Research Project: Cranberry Genetics, Physiology, and Insect Management

Location: Vegetable Crops Research

2024 Annual Report

Objectives
Objective 1: Characterize important genetic traits, developmental and biochemical pathways, and metabolic processes in cranberry including the regulation of cranberry fruit quality, yield, and abiotic stress tolerance. Goal 1.A: Characterization of cranberry phenological traits using imaging technologies and understanding genetic, environmental, and management interactions on traits that influence yield and fruit quality. Goal 1.B: Discover genes and metabolites underlying the physiological responses of cranberry to abiotic stresses (cold, flood, soil pH and heat) that influence yield through metabolic and transcriptome profiling. Goal 1.C: Soil and plant microbial dynamics associated with vine performance, fruit yield and quality for nutrient management and long-term sustainability. Objective 2: Breed improved germplasm and cultivars for cranberry production in the United States with an emphasis on superior fruit quality traits. Goal 2.A: Use next-generation sequencing (NGS) technologies to produce a cranberry pan-genome and use the resultant data to create a genotyping platform based on annotated genes. Goal 2.B: Identify cranberry genes and quantitative trait loci (QTL) that explain horticulturally important trait differences among genotypes. Goal 2.C: Horticulturally, phenotypically, and genetically characterize a cranberry diversity and breeding collection in terms of horticulturally important traits. Goal 2.D: Perform controlled crosses to improve yield and quality based on molecular and phenotypic data of a cranberry diversity and breeding collection. Objective 3: Deploy and adapt integrated pest management approaches in new cultivars for sustainable, profitable cranberry production systems with an emphasis on bio-insecticides and pheromone-based mating disruption. Goal 3.A: Design novel methods for the mass-production and deployment of two highly virulent nematode species that represent a new bio-insecticide for US cranberries. Goal 3.B: Determine ideal pheromone carrier types and deployment rates for the suppression of two major insect pests of US cranberries. Goal 3.C: Create new on-farm bee nesting structures that can be adapted to current farming practices while providing local nesting sites for a diversity of native bee fauna.

Approach
This project will address three major issues for cranberry growers. Objective 1: Biotic stress and physiology of traits. An image phenotyping system and machine algorithms (ML) will be developed to produce trait data for tracking cranberry phenology and physiological responses. Traits will be collected in different environments and under different management practices. Additionally, fundamental mechanisms underlying cranberry physiological responses to abiotic stresses (cold, flood, soil pH, and heat) using metabolic and transcriptome profiling will be identified. Tests will include response to soil pH, cold, heat, flood, and anoxia stress. Additionally, soil microbial dynamics will be identified influencing cranberry performance and physiology, fruit yield and quality, and nutrient management and long-term sustainability. Mycorrhizae and bacteria specific to cranberry will be identified and sequenced from wild and commercial cranberries, culturing conditions will be identified, and tests will be conducted of the associated organisms for improved cranberry physiological responses. Objective 2: Genetic mapping and genomic selection. We will use next-generation sequencing (NGS) technologies to mine a cranberry pan-genome and use the resultant data to create a genotyping platform based on annotated genes. Based on the developed molecular resources, cranberry genes and quantitative trait loci (QTL) that explain yield and quality differences among genotypes will be identified. A cranberry diversity and breeding collection will be characterized horticulturally, phenotypically, and genetically. Based on the molecular and phenotypic data of the cranberry diversity and breeding collection, controlled crosses to improve yield and quality will be performed and the new breeding cycle will be established in testing plots. Objective 3: Arthropod management. This project will deploy and adapt integrated pest management approaches in new cultivars for sustainable, profitable cranberry production systems with an emphasis on bio-insecticides and pheromone-based mating disruption. We will create and deploy such integrated pest management tools while protecting pollinators. Novel methods will be designed for the mass-production and deployment of two highly virulent nematode species that represent a new bio-insecticide for U.S. cranberries. A reliable mass-propagation system specific to each nematode species will be developed along with application timing and rate information for each pest target and storage of the bio-insecticide. Additionally, this project will determine ideal pheromone carrier types and deployment rates for the suppression of three major worm insect pests of U.S. cranberries. Deployment of new pheromone carrier for control of these worms will be tested in whole-farm operations. Multi-year evidence of significant mating disruption of all three pest species will be tested. Finally, this project will create new on-farm bee nesting structures that can be adapted to current farming practices while providing local nesting sites for a diversity of native bee fauna.

Progress Report
We have developed Do It Yourself (DIY) cameras using Raspberry Pi technology, incorporating solar panels and different sensors (temperature, RH, and soil moisture sensors). We are using these DIY cameras and trail cameras to take images of different cranberry cultivars in different cranberry growing regions (WI, NJ, and MA) throughout the growing season. This growing season, we are collecting second-year data to evaluate the phenological responses of cranberry cultivars to the environment (E) and management (M). Through these efforts, we will gather data on genotype (G) responses and phenological traits influenced by the E and M, which can aid in the breeding of superior cultivars. For Objective 1, Goal 1.B: The optimal soil pH range for cranberry growth is between 4.2 and 5.5, and we evaluated cranberry responses to high soil pH (> 5.5) in six different locations in central Wisconsin. We also investigated the metabolic profiles of shoots and fruits grown under high soil pH stress to understand the mechanisms. This research will assist growers in understanding the effects of abiotic stress on cranberries and management strategies for improving yield under high soil pH stress. The article was submitted to a journal and is under revision. For Objective 1, Goal 1.C: We are investigating the microbial community composition in different cranberry soils and roots. Further, we are isolating and characterizing beneficial bacteria, mycorrhizae and fungal endophytes from soil and root samples collected from wild, organic, and conventional marshes. Understanding microbial community composition will provide details on soil health in improving cranberry productivity. The beneficial microbes will aid in plant nutrient absorption and reduce the need for chemical fertilizer, allowing for more sustainable cranberry cultivation. For Objective 2, Goal 2.A: We used next-generation sequencing (NGS) technologies to produce a pan-genome using genomic information of 10 cranberry cultivars. The resultant genetic data was used to create a genotyping platform based on annotated genes consisting of 17,000 single nucleotide polymorphisms (SNPs) that will help us identify cranberry genes and quantitative trait loci (QTL) that explain horticulturally important trait differences among genotypes. For Objective 2, Goal 2.B: Based on this new genotyping platform, we are genetically characterizing a newly assembled USDA cranberry diversity collection and other bi-parental mapping populations that identify genes or QTL that control important horticultural traits. We are mapping yield-related traits and fruit quality traits, including external appearance, internal structure, firmness, chemical traits such as acids, sugars, and pigments. For Objective 2, Goal 2.C: Based on a newly assembled cranberry diversity and breeding collection, we are characterizing fruit quality traits. We are characterizing 19 varieties we collected at maturity at the Wisconsin Cranberry Research Station in 2023. To non-invasively analyze the internal structure of cranberry fruits and products, we are characterizing force–distance curves with high accuracy and precision and an X-ray micro-CT scanner. We are analyzing the correlations between various textural traits such as firmness and structural attributes such as porosity. The next step is to determine sugar, organic acid, and anthocyanin compositions using an high-performance liquid chromatography (HPLC) system and to determine the proanthocyanidins (PAC) composition based on the liquid chromatography–mass spectrometry (LC–MS) in our collaborator’s laboratory. The results will be used to select six varieties with different PAC compositions and study the effect of drying, a key processing step in sweetened dried cranberry (SDC) production, on the bioavailability of PACs. Additionally, based on a non-disclosure agreement with an SDC manufacturer, we have obtained information on the processing conditions in the SDC production. This information will be used to enable the lab-scale production of mock SDC products in our laboratory. For Objective 2, Goal 2.D: We are working towards breeding improved germplasm and cultivars for cranberry production in the United States with an emphasis on superior yield and fruit quality traits. Based on the genetic and phenotyping information, we are creating cranberry hybrids to ensure that strategic crosses are accomplished. Pedigree information is being evaluated in the light of marker information to determine the most likely genotypes or genetic pools associated with each named cultivar and their associated horticultural performance. A series of cranberry hybrids with complementary genetic pools is being created and evaluated. The materials are being planted and managed for production at the Wisconsin Cranberry Research Station in Black River Falls, Wisconsin. For Objective 3, Goal 3A: An in vivo mass-propagation system using live insects as hosts (beetle larvae) has been developed. To tailor the system to Wisconsin cranberry production, the production system is being refined for improved nematode longevity and transport to commercial cranberries. This will allow for significant scaling up of field applications, which is a critical first step to commercialization because mass-propagation throughput will dictate the rate and total acreage we can apply the nematodes in the field. In collaboration with an ARS researcher in Byron, Georgia, we have a successful fermentation system and evidence of a viable nematode storage system for our two native nematode species. One of the nematode species has proved more difficult to sustain in storage. We have focused on in vivo propagation because it preserves the nematodes’ intrinsic hunting capacity and high virulence. For Objective 3, Goal 3.B: Collaborations with Wisconsin growers and industry stakeholders have facilitated a second year of large-scale pheromone deployment on commercial acreage. The pheromone carrier in this work is a sprayable matrix, rendering applications highly efficient using standard spray equipment. Results from previous years are encouraging, and this public-private project will soon provide the evidence needed to commercialize pheromone- based mating disruption in U.S. cranberries. The project is expected to conclude in 2025. For Objective 3, Goal 3.C: This research has created a new on-farm bee nesting structure that can be manufactured in-house by farmers and adapted to current farming practices in cranberry systems. The nesting structures provide local nesting sites for a diversity of native bee fauna, facilitating bee abundance and diversity in Wisconsin cranberry marshlands.

Accomplishments
1. Development of a cranberry pangenome as a resource for future genetic studies and breeding efforts. Molecular tools to help guide cranberry breeding efforts have been relatively limited compared with those for other high-value crops. To help bridge this gap, ARS scientists in Madison, Wisconsin, in collaboration with the Vaccinium Coordinated Agricultural Project (VacCAP), leveraged previous cranberry sequencing efforts of the Stevens cultivar cranberry genome (genetic code) to sequence and annotate the genomes of ten more diverse cranberry cultivars. A pangenome (“all” genetic code) and other cranberry genetic resources were developed that were compared to other Vaccinium species (blueberry family). This genomic resource will serve as a powerful resource to guide future molecular breeding efforts and genetic studies in cranberry and other important Vaccinium species, such as blueberry and lingonberry.

2. A study on the effects of the cranberry added sugar labeling and consumers’ willingness to pay. The goal of nutrition labeling policies is to help consumers make informed food choices. Recent rules require the declaration of added sugars on packaged foods and beverages, including cranberry products. A collaboration involving ARS researchers in Madison, Wisconsin, and the Vaccinium Coordinated Agricultural Project (VacCAP) conducted a choice experiment to examine consumers’ reaction to added sugars labeling in two cranberry products (dried cranberries and cranberry juice). We found significant discounts in consumers’ willingness to pay for increases in added sugars that vary across information treatments and consumer subsamples. A positive information frame about the health benefits of cranberries was not found to consistently offset the impact of additional information on the recommended daily intake limits for added sugars. This study will serve to design mitigation strategies for the potential effect of labeling mandates and understand consumers' perceptions of cranberry products.

3. A study on the consumer acceptance of CRISPR or gene edited cranberries. Plant breeding technologies such as gene editing, specifically the clustered regularly interspaced palindromic repeats (CRISPR), offer a plausible alternative to develop cranberries with desired traits (e.g., lower acidity and increased sweetness). A collaboration involving the ARS researchers in Madison, Wisconsin, and the Vaccinium Coordinated Agricultural Project (VacCAP) estimated consumers’ willingness to pay for enhanced CRISPR cranberry products. Our study found that consumers were willing to pay a premium for cranberry products with reduced added sugar content, CRISPR-bred, and full/intense cranberry flavor relative to products with regular added sugar content, conventionally bred, and weak/bland flavor. This research underscores the importance of the conditions under which breeding technologies might gain public acceptance. This information will benefit the scientific community and industry seeking to use CRISPR to develop improved cranberry cultivars.

4. Development of lingonberry x blueberry hybrids expanding the genetic base of other Vaccinium crops. The Vaccinium genus includes species such as cranberry, lingonberry, and blueberry. It has been a long-standing dream of Vaccinium breeders to develop a bridge to cross the three species and create viable hybrids. ARS scientists in Wisconsin and New Jersey hybridized lingonberry and three blueberry species as part of a project aimed at understanding Vaccinium crossability and utilization of lingonberry in the larger Vaccinium hybridization road map. The crosses succeeded at a low level and produced eight hybrids, which were genetically verified. Hybrids were intermediate in morphology and fertile at low levels. The successful hybridizations suggest that the utilization and lingonberry in the Vaccinium hybridization road map is possible. This work provides a deeper understanding of Vaccinium species hybridization. These crosses represent a previously unrecognized hybridization path in the road map and promise to facilitate the genetic transfer of lingonberry material into usable forms for mainstream Vaccinium breeding with the potential to deliver new marketable commodities.

5. A novel method for the mass-production of highly virulent nematodes that represent a new bio-insecticide for U.S. cranberries. Soil-borne cranberry pests are often difficult to reach with conventional insecticides, and entomopathogenic nematodes represent a means to deploy predators within the soil column to find and kill pests. The work in collaboration with ARS scientists in Madison, Wisconsin, and Byron, Georgia, effectively outlines a mass propagation method for entomopathogenic nematodes of agricultural importance. This new method represents a viable, accessible way for U.S. farmers to grow their own nematode-based bioinsecticides in- house. The methods have been applied to two native Wisconsin nematode species that represent virulent new bio-control agents to help protect Wisconsin cranberries. This work provides a pesticide free control agent that can be deployed against insect pest as part of an integrated pest management program and will benefit cranberry growers, consumers, and the environment.

6. A broad re-framing of bee, microbe, and angiosperm (flowering plant) symbioses (interdependence). Globally, most agricultural and natural unmanaged ecosystems rely on insect pollinators to support plant diversity, productivity, and often the entire food-web. ARS researchers in Madison, Wisconsin, along with collaborators in Japan and Germany analyzed and synthesized the latest evidence relating to bee-microbe and microbe-angiosperm (flowering plant) symbioses (interdependence). This work not only outlines a conceptual advance for pollination ecology, but also articulates new ideas for future work relating to bee-microbe-angiosperm interactions. Understanding the importance of microbial symbioses within their three-way symbioses with bees and angiosperms will recalibrate conservation efforts to include the abundant yet invisible ‘silent third partners’ of the traditional bee-angiosperm symbiosis Pollinators are critical for fruit crops in the US and around the world fruit set, and understanding the needs of bees will help conserve pollinators, which will in turn improve agriculture and human nutrition.

Review Publications
Steffan, S.A., Dharampal, P.S., Kueneman, J.G., Keller, A., Argueta-Guzman, M.P., McFrederick, Q., Buchmann, S., Vannette, R., Edlund, A., Mezera, C.C., Amon, N., Danforth, B.N. 2023. Microbes, the ‘silent third partners’ of bee–angiosperm mutualisms. Trends in Ecology and Evolution. 39:65-77. https://doi.org/10.1016/j.tree.2023.09.001.
Hofman, C.O., Steffan, S.A., Shapiro Ilan, D.I. 2023. A sustainable grower-based method for entomopathogenic nematodes production. Journal of Insect Science. 23(5). https://doi.org/10.1093/jisesa/iead025.
Yocca, A., Platts, A., Alger, E., Teresi, S., Mengist, M., Benevenuto, J., Ferrao, L., Jacobs, M., Babinski, M., Magallanes-Lundback, M., Bayer, P., Golicz, A., Humann, J., Main, D., Espley, R., Chagne, D., Albert, N., Montanari, S., Vorsa, N., Polashock, J.J., Diaz-Garcia, L., Zalapa, J.E., Bassil, N.V., Munoz, P., Iorizzo, M., Edger, P. 2023. Blueberry and cranberry pangenomes as a resource for future genetic studies and breeding efforts. Horticulture Research. 10(11). Article uhad202. https://doi.org/10.1093/hr/uhad202.
Ma, X., Gallardo, R.K., Canales, E., Atucha, A., Zalapa, J.E., Iorizzo, M. 2024. Effects of the added sugar labeling on consumers' willingness to pay: The case of cranberry products under different nutrition-related information treatments. Journal of Agricultural and Applied Economics. https://doi.org/10.1002/jaa2.121.
Ma, X., Gallardo, R., Canales, E., Atucha, A., Zalapa, J.E., Iorizzo, M. 2024. Would consumers accept CRISPR fruit crops if the benefit has health implications? An application to cranberry products. Agricultural and Resource Economics Review. https://doi.org/10.1017/age.2023.38.
Ehlenfeldt, M.K., Luteyn, J.L., Zalapa, J.E., De La Torre, F. 2023. Triploid hybrids of 2x lingonberry (vaccinium vitis-idaea) by 2x black highbush blueberry (v. fuscatum) and 2x elliott’s blueberry (v. elliottii) as evidence of a genome balance requirement for hybridization success. Horticulturae. 9(12):1308. https://doi.org/10.3390/horticulturae9121308.