Location: Genetic Improvement for Fruits & Vegetables Laboratory
2019 Annual Report
Objectives
Objective 1: Deliver enhanced genomic resources for blueberry and cranberry breeding and genetic research including: improved genome assemblies, germplasm genotypes, mapping populations, saturated genetic linkage maps, mapping data for high value quantitative traits, and candidate gene analysis using genetic and bioinformatic approaches. [NP 301, C1, PS1A, C2, PS2A]
Subobjective 1a: Develop improved assemblies of the blueberry and cranberry genomes using long-read sequencing technologies and anchor new genome assemblies to well-saturated genetic linkage maps.
Subobjective 1b: Map QTL for cold hardiness, chilling requirement, fruiting season, disease resistances, and fruit quality traits using improved maps and well-characterized bi-parental and association mapping populations.
Subobjective 1c: Identify candidate genes for traits by their proximity to QTL, by homology to genes characterized in other systems, and by expression studies on plants with contrasting phenotypes.
Subobjective 1d: Use a systems approach to cranberry breeding and genetics that includes genetic improvement, genomics, and phenomics.
Objective 2: Develop and release new blueberry germplasm that is enhanced for prolific, indeterminate fruiting and cold tolerance by incorporating germplasm from exotic sources into the program. [NP 301, C1, PS1B]
Objective 3: Develop and release new blueberry cultivars that are enhanced for mechanical harvesting, expanded fruiting season, cold hardiness, tolerance of higher pH soils, resistance to mummy berry and fruit rot, and adaptability to changing environmental conditions. [NP 301, C1, PS1B]
Objective 4: Identify and characterize key pathogens of blueberry and cranberry and the genes that mediate plant-pathogen interactions, including stem blight (blueberry) and key pathogens in the fruit rot complex (cranberry), as well as plant-environment interactions. [NP 301, C3, PS3A; NP 303, C1, PS1, C2, 2B]
Approach
The approach entails the integration of genomic approaches with traditional breeding and plant pathology in the development of improved blueberry and cranberry cultivars. Scientists will develop enhanced genomic resources for blueberry and cranberry, including improved genome assemblies, well-saturated genetic linkage maps with anchorage to the genomes, and well-characterized bi-parental and association mapping populations, and identify quantitative trait loci (QTL) for horticulturally significant traits such as cold hardiness, chilling requirement, fruiting season, disease resistances, and fruit quality traits. In addition, scientists will carry out gene expression studies to identify the actual genes underlying significant QTL.
Scientists will also incorporate a systems approach to cranberry breeding and genetics focused on genetic improvement with supporting phenotyping and transdisciplinary research on phenomics involving plant physiology, data sciences, and engineering. Scientists will also identify key genes in blueberry and cranberry that mediate plant-pathogen interactions, including stem blight (blueberry), key pathogens in the fruit rot complex (cranberry), and plant-environment interactions. Scientists will characterize and incorporate new germplasm, and generate new blueberry cultivars that meet industry needs. Better genomic resources for these crops will enable marker development for use in marker-assisted breeding.
Scientists will also identify key genes in blueberry and cranberry that mediate plant-pathogen interactions, including stem blight (blueberry), key pathogens in the fruit rot complex (cranberry), and plant-environment interactions.
Scientists will characterize and incorporate new germplasm, and generate new blueberry cultivars that meet industry needs. Better genomic resources for these crops will enable marker development for use in marker-assisted breeding.
Progress Report
Objective 1. 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
1. 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.
2. 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.
3. 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.
4. 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.
Review 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. https://doi.org/10.3390/agriculture9060113.
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 × southern highbush cross (‘Draper’ × ‘Jewel’). Euphytica. 214:95. https://doi.org/10.1007/s10681-018-2173-8.
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. https://doi.org/10.1007/s10886-018-1043-0.
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. https://doi.org/10.3389/fevo.2019.00178.
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. https://doi.org/10.3389/fpls.2018.01310.
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. https://doi.org/10.1186/s12870-019-1650-0.
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. https://doi.org/10.21273/HORTSCI12964-18.
