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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Genetic Improvement for Fruits & Vegetables Laboratory » Research » Research Project #434363

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

Location: Genetic Improvement for Fruits & Vegetables Laboratory

2022 Annual Report

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]

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. A high-density genetic linkage map of a diploid blueberry population was aligned with blueberry genomic sequences. Genes near the map regions that control traits of chilling requirement, cold hardiness, fruit color, fruit scar size, and fruit firmness were identified. By scanning the lists of genes and searching the literature, several candidate genes for controlling each trait were identified. Ribonucleic acid (RNA) was extracted 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 is currently being used in real-time polymerase chain reaction (PCR) experiments to compare the expression of the best candidate genes for each trait in plants of opposite ends of the trait continuum. In this way, we will determine if expression of a candidate gene is associated with expression of the trait itself. Research was continued to identify genes needed for firmness in blueberry fruit. We hypothesized that a comparison of the transcriptome of soft and firm varieties of blueberry at unripe and ripe stages would reveal genes involved in maintaining firmness. Comparative transcriptome analyses of the firm-fruited ‘Cara’s Choice’ and the soft-fruited ‘Razz’ were conducted at two stages of ripeness (unripe/pink stage and ripe/blue stage). A higher number of differentially expressed genes was recovered from the soft-fruited ‘Razz’ compared to the firm-fruited ‘Cara’s Choice’. Differentially expressed genes modulated during ripening were found to be involved in a variety of pathways including hormone-signaling, secondary metabolite biosynthesis, carbohydrate synthesis, cell wall biosynthesis, and calcium-binding related genes. Comparisons between the two varieties are continuing. Quantitative trait locus (QTL) were identified for organic acid level in cranberry fruit, cranberry fruit epicuticular wax, cranberry fruit shape, and fruit rot resistance in cranberry. Markers were designed, based on single nucleotide polymorphisms (SNPs) near the QTL. Testing is in progress for some markers, but those for organic acid content and epicuticular wax work very well. A candidate gene was identified that may be associated with high fruit wax. The gene, CER1, is involved in long-chain fatty acid biosynthesis. A cranberry pre-breeding program was initiated. More than 65 crosses were performed within the last year to generate initial breeding populations. Crosses were made with the goal of exploiting novel variation in wild/unimproved cranberry while maintaining high fruit yield and quality. More than 600 first-cycle seedlings were germinated and are being propagated for initial rounds of selection. Environmentally adaptive genetic loci were mapped in cranberry. A collection of wild cranberry germplasm was used to identify genetic loci associated with local climate and soil conditions. Several loci associated with cold temperatures, low precipitation, high soil pH, and soil nitrogen content were discovered, and candidate genes were identified. Two genes, ALA3 and MOS14, are involved in cold acclimation. High-throughput cranberry phenotyping systems underwent initial testing. Two high-throughput phenotyping systems - a field-based proximal sensing cart and a postharvest berry imaging platform - were developed and underwent initial evaluation. Both systems were used to collect color images of either cranberry breeding plots (proximal sensing cart) or postharvest berry samples. Deep learning models were trained to automatically identify berries within each image, and image analysis pipelines were used to quantify berry shape, size, and color phenotypes. While phenotypic analysis using the proximal sensing cart is ongoing, image-based berry quality phenotypes collected using the postharvest imaging platform showed high concordance with traditional, low-throughput fruit quality measurements. An experiment was initiated to determine the utility of interspecific cranberry (V. macrocarpon x v. oxycoccos) hybrids for environmental resilience. As a requirement for verifying gene function in blueberry and cranberry, we have developed virus-induced gene silencing (VIGs) vectors and CRISPR-Cas9 vectors. These approaches will be tested for gene knock-out and gene editing effectiveness in cranberry first and then blueberry if successful. Objective 2. Newer germplasm, combining aspects of rabbiteye vigor, V. constablaei ’s late flowering, and highbush-like plant and fruit quality, were evaluated. A blue-fruited selection from the V. constablaei incorporation program (ARS 16-57) continues to show promise as a commercial selection and is undergoing further field testing. Another selection that has shown vivid fruit pigmentation may have promise as an ornamental variety (US 2334). An experiment containing families of varied hexaploid (V. constablaei-rabbiteye-types) x tetraploid (northern highbush) origins has been established in the field and is undergoing preliminary evaluation. ‘Nocturne’ is a productive, cold-hardy, rabbiteye-derived hexaploid blueberry cultivar with some V. constablaei ancestry. Compared to 100% rabbiteye cultivars, it is notable for its high level of self-fertility. Previously, ‘Nocturne’ was crossed to the northern highbush cultivars, Duke, Cara’s Choice, and Elliott to produce pentaploid families. Most pentaploids are not exceptionally fertile, but several of the hybrids with modest fertility were selected and used for subsequent crosses. A replicated plot containing individuals of “Nocturne × highbush” pentaploids × 4x highbush cultivars, and “Nocturne × highbush” pentaploids × 6x rabbiteye cultivars has been planted and is currently 4 years old. The field plots will undergo evaluation for a minimum of 2 years for parameters of interest: percent survival, vigor, flowering time, ripening time, productivity, fruit size, fruit quality, etc. Superior individuals will be noted and selected. New hybrids continue to be evaluated for 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 have been generated combining these 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), that has only fair fruit quality. This selection was crossed to complementary highbush cultivars with commercial fruit quality. Work has continued on the utilization of V. meridionale (formerly described as V. corymbodendron), a South American species with prolific and concentrated flowering. It was found that when one of the initial V. meridionale hybrids, 4x V. meridionale x lingonberry, was crossed to 6x rabbiteye blueberry (V. virgatum), septaploid (7x) plants were unexpectedly produced rather than the typically expected pentaploids (5x). Some of these hybrids were found to be quite vigorous and reasonably fertile and exhibited a dramatic increase in fruit size compared to the 4x V. meridionale x lingonberry parent. Explorations of blueberry / Vaccinium crossability produced a number of unique hybrids between Darrow’s blueberry (2x V. darrowii) and lingonberry (2x V. vitis-idaea). The most advanced of these hybrids was found to be fertile and holds the promise through further crosses of improving the climatic / environmental adaptation of lingonberry. A minor project has been initiated to evaluate the possibility of hybridizing with V. myrtillus (European blueberry, lowbush) with North American blueberries. There is considerable interest in V. myrtillus due to the fact that unlike highbush blueberries, V. myrtillus possesses pigmented flesh, and correspondingly high levels of antioxidants. Objective 3. 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 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. A blue-fruited selection from the V. constablaei incorporation program that shows promise as a commercial selection continues to undergo field testing (ARS 16-57). Objective 4. A new procedure for false blossom detection in cranberry was developed. False blossom is an important re-emerging disease of cranberry incited by a bacterium (phytoplasma) and transmitted by a leafhopper. Detection has traditionally been done using nested PCR. We have developed a LAMP (loop-mediated isothermal amplification) procedure that works well and saves time. The procedure also works on the insect vector of the disease and will be used for transmission experiments. Hyperspectral imaging was demonstrated to non-destructively detect some Vaccinium spp. diseases. Phenotyping can be labor intensive and time consuming. We began using hyperspectral imaging (HSI), in an effort to speed disease screening for systemic diseases and other characteristics. We have successfully classified blueberry leaves infected with scorch virus and stunt disease as well as cranberries infected with tobacco streak virus and false blossom disease.

1. A reference quality genome sequence of cranberry was completed. We published the first cranberry genome sequence in 2014, but the scaffold number was high, due to gaps. To facilitate the goals of discovering genomic regions associated with important traits, a high-quality reference genome was needed. We used a long-read sequencing platform, coupled with advanced bioinformatics procedures to complete a chromosome-scale reference quality genome of cranberry. The assembly is posted in a public database (Genome Database for Vaccinium, GDV). Our completed development of the cranberry reference quality genome and public availability directly benefits the scientific community. The public will benefit indirectly through the research that this assembly will facilitate.

2. A genetic bridge between blueberry and its exotic relatives. Numerous exotic species of blueberry have valuable traits that are of interest to breeders for improvement of the cultivated highbush blueberry. Unfortunately, genetic barriers prevent successful crosses required to transfer genes between many of these species. One of the exotic species of interest for blueberry improvement is lingonberry (V. vitis-idaea). ARS scientists in Chatsworth, New Jersey, explored other blueberry species as potential bridges between cultivated blueberry (V. corymbosum) and lingonberry. They discovered that the South American Andean blueberry (V. meridionale) could be successfully hybridized with lingonberry. This finding is significant because V. meridionale can be hybridized with cultivated blueberry and thus serve as a bridge for hybridization between blueberry and lingonberry. The first-generation hybrids between V. meridionale and lingonberry produced strong plants with notable vigor and fertility and were successfully backcrossed to both lingonberry and V. meridionale. This breakthrough is significant since these new hybrids serve as a bridge that breeders can now use for gene transfer between these distant species. This research is of practical benefit to breeders working on genetic improvement of North American Vaccinium crops.

3. Evolutionary relationships of blueberry species within the Cyanococcus section. All commercial blueberry species in North America belong to the Cyanococcus section of Vaccinium. There are many other species within this section, however, and their genetic relationships to the commercial species have not been characterized, thus hindering their practical use in breeding. ARS scientists in Beltsville, Maryland, together with a university collaborator, utilized molecular markers to examine the evolutionary relationships and genetic among 50 accessions of the different species of this section including representatives from seven diploid, six tetraploid, and two hexaploid species. Of the commercial species, tetraploid V. corymbosum (highbush blueberry) grouped most closely with the diploids V. fuscatum and V. caesariense, tetraploid V. angustifolium (lowbush blueberry) grouped with the diploids V. boreale and V. myrtilloides, and hexaploid V. virgatum (rabbiteye blueberry) grouped most closely with the diploid V. tenellum. Our results delineating these relationships have been published and made available to the scientific community. These results revealed previously unknown relationships among this germplasm and provides valuable information to public and private breeders that they can utilize to facilitate breeding among these species.

Review Publications
Neyhart, J.L., Gutiérrez, L., Smith, K.P. 2021. Optimizing the choice of test locations for multi-trait genotypic evaluation. Crop Science. 62(1):192-202.
Kawash, J.K., Colt, K., Hartwick, N.T., Abramson, B.W., Vorsa, N., Polashock, J.J., Michael, T.P. 2022. Contrasting a reference cranberry genome to a crop wild relative provides insights into adaptation, domestication, and breeding. PLoS ONE. 17(3):e0264966.
Kerkhof, L.J., Roth, P.A., Deshpande, S.V., Bernhards, C.R., Liem, A.T., Hill, J.M., Haggblom, M.M., Webster, N.S., Ibironke, O., Mirzoyan, S., Polashock, J.J., Sullivan, R.F. 2022. A ribosomal operon database and megablast settings for strain-level resolution of microbiomes. FEMS Microbes.
Edger, P.P., Iorizzo, M., Bassil, N.V., Benevenuto, J., Ferrao, L.F., Giongo, L., Hummer, K.E., Lawas, L.F., Leisner, C.P., Li, C., Munoz, P., Ashrafi, H., Atucha, A., Babiker, E.M., Canales, E., Chagne, D., DeVetter, L., Ehlenfeldt, M.K., Espley, R.V., Gallardo, K., Gunther, C.S., Hardigan, M.A., Hulse-Kemp, A.M., Jacobs, M.L., Lila, M., Luby, C.H., Main, D., Mengist, M.F., Owens, G.L., Perkins-Veazie, P., Polashock, J.J., Pottorff, M., Rowland, L.J., Sims, C.A., Song, G., Spencer, J., Vorsa, N., Yocca, A.E., Zalapa, J.E. 2022. There and back again; historical perspective and future directions for Vaccinium breeding and research studies. Horticulture Research. 9. Article uhac083.
Rowland, L.J., Ogden, E.L., Ballington, J.R. 2021. Relationships among blueberry species within the section cyanococcus of the vaccinium genus based on est-pcr markers. Canadian Journal of Plant Science.
Ehlenfeldt, M.K., Polashock, J.J., Rowland, L.J. 2022. Fertile intersectional hybrids of 4x andean blueberry (Vaccinium meridionale) and 2x lingonberry (V. vitis-idaea). HortScience. 57:525-531.
Rodriguez-Saona, C., Polashock, J.J., Kyryczenko-Roth, V., Holdcraft, R., Jimenez-Gonzalez, G., De Moraes, C.M., Mescher, M.C. 2021. Application of plant defense elicitors fails to enhance herbivore resistance or mitigate phytoplasma infection in cranberries. Frontiers in Plant Science. 12:Article 700242.