Weeding the Gems

This is a brief summary of some of the work published in 2015 by UK Strawberry researchers and it is perhaps unsurprisingly dominated by work undertaken at East Malling Research. Richard Harrison leads two studies that focus on different aspects of the diseases that effect strawberry yield while Louisa Robinson-Boyer heads an investigation into the role that arbuscular mycorrhizal fungi might play in enabling future growth of strawberries in drier condtions.
Elsewhere we introduce a study that the outlines the developement of a commercial SNP-array that will hopefully aid marker-assisted breeding and finally is a Spanish-led investigation into a class of enzymes that contribute to fruit softening.

March
Antanaviciute L, Šurbanovski N, Harrison N, McLeary KJ, Simpson DW, Wilson F, Sargent DJ, Harrison RJ (2015) Mapping QTL associated with Verticillium dahliae resistance in the cultivated strawberry (Fragaria × ananassa). Hortic Res 2:15009  http://dx.doi.org/10.1038/hortres.2015.9 Open Access

Wilt score of resistance (blue) and suspectible (purple) varieties.

Richard Harrison (East Malling) is the UK lead on this collaboration with Italian researchers in a study that investigates the QTLs that confer resistance to Verticillium dahliae, an important strawberry pathogen. A population of octoploid strawberries that was segregating for pathogen resistance was screened over three field seasons and multiple QTLs were identified. These resistance QTLs appear to be additive in this situation and their associated markers are abundant in cultivated strawberry germplasm. The authors state that genetic gain is clearly possible through marker-assisted breeding in Strawberry.

Bassil NV, Davis TM, Zhang H, Ficklin S, Mittmann M, Webster T, Mahoney L, Wood D, Alperin ES, Rosyara UR, Koehorst-Vanc Putten H, Monfort A, Sargent DJ, Amaya I, Denoyes B, Bianco L, van Dijk T, Pirani A, Iezzoni A, Main D, Peace C, Yang Y, Whitaker V, Verma S, Bellon L, Brew F, Herrera R, van de Weg E (2015) Development and preliminary evaluation of a 90 K Axiom® SNP array for the allo-octoploid cultivated strawberry Fragaria × ananassa. BMC Genomics 16:155  http://dx.doi.org/10.1186/s12864-015-1310-1 Open Access

This international collaboration includes a UK industrial contribution from researchers from AffymetrixUK and describes the development of a SNP-array to aid marker-assisted breeding of allo-octoploid cultivated strawberry. Sequence variants were identified from the sequencing of 19 varieties of available octoploid germplasm along with a diploid progenitor. Following genotyping of over 300 octoploid accessions, SNPs were analysed using Affymetrix software. Ultimately the finished array has SNPs evenly distributed across 28 chromosomes with an average density of one marker/0.5cM. The authors state that this array will allow quality genetic analysis including generation of high density linkage maps and will aid strawberry researchers to identify QTLs and provide a platform for use in future marker-assisted breeding strategies.

April
Boyer LR, Brain P, Xu XM, Jeffries P (2015) Inoculation of drought-stressed strawberry with a mixed inoculum of two arbuscular mycorrhizal fungi: effects on population dynamics of fungal species in roots and consequential plant tolerance to water deficiency. Mycorrhiza. 25(3):215-27   http://dx.doi.org/10.1007/s00572-014-0603-6

Louisa Robinson-Boyer from EMR is the corresponding author of this study that investigated the ability of cultivated strawberry to grow in drought conditions after inoculation with zero, one or two species of arbuscular mycorrhizal fungi (AMF). They showed that both species of Funneliformis AMF were able to co-colonise roots but that their relative abundance varied under different water stresses (reduced irrigation by up to 40%). The authors found that colonisation was beneficial but that single species inoculation gave similar benefits to a mixed population, irrespective of the amount of irrigation. This shows that the presence of colonisation was of principle importance. Addition of AMF to plants under drought conditions restored growth to the levels observed in plants grown with normally watering. This demonstrated that the growth of cultivated strawberries in drought conditions significantly benefits from the presence of AMF and therefore might suggest a long-term strategy to improve future production of this crop in changing climate conditions.

June
Xu X, Passey T, Wei F, Saville R, Harrison RJ (2015) Amplicon-based metagenomics identified candidate organisms in soils that caused yield decline in strawberry. Hortic Res. 2:15022  http://dx.doi.org/10.1038/hortres.2015.22 Open Access

This second paper led by Richard Harrison looks into novel soil microbial pathogens that may be a causative agent for declining strawberry yield. Multiple soil samples were collected from four sites, two that showed yield-decline and two without. The authors defined operational taxonomy units (OTU) of bacterial and fungal origin and were able to assign yield decline to four potential factors: 1- Low abundance of helpful bacterial populations, such as Bacillus and Pseudomonas 2- lack of nematophagous fungus (Paecilomyces sp) 3- high levels of two potential fungal root-rot pathogens and 4- overall wet soil conditions. The authors will use this information to identify true causative agents that contribute to strawberry yield decline. Furthermore the authors highlight that this amplicon-based metagenomics approach is useful for profiling novel soil microbiota communities.

It is an exciting time for the Harrison lab as in collaboration with The Genome Analysis Centre, they have just obtained BBSRC funding for a project to sequence the octoploid cultivated strawberry.

November
Paniagua C, Blanco-Portales R, Barceló-Muñoz M, García-Gago JA, Waldron KW, Quesada MA, Muñoz-Blanco J, Mercado JA (2015) Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening. J Exp Bot. http://dx.doi.org/10.1093/jxb/erv462 Open Access

This Spanish-led study includes UK representation from Keith Waldron at the Institute of Food Research. Softening of fruit is a significant issue when bringing strawberries to the market and is caused by an increase in the solubilization and depolymerization of pectins from cell walls. This study aimed to investigate the role that galactose-release plays in this process, a reaction that is catalyzed by β-galactosidase enzymes. One putative member of this enzyme family, FaβGal4 was identified using a novel strawberry expression array. The authors generated antisense transgenic strawberries that aimed to have reduced expression of FaβGal4. Two out of nine lines showed reduced expression of FaβGal4 (and also the related FaβGal1) that was coincident with fruits that were 30% firmer, analysed over three growing seasons. These plants have higher levels of galactose in their cell walls demonstrating that the role of the β-galactosidase enzyme was indeed inhibited in these lines. Therefore this indicates that the FaβGal4 gene represents an potential target for future breeding or transgenic strategies to generate strawberry plants that have firmer fruit.

In this Wheat Research Report we summarise some of the work published by UK wheat researchers in 2015. Given the importance of wheat for both UK and global food security there is plenty of UK work in this area that falls into a number of key areas:

– We highlight three pieces of research undertaken on the genome scale led by the Universities of Liverpool, Bristol and York, which aid to improve knowledge of the highly complex hexaploid wheat genome in order to improve future breeding efforts.

– On the phenotypic level we present studies led by both the John Innes Centre and NIAB that have identified wheat QTLs that are associated either with disease resistance or with the parameters that define grain yield.

– On the gene level researchers from Reading University investigate the role of the TIN gene on the water use efficiency associated with different levels of tillering.

– Elsewhere is a study that mixes digital imaging and field reconstructions with mathematical modeling to develop a methodology to assess the significance of the structure of the wheat canopy in final carbon gain.

– Finally we highlight a study that had high public awareness and that documents the results of a study in which transgenic wheat were generated with the aim of reducing aphid predation by production of a specific pheromone.

March

Griffiths S, Wingen L, Pietragalla J, Garcia G, Hasan A, Miralles D, Calderini DF, Ankleshwaria JB, Waite ML, Simmonds J, Snape J, Reynolds M (2015) Genetic dissection of grain size and grain number trade-offs in CIMMYT wheat germplasm. PLoS One. 10(3):e0118847. http://dx.doi.org/10.1371/journal.pone.0118847 Open Access

This is a collaboration between British, Argentinian, Mexican and Chilean researchers who investigated grain yield in a two CIMMYT varieties. Grain weight (GW) and number per unit area of land (GN) are principle components that determine grain yield and these components often show negative correlation. The CIMMYT varieties were crossed and a QTL loci was identified that showed increased GW but that did not change GN, therefore altering the usual trade off between these parameters. The authors suggest that this loci might be an attractive target for future marker-assisted breeding strategies aimed at developing higher yield wheat varieties.

June

Bruce TJ, Aradottir GI, Smart LE, Martin JL, Caulfield JC, Doherty A, Sparks CA, Woodcock CM, Birkett MA, Napier JA, Jones HD, Pickett JA (2015) The first crop plant genetically engineered to release an insect pheromone for defence. Sci Rep. http://dx.doi.org/10.1038/srep11183 Open Access

This study led by Johnathan Napier, Huw Jones and John Pickett at Rothamstead Research gained a large amount of pubilicity during its trial phase as it drew the attention of anti-GM campaigners. However for many commentators it represents an important point in the ongoing debate surrounding this technology as the public engagement undertaken by these researchers was effective in persuading the public that this was a worthwhile project. This paper reports on the findings from that study wherein hexaploid wheat was genetically engineered to produce a pheromone that aimed to reduce aphid predation. In lab experiments these pheromones were able to repel three species of aphids but in the field experiments these GM wheat showed no alteration in rate of predation. The authors suggest this might be due to erratic climatic conditions or unexpectiedly low overall insect numbers but they recognise that more work needs to be done to identify a class of pheromones that might work in this experimental system. In many ways this study is an important confirmation of the scientific process and should help persuade the general people that GM scientists are undertaking all the necessary controls before organisms with a novel genetic maekup are released into the environment.

October

Burgess AJ, Retkute R, Pound MP, Foulkes J, Preston SP, Jensen OE, Pridmore TP, Murchie EH (2015) High-Resolution Three-Dimensional Structural Data Quantify the Impact of Photoinhibition on Long-Term Carbon Gain in Wheat Canopies in the Field. Plant Physiol. 169(2):1192-204 http://dx.doi.org/10.1104/pp.15.00722 Open Access

This collaboration between crop scientists and mathematicians at the University of Nottingham and those at the University of Manchester investigates the importance of photoinhibition in the overall carbon gain of hexaploid wheat. This parameter has proven difficult to accurately assess due to the lack of data about the detailed architecture of the constantly fluctuating wheat canopy in the field. In this study they compare the canopies of three wheat species using a novel method that involves digital 3D reconstruction. This enables measurement of a range of parameters that subsequently allows an accurate assessment of light distribution throughout the canopy. This data feeds into a mathematical model that predicts that photoinhibition alone can contribute to a substantial reduction in carbon gain. The authors conclude that their model allows them to predict that even moderate changes to leaf angle at the upper levels of the wheat canopy can result in a large number of lower leaves attempting to photosynthesize in a severely light-limited location. When this information is allied to improved field phenotyping it might be possible to select for plants that have a maximal leaf arrangement. When these differences in canopy efficiency are scaled up then the changes in carbon gain will prove to be highly significant in absolute terms.

Hendriks PW, Kirkegaard JA, Lilley JM, Gregory PJ, Rebetzke GJ (2015) A tillering inhibition gene influences root-shoot carbon partitioning and pattern of water use to improve wheat productivity in rainfed environments. J Exp Bot. http://dx.doi.org/10.1093/jxb/erv457 Open Access

Peter Gregory (Reading) is the UK lead on this UK-Australian collaboration where researchers investigate the role of the wheat TILLING INHIBITION (TIN) gene during measurements of water-use in rainfed environments. They used near-isogenic lines (NILs) that varied in expression of TIN to show that the root-shoot ratio increased two-fold following tillering in plants with expression of TIN. Following a range of measurements the authors are able to conclude that water use efficiency was increased in TIN-containing lines, resulting in increases in grain yield. Although TIN was associated with increased biomass, the significant influence of both genetics and environment means that the requirement remains for careful assessment of TIN-containing progeny.

Winfield MO, Allen AM, Burridge AJ, Barker GL, Benbow HR, Wilkinson PA, Coghill J, Waterfall C, Davassi A, Scopes G, Pirani A, Webster T, Brew F, Bloor C, King J, West C, Griffiths S, King I, Bentley AR, Edwards KJ (2015) High-density SNP genotyping array for hexaploid wheat and its secondary and tertiary gene pool Plant Biotechnol J. http://dx.doi.org/10.1111/pbi.12485 Open Access

This wheat SNP genotyping project involves researchers from Bristol and Nottingham Universities alongside those from the JIC and NIAB together with industrial assistance from Affymetrix. This highly descriptive study identifies a large number of SNPs useful for subsequent genotyping of hexaploid wheat and for SNP-tracking following introgression from different genetic stocks. Following initial sequencing, markers have been validated using an ultra-high-density genome array. In the spirit of open access, all the sequence information defined by this project is available at:

http://goo.gl/e4oEhs (Putative varietal SNPs from CerealDBs)

http://goo.gl/VdN5NW (Putative SNP probes from CerealDBs)

Harper AL, Trick M, He Z, Clissold L, Fellgett A, Griffiths S, Bancroft I (2015) Genome distribution of differential homoeologue contributions to leaf gene expression in bread wheat. Plant Biotechnol J. http://dx.doi.org/10.1111/pbi.12486 Open Access

Transcript Contribution from B+D genomes. From Plant Biotech journal

Ian Bancroft at the University of York is corresponding author on this work that is a collaboration between his own lab and that of Simon Griffiths at the JIC. They have used de novo expression data together with a new genetic linkage map to develop an ordered set of nonredundant transcripts for each of the three A,B D subgenomes of hexaploid wheat (between 40K-60K unigenes from each sub-genome). An RNAseq dataset obtained from young leaf tissue was mapped to this reference sequence in order to define the relative expression of each homoeologues variant. This showed no dominance for a particular sub-genome but rather the most highly transcribed genes were distributed throughout each sub-genome. Assessment of wider genome areas was able to define regions where one genome was dominant over the other two as well as regions where one genome was repressed compared to the other two. This study offers further insights into the expression relationships between each sub-genome and adds to the other recent methylation data (outlined below) that offers a different insight into the nature of the hexaploid wheat genome.

December

Gardiner LJ, Quinton-Tulloch M, Olohan L, Price J, Hall N, Hall A (2015) A genome-wide survey of DNA methylation in hexaploid wheat Genome Biol 16(1):273 http://dx.doi.org/10.1186/s13059-015-0838-3 Open Access

Anthony Hall leads this University of Liverpool study that analyses the wheat genome by bisulfite sequencing to identify the differential methylation that occurs throughout the three sub-genomes of allohexaploid wheat. The authors correlate sub-genome specific promoter methylation with altered expression from each particular sub-genomes. They also conduct an experiment demonstrating that temperature has an impact on gene expression across all of the sub-genomes. Although there is significant variation between sub-genomes the authors show that patterns of methylation in the D-genome is conserved with regions in its diploid progenitor Aegilops tauschii.

Gordon A, Basler R, Bansept-Basler P,, Fanstone V, Harinarayan L,, Grant PK, Birchmore R, Bayles RA,, Boyd LA, O’Sullivan DM (2015) The identification of QTL controlling ergot sclerotia size in hexaploid wheat implicates a role for the Rht dwarfing alleles Theor Appl Genet. 128(12):2447-60 http://dx.doi.org/10.1007/s00122-015-2599-5

This academic-industry collaboration is led by researchers at NIAB and the University of Reading and tackles an aspect of plant biology that is of great interest to those interested in developing disease resistance wheat varieties. This study looks at infection success of Claviceps purpurea, which is fungal pathogen that infects a range of temperate grasses and is therefore economically important. Claviceps infection produces a sclerotia within the seed and, when ingested, can cause significant illness in mammals. Two varieties of winter hexaploid wheat were identified that showed more resistance to Claviceps so these were crossed to produce a doubled haploid mapping population. Following phenotypic assessment of sclerotia development, QTLs were identified that mapped to reduced height (Rht) loci and therefore indicates that semi-dwarf varieties may display resistance to Claviceps infection.

The High Value Chemicals in Plants (HVCfP) Network in a BBSRC-funded NIBB (Network in Industrial Biotechnology and Bioenergy) that is centered at the University of York. At the end of October, Cambridge Cottage at Kew Gardens hosted the HVCfP Annual Meeting that brought together over 80 interested members of the network to discuss recent research developments and their resulting commercial potential.

The schedule was comprised of invited keynote speakers as well as talks from contributors who have taken advantage of the funding opportunities provided by the network. These include the Proof of Concept (PoC) fund that are worth up to £50K as well as smaller Business Innovative Vouchers (BIV) (previously £5K, now increased to £10K). Funding opportunities are ongoing through the life of the NIBB so please check out funding (https://hvcfp.net/funding/) page for information about previous awards and current open calls.

The meeting kicked off with two keynote talks, firstly from Monique Simmons, deputy director of Kew Science who introduced the wide variety of resources associated with the Botanic Gardens. Over the past year, Kew has undergone a significant reorganization and has a renewed remit for interaction with the scientific community. Perhaps of greatest interest to GARNet readers will be the Kew DNA bank (http://apps.kew.org/dnabank/homepage.html) that contains over 40K samples from around the globe, the majority of which are available to order, either in aliquots suitable for PCR or in larger quantities if necessary.

David Hughes from Syngenta followed up with an excellent talk about the current situation surrounding the debate around Genetically Modified Crops. He gave his thoughts on why this ‘debate’ has gone on for almost 20 years even without any reliable evidence about the negative effects of developed crops. He thought the negative public response could be summed up by three emotions.

Essentialism: is the ‘essence’ of the original organism transferred to the crop

Naturalism: ‘Synthetic’ is evil, ‘natural’ is good.

Disgust: If something is not ‘100% safe’, then it must be bad.

David also advised that the power of narrative is a much more effective technique than the use of potentially bland scientific evidence, which usually falls on deaf ears amongst the public.

The day included two talks from commercial members of the HVCfP network that provided a contrast to the majority academic talks. They included a presentation from James King of Oxford Biotrans (http://oxfordbiotrans.com/), a company that is attempting to exploit biocatalytic routes to develop novel compounds. Their focus is on the use of cytochrome P450s and one of their initial challenges is to enzymatically produce nootkatone, which provides the flavor and scent from grapefruit. They aim to produce this compound via the intermediate Valecene, which can be collected from oranges. The unpredictable grapefruit harvest and the difficulties in isolation of endogeous nookatone mean that Oxford BioTrans suggest that using enzyme catalysis might be a more cost effective method for isolation of the high value product. The other SME that presented was Neem Biotech, which is located in the South Wales Valleys, a place not usually associated with biotechnology. Their major product is Vetrinol, which includes garlic extract and that is used for boosting the bovine immune system.

Cathie Martin discussing Cavity Spot

The remainder of talks featured researchers involved in PoC or BIV projects. These were all rather fascinating (especially for a researcher usually preoccupied by Arabidopsis) as they introduced novel compounds and non-model organisms that were ‘unusual’ to many in the audience. Cathie Martin (JIC) is well known for her work with purple tomatoes but here she presented her attempts to isolate the compound from Mugwort leaf extract that is responsible for reducing cavity root spot disease, which is caused by Pithium species of Oomycete. This work was performed in collaboration with ‘Root Crop Consultancy’ and resulted from an observation that carrots grown in the presence of mugwort were less susceptible to cavity spot disease.

Three talks that described research funded by PoC grants came from Gary Loake (Edinburgh), Luis Mur (Aberystwyth) and Mike Beale (Rothamstead) who each highlighted work aimed at to improve production from organisms already used to produce medicinally useful compounds. Gary Loake is hoping to develop the use of cambial meristematic cells (CMC) to improve paclitaxel biosynthesis from Taxus (Yew) (for review of CMCs http://www.sciencedirect.com/science/article/pii/S1871678415000205). The Taxus CMCs are able to produce plenty of paclitaxel so to improve this aspect they have analysed expression profiles in an attempt to identify the transcription factors that are important in this process. The ultimate aim would be to use synthetic biology approaches to potentially modify the expression of these TFs in order to maximise paclitaxel production.

Aberystwyth University has a strong tradition of research in Avena species (Oat) so Dr Mur is using these resources in order to understand and maximise the production of Avenanthramide, which has well-established medicinal benefits. The HVCfP money has funded the use of HPLC to analyse their germplasm for altered production of Avenanthramide. Broadly they have found that domesticated hexaploid Avena species have higher levels of the compound compared to wild diploid varieties. They are now conducting expression analysis in order to understand the molecular reason for these alterations.

Mike Beale is taking advantage of the National Willow Collection (NWC) to undertake a phytochemical and pharmacological screen in order to identify novel compounds. Willow (Salix) is famously the original source of aspirin so Dr Beale and colleagues are using NMR and MS in an attempt to analyse unpurified extracts from 200 species of Willow. By screening against a battery of cancer cell-lines, fungi and bacteria, they hope to identify extracts that can be further interrogated to find compounds of interest.

The other two talks resulting from BIV-funding were both focused on algae, although with organisms of different sizes! David Bailey from IOTA Pharmaceuticals outlined their plans to exploit the potential of using the macroalgae Sargassum muticum (seaweed) as a production chassis while Paul Knox (Leeds) and John Dodd (AlgaeCytes) described their early work to identify novel polysaccharides found in microalgae.

Matt Davey discussing Living Stones

The meeting ended with a series of flash presentations given by representatives of GARNet, the Liverpool GeneMill, the University of Essex Cell screening facility and finally from Matt Davey (Cambridge). His talk highlighted the incredible potential that is provided by plant species as he is developing commercial relationships to study the UV absorption properties of Living Stone species (Lithops). Initial experiments have shown that direct UV light is very successfully absorbed by Lithops species and this is of interest to the cosmetic industry.

Although much of the research presented in this meeting is in its infancy, the HVCfP Network can be pleased with the process it has made. With another set of PoC and BIV funding recently announced the network is supporting research in a wide range of organisms aimed at producing a varying array of compounds. The network has been providing funding since 2014 so the first set of PoC funding is now coming to an end. The true success of the network will be realised over the next two years, determined by the amount of follow-on funding that can be obtaining to support the research conducted in these seed-projects. We will watch this space with interest….