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

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

Location: Genetic Improvement for Fruits & Vegetables Laboratory

2020 Annual Report

Objective 1: Deliver enhanced genomic resources for blueberry and cranberry breeding and genetic research including: improved genome assemblies, germplasm genotypes, mapping populations, saturated genetic linkage maps, mapping data for high value quantitative traits, and candidate gene analysis using genetic and bioinformatic approaches. [NP 301, C1, PS1A, C2, PS2A] Expected benefits include coordinated breeding and pre-breeding for cranberrry across all production regions with the goal to enhance new cultivar development and new product development. Subobjective 1a: Develop improved assemblies of the blueberry and cranberry genomes using long-read sequencing technologies and anchor new genome assemblies to well-saturated genetic linkage maps. Subobjective 1b: Map QTL for cold hardiness, chilling requirement, fruiting season, disease resistances, and fruit quality traits using improved maps and well-characterized bi-parental and association mapping populations. Subobjective 1c: Identify candidate genes for traits by their proximity to QTL, by homology to genes characterized in other systems, and by expression studies on plants with contrasting phenotypes. Subobjective 1d: Use a systems approach to cranberry breeding and genetics that includes genetic improvement, genomics, and phenomics. Objective 2: Develop and release new blueberry germplasm that is enhanced for prolific, indeterminate fruiting and cold tolerance by incorporating germplasm from exotic sources into the program. [NP 301, C1, PS1B] Objective 3: Develop and release new blueberry cultivars that are enhanced for mechanical harvesting, expanded fruiting season, cold hardiness, tolerance of higher pH soils, resistance to mummy berry and fruit rot, and adaptability to changing environmental conditions. [NP 301, C1, PS1B] Objective 4: Identify and characterize key pathogens of blueberry and cranberry and the genes that mediate plant-pathogen interactions, including stem blight (blueberry) and key pathogens in the fruit rot complex (cranberry), as well as plant-environment interactions. [NP 301, C3, PS3A; NP 303, C1, PS1, C2, 2B]

The approach entails the integration of genomic approaches with traditional breeding and plant pathology in the development of improved blueberry and cranberry cultivars. Scientists will develop enhanced genomic resources for blueberry and cranberry, including improved genome assemblies, well-saturated genetic linkage maps with anchorage to the genomes, and well-characterized bi-parental and association mapping populations, and identify quantitative trait loci (QTL) for horticulturally significant traits such as cold hardiness, chilling requirement, fruiting season, disease resistances, and fruit quality traits. In addition, scientists will carry out gene expression studies to identify the actual genes underlying significant QTL. Scientists will also incorporate a systems approach to cranberry breeding and genetics focused on genetic improvement with supporting phenotyping and transdisciplinary research on phenomics involving plant physiology, data sciences, and engineering. Scientists will also identify key genes in blueberry and cranberry that mediate plant-pathogen interactions, including stem blight (blueberry), key pathogens in the fruit rot complex (cranberry), and plant-environment interactions. Scientists will characterize and incorporate new germplasm, and generate new blueberry cultivars that meet industry needs. Better genomic resources for these crops will enable marker development for use in marker-assisted breeding. Scientists will also identify key genes in blueberry and cranberry that mediate plant-pathogen interactions, including stem blight (blueberry), key pathogens in the fruit rot complex (cranberry), and plant-environment interactions. Scientists will characterize and incorporate new germplasm, and generate new blueberry cultivars that meet industry needs. Better genomic resources for these crops will enable marker development for use in marker-assisted breeding.

Progress Report
Objective 1. 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.

1. 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.

2. 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.

Review 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.