Research Project: Cranberry Genetics and Insect Management

Location: Vegetable Crops Research

2019 Annual Report

Objectives
Objective 1: Map and identify genes that underlie cranberry yield and quality traits, and explain the phenotypic differences between selected genotypes using genetic, genomic, and molecular approaches. Objective 2: Develop new enhanced cranberry germplasm and cultivars by integrating genetic and genomic breeding approaches with conventional cranberry breeding. Objective 3: Develop tools for the early detection and prevention of new, emerging cranberry pests (insects and mites). Sub-objective 3. Develop bio-insecticides using newly discovered, native nematode species. Objective 4: Develop new integrated pest management technologies for pest management and sustainable production of cranberry. Sub-objective 4.A. Develop a multi-species mating disruption program for the major moth pests of U.S. cranberries. Sub-objective 4.B. Investigate the biology and ecology of native pollinators to ensure the sustainable production of cranberries. Objective 5: Develop alternative cranberry production practices that improve water conservation and decrease plant disease. [NP301 C1 PS1B C2 PS2A] Expected benefits include a systems approach to cranberry production that includes genetic improvement, genomics, disease and pest mitigation, and water conservation.

Approach
Objective 1: A multi-pedigree QTL mapping approach will be used to map cranberry yield and fruit quality traits. Phenotypic trait data collection will include traditional and newly developed high-throughput methodologies to measure yield and fruit quality related traits and other horticultural measurements, including total fruit weight, percent rotten fruit, average berry weight, and other fruit quality parameters such as TAcy and firmness. A composite SSR/SNP high-resolution cranberry genetic map developed based on three half-sibling populations will be used for QTL analysis. Objective 2: This research will collaborate with cranberry growers to establish a cranberry research station in Wisconsin and to establish various sized research plots to test the horticultural needs and performance of a selection of important cranberry cultivars. Phenotypic information that will be collected will be determined based on previous research to include the best traits to measure yield and quality. Additionally, a classic inbred-hybrid system will be used based on the best performing cranberry cultivars in the industry to develop improved cranberry lines and varieties in terms of yield and quality. Prior to creating cranberry inbreds and hybrids, horticultural, genetic, and genomic information will be carefully considered to ensure that strategic crosses are accomplished. Objective 3: A novel, effective bio-insecticide will be developed for arthropod pest suppression in commercial cranberry marshes. Two highly virulent nematode species, both native to Wisconsin, will comprise the bio-insecticide, and the nematode blend will ultimately be developed such that it can be applied at large-scales using standard spray equipment. Arthropod population suppression will be assessed among pest species and non-target species alike. Objective 4: A multi-species mating disruption system will be developed to control the top three insect pests of Wisconsin cranberries. The sex pheromones of these insect species will be loaded into carriers that can be applied efficiently via standard fertilizer-application equipment. We will also examine the capacity of the cranberry plant to prime its chemical defenses after 'eavesdropping' on the pheromones of its major pests. Bee-microbe symbioses will be investigated as a means to better understand and protect the native pollinators of cranberries.

Progress Report
The progress reported relates to Objectives 1-4. We are studying the yield traits in cranberry using traditional data collection methods. We are also developing high-throughput data collection and visualization software to massively collect and understand data. Molecular tools are being applied to study and identify cranberry genes by mapping those genes found to control important cranberry traits, mainly yield traits. We have developed several high density molecular maps and a concomitant composite map that will be used to map traits of economic importance. In the future, these efforts will help breed high yielding cranberry cultivars more efficiently by allowing markers-assisted breeding. A bio-insecticide comprised of native Wisconsin nematodes has recently been shown to be highly effective when applied at large scales, and can be sprayed via standard grower spray equipment. The cranberry mating disruption program has incorporated a new pheromone carrier type (micro-encapsulated carriers) and has tested higher loading levels. Bee-microbe interactions continue to be investigated and appear to be vital for larval bee development. The Wisconsin cranberry research station is near completion. This work has been a collaboration with cranberry growers to establish a cranberry research station in Wisconsin. Various plots and experiments have been planned and established to test the horticultural needs and performance of a selection of important cranberry cultivars.

Accomplishments
1. Mapping cranberry traits. Because of its known phytochemical activity and benefits for human health, cranberry production and commercialization around the world has gained importance in recent years. Flavonoid compounds as well as the balance of sugars and acids are key quality characteristics of fresh and processed cranberry products. Additionally, fruit size and shape are particularly important characteristics that determine processing value and potential end-use products (e.g., juice vs. sweetened dried cranberries). In this study, we identified novel marker-trait associations that influence total anthocyanin content (TAcy), titratable acidity (TA), proanthocyanidin content (PAC), Brix, and mean fruit weight (MFW) in cranberry fruits. We also implemented image-based phenotyping techniques for gathering data regarding basic cranberry fruit parameters such as length, width, length-to-width ratio, and eccentricity. Using repeated measurements over the fruit ripening period, different marker-trait associations were identified at specific time points that coincide with known chemical, size, and shape changes during fruit development and maturation. Some genetic regions appear to be regulating more than one trait. In addition, we demonstrated the utility of digital imaging as a reliable, inexpensive and high-throughput strategy for the quantification of anthocyanin content and the assessment of fruit size and shape in cranberry fruits. This research will be useful to cranberry breeders seeking to improve chemical traits as well as processing traits. Cranberry color, size, and shape are attributes which can be difficult and time consuming for breeders and processors to measure, especially when relying on manual measurements and visual ratings. We provide a powerful trait phenotyping tool to determine quality parameters for fruit breeders and processors. (Objective 1).

2. Applying genomic selection in cranberry. Recently, genetic and breeding applications that use massive genomic data have become feasible in cranberry due to advances in next generation sequencing technologies. This research used genetic (molecular markers) and trait information in cranberry breeding populations to create a model to predict plant performance. We developed a prediction ability model (i.e., predicting the performance of individuals based on genomic/trait information), and demonstrated that by using multiple variable methods, we increased predictive ability and obtained better resolution to detect genetic factors responsible for traits. In addition, we found that the use of optimal molecular marker densities and close genetic relationships between populations played an important role in the magnitude of the predictive ability. This information will benefit future breeding efforts in cranberry and other fruit crops. By refining prediction ability models in cranberry, breeders will be able to evaluate more plants and more efficiently select better genotypes and half the generation time that takes to produce a cultivar. (Objective 1).

3. Sequencing and genome advancement efforts. Cranberry breeding has been hampered by the limited genetic variability observed among cultivars and the lack of genomic resources. In this study, we sequenced and assembled the nuclear and organelle genomes of cultivated cranberry and wild relatives V. microcarpum and V. oxycoccos. The sequencing efforts have resulted in an improved genome assembly for the cultivated cranberry and two additional wild species were sequenced. This research is useful to multiple cranberry stakeholders, including breeders and growers, since most of the cultivars commercially used today were derived from a few wild cranberries bred in the 1950s. In different crops, it has been shown that wild collections can be used as an important genetic resource to incorporate novel traits and increase the trait diversity of breeding materials. The two wild species of cranberries used here have been found to be cross-compatible with the cultivated cranberry, and given their adaptation to bog habitats all over the northern hemisphere, they could be useful for breeding commercial cranberries with improved cold hardiness and a range of unique fruit and vine characteristics. This research provides complete advanced genome sequences to conduct diversity and evolutionary studies as well as genomic characterization and comparative analysis, which will allow breeding to be more effective in transferring desirable traits for growers and consumers (Objective 1).

4. Cranberry breeding trait priority survey. This study investigated the relative importance of cranberry producers’ preferences for breeding traits related to fruit quality, productivity, plant physiology, and resistance to biotic and abiotic stresses. Industry responses, in general, revealed that fruit quality, in particular, firmness, fruit size and anthocyanin content, and resistance to fruit rot were the most important traits to improve in new cranberry cultivars. These traits have the potential to increase the quality standards needed to process high value sweet and dried cranberry products, positively affecting price premiums received by producers, which is critical for the economic viability of the cranberry industry. Our findings will be useful to breeders and allied scientists seeking to develop an advanced DNA based selection strategies to impact the global cranberry industry. (Objective 2).

5. Diversity in national cranberry collection. The USDA-ARS National Clonal Germplasm Repository (NCGR) houses a collection of accessions which include historical cultivars and wild selected plants that played significant roles in the domestication and crop evolution of cranberry. In this study, we genetically studied 271 plants from 77 acquisitions representing 66 named cultivars. Using molecular markers, we determined clonal purity and relatedness of cultivars within the NCGR collection. We identified 64 unique genetic individuals and observed variants in the cultivar collection. We also identified the consensus genetic constitutions for many acquisitions and cultivars. This research highlights unique genetic diversity at the NCGR collection for conservation of cranberry genetic resources and for future use by breeders, researchers, and growers. This research provides guidance to the collection for the preservation of historically relevant cultivars absent at the collection. Preserving genetic diversity in cranberry and other crops is important to provide genetic resources for improving traits for long-term sustainability of production under changing conditions. (Objective 2).

6. Bio-insecticide development. Insects are consistently ranked as the most significant threats to cranberry production. Many insect threats cannot be adequately managed using insecticides. A new bio-insecticide has been formulated using nematodes that are native to Wisconsin. Years of efficacy testing have shown these nematodes to be highly virulent against flea beetles and fruitworms, which are two significant pests of cranberries. This research study revealed that the nematodes are also highly virulent against a new invasive pest, the spotted-wing drosophila, a major threat to US fruit production which may have application to its control (Objectives 3 and 4).

7. Pollinator health. Native pollinators are extremely important to the cranberry fruit-set. Recently, bee larval development has been shown to be highly dependent on microbial protein, and in the absence of pollen-borne microbes, bee larvae endure much higher mortality. This research study revealed that conservation of pollen-borne microbes can be achieved and improve bee larval mortalityis. Knowledge of the importance of microbes to bee health will further conservation strategies (Objective 4).

Review Publications
Gallardo, R., Zhang, Q., Polashock, J.J., Rodriguez-Soana, C., Vorsa, N., Atucha, A., Zalapa, J.E., Iorizzo, M. 2018. Breeding trait priorities of the cranberry industry in the United States and Canada. HortScience. 53(10):1467–1474. https://doi.org/10.21273/HORTSCI13219-18.
Covarrubias-Pazaran, G., Schlautman, B., Diaz-Garcia, L., Grygleski, E., Polashock, J.J., Johnson-Cicalese, J., Vorsa, N., Iorizzo, M., Zalapa, J.E. 2018. Multivariate GBLUP improves accuracy of genomic selection for yield and fruit weight in biparental populations of Vaccinium macrocarpon Ait. Frontiers in Plant Science. 9:1310. https://doi.org/10.3389/fpls.2018.01310.
Schlautman, B., Zalapa, J.E., Covarrubias-Pazaran, G., Rodriguez-Bonilla, L., Hummer, K.E., Bassil, N.V., Smith, T. 2018. Genetic diversity and cultivar variants in the NCGR cranberry (Vaccinium macrocarpon Aiton) collection. Journal of Genetics. 97(5):1339–1351. https://doi.org/10.1007/s12041-018-1036-3.
Pfeiffer, V., Silbernagel, J., Guédot, C., Zalapa, J.E. 2019. Woodland and floral richness boost bumble bee density in cranberry resource pulse landscapes. Landscape Ecology. 34(5):979. https://doi.org/10.1007/s10980-019-00810-1.
Bolivar, J., Zalapa, J.E., Atucha, A., Paterson, S. 2018. Relationship between alternate bearing and apical bud development in cranberry (Vaccinium macrocarpon). American Journal of Botany. 97(2):101-111. https://doi.org/10.1139/cjb-2018-0058.
Diaz-Garcia, L., Rodriguez-Bonilla, L., Rohde, J., Smith, T., Zalapa, J.E. 2019. Pacbio sequencing reveals identical organelle genomes between American cranberry (Vaccinium macrocarpon Ait.) and a wild relative. Genes. 10(4):291. https://doi.org/10.3390/genes10040291.
Diaz-Garcia, L., Schlautman, B., Covarrubias-Pazaran, G., Maule, A., Johnson-Cicalese, J., Grygleski, E., Vorsa, N., Zalapa, J.E. 2018. Massive phenotyping of multiple cranberry populations reveals novel QTLs for fruit anthocyanin content and other important chemical traits. Molecular Genetics and Genomics. 293:1379-1392. https://doi.org/10.1007/s00438-018-1464-z.
Diaz-Garcia, L., Covarrubias-Pazaran, G., Schlautman, B., Grygleski, E., Zalapa, J.E. 2018. Image-based phenotyping for identification of QTL determining fruit shape and size in American cranberry (Vaccinium macrocarpon L.). PeerJ. 6:e5461. https://doi.org/10.7717/peerj.5461.
Steffan, S.A., Gaines-Day, H.R., Dharampal, P., Chikaraishi, Y., Takizawa, Y., Danforth, B.N. 2019. Omnivory in bees: Elevated trophic positions among all major bee families. The American Naturalist. 194(3). https://doi.org/10.1086/704281.
Ye, W., Foye, S., MacGuidwin, A.E., Steffan, S.A. 2018. Incidence of Oscheius onirici (Nematoda: Rhabditidae), a potentially entomopathogenic nematode from the marshlands of Wisconsin, USA. Journal of Nematology. 50(1):9-26. https://doi.org/10.21307/jofnem-2018-004.
Dharampal, P., Carlson, C., Currie, C., Steffan, S.A. 2019. To bee or not to bee: Larval bees require pollen-borne microbes to survive. Proceedings of the Royal Society B. 286(1904). https://doi.org/10.1098/rspb.2018.2894.
Dharampal, P., Carlson, C., Steffan, S.A. 2018. In vitro rearing of solitary bees: A tool for assessing larval risk factors. Journal of Visualized Experiments. 137(e57876). https://doi.org/10.3791/57876.
Steffan, S.A., Dharampal, P.S. 2019. Undead food-webs: Integrating microbes into the food-chain. Food Webs. 16(e00111). https://doi.org/10.1016/j.fooweb.2018.e00111.