Location: Southern Horticultural Research Unit
2018 Annual Report
Objectives
Objective 1. Develop and expand breeding pools for blueberry and woody ornamentals in the Southeast United States by identifying native germplasm resources through precision phenotyping methods for biotic and abiotic stress resistance, including in vitro screening and cytogenetic manipulation to ensure new genetic resources are sexually compatible.
Sub-objective 1.A. Introgress adaptation traits from Hibiscus moscheutos into Hibiscus syriacus by interspecific hybrids.
Sub-objective 1.B. Produce interspecific and intersectional hybrids between Vaccinium (V.) tenellum, V. pallidum, V. darrowii, and V. arboreum and produce synthetic tetraploid from V. tenellum and V. pallidum using oryzalin treatment.
Objective 2. Introduce southern adapted traits, such as tolerance to drought, high soil pH, and poor soil, into elite breeding lines by conventional and advanced genetic methods, including selectable marker associations, to increase commercial blueberry acreage and yield in the southeast United States and in other markets with similar climates.
Sub-objective 2.A. Assess the level of drought and pH tolerance in a diverse panel of 156 southern highbush genotypes (SHB) and in parents and individuals of a diploid interspecific mapping population developed from a cross between F1 #10 (Vaccinium darrowii clone FL4B x Vaccinium corymbosum clone W85-20) and Vaccinium corymbosum clone W85-23.
Sub-objective 2.B. Use capture sequencing to discover single nucleotide polymorphism (SNP) markers that can be used in association mapping and bi-parental mapping to identify genomic regions associated with drought and alkaline soil tolerance.
Objective 3. Improve blueberry and grape fruit quality (picking scar, color, firmness, sugar content, etc.), flowering, and fruit ripening timing to meet industry needs for a precise market window and increased profitability, using advanced genomic resources, including linkage mapping and genome wide associations.
Sub-objective 3.A. Develop blueberry segregating mapping populations to determine genetic and environmental effects on fruit quality traits and use SNP markers developed in objective 2 to identify quantitative trait loci (QTL) associated with fruit quality traits.
Sub-objective 3.B. Use the Genotyping-by-Sequencing (GBS) technology and bi-parental mapping populations to identify traits underlying drought and Pierce’s disease (Xylella (X.) fastidiosa) tolerance in muscadine grapes.
Approach
Sub-objective 1A: Reciprocal crosses between selections of Hibiscus (H.) moscheutos and H. syriacus will be performed and F1 seeds will be soaked in oryzalin to induce the polyploidy levels. Flow cytometry, leaves thickness, guard cell length, and cytological analysis will be used to identify the interspecific hybrids. Interspecific hybrids will be evaluated to select hybrids with winter-hardness and wide adaption to prevalent conditions in southeastern U.S.
Sub-objective 1B: F1 populations from the following reciprocal crosses Vaccinium (V.) darrowii x V. pallidum, V. darrowii x V. tenellum, and darrowii x V. arboreum will be generated. F1 seedling will be screened to select interspecific hybrids using single nucleotide polymorphism (SNP) markers and flow cytometry. Polyploidy will be induced using antimitotic chemicals colchicine and oryzalin and stomatal frequency and length, chloroplast counts, and flow cytometry will be used to screen for polyploidy, and chromosome counts will be performed on putative polyploid plants to confirm results.
Sub-objective 2A: Genome wide association mapping panel and interspecific diploid blueberry mapping population will be grown under optimal-water and water-stressed conditions. Non-destructive measures associated with drought tolerance, including carbon isotope discrimination, normalized difference vegetation index, canopy temperature, and leaf senescence rate will be evaluated. The same materials will be grown in a hydroponic system at two pH levels, 4.5 and 6.5. Stress response to changes in pH will be quantified by measuring uptake of Iron (Fe), Manganese (Mn), and Nitrogen (N) measured in leaf tissue. Based on results, the most appropriate indices for screening will be determined and used in field screening.
Sub-objective 2B: The 30,000 capture probes designed previously from the draft genome will be used to genotype the Genome wide association panel, the mapping population, and different diploid V. species. Sequence data will be used to for SNP discovery which will be used to understand the structure of the complex blueberry genome, develop a high density SNP linkage map, and confirm the interspecific hybrids in Obj. 1.
Sub-objective 3A: Parents and F1 progeny will be evaluated for blooming time, bloom-ripening interval, fruit size, sloble solids content, titratable acidity, firmness, anthocyanins content, stem scar, size, and resistance to cracking. Parents and F1 individuals will be genotyped with SNP markers developed in objective two and SNP data will be used in quantitative trait loci (QTL) analyses to identify SNP markers associated with traits of interest.
Sub-objective 3B: Crosses between V. rotundifolia cultivars, namely ‘Southern Home’, ‘Noble’, and ‘Carlos’ will be conducted. Parents and mapping populations will be inoculated with Xylella fastidiosa and the cane maturation index will be used to descriminate between resistant and susceptible genotypes. DNA will be extracted from parents and F1 progeny and used in GBS library preparation and sequencing. Polymorphic SNP markers will be used in QTL analyses to identify region(s) associated with disease resistance and fruit quality traits.
Progress Report
DNA samples extracted from three accessions each of Vaccinium (V.) darrowii, V. pallidum, V. tenellum, V. elliottii, and V. corymbosum were sent to RAPiD genomics for capture sequencing using the 30,000 capture probes designed previously from the draft genome of V. corymbosum. In addition, the same DNA samples were sent to Kyoto University, Japan, for sequencing using the Restriction site Associated DNA Sequencing technology (RAD-Seq). The sequences data generated from these activities will be used to discover species-specific single nucleotide polymorphism (SNPs) to characterize the F1 seedling and confirm the interspecific hybrids generated in objective 1 and to understand the phylogenetic relationship between these species.
For the SNPs discovery, DNA samples from three accessions each of Vaccinium darrowii, V. pallidum, V. tenellum, V. elliottii, and V. corymbosum were sent to the USDA ARS, Genomics and Bioinformatics Research Unit in Stoneville, Mississippi, for Illumina sequencing. Leaves were collected from the Vaccinium darrowii clone (NJ8807 x NJ8810) for high molecular weight genomic DNA extraction. DNA from this clone will be sent to the Genomics and Bioinformatics Research Unit in Stoneville for PacBio sequencing. The expected PacBio sequencing will help us to produce genome sequences without gaps, map the Illumina reads, and discover SNP markers for Objectives 2 and 3.
Non-Assistance Cooperative Agreement was established with the University of Southern Mississippi to conduct a preliminary study to obtain basic knowledge about the microbial community composition in roots of different Vaccinium species. Roots samples from two accessions each of V. virgatum, V. corymbosum, V. darrowii (drought tolerant), and V. arboreum (drought and high soil pH tolerant) were collected and used to extract the microbial DNA using the DNeasy PowerSoil kit. Bacterial and fungal ribosomal ribonucleic acid (rRNA) gene sequencing will be performed using Illumina bacterial 16S rRNA gene libraries and fungal 18S rRNA ITS libraries. To identify the genus and the species of most abundant microorganisms, BLASTN will be used to find homologous sequences in NCBI.
Accomplishments
1. Development of Pentaploid interspecific hybrids between V. corybosum x V. virgatum. Two pentaploid hybrids from a cross between the southern highbush cultivar ‘SunShine’ (Vaccinium corybosum 2n=4x=48) and the rabbiteye cultivar ‘Brightwell’ (Vaccinium virgatum 2n=6x=72) and a cross between ‘Powder blue’ and ‘Rebel’ were obtained. The hybrids were characterized using the flow cytometr and roots were collected for chromosome counting to confirm the finding.
2. Development of a new edible ornamental rabbiteye blueberry cultivar. "Muffin Man" is a new, non-patented edible ornamental rabbiteye blueberry cultivar developed by the USDA-ARS scientists in Poplarville, Mississippi, and deposited at the USDA ARS National Clonal Germplasm Repository, Corvallis, Oregon as PI 687223. "Muffin Man" is characterized by its heat and drought tolerance, extended berry ripening periods, notable fall coloring in leaves, numerous unique pale pink blooms that arise on long reproductive shoots, where berries developed with attractive color ranging from pink to deep blue upon ripening. "Muffin Man" is an ideal choice for edible and ornamental landscape for the USDA hardiness zones 8a thru 9a.
