Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The target audience for this research is the whole supply chain for leafy greens and the regulatory and statutory bodies engaged in developing policy regulation in this industry. The supply chain includes growers, both large, small and organic that grow leafy greens including lettuce and spring green mixes. These products are sold as whole head lettuces and more recently as chopped, whole and baby leaves. In addition to traditional field-grown agricultural operations, the target audience for this research includes hydroponic growers and those engaged in vertical agricultural indoor agricultural (vertical) systems and those developing aquaponics systems, where leafy greens are grown in combinations with animal production systems of some type. Each of these grower-types will be a target for this research although each has different needs. The drivers to improve leafy greens in the field are focused around better resource use efficiency, particularly nitrogen and water and the production of safe, healthy and nutritious leaves whilst the drivers in indoor systems are focused on quality, safety, cleanliness and improved nutrition. In addition to growers, the research is also relevant to the processing industry, where washed, bagged and prepared salads are developed and supplied to a wide range of outlets including restaurants and supermarkets. Processing of leafy greens for prepared meals and foods is also undertaken. Here the focus will be on large supermarket chains that supply much of the public, and also smaller farmers markets and organic suppliers. This research will be presented to the California Leafy Greens Research Board (Calgreens) that utilizes funds from the marketing agreement that covers lettuce (iceberg, romaine, leaf), cabbage and spring mixes (spinach and lettuce) and represents over 100 certified growers in California that produce over 80 % of leafy greens for the US. Supermarket and processor contacts will be maintained through the presentations at the post harvest short course delivered each year by the Post Harvest Technology Research and Information Center of UC Davis and regularly attracting over 100 participants. The results obtained from this research are of direct relevance to breeding and therefore to seed companies with active contacts developed and maintained through the Seed Central networking activity of the Seed Biotechnology Center at UC Davis. Seed Central organizes a monthly networking event on the campus with an informed talk and over 200 members are part of this activity including all of the major international seed companies. In this way, latest research can be presented and communicated to the major plant breeding efforts, world-wide. Changes/Problems:This reporting period has been impacted by Covid-19, since at least for the initial period of the pandemic, only critical and essential research was possible and no student interns were available to act as research assistants. Nevertheless, in general the project remains unchanged and there was a focus on paper writing and data analysis this reporting period. What opportunities for training and professional development has the project provided?Graduate training is being provided in plant breeding in both lettuce and watercress. Graduates are being trained in genomics, plant breeding, making crosses, establishing a breeding program, high throughput phenotyping, genetic analysis, plant biochemistry and analysis. Project management, field trial planning and experimentation training is being provided to post-doctoral and research scientists. Bioinformatics and genomics training is being provided too. Interactions with vertical farmers are on-going and included on-going collaborative research with Plenty, a large vertical farming company in San Francisco. At the same time, during 2020, set-up of the new vertical indoor farm on the Davis campus has engaged both graduate and undergraduate students an providing valuable training opportunities. How have the results been disseminated to communities of interest?Gail Taylor has given four talks to external audiences. She spoke as an invited speaker at the international meeting "The Future of Lettuce", March 2020 [https://g2g.ucdavis.edu/program/], on the Lettuce Leaf Microbiome. she gave two presentations to the Californian Leafy Green Research Board, in March 2020 and October 2020. She gave a seminar to report research results to the Plant Sciences group in the San Francisco company Plenty, on research done in collaboration with Plenty on vertically grown leafy greens. Annabelle Damerum has given two seminars to large audiences on the UC Davis campus - she gave a seminar to the Plant Biology Graduate group on gene edited lettuce for longer shelf life and she gave a second seminar at the Annual Plant Breeding Retreat, the mapping QTL of the lettuce leaf microbiome. Gail Taylor has give three seminars on the UC Davis campus, one to the newly initiated AI Institute for Food Systems, a collaborative venture funded by USDA and NSF. She gave two seminars in her bid to win the John B Orr Chair in Environmental Sciences, which was awarded competitively to her n November 2020. She gave a departmental seminar on her research program in November 2020. In 2020, all of these dissemination activities have been given remotely, by zoom. To compensate for the reduction in travel, and direct interaction with our research audience, we have made three videos for dissemination, that describe the set-up, use and results when deploying an indoor vertical farm for growing leafy greens. The program activities are routinely tweeted and placed on social media. Gail Taylor has interacted with four additional vertical indoor agricultural companies, providing advice and input to their commercial activities. What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will remain focused on the original five objectives. As reported last year, effort will be continued to be placed on publication activities with four more papers currently in preparation for submission in addition to the three currently accepted or published. In more detail for each objective: 1. Developing genomic resources in lettuce and watercress- We will submit for publication, one paper on the novel watercress genome. We will submit a second paper on G X E analysis of watercress, following field trials in California, US and UK and at two concentrations of nutrient input. A third paper review on the ideotype for breeding in watercress is also in preparation. We will use the new vertical farm using speed breeding to take the extended watercress bi-parental population from F5 to F8, through single seed descent, such that the traits will be fixed at the end of this period. At that time, we will collaborate with the new AI Institute for Food Systems on the Davis campus https://aifs.ucdavis.edu/, to enable AI to predict how to make better use o our limited phenotyping data to select lines for future breeding. We will screen a novel wide population of watercress sin the vertical farm, to identify the range of phenotypes possible in indoor vertical growing systems. 2. QTL mapping in lettuce and watercress - A publication on water use efficiency in lettuce is in press. We will submit a paper on the extended phenotype of QTL between leaf surface characteristics and those for the abundance and diversity of leaf surface microbes. We will complete field work in Salinas and Imperial Valley on the lettuce leaf microbiome and lettuce shelf life to confirm robust QTL for these traits. At the end of this reporting period we will have analyzed multiple QTL in multiple environments in two contrasting populations - one bi-parental and one a wide GWAS population. These are being used in a pipeline to identify genes for marker development as identified below. 3. Candidate genes and development of SNP-based markers for pre-breeding and breeding- We will continue to develop our met-analysis of candidate genes and to refine these using bioinformatics tools and to identify at least 5 gene targets per QTL-trait fir marker development, which will enable testing in the next period of research. 4. Gene editing for proof of concept for traits of interest - a publication is in progress on gene edited lettuce- We have a publication in draft and this will be submitted during the coming reporting period to demonstrate the effective use of gene editing to extend the shelf life and reduce food waste in lettuce. The focus of this research this reporting period will be on testing our gene edited lines (up to three) in two contrasting environments, in the field and in the vertical indoor farm. In this way will we test these gene edited lines for their robustness in using editing technology to extend the shelf-life of lettuce. 5. Vertical indoor farming systems- Preliminary analysis is underway on the microbiome of leaves grown in vertical indoor conditions. In this part of the project a new vertical farm is now deployed on campus and has been used to test new germplasm for suitability for indoor vertical growing systems. One trial to date has been completed and further indoor vertical trials will focus on a) speed breeding in watercress b) testing gene edited lettuce lines c) optimizing environmental conditions for long shelf-life and high nutrient watercress.
Impacts
What was accomplished under these goals?
The aim of this research project is to develop improved leafy green crops for California that are nutrient dense, including with enhanced anti-cancer properties and anti-oxidant status, have a reduced environmental footprint, particularly with respect to water consumption and a longer shelf life, thus reducing waste and are safe with reduced microbial contamination. The specific objectives are: 1. To establish novel genomic and genetic resources in lettuce and watercress enabling investigation of a wide range of traits, in particular to sequence the genome of watercress, develop the first molecular genetic map. In lettuce to identify novel sequence variants for traits of interest. Accomplishments in the reporting period: We have completed and continued to analyze the first sequenced genome of watercress using 10X, PacBio and Bionano technologies and this research is now ready for submission for publication in very advanced draft. We have developed and completed the third version of the first molecular genetic map of watercress, now informed by the whole genome sequence that was completed in our laboratory. This is highly novel and represents the first genomics resources in watercress. At the same time, the F2 mapping population has been extended from 200 to more than 800 and is now being taken to F5. We have initiated and now utilized a new technology for 'speed breeding' that reduces the generation time by 50%, with seed production completed in just a few weeks. This is a major achievement to enable future breeding targets to be met in this nutrient dense leafy green. Unfortunately covid-19 and the limited ability to host students to undertake research has delayed this part of the project but we have completed and cleaned the seed of our latest generation of watercress which will now be placed into the vertical farm for future speed breeding. 2. QTL for quality traits in lettuce and watercress Accomplishments in the reporting period: We have extended our research on QTL discovery further this reporting period and now have an extensive set of QTL in lettuce for a) shelf life, b) nutrient status using anti-oxidant potential, c) biophysical analysis using Instron testing for plasticity, elasticity and break-strength and drought tolerance traits including d) carbon isotope discrimination as a proxy for Water Use Efficiency (WUE) [now published], e) stomatal conductance and fresh and dry weight for growth in limited water supply treatments [now published]. f) This year we have focused on a new QTL mapping project on the extended phenotype between leaf surface traits and microbiome traits. We have explored the concept that leaf surface traits vary between lettuce genotypes and these differences may be significant and will determine the abundance and diversity of the leaf surface microbome. This is relevant to both human pathogen and food poisoning outbreaks and also spoilage and shelf-life. We have identified areas of the lettuce genome (QTL) where spoilage microbe abundance co-locate for leaf surface traits including leaf surface hydrophobicity, stomatal size and number and cell circularity. We have gained these data sets by growing both a bi-parental population supplied by the Michelmore group, where we have sequenced genome data available for the lettuce and also because we have now sequenced the surface DNA from each of the F9 unique genotypes. We are now also performing these studies in the wide GWAS population supplied by Simko at USDA. They have been grown in multiple field trials during the reporting period. Previous research in the laboratory of Gail Taylor has utilized the core lettuce mapping population and resources (Truco et al., 2013) developed by the Michelmore laboratory to identify Quantitative trait loci (QTL); regions of the genome associated with the phenotype of interest. This is a population of recombinant inbred lines (RILs) developed from a cross between the Salinas cultivar and the expected wild progenitor of lettuce, Lactuca serriola. 3. Characterize genes underlying QTL, develop robust molecular markers for the traits of interest Accomplishments in the reporting period: We have identified a set of candidate genes for extended shelf life, water use efficiency and nutritional quality (polyphenolic content) in lettuce and this year, we have added leaf surface traits and microbial abundance and diversity for a bi-parental mapping population. These have emerged from the QTL pipeline described in 2. above. From this approach we have now several refined areas of the lettuce genome that are "hotspots" for each of these traits. Identification of candidate genes from the GWAS research is continuing. Molecular markers boarding these QTL regions located in the lettuce genome and all predicted genes are being retrieved using the lettuce genome resource http://lgr.genomecenter.ucdavis.edu/. Genes were annotated to determine predicted gene function by using the Basic Local Alignment search Tool (BLAST) against the NCBI database. Our long-list of candidate genes is being refined using literature review and also by checking the genotyping of particular lines of interest to determine the best approach to develop markers. In this approach we are suing high resolution PCR marker development that can differentiate single base changes in genes fragments of interest in PCR assay. These candidate gene markers will be tested in a wide panel of lettuce genotypes. 4. Develop proof-of-concept CRISPR-Cas approaches in lettuce to test traits of interest. Accomplishments in the reporting period: During the reporting period we have harvested, cleaned and inventoried seeds from our F3 lines, where we have several constructs that are gene edited using CRISPR Cas9 to modify the six XTH genes that were targeted from within one QTL as reported previously. The next steps for this research are to test the gene edited lettuce lines in the field. Our research this period has shown that shelf life has been extended in the lines by up to 2 days and that leaf cells in one line are smaller with a higher break strength. These are the results that we would predict from out QTL work highlighted in 2., above. However, the lines vary in the phenotypes and we are keen to test the lines in a number of contrasting environments. This research was somewhat delayed because of covid-19, but seed is now available for field and vertical farm work to start in march 2021. 5. Work in vertical agricultural systems to determine the best phenotypes for indoor agriculture with optimized nutrition and WUE. Accomplishments during the reporting period: The UC Davis campus has invested in a new 40 foot container that is an indoor vertical farm, from freight farms. This has been procured, with $200,000 of campus investment to get the site ready for delivery of the $110,000 vertical farm. A project with UAE was also submitted in August 2020 and a decision on funding is still awaited. At the same time, we have commissioned the new vertical farm on the UC Davis campus and completed the first trial on watercress where we have identified the best conditions for watercress growth in the farm. Watercress is a crop that is highly suited to indoor vertical growing and we have established the conditions for speed breeding. We have completed a site analysis in collaboration with the company Plenty and have characterized the microbiome of lettuce and arugula grown in indoor vertical conditions, where we have been able to show that microbiome traits different with leafy green crop (lettuce versus arugula), between root and shoot and also as the crop moves through the vertical system, from seedling to harvest. We have also shown in preliminary data that the abundance of microbes on the leaf surface is significantly reduced for indoor versus field grown leafy green crops, although the composition of the microbiome appears to be similar.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Damerum A, Chapman MA, Taylor G (2020). Innovative breeding technologies in lettuce for improved post-harvest quality. Post Harvest Biology and Technology, 168: 111266, October 2020
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
Damerum H, Smith H, Clarkson G, Truco MJ, Michelmore RW and Taylor G. The genetic basis of water-use efficiency and yield in lettuce.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
Voutsina N, Hancock RD, Becerra F, Taylor G. Characterization of a new dwarf watercress "Boldrewood" in commercial trails reveals a consistent increase in chemopreventive properties in a longer-grown crop
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Progress 04/18/19 to 09/30/19
Outputs
Target Audience:The target audience for this research is the whole supply chain for leafy greens and the regulatory and statutory bodies engaged in developing policy regulation in this industry. The supply chain includes growers, both large, small and organic that grow leafy greens including lettuce and spring green mixes. These products are sold as whole head lettuces and more recently as chopped, whole and baby leaves. In addition to traditional field-grown agricultural operations, the target audience for this research includes hydroponic growers and those engaged in vertical agricultural indoor agricultural (vertical) systems and those developing aquaponics systems, where leafy greens are grown in combinations with animal production systems of some type. Each of these grower-types will be a target for this research although each has different needs. The drivers to improve leafy greens in the field are focused around better resource use efficiency, particularly nitrogen and water and the production of safe, healthy and nutritious leaves whilst the drivers in indoor systems are focused on quality and improved nutrition. In addition to growers, the research is also relevant to the processing industry, where washed, bagged and prepared salads are developed and supplied to a wide range of outlets including restaurants and supermarkets. Processing of leafy greens for prepared meals and foods is also undertaken. Here the focus will be on large supermarket chains that supply much of the public, and also smaller farmers markets and organic suppliers. This research will be presented to the California Leafy Greens Research Board (Calgreens) that utilizes funds from the marketing agreement that covers lettuce (iceberg, romaine, leaf), cabbage and spring mixes (spinach and lettuce) and represents over 100 certified growers in California that produce over 80 % of leafy greens for the US. Supermarket and processer contacts will be maintained through the presentations at the post harvest short course delivered each year by the Post Harvest Technology Research and Information Center of UC Davis and regularly attracting over 100 participants. The results obtained from this research are of direct relevance to breeding and therefore to seed companies with active contacts developed and maintained through the Seed Central networking activity of the Seed Biotechnology Center at UC Davis. Seed Central organizes a monthly networking event on the campus with an informed talk and over 200 members are part of this activity including all of the major international seed companies. In this way, latest research can be presented and communicated to the major plant breeding efforts, world-wide. Changes/Problems:No changes have been made to the original plan. What opportunities for training and professional development has the project provided?Graduate training is being provided in plant breeding in both lettuce and watercress. Graduates are being trained in genomics, plant breeding, making crosses, establishing a breeding program, high throughput phenotyping, genetic analysis, plant biochemistry and analysis. Project management, field trial planning and experimentation training is being provided to post-doctoral and research scientists. Bioinformatics and genomics training is being provided too. Interactions with vertical farmers are on-going and included planned collaborative research with Plenty and other companies. How have the results been disseminated to communities of interest?Gail Taylor gave a presentation at the international Plant and Animal Genome meeting, 2020 and followed up with more than 600 views of this presentation on the watercress genome and watercress breeding on linkedin. She presented this research project at the Leafy Green Annual research meeting that routinely attracts 200 people from across the leafy green industry including growers, processors and seed companies engaged in breeding. The research was also presented by Dr Annabelle Damerum to the leafy green research board mid-term review in Salinas. Dr Annabelle Damerum also presented a paper at the Plant and Animal Genome meeting on lettuce gene editing for enhanced shelf life. Graduate student Yufei Qian presented a poster at the Plant and Animal Genome meeting 2020 on new genomic resources. The project was demonstrated to the wider public at picnic day, April 2019, where more than 15,000 visitors were interacting on the UC Davis campus. Over 1,000 plants were given away to the public alongside information leaflets on on-going research on leafy greens. What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will remain focused on the original five objectives, however, overall there will be more effort placed on publication activities with four papers currently in preparation for submission and two in submitted. In more detail: 1. Developing genomic resources in lettuce and watercres 2. QTL mapping in lettuce and watercress 3. Candidate genes and development of SNP-based markers for pre-breeding and breeding 4. Gene editing for proof of concept for traits of interest 5. Vertical indoor farming systems In this part of the project a new vertical farm will be depoyed on campus and used to test new germplasm for suitaility for indoor vertical growing systems.
Impacts
What was accomplished under these goals?
The aim of this research project is to develop improved leafy green crops for California that are nutrient dense, including with enhanced anti-cancer properties, have a reduced environmental footprint, particularly with respect to water consumption and a longer shelf life, thus reducing waste and are safe with reduced tendency for microbial contamination. The specific objectives are: 1. To establish novel genomic and genetic resources in lettuce and watercress enabling investigation of a wide range of traits, in particular to sequence the genome of watercress, develop the first molecular genetic map. In lettuce to identify novel sequence variants for traits of interest. Accomplishments in the reporting period: We have completed the first sequenced genome of watercress using 10X, PacBio and Bionano technologies - all during the reporting period - and this research is now being prepared for publication. We have developed and completed the first molecular genetic map of watercress. This is highly novel and represents the first genomic resources in watercress. At the same time, the F2 mapping population has been extended from 200 to more than 800 and is now being taken to F3, F4 and F5. We have initiated a new technology for 'speed breeding' that reduces the generation time by 50%, with seed production completed in just a few weeks. This is a major achievement to enable future breeding targets to be met in this nutrient dense leafy green. 2. QTL for quality traits in lettuce and watercress Accomplishments in the reporting period: We have extended our research on QTL discovery and now have an extensive set of QTL in lettuce for a) shelf life , b) nut rient status using anti-oxidant potential, c) biophysical analysis using Instron testing for plasticity, elasticity and break-strength and drought tolerance traits including d) carbon isotope discrimination as a proxy for Water Use Efficiency (WUE), e) stomatal conductance and fresh and dry weight for growth in limited water supply treatments. We have gained these data sets by growing both a bi-parental population supplied by the Michelmore group and also a wide GWAS population supplied by Simko at USDA. They have been grown in multiple field and greenhouse trials during the reporting period. Previous research in the laboratory of Gail Taylor has utilized the core lettuce mapping population and resources (Truco et al., 2013) developed by the Michelmore laboratory to identify Quantitative trait loci (QTL); regions of the genome associated with the phenotype of interest. This is a population of recombinant inbred lines (RILs) developed from a cross between the Salinas cultivar and the expected wild progenitor of lettuce, Lactuca serriola. Using our extensive phenotype data on this population collected from field trials, we conducted analyses to identify candidate genes for shelf life and nutritional quality traits. QTL from each of the trials were mapped together to identify genomic regions of interest. Positions where QTL for shelf life measured in different environments and estimated to control a high percentage of the phenotypic variation were selected for analyses . 3. Characterize genes underlying QTL, develop robust molecular markers for the traits of interest Accomplishments in the reporting period: We have identified a set of candidate genes for extended shelf life in lettuce. These have emerged from the QTL pipeline described in 2. above. Identification of candidate genes from the GWAS research is on-going. Field work has been completed, samples are curretly being processes and QTL analysis and bioinformatics will be completed during the forthcoming reporting period. Molecular markers boarding these QTL regions were located in the lettuce genome and all predicted genes were retrieved using the lettuce genome resource (LGR: http://lgr.genomecenter.ucdavis.edu/, Genes were annotated to determine predicted gene function by using the Basic Local Alignment search Tool (BLAST) against the NCBI database. Genes involved in cell wall biosynthesis, carbohydrate metabolism and transportation, cellular senescence, abiotic or biotic stress response or hormone response were selected as candidates for shelf life. 4. Develop proof-of-concept CRISPR-Cas approaches in lettuce to test traits of interest. Accomplishments in the reporting period: During the reporting period we have largely completed the phenotypic and genotypic analysis of our novel lines. CRISPR/Cas9 genome editing was previously utilized in collaboration with the Michelmore laboratory to develop lettuce lines with gene knockout mutations in six shelf life candidate genes. These genes encode xyloglucan endotransglucosylase/hydrolase (XTH) enzymes, which function in modifying, through extending and shortening, xyloglucan tethers between cellulose microfibrils in the cell wall. This extension of these xyloglucan chains leads to cell expansion, modifying cell wall strength and plastic and elastic properties. The six XTH genes targeted were found within QTL for shelf life on chromosomes 4 and 8 in previous candidate gene mining studies, as described in Objective 1. Guide RNAs (gRNAs), 20bp in length, guided the Cas9 double-stranded DNA endonuclease to regions within the first exon of each gene. Each gRNA was cloned into a custom construct (modified from Fauser et al., 2014, Plant Journal, by the Brady laboratory, UC Davis), containing genes encoding Streptococcus pyogenes Cas9 driven by the constitutive Ubiquitin 4-2 promoter and terminated by Arabidopsis Heat Shock Protein 18, and a kanamycin resistance gene as a selectable marker, controlled by the nopaline synthase promoter and terminator. Three gRNAs were targeted to each gene, in an effort to maximize the efficiency of gene knockout. Constructs were transformed into Agrobacterium tumefaciens and then into lettuce cultivar Salinas by the UC Davis Plant Transformation Facility (https://ptf.ucdavis.edu/). Mutant lines were identified by amplicon sequencing of the primary transformants. 5. Work in vertical agricultural systems to determine the best phenotypes for indoor agriculture with optimized nutrition and WUE. Accomplishments during the reporting period: Over the past year, several projects related to indoor vertical agriculture have been initiated. Firstly the UC Davis campus has invested in a new 40 foot container that is an indoor vertical farm, from freight farms. This has been procured, with $200,000 of campus investment to get the site ready for delivery of the $110,000 vertical farm. A project with the Singapore Food Agency is in discussion. A project with UAE is discussion for submission in April 2020.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
The potential to improve culinary herb crop quality with deficit irrigation
LS Rowland, HK Smith, G Taylor (2019). The potential to improve culinary herb crop quality with deficit irrigation. Scientia Horticulturae 242, 44-50
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