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

Location: Genetic Improvement for Fruits & Vegetables Laboratory

2021 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. By aligning the genetic map of blueberry with the blueberry genome sequence, genes were identified in the map regions that control the traits of chilling requirement, cold hardiness, fruit color, fruit scar size, and fruit firmness. By scanning the lists of genes and searching the literature, the best candidate genes were identified for controlling each trait. Efforts were begun to extract RNA from the 3-5 plants of the mapping population at the extreme ends of each trait continuum, for example, plants with black-colored fruit versus light blue-colored fruit and plants with extreme cold sensitivity versus plants with extreme cold hardiness. The RNA will be used to compare the expression of the best candidate genes for each trait in plants at opposite ends of the trait continuum. In this way, we will determine if an expression of a candidate gene for a particular trait is associated with the expression of the trait. A study has been underway to analyze fruit firmness in blueberry using three different methods. Varieties with firm fruit are needed by the blueberry industry because they are better for mechanical harvesting and because consumers prefer the texture of firm, crispy fruit over soft fruit. This study will be helpful in determining optimal methods for measuring firmness and identify varieties with contrasting firmness levels (firm and soft-fruited) to use in gene expression studies to identify genes associated with firm fruit. The study will also identify varieties that retain their firmness longer after ripening. The fruit was collected at unripe (pink), ripe (blue), and overly ripe stages from twelve blueberry varieties representing a range of different firmness levels. Firmness was measured in three ways, one using a texture analyzer and a probe that measures the force to press the fruit, one using a texture analyzer and a probe that punctures the fruit skin, and one using a Firmtech machine, which also presses the fruit. Statistical analysis was performed on the data to examine the accuracy and the precision of the three methods. The ability of the three methods to accurately measure firmness appeared fairly consistent when ranking varieties by firmness means. ‘Cara’s Choice’ and ‘Reveille’ were two of the firmest varieties, while ‘Razz’ and ‘Herbert’ were two of the least firm varieties at the ripe stage across all methods. How variable a firmness measurement was relative to its mean firmness score provided a measure of each method’s precision. The FirmTech method exhibited the least variability of the three methods. Analyses are still underway to determine which varieties retain their firmness longer after ripening. A cranberry pre-breeding program was initiated with the goal of investigating and improving fruit quality, disease resistance, and abiotic stress tolerance traits through an integrated genomic-phenomic approach. The first round of selective crosses was performed to generate foundational plant material for this program. Additionally, we have started three initial studies to support the goals of the program. First, we have begun building and evaluating a proximal sensing cart for high-throughput phenotyping of cranberry breeding beds. This cart will enable routine image-based measurements of plant vigor and fruit ripening throughout the growing season. Second, we analyze an interspecific population created via hybridization between cultivated cranberry (Vaccinium macrocarpon) and small-fruited cranberry (Vaccinium oxycoccos). We have phenotyped the population for flowering time, pollen viability, and leaf shape and size. We are currently genotyping the population for single nucleotide polymorphism (SNP) markers, which will be used to determine the genomic regions influencing these fitness-related traits. Small-fruited cranberry possesses characters that may be favorable for improving cultivated cranberry. Our analysis will help determine any interspecific incompatibilities that might prohibit the transfer of desirable traits. Third, we have initiated an environmental association analysis in a V. macrocarpon diversity collection to identify genomic regions that putatively confer adaptation to different climatic conditions. This analysis will help us identify individuals that may possess tolerance to critical abiotic stresses such as heat. Objective 2. Germplasm, combining aspects of rabbiteye vigor, V. constablaei’s late flowering, and highbush-like plant and fruit quality, continue to be evaluated. A blue-fruited selection from the V. constablaei incorporation program shows promise as a commercial selection and is undergoing field testing (ARS 16-57). Another selection has shown vivid fruit pigmentation and may have promise as an ornamental variety (US 2334). New hybrids continue to be generated of highbush blueberry with the section Hemimyrtillus species, V. padifolium, V. cylindraceum, and V. arctostaphylos. These species carry genes for indeterminate flowering and fruiting not currently available in highbush germplasm. Tri-specific hybrids were generated combining all three species, and additional backcross hybrids to highbush blueberry were made. Selections were made from field-grown populations of backcross (2nd generation) V. padifolium hybrids that expressed good vigor, adaptation, and reasonable yield. Several of these selections are being propagated for further testing. A repeat/continuous flowering selection was identified (US 2158), but has only fair fruit quality. Selves of this selection are being evaluated for improved genotypes. The selection is also being crossed to complementary highbush cultivars with commercial fruit quality. Work has continued on the utilization of V. meridionale, a South American species with prolific and concentrated flowering. Tetraploid hybrids utilizing V. meridionale hold the promise of facilitating hybridization and gene transfer among blueberry, cranberry, and lingonberry germplasm. We currently have fertile hybrids of V. meridionale with lingonberry, blueberry, and cranberry. The hybrids with lingonberry and cranberry are particularly unprecedented. These hybrids are in the process of being back-crossed to their respective crop species. The initial hybrids also successfully back-cross, at low levels, to all three commercial Vaccinium crops, facilitating inter-crop gene transfer. Objective 3. Research continues to evaluate several crosses utilizing the southern highbush cultivar ‘Reveille’ that yield populations with high numbers of firm-fruited progeny, although it was delayed by COVID-19 limitations. Progeny from another cross that produces a low frequency of hybrids with outstanding firmness in northern highbush blueberry also continues to be evaluated. A clone from this population has been propagated for advanced 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. Objective 4. Several stem pathogens of blueberry have been identified that have a negative impact on production. Some of the pathogens in New Jersey were previously described in the southeast. We suspect that climate change is allowing these pathogens to move north. The complex of pathogens causing fruit rot in cranberry has remained somewhat stable. However, a new leaf pathogen that can kill untreated cranberry plants has been tentatively identified as a Colletotrichum sp. We have a field population that is segregating for resistance to this disease. Genotyping and phenotyping have been completed, and the data are being analyzed to determine QTL associated with resistance/susceptibility. A different approach to microbiome identification, called stable isotope probing, is being used to determine the active soil microbes associated with blueberry decline. We have also noted gaps in the currently available databases for strain-level identification of soil microbes and initiated the development of our own database. The database will be publicly available. Utilizing a population resulting from an interspecific cross in cranberry, we examine traits associated with response to climate change. We have identified changes in cranberry gene expression associated with the complex tri-trophic interaction of false cranberry blossom, a bacterium-incited disease, the leafhopper insect vector of the disease, and natural predators of the leafhoppers. Affected plants flower but do not produce fruit, thus reducing yield. Plants have natural defenses against disease and insect predation. These natural defenses can be boosted by application of certain chemicals called elicitors. In this study, scientists with the USDA-ARS, in Chatsworth, New Jersey, hypothesized that boosting plant defenses using elicitors would slow the spread of false blossom in cranberry fields by a combination of decreasing the number of bacteria in the plant and discouraging feeding by the insect vector (leafhoppers) of the disease. Instead, we found that the tested elicitors had no effect on the amount of the bacterial pathogen in the plant and increased the performance and feeding of the vector. These data suggest that boosting plant defenses with elicitors is not a viable approach to controlling this disease and the insects that feed on cranberry.

Accomplishments
1. A high-density map and markers for chilling requirement, cold hardiness, and fruit quality traits in blueberry. Breeding new blueberry varieties is a slow process but can be accelerated and made more efficient if breeders could quickly identify genes in breeding lines for horticulturally important cultivar attributes. ARS scientist in Beltsville, Maryland, together with university collaborators, developed a high-density genetic map of blueberry comprised of 17,468 deoxyribonucleic acid (DNA) markers. We demonstrated utility of the map for public and private breeders via identification and mapping of markers associated with chilling requirement, cold hardiness and fruit quality attributes. The completed map and new markers are genetic tools and are available to blueberry breeders and geneticists worldwide to develop new cultivars more efficiently with desirable traits.

2. Genetic control of organic acid content in cranberry fruit. Cranberry fruit is high in organic acids as compared to most other fruits. This causes cranberry fruit and fruit products to be very tart, thus requiring large amounts of added sugar to be more palatable. Scientists in Chatsworth, New Jersey, took advantage of natural variation in organic acid content in cranberry germplasm to study the genetic basis of acid accumulation. We identified chromosomal regions in cranberry associated with citric and malic acid production and accumulation. We successfully designed markers for this (low acid) trait. These genetic tools are now available for public and private breeders to develop new cranberry cultivars that can be readily utilized for lower added sugar cranberry products, which will be more desirable to consumers.

3. Release of ‘Talisman’, a northern highbush blueberry. Blueberry cultivars that are high-yielding and suitable for machine harvesting are needed by the industry to meet the demand for high-quality blueberry fruit. ARS scientists in Chatsworth, New Jersey, developed and released a productive, mid-late ripening northern highbush blueberry selection, ‘Talisman’ (ARS 05-171), as a public domain variety. 'Talisman' produces firm high-quality fruit and is expected to be suitable for machine harvesting. Release as a public domain variety will benefit growers and smaller nurseries that are often unable to meet the business requirements for patented varieties. Propagules of this new ARS variety are available upon request.

Review Publications
Rowland, L.J., Ogden, E.L., Vinyard, B.T. 2020. Phenotypic evaluation of a hybrid diploid blueberry population for plant development and fruit quality traits. Agronomy. 10(8), 1067. https://doi.org/10.3390/agronomy10081067.
Drummond, F.A., Rowland, L.J. 2020. The ecology of autogamy in wild blueberry (Vaccinium angustifolium Aiton): Does the early clone get the bee?. Agronomy. 10(8):1153. https://doi.org/10.3390/agronomy10081153.
Die, J.V., Jones, R.W., Ogden, E.L., Ehlenfeldt, M.K., Rowland, L.J. 2020. Characterization and analysis of anthocyanin-related genes in wild-type blueberry and the pink-fruited mutant cultivar 'Pink Lemonade': New insights into anthocyanin biosynthesis. Agronomy. https://doi.org/10.3390/agronomy10091296.
Benevenuto, R., Seldal, T., Polashock, J.J., Moe, S.R., Rodriguez-Saona, C., Gillespie, M.A., Hegland, S.J. 2020. Molecular and ecological plant defense responses along an elevational gradient in a boreal ecosystem. Ecology and Evolution. https://doi.org/10.1002/ece3.6074.
Fong, S.K., Kawash, J., Wang, Y., Johnson-Cicalese, J., Polashock, J.J., Vorsa, N. 2021. A low malic acid trait in cranberry fruit: genetics, molecular mapping, and interaction with a citric acid locus. Tree Genetics and Genomes. 17(1):1-14. https://doi.org/10.1007/s11295-020-01482-8.
Ehlenfeldt, M.K., Luteyn, J.L. 2021. Intersectional F1 hybrids of 4x vaccinium meriodionale (swartz) (section pyxothamnus) and highbush blueberry, V. corymbosum (section cyanococcus). HortScience. 56(3):318–323. https://doi.org/10.21273/HORTSCI15523-20.
Ehlenfeldt, M.K. 2021. Production of dwarfs in rabbiteye blueberry (V. virgatum Aiton) crosses. Journal of American Pomological Society. 75:31-37.
Ehlenfeldt, M.K. 2020. ‘Talisman’ northern highbush blueberry - A productive late-season cultivar with concentrated ripening, suitable for mechanical harvest. HortScience. 56(1):101–103. https://doi.org/10.21273/HORTSCI15321-20.
Qi, X., Ogden, E.L., Boston, H., Sargent, D.J., Ward, J., Gilbert, J., Orizzo, M., Rowland, L.J. 2021. High-density linkage map construction and QTL identification in a diploid blueberry mapping population. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2021.692628.