinzer B, Boardman R, Sinclair I, Marchant I, Roose T, (2013), High resolution synchrotron imaging of wheat root hairs growing in soil, and image based modelling of phosphate uptake, New Phytologist, Volume 198, Issue 4, pages 1023 - 1029
Smith

Ollie Smith: Archaeological barley stripe mosaic virus: insights into evolution, agriculture, trades and crusades

Following the transition of humans from hunter-gatherers to farmers, the domestication and cultivation of staple food crops is arguably one of the cornerstones of society. Poaceae were the first to be truly domesticated and are still the major family of global food production in terms of economics, calories and distribution. Barley (Hordeum vulgare) was one of the first domesticated crops, becoming so around 10,000 years ago in the fertile crescent of the Near East, and formed the major staple crop of pre- and post-dynastic Egypt. With domestication however comes vulnerability from reduced genetic diversity and growing conditions, leading to the rise of pathogens. Estimates of molecular evolution suggest a recent emergence (within a few hundred years) of viral pathogens. Presented here is the first study to completely reconstruct an archaeological RNA virus, barley stripe mosaic virus, suggesting emergence significantly earlier than molecular estimates predict.

Ancient DNA has been increasingly prominent in recent years, as entire genomes of extinct fauna and historically important plants have been sequenced. This is in no small part due to the technical advances overcoming breakdown, or diagenesis, inherent to DNA. RNA on the other hand is less studied, due to its increased breakdown rate (around 50 times that of DNA) and less obvious use when constructing phylogenies or evolutionary models. RNA does however survive in the archaeological record when conditions are right; in this case, the extreme aridity of Qasr Ibrim, a multi-period Egyptian hill fort near the Nile, allowed survival of ancient RNA in archaeological barley up to 2,500 years old.

Originally, our task was to ascertain the persistence of small RNA in a sample of ~750 year-old barley grain. This was successful, and showed signs of significant stress-response in the barley. Although swamped by broken down fragments of transcript, ribosomal, transfer, and other RNA, a preliminary sweep of these 'other' sequences identified several belonging to BSMV. We performed a more intensive search and to our surprise, the total fragments were enough to reconstruct the genome to 99.4%; all but 60 bases were accounted for, which when inferred from existing topology of 6 extant reference sequences, gave us the first complete draft ancient RNA genome; almost entirely by accident.

Cursory phylogenetic analysis using the Neighbour-joining method indicated that the archaeological strain was basal to all the extant strains. A more detailed analysis using a Bayesian inference method, combined with out-groups, showed again the archaeological virus to be basal to the rest. More interesting however was the estimation of BSMV's emergence being around 2,000 years ago; much earlier than previously estimated. Based on the genetic diversity of extant strains, we hypothesized that the virus emerged in the Near East, near its host's domestication centre, and spread to Europe and East Asia during the middle ages via established trade routes (following the re-opening of the Silk Road by the Mongol Empire) and possibly military expansion (the node basal to the European group coincides with the 7th crusade of Louix IX), where it underwent rapid evolution in response to host and environmental diversity. Further evolution occurred during the 20th century where the virus spread to North America.

The work is of importance for two reasons. First, it highlights the utility of RNA in the archaeogenetic field as a viable molecule with relevant information share in terms of both phylogenetic research and in vivo processes of archaeological material. RNA, and its effects on its own and other genomes, potentially adds a new layer of investigation to archaeogenetic study; to uncover ancient genomes did as opposed to simply what they were. Secondly, our phylogenetic estimates show a slower, more variable rate of evolution in viruses than previously thought. This may well have future relevance in terms of combating plant pathogens, where evolutionary rate appears to be related to genetic diversity of the host, and again adds further consideration to effects of selection pressure on the evolution of different organisms.

ElWasi

Mohamed ElWasi: University of Warwick - Awaiting Statement:

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