Final Report Summary - MAX-CROP (Max-imising the potential of CROP researchers)
Max-CROP has trained four early career stage researchers (ESRs) to the level of a PhD degree in areas of science that impact on crop production. The training provided has maximised the potential of this next-generation of crop researchers, producing individuals that understand the problems facing the agricultural industry, including crop breeding, agronomy and production, while being able to apply advanced scientific understanding and modern technologies to solve these problems. The ESRs have received Max-CROP training in crop genomics and biology, crop pathology and biostatistics, and entrepreneurship at the Cambridge University, Judge Business School, while complementary and transferable skills has been provided by Wageningen University (WU). They have participated in many stakeholder and pubic engagement activities hosted by NIAB Trading. Along with the research training undertaken for their PhDs, this training will enable them to make significant contributions to crop production in their subsequent careers. The research projects have covered four crops, wheat, barley, oil seed rape and potato. Max-CROP has made careers in agriculture an attractive choice for a new generation of researchers, opening up new opportunities and improving career prospects for the ESRs. Max-CROP has also established new collaborations between NIAB Trading, Cambridge, UK and the academic partner, WU, Netherlands.
ESR1 has confirmed that two barley receptor-like kinases identified in transient assays confer non-host resistance (NHR) in barley to the wheat-adapted pathogen of powdery mildew, Blumeria graminis f. sp. tritici. ESR1 has also narrowed down a region on the barley genome that confers NHR to the wheat leaf rust pathogen, Puccinia triticina, to a genetic region of 0.6cM. ESR2 has identified mutations within the α- γ- and or ω-gliadins in wheat cvs Paragon and Fielder. They have developed protein and quantitative DNA assays by which to identify these mutations. ESR4 has confirmed that the UK Verticillium longisporum population is genetically more diverse than other V. longisporum populations studied. Field assessment of oil seed rape varieties to V. longisporum identified variation in resistance. However, the impact of V. longisporum on yield was inconsistent. ESR 4 has produced a genome sequence of V. longisporum which shows that V. longisporum has a mosaic genome structure due to recombination between parental chromosome sets, and that V. longisporum genes have generally a more divergent evolution than genes from non-hybrid Verticillium species. ESR4 has also shown that the parental mitochondrial genomes have recombined, resulting in a mosaic structure of the V. longisporum mitochondrial genome. ESR5 has shown that only large-spored Alternaria species; A. solani, A. grandis, A. linariae (syn. A. tomatophyla) and A. protenta, cause early-blight symptoms on potato. DArT-Seq SNP markers were used to identify individual Alternaria isolates, showing the incursion of isolates into experimental field trials, and that certain isolates were able to persist over years.
ESR Project 1: “Would non-host resistance (NHR) in barley prove a source of durable, race-nonspecific resistance for wheat”
ESR1 consisted of two parts: (i) Fine mapping of a QTL region in the barley variety Vada that conferred resistance to four heterologous rusts; Puccinia hordei murini (Phm), P. hordei secalini (Phs), P. triticina (Pt) and P. graminis lolii (Pgl), and (ii) Cloning of two barley receptor-like kinases (RLK) that conferred NHR to the wheat-adapted pathogen, Blumeria graminis f. sp. tritici (Bgt), followed by over-expression of these barley RLKs in the wheat. Through repeated rounds of selfing recombinant plants were obtained that allowed fine mapping of three QTLs; sub-region 1 (Phm and Phs NHR), sub-region 2 (Pt NHR), and sub-region 3 (Pgl NHR). The intervals identified from previous research for Phm and Pgl could not been confirmed, therefore, subsequent work focused only of sub-region Pt. The Pt sub-region has been mapped to a genetic distance of 0.6cM representing 20 Mb on the barley Morex genome reference. Two barley RLKs have been cloned for variety Morex and over-expressed in the wheat variety Fielder. The NHR phenotype has been confirmed in barley, variety Golden Promise, by siRNA. However, despite extensive testing, the host resistance seen in transient over-expression studies in the wheat variety Kanzler could not be duplicated in the Fielder genetic background.
ESR Project 2: “Development of wheat varieties with reduced gluten toxicity, increasing safety for celiac disease patients”
In bread wheat the three gliadin protein families, α, β and γ harbour immunogenic epitopes which can trigger Coeliac disease (CD) in genetically predisposed humans. In this project, we used two mutagenesis approach to modify immunogenic gliadin epitopes: gamma-irradiation TILLING and CRISPR/Cas9 gene editing. A gamma-irradiated population in the wheat variety Paragon was screened for changes in the gliadin protein profiles. The ESR also performed targeted mutagenesis against the α- and γ-gliadin genes using CRISPR/Cas9, designing five guide RNA, all transformed into the wheat variety Fielder. Four different methods were optimised and implemented to screen gliadin differences in potential mutant plants compared to the original wild type (WT) plants. Paragon gamma-irradiated mutants, and Fielder-CRISPR lines, were found with α- γ- and or ω-gliadins proteins missing or altered compared to their respective WT. A reduction in α-gliadin gene copy number was found in both Paragon mutant and Fielder-CRISPR lines. To identify the mutations in gliadin genomic sequences we have developed GlutEnSeq, an exome capture system that enriches for gluten genes.
ESR Project 4: “Population and pathogenicity dynamics of the Brassica pathogen Verticillium longisporum”
A global V. longisporum collection, comprised of 88 isolates from nine different countries was constructed. A population study revealed that the UK population was genetically more diverse than V. longisporum populations from different countries. Consequently, the recent emergence of Verticillium stem striping disease on oilseed rape in the UK is likely caused by a longer established V. longisporum population in the UK (Depotter et al. 2017a). The virulence of UK V. longisporum isolates was therefore accessed, however UK isolates were as virulent on Brassica host as previously characterized V. longisporum isolates from the A1/D1 lineage (Depotter et al. 2017b). Field assessment of oil seed rape varieties to V. longisporum identified variation in resistance. However, the impact of V. longisporum on yield was inconsistent (Depotter et al. 2018a).
ESR 4 sequenced the genomes of two V. longisporum isolates (Depotter et al. 2017a). It was found that V. longisporum has a mosaic genome structure due to recombination between parental chromosome sets (Depotter et al. 2018b), and that V. longisporum genes have generally a more divergent evolution than genes from non-hybrid Verticillium species. Expression patterns of the two V. longisporum sub-genomes show remarkable similarity, in particular expression levels of genes encoding secreted proteins homogenize upon plant colonization. The parental mitochondrial genomes recombined, resulting a mosaic structure of the V. longisporum mitochondrial genome (Depotter et al. 2018b).
ESR Project 5: “Emerging plant diseases: reducing the impact of Alternaria species on potato production”
Different Alternaria spp. were examined for their ability to cause early-blight of potato, focusing on detached leaf and whole plant field assays. The novelty of this work is that it systematically tested all species of Alternaria that have been “suggested” to cause early-blight symptoms on potato plants. It was found that small-spored Alternaria species are significantly different from the large-spored species, with small-spored species such as A. alternata (syn A. tenuissima), A. infectoria, and A. arborescens being unable to cause early-blight symptoms in our tests. We also investigate the possibility that small spore species may interact with large-spored species; A. solani, A. grandis, A. linariae (syn. A. tomatophyla) and A. protenta. However, the presence of small spore species did not enhance infection by large spores species, their presence in an Early-Blight epidemic having no effect on disease progress. This has profound implications on disease monitoring and control.
To gain insights into the Alternaria inoculum flow samples were collected from fields showing signs of early blight infection, fields up to 1.3 Km away and fields were early blight was found in previous years. The UK potato growers showed a lot of interest in this project, and supported the work by providing samples from their own potato fields from across the UK. Alternaria isolates from the Westerdijk Fungal Biodiversity Centre in the Netherlands were also included, and analysed along with the isolates collected in the UK. DArT-Seq DNA SNP markers were used to analyses these isolates. Incursion of isolates into experimental field trials was observed, while certain isolates were able to persist over years.
Functional Structural Plant Modelling is being used to answer questions such as: (1) To what extent can early blight affect the yield of potato crops? (2) At what point in the potato cropping cycle does Alternaria have its biggest effect? Data has been collected on disease progress from 3 years of field experiments, and this is being used to fit a model of disease progress on three different canopy levels of the potato crop.
The Max-CROP project website can be found at http://www.niab.com/max-crop
ESR1 has confirmed that two barley receptor-like kinases identified in transient assays confer non-host resistance (NHR) in barley to the wheat-adapted pathogen of powdery mildew, Blumeria graminis f. sp. tritici. ESR1 has also narrowed down a region on the barley genome that confers NHR to the wheat leaf rust pathogen, Puccinia triticina, to a genetic region of 0.6cM. ESR2 has identified mutations within the α- γ- and or ω-gliadins in wheat cvs Paragon and Fielder. They have developed protein and quantitative DNA assays by which to identify these mutations. ESR4 has confirmed that the UK Verticillium longisporum population is genetically more diverse than other V. longisporum populations studied. Field assessment of oil seed rape varieties to V. longisporum identified variation in resistance. However, the impact of V. longisporum on yield was inconsistent. ESR 4 has produced a genome sequence of V. longisporum which shows that V. longisporum has a mosaic genome structure due to recombination between parental chromosome sets, and that V. longisporum genes have generally a more divergent evolution than genes from non-hybrid Verticillium species. ESR4 has also shown that the parental mitochondrial genomes have recombined, resulting in a mosaic structure of the V. longisporum mitochondrial genome. ESR5 has shown that only large-spored Alternaria species; A. solani, A. grandis, A. linariae (syn. A. tomatophyla) and A. protenta, cause early-blight symptoms on potato. DArT-Seq SNP markers were used to identify individual Alternaria isolates, showing the incursion of isolates into experimental field trials, and that certain isolates were able to persist over years.
ESR Project 1: “Would non-host resistance (NHR) in barley prove a source of durable, race-nonspecific resistance for wheat”
ESR1 consisted of two parts: (i) Fine mapping of a QTL region in the barley variety Vada that conferred resistance to four heterologous rusts; Puccinia hordei murini (Phm), P. hordei secalini (Phs), P. triticina (Pt) and P. graminis lolii (Pgl), and (ii) Cloning of two barley receptor-like kinases (RLK) that conferred NHR to the wheat-adapted pathogen, Blumeria graminis f. sp. tritici (Bgt), followed by over-expression of these barley RLKs in the wheat. Through repeated rounds of selfing recombinant plants were obtained that allowed fine mapping of three QTLs; sub-region 1 (Phm and Phs NHR), sub-region 2 (Pt NHR), and sub-region 3 (Pgl NHR). The intervals identified from previous research for Phm and Pgl could not been confirmed, therefore, subsequent work focused only of sub-region Pt. The Pt sub-region has been mapped to a genetic distance of 0.6cM representing 20 Mb on the barley Morex genome reference. Two barley RLKs have been cloned for variety Morex and over-expressed in the wheat variety Fielder. The NHR phenotype has been confirmed in barley, variety Golden Promise, by siRNA. However, despite extensive testing, the host resistance seen in transient over-expression studies in the wheat variety Kanzler could not be duplicated in the Fielder genetic background.
ESR Project 2: “Development of wheat varieties with reduced gluten toxicity, increasing safety for celiac disease patients”
In bread wheat the three gliadin protein families, α, β and γ harbour immunogenic epitopes which can trigger Coeliac disease (CD) in genetically predisposed humans. In this project, we used two mutagenesis approach to modify immunogenic gliadin epitopes: gamma-irradiation TILLING and CRISPR/Cas9 gene editing. A gamma-irradiated population in the wheat variety Paragon was screened for changes in the gliadin protein profiles. The ESR also performed targeted mutagenesis against the α- and γ-gliadin genes using CRISPR/Cas9, designing five guide RNA, all transformed into the wheat variety Fielder. Four different methods were optimised and implemented to screen gliadin differences in potential mutant plants compared to the original wild type (WT) plants. Paragon gamma-irradiated mutants, and Fielder-CRISPR lines, were found with α- γ- and or ω-gliadins proteins missing or altered compared to their respective WT. A reduction in α-gliadin gene copy number was found in both Paragon mutant and Fielder-CRISPR lines. To identify the mutations in gliadin genomic sequences we have developed GlutEnSeq, an exome capture system that enriches for gluten genes.
ESR Project 4: “Population and pathogenicity dynamics of the Brassica pathogen Verticillium longisporum”
A global V. longisporum collection, comprised of 88 isolates from nine different countries was constructed. A population study revealed that the UK population was genetically more diverse than V. longisporum populations from different countries. Consequently, the recent emergence of Verticillium stem striping disease on oilseed rape in the UK is likely caused by a longer established V. longisporum population in the UK (Depotter et al. 2017a). The virulence of UK V. longisporum isolates was therefore accessed, however UK isolates were as virulent on Brassica host as previously characterized V. longisporum isolates from the A1/D1 lineage (Depotter et al. 2017b). Field assessment of oil seed rape varieties to V. longisporum identified variation in resistance. However, the impact of V. longisporum on yield was inconsistent (Depotter et al. 2018a).
ESR 4 sequenced the genomes of two V. longisporum isolates (Depotter et al. 2017a). It was found that V. longisporum has a mosaic genome structure due to recombination between parental chromosome sets (Depotter et al. 2018b), and that V. longisporum genes have generally a more divergent evolution than genes from non-hybrid Verticillium species. Expression patterns of the two V. longisporum sub-genomes show remarkable similarity, in particular expression levels of genes encoding secreted proteins homogenize upon plant colonization. The parental mitochondrial genomes recombined, resulting a mosaic structure of the V. longisporum mitochondrial genome (Depotter et al. 2018b).
ESR Project 5: “Emerging plant diseases: reducing the impact of Alternaria species on potato production”
Different Alternaria spp. were examined for their ability to cause early-blight of potato, focusing on detached leaf and whole plant field assays. The novelty of this work is that it systematically tested all species of Alternaria that have been “suggested” to cause early-blight symptoms on potato plants. It was found that small-spored Alternaria species are significantly different from the large-spored species, with small-spored species such as A. alternata (syn A. tenuissima), A. infectoria, and A. arborescens being unable to cause early-blight symptoms in our tests. We also investigate the possibility that small spore species may interact with large-spored species; A. solani, A. grandis, A. linariae (syn. A. tomatophyla) and A. protenta. However, the presence of small spore species did not enhance infection by large spores species, their presence in an Early-Blight epidemic having no effect on disease progress. This has profound implications on disease monitoring and control.
To gain insights into the Alternaria inoculum flow samples were collected from fields showing signs of early blight infection, fields up to 1.3 Km away and fields were early blight was found in previous years. The UK potato growers showed a lot of interest in this project, and supported the work by providing samples from their own potato fields from across the UK. Alternaria isolates from the Westerdijk Fungal Biodiversity Centre in the Netherlands were also included, and analysed along with the isolates collected in the UK. DArT-Seq DNA SNP markers were used to analyses these isolates. Incursion of isolates into experimental field trials was observed, while certain isolates were able to persist over years.
Functional Structural Plant Modelling is being used to answer questions such as: (1) To what extent can early blight affect the yield of potato crops? (2) At what point in the potato cropping cycle does Alternaria have its biggest effect? Data has been collected on disease progress from 3 years of field experiments, and this is being used to fit a model of disease progress on three different canopy levels of the potato crop.
The Max-CROP project website can be found at http://www.niab.com/max-crop