Progress 03/20/18 to 03/19/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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 ÿ highbush pentaploids ÿ 4x highbush cultivars, and ⿿Nocturne ÿ highbush pentaploids ÿ 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. Artificial Intelligence (AI)/Machine Learning (ML) Our image-based postharvest berry phenotypic workflow uses a custom- trained deep learning model. This model can separate berries from background using standard color images taken using a readily available DSLR camera. Our computer vision workflow uses the output of this deep learning model to measure the shape, size, and color of individual berries; we are also able to quantify the heterogeneity of shape, size, and color within a sample of cranberries. Color attributes are highly predictive of total anthocyanin content, an important trait for stakeholders. Capturing images and extracting traits from those images can be accomplished in less than 1 minute per sample, while providing more information about fruit quality than the standard, low-throughput method that may require 15 minutes per sample. We have generated a pipeline for the high throughput classification of fruit rot using hyperspectral imaging. This method utilizes regression trees where the intensity values given through the camera act as the features. Combining this pipeline with a conveyor system under the camera has resulted in a near limitless potential for high throughput phenotyping. The data collected from the camera is processed using a python script on a local compute server that is readily available on-site. Though once the model for classification is generated, a compute node is not necessary, and classification can be performed on a high performance desktop. ACCOMPLISHMENTS 01 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. 02 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. 03 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.
Impacts
(N/A)
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, K.R., 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) ÿ lingonberry (V. Vitis-idaea) Hybrid. Plants. 11(22):3152. https://doi.org/10.3390/plants11223152.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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 ÿ highbush pentaploids ÿ 4x highbush cultivars, and ⿿Nocturne ÿ highbush pentaploids ÿ 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. ACCOMPLISHMENTS 01 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. 02 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. 03 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.
Impacts
(N/A)
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. https://doi.org/10.1002/csc2.20657.
- 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. https://doi.org/10. 1371/journal.pone.0264966.
- 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. https://doi.org/10.1093/femsmc/xtac002.
- 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. https://doi.org/10.1093/hr/ 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. https://doi. org/10.1139/cjps-2021-0221.
- 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. https://doi.org/ 10.21273/HORTSCI16285-21.
- 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. https://doi.org/10.3389/fpls.2021.700242.
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Progress 10/01/20 to 09/30/21
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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. Record of Any Impact of Maximized Teleworking Requirement: The maximized telework posture has had a negative impact on the research project as a whole. Regarding the blueberry mapping projects, we have not been able to begin phenotyping plants of the association panel. The plants for this project were located at a grower⿿s farm. Last year we had no access to the farm due to the pandemic, and the plants were not being properly maintained by the farm. All of the plants were moved to the research farm at the Chatsworth Center on 3/15/21. We are trying to begin phenotyping for some traits (such as flowering time) this season. We will not be able to begin phenotyping for other traits until next year. This means that all downstream projects requiring these data are delayed for approximately two years. Telework has also delayed the annual field planting of blueberry seedling families from the breeding program. This will set breeding efforts back by one year. Field evaluations of older materials were similarly delayed. Some other collaborative projects, such as the project studying the blueberry microbiome associated with replant disease, have been delayed somewhat because the collaborator⿿s lab at Rutgers University has also been shut down due to the pandemic and has only recently partially reopened. Hiring postdocs for the project as a whole has been delayed because we cannot bring in new personnel to conduct field and lab work when we are still operating only at 25% capacity. Also, since we cannot currently hire foreign nationals as postdocs, we have not received many applications for the positions we have so far advertised. This is affecting our ability to analyze RNA-seq data that has already been generated to study changes in gene expression associated with fruit softening in blueberry and other traits in both blueberry and cranberry. ACCOMPLISHMENTS 01 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. 02 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. 03 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.
Impacts
(N/A)
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.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Objective 1. Long read sequencing technology, coupled with our existing short read data, allowed the assembly of cranberry (Vaccinium macrocarpon) genome to near-chromosome scale. This has allowed more precise anchoring of our genome data onto genetic maps. This assembly was utilized to better resolve previously identified regions of quantitative trait loci (QTL) for genes associated with fruit acidity. We further developed single nucleotide polymorphism (SNP)-based competitive allele-specific (KASP) markers for marker-assisted selection (MAS). The markers developed were successfully used to screen seedlings and identify those likely to express the low acid trait. We also sequenced and assembled the genome of the small-fruited cranberry (Vaccinium oxycoccos) using Oxford nanopore long-read sequencing technology. The two species of cranberry have important differences that can be exploited to improve cultivated cranberry (V. macrocarpon). We demonstrated that the two species readily hybridize and produce fertile offspring. Whole-genome comparisons are in progress. Three populations of blueberry were genotyped using a commercial Capture- Seq platform for 30,000 SNPs. Plants will be phenotyped for fruit quality in mid-June 2020. The SNP data and the phenotypic data will be used for GWAS (genome-wide association mapping). This will allow the identification of genomic regions associated with key traits. A high-density genetic linkage map of a diploid blueberry mapping population, based on single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers, was used to correct mistakes in a chromosome-level genome assembly of blueberry. The map was also used to identify regions (QTL) that control significant horticultural traits. Highly significant QTL were identified for chilling requirement, cold hardiness, fruit color, fruit scar, timing of early green fruit, timing of full bloom, and timing of petal fall. A study is underway to identify genes associated with fruit firmness in blueberry and cranberry. Firm fruit are desirable for mechanical harvesting. The study utilizes fruit collected at unripe (pink for blueberry and white for cranberry) and ripe (blue for blueberry and red for cranberry) stages from a collection of four blueberry and two cranberry cultivars that represent a range of different firmness levels. Fruit samples were used for RNA extractions and complementary DNA (made from the RNA) was sequenced in an effort to identify genes whose expression is associated with firm fruit. Analyses of the sequencing data are underway to identify genes that are expressed differently across the different stages of fruit development and among the different cultivars (firm versus soft) and species. A cranberry population segregating for waxy bloom on the fruit was genotyped using SNPs. QTL analysis identified a region associated with the trait. The QTL region only had one gene that appears to be associated with fruit wax deposition. Expression studies (RT-qPCR) of developing fruit to support the association of putative casual gene with the waxy trait are scheduled for the summer of 2020. Objective 2. Desirable new 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 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), that has only fair fruit quality. This selection is being crossed to complementary highbush cultivars with commercial fruit quality. Work has continued on the utilization of V. corymbodendron, a South American species with prolific and concentrated flowering. Tetraploid hybrids utilizing V. corymbodendron hold the promise of facilitating hybridization and gene transfer among blueberry, cranberry, and lingonberry germplasm. We currently have fertile hybrids of V. corymbodendron 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. Blueberry progeny from wide crosses often have unique phenotypes, but proving that the putative hybrids are indeed the result of hybridization can be challenging. We used genotyping by sequencing (GBS) to genotype hybrids resulting from crosses of highbush blueberry (V. corymbosum) and V. padifolium. The data collected were used to show that the putative hybrids share unique SNP markers with both parents, confirming that they are indeed true hybrids. Objective 3. A northern highbush selection, ARS 05-171 (⿿Talisman⿿), has been designated for release pending a release committee review. Another northern highbush selection, ARS 99-72, continues to be evaluated. Both selections have characteristics that make them suitable for mechanical harvesting. Research continues to evaluate several crosses utilizing the southern highbush 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 is undergoing field testing (ARS 16-57). Objective 4. Blueberry stem blight was previously demonstrated to be caused by a complex of pathogenic fungi. We have shown that the fungi vary dramatically in the severity of symptoms caused on test blueberry plants. This is likely to be the underlying cause of conflicting virulence data in the literature. Some of the fungal species are more predominant and it is possible that the predominant isolates are fungicide resistant- a possibility we are exploring. We have placed key isolates taxonomically based on sequences of targeted genes coupled with similarity comparisons to vouchered specimens in public databases. Plant defenses as influenced by climate. ARS scientists in Chatsworth, New Jersey, and cooperators at the Western Norway University of Applied Sciences induced plant defenses in European blueberry plants along an elevational temperature gradient, as a proxy for climate change. Differences in plant responses and changes in gene expression were monitored. In general, defense-induced plants reduced growth and reproduction while increasing resistance, but the response varied by temperature. Our results suggest that plant defense responses at both the molecular and ecological levels are modulated by the combination of climate and herbivory pressure, such that plants under different environmental conditions differentially direct the resources available to specific anti-herbivore strategies. Our findings are important to scientists trying to understand the complex impact of future climate changes on plant⿿herbivore interactions, as this is a major driver of ecosystem functioning and biodiversity. Accomplishments 01 A marker for low-acidity in cranberry. Cranberry fruit contain very little natural sugar and are highly acidic. Since a sugar-acid balance is required for palatability, current cranberry products require large amounts of ⿿added sugar⿿. However, consumption of such high amounts of sugar is considered unhealthy. ARS scientists in Chatsworth, New Jersey, together with cooperators at Rutgers University utilized a cranberry germplasm screen to identify lines that are naturally low in citric acid, one of the main contributors to acidity in cranberry. We showed that the low acid trait is heritable. We mapped the location of the responsible genes and made markers to identify the region in offspring. It was demonstrated that we can screen seedlings and predict those that will have the low acid trait. This will help breeders, by dramatically speeding up the breeding process for development of low acid cranberry varieties. Release of a low acid cranberry variety that requires much lower amounts of added sugar will be useful in making cranberry products more healthful for consumers. 02 Blueberry population with genes for fast growth and better fruit quality. A blueberry population of about 120 plants, evaluated by ARS scientists in Beltsville, Maryland, and Chatsworth, New Jersey, for growth and fruit quality traits, showed growth qualities that are important for expanding the harvest season by developing earlier and later fruiting varieties. The improved qualities are the timing of flower bud break, full bloom, early fruit set, ripe fruit, and better berry firmness, size, weight, color, and flavor. This new population was developed to aid researchers in mapping genes for early and late season maturity and fruit quality attributes. This population will be useful to blueberry scientists developing new blueberries varieties.
Impacts
(N/A)
Publications
- Qi, X., Ogden, E.L., Die, J.V., Ehlenfeldt, M.K., Polashock, J.J., Darwish, O., Alkharouf, N., Rowland, L.J. 2019. Transcriptome analysis identifies genes associated with the waxy coating on blueberry fruit in two northern- adapted rabbiteye hybrid breeding populations. Biomed Central (BMC) Plant Biology.
- Rowland, L.J., Ogden, E.L., Bell, D.J., Drummond, F.A. 2020. Pollen- mediated gene flow in managed fields of lowbush blueberry. Canadian Journal of Plant Science.
- Qi, X., Ogden, E.L., Ehlenfeldt, M.K., Rowland, L.J. 2019. Dataset of de novo assembly and functional annotation of the transcriptome of blueberry (Vaccinium spp.). Data in Brief.
- Fong, S.K., Kawash, J., Wang, Y., Johnson-Circalese, J., Polashock, J.J., Vorsa, N. 2020. A low citric acid trait in cranberry fruit: genetics, molecular mapping and relationship to titratable acidity. Tree Genetics and Genomes.
- Bassil, N.V., Bidani, A., Nyberg, A.M., Hummer, K.E., Rowland, L.J. 2020. Microsatellite markers confirm identity of blueberry plants in the USDA- ARS National Clonal Germplasm Repository collection. Genetic Resources and Crop Evolution. 67:393-409.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): 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] 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 (from AD-416): 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. Objective 1. Oxford nanopore long-read sequencing technology was used to obtain a better genome reference assembly for the large-fruited cranberry (Vaccinium macrocarpon). The new assembly is pseudo-chromosome scale. This assembly has already been utilized to better resolve previously identified regions (quantitative trait loci or QTL) for key traits, including fruit rot resistance. The increased resolution was used to select candidate genes associated with various traits and to develop markers for marker-assisted selection (MAS). The markers developed were successfully used to screen seedlings and identify those likely to express the desired traits. We also sequenced and began assembling the genome of the small-fruited cranberry (Vaccinium oxycoccos) using Oxford nanopore long-read sequencing technology. Although still in progress, we expect the new assembly to be at the pseudo-chromosome scale also. The two species of cranberry have important differences that can be exploited to improve cultivated cranberry (V. macrocarpon). The next step is to perform whole-genome comparisons. Three populations of blueberry are currently being genotyped with molecular markers to ultimately identify markers associated with traits important for blueberry breeding. A commercial platform is being used to genotype the progeny from each of the three crosses. A high-density genetic linkage map of a diploid blueberry mapping population has just been completed. The map is based on single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers. This saturated map is currently being used to correct mistakes in a chromosome- level genome assembly of blueberry. The map is also being used to identify regions (QTL) that control significant horticultural traits including chilling requirement, cold hardiness, and various plant and fruit quality traits. The diploid blueberry mapping population was evaluated for another year at Beltsville, Maryland, for firmness using a texture analyzer. This work is helping to identify regions of the genome (QTL) that control this trait. The diploid blueberry mapping population was also established at a second ARS location in Poplarville, Mississippi. This will allow future QTL mapping over multiple locations, in addition to multiple years. A study is underway to identify genes associated with fruit firmness in blueberry. Firm fruit are desirable for mechanical harvesting. The study utilizes fruit collected at unripe (pink) and ripe (blue) stages from a collection of blueberry cultivars that represent a range of different firmness levels. Firmness measurements were taken for a third year for two of the firmest and two of the softest blueberry cultivars and for the first year for two cranberry cultivars (at white and red stages). Cranberry cultivars were included this year for comparison to blueberry cultivars, because cranberry fruit does not soften during ripening like blueberry fruit does. Efforts are underway to extract RNA from samples and use the RNA to identify genes whose expression is associated with fruit softening. Efforts to identify genes that are expressed differently across the different stages of fruit development and among the different cultivars (firm versus soft) and species are being made. A cranberry population segregating for waxy bloom on the fruit was genotyped using SNPs. QTL analysis identified a region associated with the trait. Genes in the QTL region that appear to be associated with wax biosynthesis, transport, or deposition will be used in expression studies (RT-qPCR) of developing fruit to support their association with the waxy trait. Objective 2. Desirable new germplasm, combining aspects of rabbiteye vigor, V. constablaei⿿s late flowering, and highbush plant and fruit quality, were evaluated. A blue-fruited selection from the V. constablaei incorporation program shows promise as a commercial selection and is undergoing field testing. Another selection has shown vivid fruit pigmentation and may have promise as an ornamental variety. 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. This year trispecific 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) hybrids that expressed good vigor, adaptation, and reasonable yield. These selections are being propagated for further testing. Work has expanded on the utilization of V. corymbodendron a South American species with prolific and concentrated flowering. Tetraploid hybrids utilizing V. corymbodendron hold the promise of facilitating hybridization and gene transfer among blueberry, cranberry, and lingonberry germplasm. We currently have fertile hybrids of V. corymbodendron with lingonberry and blueberry, and have crosses made of V. corymbodendron with cranberry. These hybrids successfully back-cross, at low levels, to all three commercial Vaccinium crops. Objective 3. A northern highbush selection, ARS 05-171, has been designated for release pending patenting committee review. Another northern highbush selection, ARS 99-72 continues to be evaluated. Both selections have characteristics that make them suitable for mechanical harvesting. Research continues to evaluate several crosses utilizing the southern highbush 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. 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 is undergoing field testing. Objective 4. Blueberry stem blight was demonstrated to be caused by a complex of pathogenic fungi. The fungi vary dramatically in the severity of symptoms caused on test blueberry plants. It is likely that several of the fungi isolated are previously undescribed species. Representative genes from these fungi are being sequenced and morphological characters recorded to determine proper taxonomic placement. Accomplishments 01 A gene for waxy coating on blueberry fruit. The presence of a waxy coating or ⿿bloom is responsible for the dusty light blue color of blueberry fruit that is desirable for berries destined for the fresh market. The waxy coating also provides protection against desiccation and deterioration. ARS scientists in Beltsville, Maryland, combined two molecular biology-based techniques to identify genes that are differentially expressed between progeny that have the waxy coating and progeny that do not. This work identified a gene, FatB, whose expression is associated with the waxy trait and is involved in the biosynthesis of long-chain fatty acids and wax production. This gene is being tested by molecular geneticists and breeders to determine if it is also associated with waxy fruit in cranberry, a relative of blueberry, and its utility as a selectable marker in breeding for waxy fruit. 02 Unique defense response of European blueberry. European blueberry is one of the most abundant wild berries in the Northern Europe and is recognized for producing compounds involved in natural defenses against insect feeding. Understanding the plants defense response to insect attack will help develop sustainable approaches to pest management in cultivated blueberry. ARS scientists in Chatsworth, New Jersey, compared differences in gene expression between plants with defenses ⿿turned on⿿ and those growing under normal conditions. Expression of genes associated with growth and reproduction were repressed when defenses were ⿿turned on⿿. This demonstrates that there is metabolic ⿿cost⿿ for the plants to defend themselves. Thus, in developing new pest resistant blueberry cultivars and pest management practices, the ⿿cost⿿ of utilizing the plant⿿s natural defenses against insect feeding must be balanced with the resources needed for growth and fruit production. 03 Cranberry False Blossom disease induces attractiveness to pests. False blossom disease (FBD) of cranberry poses a serious threat to the cranberry industry. To better understand why the disease is spreading, ARS scientists in Chatsworth, New Jersey, characterized differences in gene expression of infected vs. healthy plants. We discovered that FBD infection increased the expression of plant genes associated with nutrient metabolism, while suppressing genes associated with defensive pathways. These changes made the plant more attractive to insects, thus promoting disease transmission. These results provide a new strategy to breed for FBD management whereby breeders can select for response insensitive genes in FBD challenged plants, thus facilitating identification of plants that are less attractive to insect vectors that spread FBD. 04 Cranberry plants produce volatiles to combat insect pests. Plant eating insects are an important problem in cranberry production and their control requires application of chemical pesticides. Cranberry plants have natural defense mechanisms that can be exploited for development of reduced-pesticide insect control. ARS scientists in Chatsworth, New Jersey, focused on cranberry plant volatiles, which are attractive to predators of plant-eating insects. Nine cranberry varieties were tested for expression of genes involved in volatile biosynthesis and/or emission. The results showed that different cranberry genotypes vary in their emission of volatiles. This finding is being used by scientists to develop new cranberry varieties with superior natural defense against insect feeding and attractiveness to natural predators.
Impacts
(N/A)
Publications
- Beers, L., Rowland, L.J., Drummond, F.A. 2019. Genetic diversity of lowbush blueberry throughout the United States in managed and non-managed populations . Agriculture.
- Hancock, J.F., Olmstead, J.W., Itle, R.A., Callow, P.W., Neils-Kraft, S.P., Wheeler, E.J., Mangandi, J., Sooriyapathirana, S.S., Rowland, L.J., Mackey, T.A., Bassil, N.V., Finn, C.E. 2018. Performance of an elite, hybrid family of a northern ÿ southern highbush cross (⿿Draper⿿ ÿ ⿿Jewel⿿). Euphytica. 214:95.
- Delange, E.S., Salamanca, J., Polashock, J.J., Rodriguez-Saona, C. 2019. Genotypic variation and phenotypic plasticity in gene expression and emissions of herbivore-induced volatiles, and their potential tritrophic implications, in cranberries. Journal of Chemical Ecology. 45(3):298-312.
- Pradit, N., Rodriguez-Saona, C., Kawash, J., Polashock, J.J. 2019. Phytoplasma infection influences gene expression in American cranberry. Frontiers in Ecology and Evolution. 7:178.
- 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.
- Fonseca Benevenuto, R., Seldal, T., Hegland, S.J., Rodriguez-Saona, C., Kawash, J., Polashock, J.J. 2019. Transcriptional profiling of methyl jasmonate-induced defense responses in bilberry (Vaccinium myrtillus L.). Biomed Central (BMC) Plant Biology. 19:70.
- Gallardo, R.K., Zhang, Q., Klingthong, P., Dossett, M., Polashock, J.J., Rodriguez-Saona, C., Vorsa, N., Edger, P., Scherm, H., Ashrafi, H., Babiker, E.M., Finn, C.E., Iorizzo, M. 2018. Breeding trait priorities of the blueberry industry in the United States and Canada. HortScience. 53(7) :1021-1028.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): 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] 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. 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 (from AD-416): 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 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. Objective 1. Pacific Biosciences� (PacBio) and other long-read sequencing technologies are being used to improve the assemblies of the diploid blueberry and large-fruited cranberry genomes. The whole genome of a small-fruited cranberry (Vaccinium oxycoccos) has been sequenced as well, and assembly is in progress. This will add to the Vaccinium spp. genomes we already have sequenced and will allow expansion of comparative genome analyses. A technology (exome capture) is being used to add tens of thousands of markers to the genetic map of diploid blueberry. These kinds of saturated maps will be used to anchor the improved genome assemblies of blueberry and cranberry. The diploid blueberry mapping population was evaluated for another year at Beltsville, Maryland, for firmness using a texture analyzer and for soluble solids. This work is helping to identify regions of the genome (QTL) that control this trait. Studies were carried out to identify the actual gene(s) themselves associated with fruit quality traits of firmness and presence of a waxy coating on blueberry fruit. Firm fruit are desirable for mechanical harvesting. A study was begun utilizing fruit collected at unripe, ripe, and overly ripe stages from a collection of 10 blueberry cultivars that represent a range of different firmness levels. Efforts are underway to measure expression levels of several genes known to be involved in fruit softening across the different stages of fruit development and among the different cultivars. Another study to identify the gene(s) responsible for the protective waxy coating on blueberry fruit and leaves was continued. This study utilizes unique germplasm families resulting from crosses made in the blueberry breeding program that segregate for the presence/absence of the waxy coating. The presence of the waxy coating is responsible for the desirable dusty light blue coating on blueberry fruit. A technique called RNA-seq was combined with a bulked-segregant analysis (BSA) to identify genes that are differentially expressed between progeny that have the waxy coating and progeny that do not. Differential expression of candidate genes was then confirmed by another technique, real-time PCR. This work has identified an excellent candidate gene responsible for the waxy trait in these populations. We are currently sequencing the gene to compare the gene structure in waxy and non-waxy individuals. Objective 2. Desirable new germplasm, combining aspects of rabbiteye vigor, V. constablaei�s late flowering, and highbush plant and fruit quality, were evaluated. A blue-fruited selection from the V. constablaei incorporation program shows promise as a commercial selection and is undergoing field testing. Another selection has shown vivid fruit pigmentation and may have promise as an ornamental variety. New hybrids were 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. Seedlings of the second generation of these hybrids are being evaluated in the field. First and second generation hybrids of 6x rabbiteye-type hybrids x 6x V. smallii were created. Hexaploid species are relatively rare, and V. smallii may broaden the 6x genepool of rabbiteye blueberry. Germplasm incorporation and evaluation work continues to evaluate germplasm from a few other species of potential value to commercial blueberry. Most notable from this work has been the production of tetraploid hybrids utilizing V. corymbodendron that hold the promise of facilitating hybridization and gene transfer among blueberry, cranberry, and lingonberry germplasm. Objective 3. Two northern highbush selections, ARS 05-171 and ARS 99-72, that have characteristics that make them suitable for mechanical harvesting were selected and are considered likely to be released under the current project. These selections are in the latter stages of evaluation prior to a decision on release. Research also identified several crosses utilizing the southern highbush cultivar �Reveille� that yield populations with 100% firm-fruited progeny. Another cross was identified that produces a low frequency of hybrids with outstanding firmness in northern highbush blueberry. These populations are being further explored and expanded to generate populations for molecular studies and also to generate selections for machine harvest testing. A blue-fruited selection from the V. constablaei incorporation program that shows promise as a commercial selection is undergoing field testing. A gene expression study (RNAseq project) was completed to better understand the interaction of the mummy berry pathogen (Monilinia vaccinii-corymbosi) with resistant and susceptible blueberry. Further analyses of the differentially expressed genes are in progress. Objective 4. Blueberry stem blight was found to be caused by at least three different pathogenic fungi. Further characterization of the fungi as well as virulence testing on representative blueberry plants is in progress. A gene expression study (RNAseq project) was continued to determine how soluble cranberry flower extracts affect the growth and development of a key pathogen (Colletotrichum fioriniae) in the cranberry fruit rot complex. A study was initiated to catalog the microorganisms (the microbiome) associated with the roots of blueberry and cranberry. The myriad of organisms associated with the root system of plants can directly and/or indirectly affect plant health. Thus, it is important to determine the �core� microbiome associated with these crops.
Impacts
(N/A)
Publications
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