Seeds are the start and end point for the vast majority of human agriculture. The annual global seed trade is currently valued at over £34 billion, and the production and sale of high quality seeds which germinate uniformly and rapidly underpin this industry. Seeds experience a range of stresses in the field prior to crop establishment. These include low water stress and mechanical impedance from compact soils. Seed vigour refers to the ability of seed to germinate and establish seedlings across a wide range of environmental conditions, and defines the success of crop establishment in the field. This is a key determinant of yield as the absence of a plant leads to no end product to harvest. Improving this trait in crops is a primary goal of the agricultural industry, however the underlying mechanisms of vigour remain poorly understood. The growth of plant cells is a mechanical process driven by internal turgor pressure pushing against the surrounding cell wall. Cells get bigger when the surrounding cell wall is weakened and yields in response to internal turgor. Genes which encode proteins that are secreted to the cell wall and modify its structural composition and strength have been identified. Once such protein is named expansin, and acts to loosen cell wall structures, permitting cell growth. The seed to seedling transition is driven exclusively through cell expansion in the absence of cell divisions. The ability to generate of mechanical force sufficient to counteract external stresses defines the ability of a seedling to establish across a wide range of environmental conditions, and hence be vigorous. Increasing the expression of expansin enables seedling establishment under stress conditions which normally limit this process. Seed vigour may therefore be considered a mechanically driven agronomic trait and the control of expansin expression a target. This project takes an interdisciplinary approach to uncover the genetic factors and mechanical basis of the seed to seedling transition, and seed vigour. We previously identified proteins which represent high confidence candidate regulators of expansin gene expression. Increasing expansin gene expression can increase seed vigour making these genetic targets to enhance seed vigour. These genes will be explored in the model plant system Arabidopsis. These findings will be extended to enhance seed vigour in the crop species Brassica oleracea. Mutations within newly characterized vigour genes will be identified in different Brassica plants. Together with industrial partner Syngenta, the vigour of these new Brassica seeds will be characterized. This will lead to the identification of varieties which can be used directly in breeding programs to enhance seedling establishment, field crop performance and yield. We have previously shown that the size, shape and arrangement of cells can influence the early stages of seed germination in response to growth-promoting gene expression, such as expansin. This observation highlighted the presence of mechanical constraints on plant growth. How these constraints affect the growth of seedlings however remains unknown. Understanding the mechanical basis of the seed to seedling transition is of central importance to understanding the establishment of crops in the field and seed vigour. Using a combination of 3D image analysis and mechanical modelling, the relationship between growth promoting gene expression and seedling growth will be established. In this way the mechanical basis of seedling establishment and seed vigour will be uncovered. Enhancing Brassica seed vigour will increase both crop yields and food security during this period of rapid climate change. The findings in this project may in turn may in turn be extended to other crop species.

To grow crops successfully, the farmer has to establish the correct number of seedlings from the seeds they sow. Too many or too few seedlings can have a devastating effect on the profitability of the crops they produce. There is also an environmental consequence, because chemical crop treatments like insecticides, herbicides and fertilisers are not used efficiently. The soil seedbed can be a very harsh environment in which to germinate and therefore seeds with high vigour are required to consistently provide the same number of seedlings following sowing. However, in practice seed vigour is variable. The problem is, that is it not fully understood what makes seeds vigorous and consequently this characteristic is not included in breeding programmes for new crop cultivars. The environment in which seeds are produced can affect seed vigour and seed companies have been addressing this using a range of basic technologies, but despite this seed vigour remains variable. However, we have shown that differences in seed vigour can also have a genetic basis and so there is a further opportunity for improving seed vigour. In this proposal we aim to gain a greater understanding of what underlies these genetic differences. To do this, we will identify the gene or genes which are responsible for arguably the most important aspect of seed vigour, how fast they germinate. During this work we will gain insight and understanding into how these genes regulate processes within the seed that affect germination and this will help companies develop methods to improve seed vigour. Different plant lines can germinate at different rates, and this may be due to only small differences (sequence differences) within the same gene. By looking at the differences in the sequence of our selected gene in a range of lines which are known to germinate at different rates we aim to see how these differences in sequence are linked to differences in germination rate. A further complication is that some crops contain more than one copy of many genes. It is likely that not all copies of the genes will have the same impact on your trait of interest (e.g. germination rate), and so we must also determine which of the gene copies are significantly affecting germination rate. With this type of information it is possible for plant breeders to breed and select for faster germination (enhanced vigour) in the new cultivars they produce. We will carryout the proposed work in the crop species Brassica oleracea which contains familiar vegetables like cabbage, cauliflower and sprouts, but will also work with oil Seed Rape (Brassica napus) which is an increasingly important arable crop. However, in the long term the work has relevance to many other crops and even ornamentals such as bedding plants.

It has been shown that the isoform of the eukaryotic translation initiation factor 4E (eIF(iso)4E) protein of Arabidopsis binds with the viral protein linked to the genome (VPg) of Turnip mosaic virus (TuMV; a member of the Potyvirus genus) in yeast 2-hybrid experiments. The interaction has also been demonstrated in vivo in brassica and a mutation in the interaction domain of the TuMV VPg completely abolished the interaction. In other examples of recessive plant resistance to potyviruses, it has been shown that certain natural mutations in the eIF(iso)4E or eIF4E genes results in the VPg no longer being able to bind to the proteins and it is assumed that this is the mechanism underlying the resistance, where lack of binding means the viruses cannot translate their RNA in to protein and hence cannot replicate. We have shown that broad-spectrum resistance to TuMV in Brassica rapa is controlled by two genes, one of which is recessive (retr01) and the other dominant (ConTR01). retr01 corresponds to eIF(iso)4E and ConTR01 corresponds to eIF4E. The student will utilise the yeast 2-hybrid system to determine whether the brassica eIF4E and eIF(iso)4E alleles conferring resistance to TuMV bind to the TuMV VPg. He / she will also investigate whether the protein from alleles of these genes at other loci, particularly the other loci of the Syngenta lines in to which the resistance is being introgressed, interact with the TuMV VPg. If as in other plant - potyvirus interactions, the presence or absence of interaction correlates with susceptibility and resistance respectively, the yeast 2-hybrid system and sequencing will be used to search for novel / superior alleles of eIF4E and eIF(iso)4E in a range of different brassica species with a view to identifying new opportunities to produce sources of resistance in these different brassica types. We have a large collection of over 200 TuMV isolates obtained from different plant species growing in many different parts of the world. Many of the isolates have been characterised in terms of their pathotype, serotype and genetic group. The VPg will be cloned from TuMV isolates from distinctly different genetic groups and their interaction with eIF4E and eIF(iso)4E alleles conferring resistance to TuMV will be determined in the yeast 2-hybrid system in order to investigate the potential durability of such resistance. Syngenta Netherlands was selected as the partner for the project as it is Syngenta's site responsible for vegetable breeding and has all the infrastructure and expertise to compliment the academic partner and take the project and materials forward for commercialisation, whilst providing the student with excellent industrial experience. The student will work with Syngenta Netherlands on the introgression of the broad-spectrum resistance to TuMV into commercial varieties of B. rapa using markers for the alleles of eIF(iso)4E and eIF4E that confer resistance to TuMV. They will verify the ability of the markers to discriminate the alleles conferring resistance from those alleles conferring susceptibility. He / she will also amplify and sequence the other copies of eIF4E and eIF(iso)4E present at other loci in the B. rapa lines in to which the resistance will be introgressed in order to determine whether they are likely to interfere with the resistance. The student will also phenotype plant lines produced during introgression of the resistance in to commercial B. rapa types, to determine whether they are resistant or susceptible to TuMV and thereby verify genotype determined using the markers. The susceptibility / resistance status of any brassica lines possessing potentially superior alleles of eIF4E and / or eIF(iso)4E will also be determined. With a four year studentship, there is sufficient time and flexibility for the student to develop their own ideas and approaches and pursue complimentary aspects to the project whilst achieving the goals outlined above.

The virus called turnip mosaic virus (TuMV) is an important pathogen infecting many crop plant types, reducing yields and making them unmarketable. In order to reproduce, the virus has to use certain proteins in plants. Without these proteins it cannot reproduce. One such plant protein is called the eukaryotic initiation factor iso4E [eIF(iso)4E]. We have shown that in Chinese cabbage (Brassica rapa sub-species pekinensis) which has three copies of eIF(iso)4E, if one of the copies is missing, the virus cannot reproduce, but the plant is unaffected. Chinese cabbage varieties with this resistance (that was identified and characterised at the University of Warwick) are currently being developed by an international seed company (Syngenta). Chinese cabbage is the most important vegetable brassica crop worldwide. We have been unable to identify any resistance to TuMV in the related and important plant species Brassica oleracea, which includes broccoli, cabbage, cauliflower, kale, Brussels sprouts etc. In collaboration between the Elizabeth Creak Horticultural Technology Centre and the Plant Virology Group at the University of Warwick, NIAB and the commercial plant breeding company Syngenta, we aim to knock out the copy of eIF(iso)4E that TuMV needs in order to reproduce in B. oleracea utilising gene editing technology, thereby establishing a technique to rapidly develop virus-resistant varieties of the different B. oleracea types. In collaboration with the commercial seed company Syngenta, we also aim to move the copy of eIF(iso)4E that the virus cannot use from Chinese cabbage into B. oleracea by conventional crossing, in order to develop virus-resistant plants by this alternative route.

The evolution of insect resistance to insecticides represents a growing threat to the sustainable control of many important insect crop pests and disease vectors, threatening global food security and human health. To effectively combat resistance, it is critically important to understand its underlying genomic architecture, including the genetic variants underpinning resistance and their affect on phenotype. Advances in genome sequencing technology over the last two decades have greatly facilitated investigation of the genetic basis of insecticide resistance. However, key knowledge gaps remain on the type and number of genetic variants affecting resistance and their relative contribution to phenotype. Many of these deficits in understanding relate to the current paradigm of using a single reference genome as the starting point for most genomic analyses. This approach means that certain types of genetic variation not present in the reference typically remain undiscovered. This problem is particularly acute for structural variation (SV) encompassing presence-absence variation (PAV), copy number variation (CNV) and chromosomal rearrangements, which, as a consequence, have been referred to as the 'dark matter' of the genome. The failure to effectively characterise SV is important, as in humans SVs have been shown to affect more of the genome per nucleotide change than any other class of sequence variant. Furthermore, research by ourselves and others has provided clear evidence that SV can be a key source of genetic variation in the evolution of insecticide resistance. The overarching objective of this project is to develop a new paradigm for the genomic analysis of adaptive traits such as insecticide resistance in pest insects. We will leverage recent advances in sequencing technology, in combination with an exceptional biological resource comprising a living library of globally sampled clones of the damaging aphid crop pest Myzus persicae, to assemble the pangenome, the collection of all the DNA sequences that occur in a species, of this pest. This unprecedented resource will allow us to characterise the complete spectrum of genetic variation in a global crop pest species for the first time, and address multiple key knowledge gaps on the insect pangenome and the role of SV in adaptive evolution. These include understanding the percentage of the pangenome that is structurally variant in pest populations and how this differs across different SV types, how often and how many SVs lead to changes in gene function or expression, and crucially, their overall relevance and contribution to key phenotypic traits such as insecticide resistance. Our analyses will focus on three types of SV that have been frequently implicated in insecticide resistance, comprising CNV, PAV and chromosomal rearrangements. We will then test for association between the full spectrum of genetic variation identified in our aphid clone library and insecticide resistance. Finally, we will examine the impact of different types of SVs on gene expression and gene function, and validate the role of a selection of candidate SVs in resistance using functional approaches. Together, these analyses will allow us to fundamentally and systematically interrogate the genetic determinants of insecticide resistance in a way that has never been previously possible. The knowledge and tools generated in this project will provide both fundamental advances in our understanding of the genetic variation that provides the substrate for natural selection in insects, and powerful resources to develop strategies for the sustainable control of highly damaging, globally distributed crop pests.