Location: Southern Horticultural Research Unit
2023 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
This is the final report for this project. Refer to new project 6062-21000-011-000D, "Genetic Improvement of Small Fruits and Ornamental Plants to Enhance Traits Desirable to Consumers and Tolerance to Biotic and Abiotic Stress" for additoinal information.
Significant progress has been made on all three objectives and their subobjectives. Under Objective 1, seeds from interspecific crosses between different Vaccinium species were obtained for in vitro ploidy manipulation. Under Objective 2, a diverse panel of southern highbush blueberry and two bi-parental mapping populations were characterized for phenological and fruit quality traits using high throughput phenotyping platforms. A new high-throughput genotyping platform for blueberries with a 3,000 SNPs using the Diversity Arrays Technology (DArT) was developed in collaboration with Breeding insight. The diverse panel and the two mapping populations were genotyped using the DArt array. In addition, a chromosomal-level genome of muscadine grape, has been assembled and used in combination with transcriptome sequencing to identify genes involved in resistance to Pierce’s disease. Under Objective 3, a new, non-patented early rabbiteye blueberry cultivar ‘USDA-Spiers’ was released for growers in the USDA hardiness zones 8a thru 9a. ‘USDA-Spiers’ produces an abundance of medium to large, attractive light blue and very firm berries with resistance to rain-induced splitting. Mature ‘USDA-Spiers’ bushes are moderately spreading with narrow crowns, and compared to most rabbiteye blueberry cultivars, grow more slowly, and require little pruning to manage excessive vegetative growth.
Accomplishments
1. Assembled a new, long-read genome for ‘Carlos’, a cultivar of muscadine and identified genes involved in resistance to Pierce’s disease. Muscadine grape is resistant to many of the pathogens that negatively impact the production of common grape, including the bacterial pathogen Xylella fastidiosa, which causes Pierce’s Disease. ARS researchers in Poplarville, Mississippi, in collaboration with University of Tennessee have sequenced, assembled, and annotated the muscadine grape genome. Comparison of resistance gene content between the ‘Carlos’ and common grape genomes indicates an expansion of resistance (R) genes in ‘Carlos.’ The group further identified 234 genes involved in Xylella fastidiosa response by transcriptome sequencing ‘Carlos’ plants inoculated with the bacteria. The most differentially expressed genes in the muscadine response were not found in common grape, and three of the R genes identified as differentially expressed in muscadine do not have an ortholog in the common grape genome.
2. ‘USDA-Spiers’ rabbiteye blueberry. ‘USDA-Spiers’ is a new, non-patented early rabbiteye blueberry cultivar developed by the ARS researchers in Poplarville, Mississippi. ‘USDA-Spiers’ derived from a series of hybridizations among rabbiteye blueberry cultivars and selections. ‘USDA-Spiers’ produces an abundance of medium to large, attractive light blue and very firm berries with resistance to rain-induced splitting. Mature ‘USDA-Spiers’ bushes are moderately spreading with narrow crowns, and compared to most rabbiteye blueberry cultivars, grow more slowly, and require little pruning to manage excessive vegetative growth. ‘USDA-Spiers’ is an ideal choice for the USDA hardiness zones 8a thru 9a.
3. Assessment of the southern highbush blueberry germplasm collection for fruit quality attributes. Breeding new blueberry cultivars with enhanced fruit quality requires simple, accurate and cost-effective assays to select individual from segregating population. ARS researchers in Poplarville, Mississippi, characterized the southern highbush blueberry collection for berry diameter, berry weight, firmness, pH, total polyphenol, total acids, glucose, fructose, total glucose, and total sugars content using high throughput phenotyping tools. A marked degree of diversity for tested traits was observed and glucose and fructose were the predominant sugars in tested genotypes. Further, 27-39% of the tested genotypes displayed a firmness value of 160 g/mm or above which is considered as superior value. Information obtained from this study is critical to identify superior genotypes for future crosses and advance evaluation. In addition, the firmness tester and the discrete analyzer used in this study have shown numerous potentials to improve the efficiency and precision of phenotyping.
Review Publications
Stringer, S.J., Draper, A.D., Babiker, E.M., Sampson, B.J., Sakhanokho, H.F. 2023. 'USDA-Spiers' rabbiteye blueberry. HortScience. 58(3):338–340. https://doi.org/10.21273/HORTSCI17042-22.
Babiker, E.M., Stringer, S.J., Sakhanokho, H.F., Munoz, P. 2023. Combining high throughput phenotyping and multivariate analysis to assess fruit quality traits in southern highbush blueberry (V. corymbosum interspecific hybrids) germplasm collection. HortScience. 58(7):750-755. https://doi.org/10.21273/HORTSCI17185-23.
Werle, C.T., Babiker, E.M., Adamczyk Jr, J.J. 2022. Seasonal abundance and diversity of potential Xylella (Xanthomonadales: xanthomonadaceae) vectoring leafhoppers in Mississippi muscadine vineyards. Subtropical Agriculture and Environments. 73:1-8.
Bowden, A.T., Knight, P.R., Ryals, J.B., Coker, C.E., Langlois, S.A., Broderick, S.R., Blythe, E.K., Sakhanokho, H.F., Babiker, E.M. 2022. Evaluation of one-time applications of foliar applied auxin co-applied with surfactant for use in commercial cutting propagation. Agronomy Journal. 12(10):2243. https://doi.org/10.3390/agronomy12102243.
Huff, M., Babiker, E.M., Hulse-Kemp, A.M., Scheffler, B.E., Youngblood, R.C., Simpson, S.A., Staton, M. 2023. Long-read, chromosome-scale assembly of Vitis rotundifolia cv. Carlos and its unique resistance to Xylella fastidiosa subsp. fastidiosa. BMC Genomics. https://doi.org/10.1186/s12864-023-09514-y.
