Research Project: Genetic Improvement of Blueberry and Cranberry Through Breeding and Development/Utilization of Genomic Resources

Location: Genetic Improvement for Fruits & Vegetables Laboratory

2023 Annual Report

Objectives
Objective 1: Deliver enhanced genomic resources for blueberry and cranberry breeding and genetic research including: improved genome assemblies, germplasm genotypes, mapping populations, saturated genetic linkage maps, mapping data for high value quantitative traits, and candidate gene analysis using genetic and bioinformatic approaches. [NP 301, C1, PS1A, C2, PS2A] Expected benefits include coordinated breeding and pre-breeding for cranberrry across all production regions with the goal to enhance new cultivar development and new product development. Subobjective 1a: Develop improved assemblies of the blueberry and cranberry genomes using long-read sequencing technologies and anchor new genome assemblies to well-saturated genetic linkage maps. Subobjective 1b: Map QTL for cold hardiness, chilling requirement, fruiting season, disease resistances, and fruit quality traits using improved maps and well-characterized bi-parental and association mapping populations. Subobjective 1c: Identify candidate genes for traits by their proximity to QTL, by homology to genes characterized in other systems, and by expression studies on plants with contrasting phenotypes. Subobjective 1d: Use a systems approach to cranberry breeding and genetics that includes genetic improvement, genomics, and phenomics. Objective 2: Develop and release new blueberry germplasm that is enhanced for prolific, indeterminate fruiting and cold tolerance by incorporating germplasm from exotic sources into the program. [NP 301, C1, PS1B] Objective 3: Develop and release new blueberry cultivars that are enhanced for mechanical harvesting, expanded fruiting season, cold hardiness, tolerance of higher pH soils, resistance to mummy berry and fruit rot, and adaptability to changing environmental conditions. [NP 301, C1, PS1B] Objective 4: Identify and characterize key pathogens of blueberry and cranberry and the genes that mediate plant-pathogen interactions, including stem blight (blueberry) and key pathogens in the fruit rot complex (cranberry), as well as plant-environment interactions. [NP 301, C3, PS3A; NP 303, C1, PS1, C2, 2B]

Approach
The approach entails the integration of genomic approaches with traditional breeding and plant pathology in the development of improved blueberry and cranberry cultivars. Scientists will develop enhanced genomic resources for blueberry and cranberry, including improved genome assemblies, well-saturated genetic linkage maps with anchorage to the genomes, and well-characterized bi-parental and association mapping populations, and identify quantitative trait loci (QTL) for horticulturally significant traits such as cold hardiness, chilling requirement, fruiting season, disease resistances, and fruit quality traits. In addition, scientists will carry out gene expression studies to identify the actual genes underlying significant QTL. Scientists will also incorporate a systems approach to cranberry breeding and genetics focused on genetic improvement with supporting phenotyping and transdisciplinary research on phenomics involving plant physiology, data sciences, and engineering. Scientists will also identify key genes in blueberry and cranberry that mediate plant-pathogen interactions, including stem blight (blueberry), key pathogens in the fruit rot complex (cranberry), and plant-environment interactions. Scientists will characterize and incorporate new germplasm, and generate new blueberry cultivars that meet industry needs. Better genomic resources for these crops will enable marker development for use in marker-assisted breeding. Scientists will also identify key genes in blueberry and cranberry that mediate plant-pathogen interactions, including stem blight (blueberry), key pathogens in the fruit rot complex (cranberry), and plant-environment interactions. Scientists will characterize and incorporate new germplasm, and generate new blueberry cultivars that meet industry needs. Better genomic resources for these crops will enable marker development for use in marker-assisted breeding.

Progress Report
Objective 1. Through collaborative projects a Vaccinium pangenome is nearly complete. We also collaborated to develop a 3,000-marker Diversity Arrays Technology (DArT) panel and a 15,000-marker Flex-seq marker array for cranberry genotyping. QTL were identified and markers developed for cranberry organic acids, fruit epicuticular wax, fruit shape, and fruit rot resistance. Markers developed for organic acid content and epicuticular wax work very well. Markers for fruit rot resistance (FRR) in cranberry are still being tested. A study to determine the utility of interspecific cranberry (V. macrocarpon x V. oxycoccos) hybrids for environmental resilience was continued. Transcriptome sequencing was completed, and differential expression assessment is in progress. We continued development of virus-induced gene silencing (VIGs) vectors and CRISPR-Cas9 vectors for Vaccinium spp. These approaches were tested for gene knock-out and gene editing effectiveness in cranberry. The cranberry pre-breeding program made crosses to generate initial breeding populations. A total of 85 crosses have been made using accessions of wild, landrace, and improved genotypes, generating more than 2,000 first-cycle seedlings for genotypic and/or phenotypic evaluation. These crosses were made based on multi-trait indices, environmentally adaptive genetic loci, or putative disease resistance. A generalized image analysis workflow for high-throughput cranberry phenotyping systems, based on training custom deep learning models, was published. This workflow measures berry shape, size, and total anthocyanin content in postharvest samples of cranberry using images captured in a custom-designed lightbox. Image-based traits extracted using this workflow are highly predictive of traits measured using standard, low-throughput methods. Objective 2. Germplasm, combining rabbiteye vigor, V. constablaei’s late flowering, and highbush-like fruit quality, were evaluated. Selections (ARS 16-57) show promise for commercial use and another vivid fruit pigmentation has promise as an ornamental variety (US 2334). Families of varied hexaploid (V. constablaei X rabbiteye) x tetraploid (northern highbush) ancestry are undergoing a second year of field evaluation. ‘Nocturne’ is a productive, cold-hardy, rabbiteye-derived hexaploid blueberry cultivar. ‘Nocturne’ was crossed to the northern highbush cultivars, Duke, Cara’s Choice, and Elliott to produce pentaploid families. A replicated plot containing individuals of “Nocturne × highbush” pentaploids × 4x highbush cultivars, and “Nocturne × highbush” pentaploids × 6x rabbiteye cultivars is currently 5 years old. The field plot will undergo evaluation for a minimum of 3 years. Preliminary selections have been made. Tetraploid hybrids, utilizing V. meridionale, a South American species with prolific and concentrated flowering, may facilitate hybridization and gene transfer among blueberry, cranberry, and lingonberry germplasm. We produced fertile hybrids of V. meridionale with lingonberry, blueberry, and cranberry. The hybrids with lingonberry and cranberry are particularly unprecedented. We are evaluating the capabilities of the initial hybrids to backcross to their parental crop species and to cross to alternate crop species to facilitate inter-crop gene transfer (example [V. meridionale x highbush blueberry] x lingonberry). This information will produce a crossability “road map” as a guide for future exploitation of such crosses. Plants of V. meridionale x lingonberry genotypes and V. meridionale x highbush genotypes are being propagated for distribution to cooperators to evaluate regional adaptation. Explorations of blueberry species crossability produced a set of unique hybrids between Darrow’s blueberry (2x V. darrowii) and lingonberry (2x V. vitis-idaea). These hybrids hold promise through further crosses of improving the climatic/environmental adaptation of lingonberry. Further generation of similar hybrids has been completed. A project was initiated to evaluate the possibility of hybridizing V. myrtillus (European blueberry) with North American blueberries and other Vaccinium spp. There is considerable interest in V. myrtillus due to its high levels of pigmentation and correspondingly high levels of antioxidants. V. myrtillus also expresses numerous fruit volatiles. Objective 3. A population of blueberry hybrids that segregates for fruit color (blue vs. white) was phenotyped and genotyped using bulk-segregant analysis. Analysis is in progress for QTL/gene discovery associated with fruit pigmentation. Research continues to evaluate several crosses utilizing the southern highbush blueberry cultivar Reveille that yield populations with high numbers of firm-fruited progeny. Progeny from another cross that produces a low frequency of hybrids with outstanding firmness in northern highbush blueberry continue to be evaluated. A clone from this population has been in initial replicated testing (ARS 15-59). These populations are being further explored and expanded to generate populations for molecular studies and to generate selections for machine-harvest testing. A blue-fruited selection from the V. constablaei incorporation program that shows potential promise as a commercial selection continues to undergo field testing (ARS 16-57). In 2020 and 2021, COVID-19 and its associated work restrictions hampered the evaluation of advanced selections. Seedling families were evaluated, and primary selections were made in 2022. Backcross hybrid seedling families comprised of ¼ V. meridionale and ¾ highbush (V. corymbosum) were planted in the field. These will undergo evaluation for cultivar potential in the forthcoming years. Objective 4. Colletotrichum spp. (fungal pathogens) were collected from fruit and vegetative tissues and sequenced for species identification/confirmation and fungicide resistance. Resistant isolates were detected and are being further characterized. We demonstrated, using hyperspectral imaging, that certain systemic diseases of blueberry and cranberry could be accurately detected in infected leaf tissue that looks healthy to the naked eye. We further developed a system to rapidly determine the level of fruit rot in heterogeneous cranberry fruit samples. We custom built a cart for in-field phenotyping using both hyperspectral imaging and RGB-based imaging. Fungi associated with stem blight symptoms in blueberry were isolated in previous seasons. New species have been implicated in stem blight disease and a pathogen normally associated with minor twig blight (Phomopsis vaccinii) has been determined to be mixed, but closely related, species that are causing more severe symptoms. We are near publication of characterizing the whole draft genome and population genetics of Valdensia heterodoxa, the causal agent of Valdensia Leaf Spot disease of lowbush blueberry. A paper describing the rhizosphere microbiome and potential pathogens therein was just published as a prerequisite for understanding blueberry and cranberry decline and replant disease. Summary of Progress Over the 5-yr Life of the Project Plan - High quality genome and transcriptome data were produced and published for blueberry and cranberry. - High density genetic maps were published for blueberry and cranberry. - QTL were discovered for important traits in blueberry and cranberry. Some of which were published (e.g. organic acids, fruit rot resistance, yield, fruit size, fruit shape). - Markers, based on discovered QTL, were designed, tested and verified for use in Marker-assisted Selection. Some markers were published. - High throughput genotyping was improved by development of DArt and Flex-seq arrays. - High throughput phenotyping was developed using advanced imaging techniques coupled with new approaches to analyze imaging data. - Better analysis methods for QTL discovery were developed, using machine learning. - New pathogens of blueberry and cranberry were characterized. - Blueberry and cranberry rhizosphere microbial communities were characterized and published. - A patent was issued (US-10815491-B2) with collaborators at Oxford, MS describing Sorghum-derived transcription regulatory elements predominantly active in root hair cells and uses thereof‘. - Publications documented that plants produce volatiles that affect insect herbivores and non-herbivores in different ways (plant-pathogen-insect tri-trophic interactions). - Demonstrated that phytoplasma infection affects gene expression in cranberry. This has implications for how vector insects interact with infected plants. - QTL were discovered and reliable markers were developed for ow organic acids (citric and malic) in cranberry. This research has the potential to make cranberry products that require much less added sugar. - We found that natural volatile defenses in bilberry are inducible by herbivory. This work will potentially lead to improved methods of insect pest control in plants. - ‘Talisman’, a late- midseason blueberry cultivar suitable for machine harvesting was released. - Hybrids of Andean blueberry (V. meridionale) with Highbush blueberry (V. corymbosum), American cranberry (V. macrocarpon), and lingonberry (V. vitis-idaea) were generated. These unprecedented hybridizations form the first steps for the transfer of genetic material among these Vaccinium species. - An accession from a rabbiteye blueberry (V. virgatum hybrid) that generates high frequencies of dwarf phenotypes was identified. Dwarfs have the potential to be productive while having a reduced and more manageable stature. - Completed transcriptome analysis that identified genes involved in the production of the waxy coating on blueberry fruit in rabbiteye blueberry (V. virgatum). - Completed characterization and analysis of anthocyanin-related genes in blueberry and a pink-fruited cultivar. This research provided new insights into anthocyanin production in blueberry.

Accomplishments
1. First known hybrids between the South American Andean blueberry (V. meridionale) and the American cranberry (V. macrocarpon) created. The South American blueberry species V. meridionale has shown value as a bridge between taxonomic sections and ploidies in Vaccinium as either a first-generation or second-generation parent. It has already been hybridized with highbush blueberry (V. corymbosum). Further explorations were undertaken by ARS scientists in Chatsworth, New Jersey, to facilitate germplasm transfer in more distant Vaccinium species. The V. meridionale x cranberry crosses produced strong plants with notable vigor and moderate fertility. The first-generation hybrids were successfully sib-mated and backcrossed to cranberry. These tetraploid hybrids provide breeders with new opportunities to significantly enlarge the genepool of cranberry and introgress cranberry germplasm into highbush blueberry. This research will benefit breeders of any of the three commercial North American Vaccinium crops.

2. New reproductive behavior discovered in blueberry to circumvent crossing barriers. USDA scientists at Chatsworth, New Jersey, documented asymmetric crossing behavior of a V. meridionale x lingonberry hybrid, US 1930. In US 1930 x V. corymbosum crosses, hexaploid offspring were produced. Reciprocally, in V. corymbosum x US 1930 crosses, tetraploid offspring were produced. This asymmetric behavior is unique in that it allows introgression of V. meridionale/lingonberry germplasm to two different ploidy levels, and two different germplasm pools, highbush blueberry (V. corymbosum) and rabbiteye blueberry (V. virgatum). This asymmetric crossing behavior offers insights about genome dosage effects in hybridization. This research will benefit breeders of both highbush and rabbiteye blueberry to transfer genes for crop improvement between previously inaccessible germplasm.

3. New database for microbiome of blueberry and cranberry. USDA scientists in Chatsworth, New Jersey, documented the diversity of rhizosphere (root zone) microbiome (bacteria and fungi) in commercial blueberry and cranberry fields. These baseline data are required to begin understanding the complex characteristics of soil health and crop decline over time. Organisms were detected that included many plant pathogens as well as beneficial organisms. This fundamental research will benefit all aspects of Vaccinium species crop health and diverse disciplines with soil scientists, horticulturalists, plant pathologists, geneticists, and extension personnel active in Vaccinium crop health and management.

Review Publications
Ehlenfeldt, M.K., Polashock, J.J., Vorsa, N., Zalapa, J.E., De La Torre, F., Luteyn, J.L. 2023. Fertile Intersectional F1 Hybrids of 4x Andean Blueberry (Vaccinium meridionale) and 4x American Cranberry (Vaccinium macrocarpon). HortScience. 58(2):234-239. https://doi.org/10.21273/HORTSCI16824-22.
Erndwein, L., Kawash, J.K., Johnson-Cicalese, J., Vorsa, N., Polashock, J.J. 2023. Cranberry fruit epicuticular wax benefits and identification of a wax-associated molecular marker. BMC Plant Biology. https://doi.org/10.1186/s12870-023-04207-w.
Schwanitz, T.W., Polashock, J.J., Stockton, D.G., Rodriguez-Saona, C., Sotomayor, D., Loeb, G.M., Hawkings, C. 2022. Molecular and behavioral studies reveal differences in olfaction between winter and summer morphs of Drosophila suzukii. PeerJ. 10. Article e13825. https://doi.org/10.7717/peerj.13825.
Mengist, M., Bostan, H., De Paola, D., Teresi, S., Platts, A., Cremona, G., Qi, X., Mackey, T.A., Bassil, N.V., Ashrafi, H., Giongo, L., Jibran, R., Chagne, D., Bianco, L., Lila, M., Rowland, L.J., Iovene, M., Edger, P.P., Iorizzo, M. 2022. Autopolyploid inheritance and a heterozygous reciprocal translocation shape chromosome genetic behavior in tetraploid blueberry (Vaccinium corymbosum). New Phytologist. 237(3):1024–1039. https://doi.org/10.1111/nph.18428.
Iorizzo, M., Lila, M., Perkins-Veazie, P., Luby, C.H., Vorsa, N., Edger, P., Bassil, N.V., Munoz, P., Zalapa, J.E., Gallardo, R.K., Atucha, A., Main, D., Giongo, L., Li, C., Polashock, J.J., Sims, C., Canales, E., DeVetter, L., Coe, M., Chagne, D., Colonna, A., Espley, R. 2023. VacciniumCAP, a community-based project to develop advanced genetic tools to improve fruit quality in blueberry and cranberry. Acta Horticulturae. 1362:71-80. https://doi.org/10.17660/ActaHortic.2023.1362.11.
Wang, D.R., Kantar, M.B., Murugaiyan, V., Neyhart, J.L. 2023. Where the wild things are: Genetic associations of environmental adaptation in the oryza rufipogon species complex. Genes, Genomes, Genetics. https://doi.org/10.1093/g3journal/jkad128.
Ehlenfeldt, M.K., Rowland, L.J., Ogden, E.L., Luteyn, J.L. 2022. Asymmetric reciprocal crossing behavior of an andean blueberry (V. Meridionale) × lingonberry (V. Vitis-idaea) Hybrid. Plants. 11(22):3152. https://doi.org/10.3390/plants11223152.