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Vegetable Genetic Improvement Network for leafy and field vegetable 2 - CH0101

Horticulture is a highly significant economic sector in the UK with a production value in 2012 of approximately £3.0 billon. The UK is well suited to growing vegetables, but currently only supplies 60% of UK requirements and exports less than 10% of home production. Our Ministers have highlighted the potential for growth in this sector, but vegetable farmers face severe challenges in growing the high quality produce required by the market economically.
The future success of this industry rests in the possibility of introducing new varieties that are tailored to UK market requirements as well as more resilient to unpredictable weather patterns and/or resistant to pests, diseases and other threats. This project is the continuation of two previous Defra projects (IF0157 and IF0158), which developed varieties that are more adaptable to variable weather conditions (carrot), pest (cabbage) and disease resistant (onion and lettuce) and abiotic stresses tolerant (brassicas).
The previous projects have made a difference, but there is much more to be done to support and increase the sustainable production of leafy vegetables, carrot and onion through pre-breeding, which is based on the development of genetic resources to understand and control key traits. The new phase of the project aims to create a set of plants tailored to enhance British horticulture competitiveness and to help managing risks and contingencies in the UK's changing rural economy and environment. This addresses a continued market failure caused by the fragmented nature of the industry, where no player is big enough on its own to want to take the financial risk of R&D projects of this nature.
The new phase builds on and uses the existing strategic network of stakeholders encompassing the main academic and industrial interests (including HDC), to identify the traits that should be priorities and to provide a route for market delivery of R&D outcomes. This project will also utilise the legacy of Link (industry partnership) collaboration that Defra has supported. The novel traits taken forward in this project will be the ones highlighted by stakeholders as valuable for the industry and UK market demands as well as the ones with potential to be uptaken in new cross government strategies such as TSB-SAFIP and Agri-tech.

1a. Seed supply: Future projects and collaborations will require seed of the DFFS lines to be available and bulking these is costly and time-consuming. This is a continuation of the Care and Maintenance funding April to August 2013.
• Brassica: We will establish up to three mature plants per line of the B. oleracea and wild C genome species DFFS DH lines with low seed levels (85 lines with less than 100 seeds) for seed multiplication during 2014/2015 (this activity will extend beyond September 2014). We will also produce seed for further microspore-derived DH plants generated in VeGIN that are currently growing but have not set seed yet (~70 plants), but will make a valuable contribution to the total number of DH lines available for the community.
• Lettuce: good seed stocks were generated for all lines in VeGIN, further regeneration is currently not required.
• Onion: stocks of seeds for half-sib families from several lines are running low and hence further diversity set seed production is required. Bulbs will be produced for selected half sib families from each of the 96 lines to be harvested in autumn 2014 with the aim that seed can be regenerated from flower production in 2015. Short day onions do not produce good bulbs under natural UK conditions. 10 plants of 10 lines of SD onions from the diversity sets will be grown in 12 h days in the Phytobiology Facility to obtain bulbs that will support flowering and seed production.
• Carrot: Fifteen carrot lines have low seed stock levels. We will sow seed to obtain roots for these lines for seed production in 2015.

1b. Refining Diversity Sets: In VeGIN, diversity sets of approximately 100 lines were used for trait screening and SNP analysis. A diversity set reduced in size without significantly reducing the overall range of genetic variation would lead to more cost-effective screening, including for multiple traits, both internally and externally. SNP and DArT marker data obtained in VeGIN will be used to analyse the genetic structure of the diversity sets with the aim of removing significant redundancy and defining a reduced set for future work. Existing data on trait screening in VeGIN will act as a validation check on this approach. For the Brassica C genome DFFS this will involve identifying the minimum set of DH lines for each of the 53 crosses that maximize representation of the donor wild species line.

1c. Expanding diversity set information:
• BolDFFS: Under BBSRC funding to a Lola led by Ian Bancroft, University of York, sequence analysis of a large population of BolDFFS is being generated in a collaborative project with Warwick. This will be very informative to expand our knowledge of the allele variation in the B oleracea diversity set.
• C genome wild species DFFS: Although 140 lines were genotyped using approx. 1500 KASP markers, the number of markers that were informative in each of the 53 DHSL150 x wild species crosses that DH lines have been derived from was in the range 100-500. Unfortunately, we find that this does not provide sufficiently dense information to fully resolve the locations of the wild species introgressions in the DHSL150 recurrent background. The most cost-effective way to address this is to use genotype by sequencing. We expect this to provide several thousand polymorphic makers per cross and give a considerably more detailed understanding of the introgressions. We will establish the methodology for this technique at Warwick and use it to genotype the majority of the DH lines (up to 235) and the 53 parent lines they were derived from as well as some additional valuable mapping lines. We will use the data to define the wild species introgressed segments for all the DH lines. This data will also deliver to the task 1b.
• Genotyping onion diversity set: genotyping of the mother plant from one half-sib family from each of the 96 onion lines has already been done using a KASP assay for 892 SNPs. Further genotyping is also required for the other 9 mother plants of selected half-sib families to assess levels of polymorphism, and in particular to compare this for mother plants derived from F1 hybrids with those from conventional breeding lines.
• Genotyping analysis: The original genotyping data for single mother plant from each of the 96 lines needs to be analysed to determine which SNPs are polymorphic and hence useful for distinguishing different onion lines, the phylogenetic relationships between different lines and also any relationship between the SNP data and the traits that have been assessed so far (Fusarium resistance, seed vigour and response to mycorrhiza). The SNP analysis will allow us to identify a maximally informative subset of lines of the diversity set by excluding those which are similar to each other and in the same phylogenetic clade. This will help reduce the resource required to carry out future trait work while maintaining an extensive diversity.

1d. Expanding value of the B. oleracea integrated map
Having common markers across several populations should allow considerable refinement of QTL identified in each individual population. Oil content within the oleracea DFFS has been shown to be extremely diverse and we will use this as an easily quantifiable trait to test the integrated map. We have previously mapped QTL in the AG population for key enzymes in the oil synthesis pathway though mapping the amounts of individual fatty acids. Fatty acid analysis will be carried out on the MG and TB populations (100 lines per population). Mapping of the oil traits will then be carried out using the new integrated map and data compared to the results obtained with each mapping population alone to indicate the added value of the integrated map.

Milestone 1a.1: Establish mature plants for seed regeneration of C genome DFFS DH lines with low seed stocks during 2014/2015 (Sept 2014 and ongoing).
Milestone 1a.2: Raise transplants from half-sib families for 96 onion lines (20 plants / line), plant out and harvest bulbs. (Sept 2014)
Milestone 1b.1: Refine diversity sets, selection of reduced sets, maximizing diversity (March 2014)
Milestone 1c.1: Genotyping of wild species Brassica DH to improve resolution of introgressed genome segments (Sept 2014)
Milestone 1c.2: KASP genotyping analysis of up to 96 mother plants from selected onion lines. (Sept 2014)
Milestone 1c.3: Analysis of SNP data from approx. 192 DNA samples from mother plants and half sib families of selected onion lines. (Sept 2014)
Milestone 1d.x: Refine the B oleracea genetic map using trait data for all 3 populations (March 2014)


Screening the diversity sets in the first VeGIN project identified lines that carry beneficial phenotypic variation in traits of potential value to improvement programmes. An important objective in the first year will be to safeguard these lines through seed production and / or making crosses to facilitate the future transfer of the trait to commercial material, supported where possible with linked genetic markers.
2a: Resistance to Nasonovia ribisnigri in lettuce (Warwick)
Currant-lettuce aphid (Nasonovia ribisnigri) is the most important pest aphid of lettuce crops in temperate regions. Host-plant resistance in lettuce to N. ribisnigri has already been exploited effectively, but in the last few years this single-gene, complete resistance has failed as a result of selection for strains of N. ribisnigri that can overcome the resistance mechanism. We have laboratory cultures of one wild type and two independently isolated resistance-breaking (Rb) strains at Warwick and in VeGIN we identified several wild lettuce species and a cultivated lettuce accession that showed significant resistance to the Rb strain. To develop these resistance resources for subsequent project proposals we will perform initial crosses to start the production of segregating populations. In addition, it is important to understand the genetic basis of how the aphid overcomes the lettuce resistance gene. This knowledge will help inform how to manage the deployment of these resistance loci.
Proposed work
• Perform crosses between the resistant cultivated lettuce line and a susceptible line (selected to maximize the number of traits that also segregate in the crosses).
• Attempt to cross one of the wild species resistant lines (L. virosa) to a susceptible line.
• If cross is successful - Initiate selfing from the F1 plants for F2 seed generation after Sept 2014.
• For the wild species Rb resistance source perform crosses to the original Nr resistance accession to produce F1 seed, with the aim of examining allelism between the two resistance loci in the F2 generation in subsequent work.
• Perform transcriptome sequencing of up to three Rb and three WT aphid strains to identify expressed genes specific to the Rb aphids. Although there is currently no genomic information available for N. ribisnigri, the genome of the closely related Pea aphid (Acyrthosiphon pisum) has recently been published and will facilitate analysis of the sequence data. This work will complement ongoing work at Warwick where we are attempting to cross the WT and Rb strains to generate resources to map the Rb locus (or loci) in the aphid genome.

Milestone 2a.1: Obtain transcriptome sequence for aphid lines (March 2014)
Milestone 2a.2: Perform crosses between lettuce lines resistant and susceptible to Nasonovia (Sept 2014)

2b: Diamond back moth resistance in Brassica (Warwick)
The diamond-back moth (DBM) (Plutella xylostella) is one of the most important pests of cruciferous crops worldwide. Its global distribution is currently restricted by its inability to over-winter in cooler climates such as northern Europe. It is already a major migratory pest in the UK in warm summers, and caused significant problems in eastern England/Scotland in 2013. Climate projections for the UK suggest that warmer winter months may allow over-wintering which, in combination with short generation times in warmer summer conditions, will rapidly increase the threat from this species. Diamond-back moth is difficult to control with insecticides (due mainly to selection for insecticide resistance) and it is imperative to find sources of resistance that can be incorporated into breeding programmes to produce varieties for general release over the next decade. In VeGIN we identified potential sources of resistance to DBM in our DFFS lines. These will be analysed further by the generation of new mapping populations followed by SNP marker analyses and QTL mapping (in subsequent projects).
Proposed work
• Crosses will be made between parental lines that exhibit clear differences in resistance to DBM in preparation for subsequent genetic analysis. For one line we already have F1 seed and will grow this up to generate segregating F2 seed (completion after Sept 2014).
Milestone 2b.1: Perform crosses between Brassica lines resistant and susceptible for DBM. (Sept 2014)


The genetically fixed Diversity sets should be exploited to identify novel sources of alleles for beneficial traits. We propose to undertake preliminary work to develop methods for this purpose. This will enable follow on projects exploiting VeGIN resources to be initiated without a long period of preparatory work to establish material and methods. Specifically, we propose to develop screening methods for the following valuable traits.
3a. Whitefly Resistance in Brassica (Warwick)
Cabbage whitefly (Aleyrodes proletella) is an increasingly important pest of Brassica crops and particularly those such as Brussels sprout and kale that are eaten during the winter. Populations of whitefly are difficult to control with insecticides, some have developed resistance to widely used pyrethroid insecticides and, although there is now one relatively new effective active ingredient available, there are limits on the number of permitted applications, which means that it cannot be used to provide season-long control. In addition, reliance on one insecticide increases the risk of the cabbage whitefly evolving resistance to it. At Warwick we have established rearing methods and maintain a laboratory culture of this pest.
Proposed work
• Cabbage white fly will be reared to obtain large numbers of adults at a defined stage of development to be used in the screens.
• A phenotyping assay for cabbage white fly resistance will be refined using a small number of key lines which will include diverse members of the DFFS.
Milestone 3a.1 – Develop methods for whitefly resistance assessment (Sept 2014)

3b. Abiotic stress tolerance in Brassica and lettuce (Warwick and Harper Adams)
Improved tolerance to combined abiotic stresses is a key target for future successful crop production under scenarios of sustainable intensification (with less input) and unpredictable weather patterns; this is particularly important for field vegetables where maintenance of quality and growth rate is paramount to marketability We will develop methods to assess and quantify abiotic stress response in brassica and lettuce.
Brassica. We will develop methodology for phenotyping genotypes ( e.g. DDFS lines, mapping populations, commercial cvs) for tolerance to abiotic stress. A current PhD student is investigating methods for inducing stress in Brassica and has identified levels of heat, cold and salt that confer an obvious but not extreme change in growth patterns. Gene expression analysis of plants following these treatments will be carried out to identify stress related gene expression, overlaps between stresses and source potential Arabidopsis stress gene orthologues.
We will extend this analysis to develop methods for quantification of phenotypes and define conditions and timings that will allow assessment of subtle differences between lines and investigate the longevity of such effects. This will be done in glasshouse and CE at HAU and Warwick initially using commercial cvs but then validated using a subset (10?) DFFS lines selected to represent significant diversity, choice of lines will also be informed by seed availability. Treated plants will be measured for growth characteristics to determine effects of the stress.
Development of a robust screening methodology and demonstration of variation for a general stress tolerance will support an application for funding for further work from the BBSRC HAPI programme to investigate variation in the DFFS collections to identify lines for future genetic analysis using a combined QTL and candidate gene approach.
Proposed work
• Develop robust growth assays for quantification of responses of brassicas to several different environmental stresses including salt treatment, heat and cold.
• RNAseq analysis will be carried out on Brassica control and stressed plants (3 different stresses) to identify stress related gene expression and compare to Arabidopsis gene expression.
Milestone 3b.1 – Develop methods for stress phenotyping in Brassica, test on DFFS (Sept 2014)
Milestone 3b.2 - Transcriptome analysis of stress treated Brassica (March 2014)

Lettuce: We will investigate the stress response of ‘tip burn’ in lettuce. These are areas of necrosis around the growing edge of a lettuce leaf. This phenomenon reduces quality of lettuce crops and can lead to a crop being ploughed in if severe. Once tipburn has occurred bacterial breakdown often follows producing unacceptable sliminess with subsequent crop rejection, waste and loss to growers. The underlying physiology of tip burn is not well studied but has been shown to be correlated with nutrition, especially calcium. Calcium is translocated to the growing margin within the xylem so environmental stresses that limit translocation are associated with tip scorch. Field conditions that reportedly lead to tip burn are low light, overcast, and cool temperatures with high relative humidity as may be experienced in early and late seasons of crop production. Symptoms are particularly marked where there are large fluctuations in temperature and an increase in the frequency of extreme weather events is likely to increase the incidence of tip burn. Lettuce tip burn also occurs in hydroponic production systems and has been associated with rapid growth rates outstripping nutrient supply in high RH environments. The role of light levels is not clear.
Genotypic differences for the incidence of tip burn have been reported, with some iceberg varieties being reportedly more prone to tip burn. Different crop types also differ with Cos/Romaine types generally more prone to tip burn. There is therefore an opportunity to use a breeding approach to reduce tip burn sensitivity. However, the unpredictability of the problem means it is difficult to carry out field selection. A marker selection approach would be beneficial but lettuce breeders are hindered in their ability to do this by a lack of knowledge of the underlying genetics. A QTL analysis of tip burn insensitivity would provide molecular markers and develop a better understanding of the underlying genetics of the disorder facilitating the breeding of improved varieties for UK lettuce growers. However, the prerequisite initial step in this approach is the development of a robust screen for tip burn insensitivity.
We will develop methodology for a reliable screen for tip burn in lettuce utilising a hydroponic system allowing year round study of the symptoms. The screening methodology will then be available for future work to screen the VeGIN lettuce diversity set for sources of resistance and to work with UK breeding company partners in an application to the AgriTech catalyst fund.
Proposed work:
To develop a screening methodology to identify tolerance to tip burn
• We will use a minimum of 3 genotypes – Saladin and Iceberg (parents of the VeGIN mapping population) and an industry advised variety.
• Initial work in glasshouse during early summer to find conditions that consistently induce tip burn in a hydroponic system.
• Validate findings in a polytunnel in autumn to develop an assay suited to screening up to 100 genotypes
Milestone 3b.3 – Develop method for tipburn assessment (Sept 2014)

3c. Cabbage Root Fly in Brassica (Warwick)
Cabbage root fly (Delia radicum) is a common and serious pest of Brassica crops in northern Europe and North America. The adult stage is hard to control (fine mesh netting is used on root crops) and the larvae are susceptible to a small number of insecticides. The most widely used insecticide is soon to be withdrawn. Treatments are applied prophylactically. At Warwick we maintain a laboratory culture of cabbage root fly, and have developed plant phenotyping methodology.
Proposed work
• 50 crop type Brassica lines, 50 wild species parents and 100 wild species DH lines will be screened for resistance to cabbage root fly using the established assay.
• Crosses will be performed between any resistant lines and a standard susceptible line to generate segregating material to map the resistance.
Milestone 3c.1 – Screen subset of Brassica DFFS for cabbage root fly resistance (March 2014)
Milestone 3c.2 – Establish mature plants to initiate crosses between Brassica lines resistant and susceptible to cabbage root fly (Sept 2014 and ongoing)

3d. Sclerotinia resistance in Lettuce (Warwick)
The fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea have been identified by the VeGIN stakeholder group as having significant economic impact on lettuce production. Both are necrotrophic pathogens with wide host ranges, are closely related and have similar infection strategies. S. sclerotiorum can result in up to 50% crop loss pre-harvest while B. cinerea can be a particular problem post-harvest. Chemical control is problematic as few actives are available, the number of sprays is restricted and timing is difficult. Moreover, the fungicides are medium to high risk for development of resistance. The development of durable resistance in the crop is a more sustainable solution, but this has been an intransigent problem for breeders and hence there are no resistant cultivars available. Given the similarity between the two pathogens it is possible that similar resistance mechanisms exist. A leaf-based assay was developed for B. cinerea at Warwick and a preliminary screen of 26 DFS lines using a single isolate identified differences in resistance. The most resistant lines from this screen have been crossed to Saladin and Iceberg (genetic map parents) to initiate new mapping populations for disease resistance. We propose to screen the same subset of the lettuce DFS to identify sources of resistance to S. sclerotiorum following the development of a similar robust high-throughput screen. This will provide evidence that the same lettuce lines may confer resistance to both Botrytis and Sclerotinia and lay the groundwork for the screening of the full lettuce DFS lines for resistance to both pathogens in the future.
Knowledge of lettuce defence responses to Sclerotinia and Botrytis will enhance our ability to identify candidate genes underlying potential QTLs for resistance and drive novel strategies for durable disease resistance using systems biology approaches (translating methodologies from our work in Arabidopsis with B. cinerea). We therefore propose to profile the lettuce transcriptome following infection with S. sclerotiorum. This data set will be used as a basis for funding proposals in collaboration with industry. We are currently developing a HAPI proposal with the support of Tozer and HDC to build on the lettuce data already collected and the genetic resources developed at Warwick. Tozer will generate F2 seed for new mapping populations this year.
Milestone 3d.1: Develop a leaf assay for S. sclerotiorum resistance (optimising spore concentration) using a standard lettuce line (Saladin) and single preliminary screen of selected DFS lines (March 2014)
Milestone 3d.2: Transcriptome profiling of lettuce leaves infected with S. sclerotiorum (March 2014)
Milestone 3d.3: Produce spore inoculum of S. sclerotiorum for standard isolates for DFS screening (July 2014)
Milestone 3d.4: Screen 26 lettuce DFS lines for resistance to S. sclerotiorum (September 2014).
Milestone 3d.5: Generate F2 seed from crosses of Saladin/Iceberg to lines showing most resistance to B. cinerea

3e. Downy mildew in onion (Peronospora destructor) resistance: (Warwick)
Downy mildew is one of the major foliar diseases of onion where fungicides are intensively targeted and hence sources of resistance would be beneficial for a more integrated approach to control in the future. We have previous experience with this pathogen and contemporary isolates would need to be collected and assessed for pathogenicity in preliminary experiments. As P. destructor is an obligate pathogen, specific resources are required to maintain multiple isolates.
Milestone 3e.1: collect, characterise and store contemporary isolates of P. destructor (August 2014)
Milestone: 3e.2: develop and optimise inoculation method on onion for future screening of onion diversity set. (September 2104)

3f. Susceptibility to cavity spot (Pythium violae) and other traits in carrot (Warwick)
Cavity spot caused by Pythium violae has been identified by the VeGIN stakeholder group as one of the major disease problems in carrots. We will carry out a preliminary trial to assess current inoculum levels at Wellesbourne in a field previously known to have high levels of P. violae with a standard susceptible commercial variety (Nairobi). We will also compare disease levels in this standard cultivar with at least two additional commercial cultivars with some reported resistance to P. violae as well as some selected lines from the carrot diversity set which are being assessed for additional traits. This will lay the groundwork for assessing the full carrot diversity set for cavity spot resistance in the future. A field trial of 80 lines, including selected lines from the Carrot Diversity Set and current commercial varieties, will be carried out to investigate whether any differences exist in reaction to P. violae in terms of disease symptoms. Growing most of the Diversity Set lines in the field will also allow phenotyping of root traits and flowering time to obtain phenotype data in an additional environment for extra replication of existing datasets. Data collected previously in 2011 will enable selection of the most informative material to trial.
Milestone 3f.1: carry out field experiment to assess levels of cavity spot at Wellesbourne and assess different carrot cultivars and lines from the diversity set for resistance (September 2014).
Milestone 3f.2: assess diversity set lines for other traits including root morphology and flowering time (September 2014)


Industrial engagement and interaction is key for us to continue to ensure the sustainability of the horticulture sector within the UK despite foreign competition and to grow the rural economy through enabling the industry to apply technological developments and providing access to enhanced pre-breeding material. The proposed collaboration between Warwick and Harper Adams will enable us to strengthen the initial Network. We will hold a stakeholder event towards the end of the 7 month funded time and seek to widen participation through active dialogue with the newly formed Horticulture Innovation Partnership (HIP). It is important to maintain stakeholder involvement with defining the aims of VeGIN and to obtain stakeholder feedback and guidance on the activities we are proposing (e.g. what are the most important problems in their specific crops, how can developments in production or retail trends help defining targets for crop improvement).
Milestone 4.1: VeGIN website will be updated and maintained
Milestone 4.2: A VeGIN stakeholder meeting will be held in September 2014
Project Documents
• EVID4 - Final project report : Evid4 CH0101 Final report   (1359k)
Time-Scale and Cost
From: 2014

To: 2014

Cost: £296,805
Contractor / Funded Organisations
University of Warwick
Biotech-non GM              
Disease Control