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]
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.