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ARS Home » Northeast Area » Washington, D.C. » National Arboretum » Floral and Nursery Plants Research » Research » Research Project #434613

Research Project: Genetic Improvement of Nursery Crops through Functional Genomics and Breeding

Location: Floral and Nursery Plants Research

2019 Annual Report

Objective 1: Establish long-term experimental hydrangea populations, and develop information management systems and standards for hydrangea phenotyping and genetic mapping to accelerate the development of disease and stress resistant hydrangea cultivars with superior product quality. [NP301, C1, PS1A; NP301, C4, PS4A] Sub-objective 1.A. Establish and phenotype Hydrangea macrophylla experimental populations for identification of disease resistance and ornamental traits. Sub-objective 1.B. Establish, genotype, and phenotype a range-wide Hydrangea quercifolia collection for natural genetic variation for disease resistance and ornamental traits. Sub-objective 1.C. Develop new phenotyping methods for important traits and create associated data management systems for genomic and phenotypic data. Objective 2: Develop, evaluate, and release improved germplasm of hydrangea and other high-value, native, and underrepresented nursery crop species to expand market opportunities for producers, stimulate consumer interest, and mitigate the environmental footprint of nursery production. [NP301, C1, PS1B] Sub-objective 2.A. Accelerate the development of superior Hydrangea germplasm using ploidy manipulations, and wide hybridization. Sub-objective 2.B. Evaluate germplasm including Hydrangea, Osmanthus, and Hamamelis for nursery production and breeding improvement potential.

During the next five years, Hydrangea and Osmanthus selections from previous breeding cycles will be progagated and distributed to cooperators. Germplasm evalutations of new species and cultivars will ensure a broad base for future cultivar development. We will confirm the three major powdery-mildew resistance SNPs in the two most resistant and two most susceptible cultivars using real-time PCR with TaqMan probes. Controlled crosses between the most powdery mildew-resistant and most susceptible cultivar are underway. This F1 mapping population will be genotyped via RT-PCR in the same manner as the mapping parents. This population will be planted in the field for a minimum three-year observation period. During this time, natural powdery mildew incidence and/or induced powdery mildew infection will be recorded. Digital images of each plant will be obtained in regular intervals throughout the growing season. Image analysis will be used to monitor the progression of disease infection and the number and size of new inflorescences. Together, the genotype and phenotype data will be used to refine the genome-wide association analysis, determine the efficacy of marker-assisted selection for powdery mildew resistance at the seedling stage in Hydrangea, and identify plants for further gene expression analysis. For genetic and horticultural analysis of oakleaf hydrangea, we will sample up to 20 populations within each state on a longitudinal/latitudinal cline, as well as representing the full diversity of environments in which the species occurs. Within each of these populations we will sample up to 20 plants. Three to five recently expanded leaves will be collected from selected plants, stored flat in Ziploc bags and stored in coolers with unique identifiers linked with each bag. Plant morphology, location, and other notes will be recorded using the EpiCollect5 web application. Leaves will be shipped overnight via UPS to the USDA-ARS in McMinnville, TN for processing, DNA isolation, and storage. Genotyping-by-sequencing will be performed by the University of Minnesota Genomics core facility. Bioinformatic support will be provided by the ARS NEA Statistics Group. Ten populations per state on a longitudinal/latitudinal cline, as well as representing the full diversity of environments in which the species occurs, will be evaluated for current generation estimates of genetic diversity and levels of inbreeding and for common garden horticultural characterizations in Minnesota, Mississippi, and Tennessee. Each location will evaluate 25-50 individuals of each population (60 populations total) for growth, form, floral, and disease resistance traits. Over 1500 F2 seedlings derived from hybridizations between 'Snow Queen' and the compact cultivars Pee Wee and Sikes Dwarf were evaluated for compact plant habit, quality and quantity of inflorescences, disease resistance and fall foliage color. Seven plants from this population have been selected for compact form and superior floral display. Seed and seedlings from these selected plants will be used for development of disease-screening methods.

Progress Report
This is the first report for Project 8020-21000-072-00D, which began in April 2018. Significant progress was made on both objectives, which fall under NP301. Under Objective 1.A., we produced and phenotyped a large F2 mapping population of Hydrangea macrophylla using genetically well-characterized parents. We established a permanent cultivar panel in-ground consisting of more than 60 Hydrangea cultivars. We completed a genome-wide association study of flowering traits using a hydrangea cultivar panel. These well-characterized populations will be used for genetic mapping and determining gene-trait associations. Under Objective 1.B., we used genotyping-by-sequencing to genotype oakleaf hydrangea leaf samples from a range-wide collection. We collected seed from previously identified oakleaf hydrangea populations across the southeast and germinated the seed in preparation for a range-wide field trial. Under Objective 1.C., we tested two methods for screening oakleaf hydrangea seedlings for root-rot susceptibility. Both methods failed to produce significant disease symptoms in the seedling populations. New cultural methods and inoculation techniques are being tested. We developed two different protocols for hydrangea SNP genotyping, including a CAPS method for low-to-medium throughput and a KASP method for high-throughput SNP genotyping of inflorescence type. Under Objective 2.A., we completed a study of pollen tube growth among interploid hydrangea crosses, noting that crosses between diploids and triploids and among triploids resulted in pollen tube growth equal to that of diploid crosses. Interploidy crosses were initiated with the goal of recovering unique ploidy levels or phenotypes in the offspring. Evaluation of powdery mildew infection among ploidy levels concluded. Controlled growth chambers were used to manipulate the number of unreduced gametes produced by two hydrangea cultivars. Under Objective 2.B., blueing studies of Dichroa and hybrids were concluded, indicating that Dichroa has a similar aluminum accumulation mechanism as hydrangea. Crosses were made using Dichroa of differing ploidy levels and hydrangeas of differing ploidy levels with the goal of producing hybrids with phenotypes closer to the hydrangea parent. Under Objective 2.C., collections of Osmanthus, Hamamelis, and Callicarpa were phenotyped in a multi-year field trial. Germination studies using Chionanthus and Osmanthus seed revealed a regime of warm treatment followed by cold treatments was sufficient to break both root and shoot dormancy of both species. Seed representing novel Osmanthus hybrids was germinated for multi-year evaluation. A rooting and finishing study using Hamamelis cultivars was initiated.

1. A single molecular marker identifies bigleaf hydrangea inflorescence type with 100% accuracy. Inflorescence type is a valuable trait in bigleaf hydrangea (Hydrangea macrophylla) and is recessively inherited. Molecular marker-assisted selection (MAS) can greatly reduce the time necessary to breed cultivars with the desired inflorescence type. An ARS scientist in McMinnville, Tennessee, conducted a genome-wide association study (GWAS) using 5,805 single nucleotide polymorphisms (SNPs) using a panel of 82 bigleaf hydrangea cultivars. One SNP locus associated with inflorescence type was converted to a cleaved amplified polymorphic sequence (CAPS) marker that showed absolute identification accuracy (100%) of inflorescence type in a validation panel consisting of eighteen H. macrophylla cultivars. This marker will increase the efficiency of breeding novel hydrangea cultivars by reducing time-to-market for new cultivars.

2. A multi-year evaluation of Osmanthus species provides recommendations to growers. Production and use of Osmanthus as a landscape plant is currently limited to USDA Hardiness Zones 7 to 10, although nursery growers wish to extend the range of Osmanthus species into colder climates. In order to provide recommendations to growers and landscapers and inform breeding efforts for cold-hardiness improvement, ARS scientists in McMinnville, Tennessee, conducted a replicated Osmanthus trial in a USDA Hardiness Zone 6b/7a transition zone. Sixteen Osmanthus cultivars representing four species were evaluated for growth, stem necrosis, and flowering in a pot-in-pot production system from 2015 – 2017. The most-cold tolerant cultivars were O. heterophyllus ‘Kaori Hime’, ‘Hariyama’, ‘Shien’, and ‘Head-Lee Fastigate’; O. xfortunei ‘San Jose’, O. heterophyllus ‘Rotundifolius’, and O. armatus ‘Longwood’. Of these cultivars, O. heterophyllus ‘Kaori Hime’, O. xfortunei ‘San Jose’, and O. armatus ‘Longwood’ flowered under winter-exposed conditions, while Osmanthus fragrans cultivars were generally not hardy. Plants selected and produced in transition zones are often suitable over a wide geographic and climatic area, and these results will be used by growers and landscapers to inform choice of Osmanthus cultivars to plant and/or sell.

Review Publications
Alexander, L.W. 2019. Optimizing pollen germination and pollen viability estimates for Hydrangea macrophylla, Dichroa febrifuga, and their hybrids. Scientia Horticulturae. 246:244-250.
Alexander, L.W. 2019. Growth, cold-hardiness, and flowering of Osmanthus in Tennessee. HortTechnology. 29:78-84.