Location: Genetic Improvement for Fruits & Vegetables Laboratory
2024 Annual Report
Objectives
Objective 1. Enhance genomic resources for blueberry and cranberry by increasing the number of reference genomes in these crops and related species, leveraging evolving genotyping platforms to develop well-saturated genetic maps, and mapping and utilizing QTL for marker development and gene discovery of selected traits.
Sub-objective 1a. Develop new reference genomes.
Sub-objective 1b. Develop a consensus genetic-physical map for cranberry.
Sub-objective 1c. Map QTL for selected traits in blueberry and cranberry.
Sub-objective 1d. Develop markers for key QTL and identify trait-associated candidate genes.
Sub-objective 1e. Develop and use virus-induced gene silencing (VIGs) and CRISPR gene editing to test target gene function in blueberry and cranberry.
Objective 2. Develop improved cranberry/blueberry pre-breeding and breeding methods that exploit high-dimensional genomic, phenomic, and environmental data, leading to the development of genetic stocks, improved breeding lines, and elite cultivars.
Sub-objective 2a. Develop and evaluate methods to effectively and reciprocally introgress rabbiteye (V. virgatum) and highbush (V. corymbosum) germplasm to produce improved and environmentallyadapted selections and cultivars.
Sub-objective 2b.Develop and evaluate methodology to utilize V. meridionale (Andean blueberry) to improve highbush blueberry (V. corymbosum), and to cross-transfer blueberry (V. corymbosum / V. virgatum), cranberry (V. macrocarpon), and lingonberry (V. vitis-idaea) germplasm.
Sub-objective 2c. Develop, validate, and implement genomic selection models to initiate a rapid recurrent selection cranberry pre-breeding pipeline.
Sub-objective 2d. Develop and deploy systems for image-based high-throughput phenotyping and trait discovery in cranberry and blueberry.
Objective 3. Apply ‘omics technologies to better understand fundamental cranberry/blueberry processes and plant-microbe interactions such as disease resistance and environmental stress tolerance.
Sub-objective 3a. Develop hyperspectral imaging techniques for the collection of phenotypic data of blueberry and cranberry, such as disease status, fruit wax, and stress response.
Sub-objective 3b. Use transcriptomics to characterize response of blueberries and cranberries to various treatments such as temperature stress and during various other processes/developmental stages.
Approach
Uncover genome variation in Vaccinium crops and related species, using long-read sequencing and advanced bioinformatics tools. Evaluate V. meridionale by highbush blueberry, by lingonberry, and by cranberry F1 progeny for quality traits and as pre-breeding material for the development of superior lines. Develop genomic selection (GS) models to enhance prediction accuracy for selection and breeding. Increase the use of imaging for high-throughput phenotyping and the procedures for image analysis. Employ RNAseq experiments to identify important target genes and create markers for their selection in progeny.
Progress Report
In support of Objective 1, Sub-objective 1a, new accessions of cranberry have been fully sequenced. These will be processed and added to our growing list of reference genomes.
In support of Objective 1, Sub-objective 1b, linkage maps have been constructed from previous cranberry mapping population data and from new mapping populations using the improved physical reference genomes. Linkage maps will be combined to create a consensus linkage map.
In support of Objective 1, Sub-objective 1c, annotation of our published reference genomes of Vaccinium macrocarpon and V. oxycoccos were improved and updated. Several traits have been mapped in cranberry.
In support of Objective 1, Sub-objective 1c, as part of our leaf drop work in cranberry (caused by Colletotrichum spp.), we have isolated the casual agent(s) and long-read sequenced representative isolates. These data will help identify, to species level, the casual pathogen(s), a first step toward mapping cranberry for resistance to this pathogen.
In support of Objective 1, Sub-objective 1d, QTL for many traits (e.g. citric acid, malic acid, benzoic acid, Brix, fruit weight, yield, etc.) have been identified. Those traits for which markers have been made and tested are epicuticular wax (2), malic acid (1), citric acid (1), and fruit rot resistance (1).
In support of Objective 2, Sub-objective 2a, a third year of evaluation is being done in populations combining rabbiteye (V. virgatum) and highbush (V. corymbosum). Selections are being made of superior genotypes. These selections will be the foundation for further cycles of crossing and introgression. New cycles of crossing to produce similar progeny with other diversified parents have been conducted.
In support of Objective 2, Sub-objective 2a, crosses using ‘Talisman’ blueberry are being fast-tracked for potential cultivar evaluation. ‘Talisman’ has demonstrated itself to be an excellent parent for fruit quality in the correct combinations.
In support of Objective 2, Sub-objective 2b, 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 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 × highbush blueberry] × lingonberry). This information is producing a crossability “road map” as a guide for future exploitation of such crosses. Plants of V. meridionale × lingonberry genotypes and V. meridionale × highbush genotypes are being propagated for distribution to cooperators to evaluate regional adaptation. Selected genotypes of Highbush blueberry (V. corymbosum) x Andean blueberry (V. meridionale) and genotypes of Andean blueberry (V. meridionale) x lingonberry (V. vitis-idaea) are being propagated for distribution to cooperators to allow evaluation of environmental adaptation; however, propagules have been slow to establish.
In support of Objective 2, Sub-objective 2b, hybrids have been created between diploid blueberry (V. fuscatum) and small-fruited cranberry (V. oxycoccos). These crosses produced multiple hybrids and represent an advance in the potential to transfer germplasm directly between blueberry and cranberry.
In support of Objective 2, Sub-objective 2b, hybrids have been created between Box Huckleberry and Lingonberry. This is the first known intergeneric hybrid between huckle berry and blueberry germplasm and represents a significant advance in broadening the accessible germplasm base of blueberry.
In support of Objective 2, Sub-objective 2b, Hybrids and backcross hybrids have been created between Box Huckleberry and Dwarf Huckleberry. This project was initiated with USDA Floral and Nursery Crops lab to produce an improved native species groundcover. Backcross hybrids are being evaluated for potential release.
In support of Objective 2, Sub-objective 2c, Genomic prediction models were trained and evaluated using historical phenotypic data and genotype data (whole-genome genotyping-by-sequencing) from diverse cranberry germplasm. Field plantings of additional diverse cranberry germplasm, along with first-cycle pre-breeding material, were established to refine and validate genomic prediction models. These new plantings will be phenotyped starting in FY26.
In support of Objective 2, Sub-objective 2d, Software pipelines were developed to measure fruit size and color traits from images of cranberry fruit post-harvest. This pipeline i) determines sample identification by reading QR codes, ii) corrects color values using standards, and iii) segments berries using deep neural networks and AI. The pipeline measures the average and uniformity of fruit size, shape, and color from a sample. The first year of in-field RGB and thermal images of experimental cranberry germplasm were collected. These images will be used to estimate in-field fruit yield, color, and percent rot, along with heat stress tolerance.
In support of Objective 3, Sub-objective 3a, Hyperspectral imaging was used to successfully classify blueberry and cranberry with certain systemic diseases (viruses and bacteria).
In support of Objective 3, Sub-objective 3b, Cranberry plants were exposed to short term heat (42 C) and cold stress (10 C) and their RNA extracted for gene expression analyses. The responses (changes in gene expression) have been determined.
Accomplishments
1. Blueberry and cranberry pangenomes completed. A pangenome is a composite reference genome designed to capture all of the genes in a given species. Pangenomes for minor crops, such as blueberry and cranberry, are rare. This work by USDA-ARS scientists in Chatsworth, New Jersey, expands the availability of genomic resources for blueberry and cranberry and was used to build high-resolution marker arrays. It will be of great value to researchers working on gene discovery and utilization for improvement of Vaccinium species such as blueberry and cranberry.
2. Database for rhizosphere communities in’ berryland’ soils. Blueberries and cranberries grow in unique, high acid, ‘berryland’ soils. The microbes in these soils, many of which impact crop health, are not well characterized compared to more typical farm soils. To understand the complex ecology of these soils, USDA-ARS scientists in Chatsworth, New Jersey, we determined the microbial communities associated with the roots of blueberry and cranberry. Considerable diversity for bacterial and fungal genera were identified and a database was developed to catalog this information. This data detailing the microbial diversity in acidic ‘berryland’ soils will be used by researchers to address crop health and plant pathogen management and improve sustainability of berry crop production.
3. Production of intersectional hybrids between blueberry and lingonberry. Utilization of genes in divergent crop species is often difficult due to crossing barriers that prevent transfer of genes between species. Lingonberry and the cultivated blueberry are two such genetically distant species. USDA-ARS Researchers in Chatsworth, New Jersey, have hybridized Darrow’s Blueberry, a blue-fruited blueberry and Lingonberry. This is the first well- documented intersectional hybrid between a blue-fruited highbush-compatible blueberry species and the red-fruited lingonberry. Modest fertility was found in the offspring, and several secondary hybrids were produced. These hybrids have the potential of improving the heat adaptation of lingonberry, and the potential of introducing new and useful germplasm into highbush blueberry. This new germplasm is of great interest to blueberry and lingonberry growers and breeders.
4. Affordable targeted genotyping platform validated for cranberry. Implementing genomics-enabled breeding methods relies on having affordable options for high-throughput genotyping of molecular DNA markers. In collaboration with Breeding Insight, USDA-ARS scientists in Chatsworth, New Jersey, validated a 3,000 single nucleotide polymorphism (SNP) array designed to target markers across the cranberry genome. We genotyped 564 diverse cranberry accessions from the Rutgers University germplasm collection using this array. We characterized the genetic diversity in these collections and identified many genetically duplicated accessions. We also genotyped 470 first-cycle breeding clones from the cranberry pre-breeding program, which will enable early-stage selection of these clones for potentially novel yield, fruit quality, and abiotic stress tolerance traits.
5. Genetic signatures for local adaptation in cranberry. Natural selection shapes wild or landrace germplasm of modern crops through adaptation to local environments. This plant material is a valuable source of potential genetic tolerance to environmental stresses. Using novel environmental association methodology, USDA-ARS scientists in Chatsworth, New Jersey, discovered genomic regions in cranberry that are associated with local climate or soil conditions. These genetic signatures have already been applied to identify germplasm in the cranberry collection and in breeding programs to improve heat and cold tolerance in cultivated cranberry. These genetic signatures have been utilized by stakeholders to make informed decisions for new exploration trips to collect stress tolerant cranberry germplasm.
Review Publications
Ehlenfeldt, M.K., Bassil, N.V., Zalapa, J.E., De La Torre, F., Luteyn, J.L. 2024. Intersectional hybrids between darrow’s blueberry (V. Darrowii camp) and lingonberry (V. vitis-idaea L.). Plants. 13(11). Article e1572. https://doi.org/10.3390/plants13111572.
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.
Kawash, J.K., Oudemans, P., Erndwein, L.C., Polashock, J.J. 2023. Assessment and comparison of rhizosphere communities in cultivated Vaccinium spp. provide a baseline for study of causative agents in decline. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2023.1173023.
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.
Ewing, P.M., Kantar, M.B., Killian, E., Neyhart, J.L., Sherman, J., Williams, J., Lachowiec, J., Eberly, J. 2024. Local adaptation and broad performance are synergistic to productivity in modern barley. Crop Science. 64(1):192-199.
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.
Geng, P., Harnly, J.M., Sun, J., Chen, P. 2024. Variability and determinants of secondary metabolite profiles in cranberries (Vaccinium macrocarpon) from key cultivation states. Journal of Agriculture and Food Research. 15:100983. https://doi.org/10.1016/j.jafr.2024.100983.
Kawash, J.K., Erndwein, L.C., Johnson-Cicalese, J., Knowles, S., Vorsa, N., Polashock, J.J. 2024. Quantitative trait loci analysis and marker development for fruit rot resistance in cranberry shows potential genetic association with epicuticular wax. Phytopathology. 114:1366-1372. https://doi.org/10.1094/PHYTO-12-23-0477-R.