Research Project: Blueberry and Woody Ornamental Plant Improvement in the Southeast United States

Location: Southern Horticultural Research Unit

2022 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
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 tetraploid mapping populations were characterized for phenological and fruit quality traits. The diverse panel was genotyped using double-digest restriction site-associated DNA sequencing. Genome wide association analyses of phenology-related traits were performed and one genomic region for chilling requirement was detected on chromosome 4. Further, a chromosomal-level genome of V. darrowii, a progenitor species for the commercially grown southern highbush blueberry, has been assembled. Results from these activities have been used by the Vaccinium research community to develop a genotyping platform for blueberry. Under Objective 3, a plant invention disclosure has been submitted to release a new rabbiteye blueberry (MS 794). MS 794 has several advantages for growers in the Southeastern U.S. Among these are early ripening period, a compressed plant architecture in comparison to most other rabbiteye cultivars, high yield potential, very large firm berries, and excellent flavor.

Accomplishments
1. Identification of genetic markers associated with chilling requirement in blueberry. Genetic markers enable plant breeders to efficiently and affordably screen thousands of individuals and select a subset of individual plants with the desired traits. ARS researchers in Poplarville, Mississippi, detected DNA markers for chilling requirement in blueberry. DNA sequences generated through this activity has been used by the Vaccinium research community to develop a targeted capture genotyping platform for blueberry

2. Molecular markers for ornamental cultivars of Weigela. Weigela is an ornamental plant popular for its flower and leaf colors, but currently there is only limited information on its genetic diversity. ARS researchers in Poplarville, Mississippi, in collaboration with scientists at University of Tennessee have sequenced the whole genome of Weigela cultivar Spilled Wine® and developed 20 genomic simple sequence repeat (gSSR) markers. The gSSR markers were used to assess the genetic diversity of 18 Weigela cultivars. Markers developed in this study can be used in future genetic studies involving Weigela germplasm and possibly closely related taxa.

3. Diversity and seasonal abundance of potential Xylella vectoring leafhoppers in Muscadine vineyards. Pierce’s disease remains an important economic threat to American wine, table, and raisin grape production. Although muscadine grapes exhibit resistance, when stressed the vines can become susceptible. Vector management may be a useful strategy in limiting the pathogen spread, but knowledge of leafhopper species composition and their Xylella-vectoring potential remains scant. ARS researchers in Poplarville, Mississippi, have used sticky traps to identify potential leafhopper vectors and estimate their abundance and diversity in two muscadine grape vineyards. While the glassy-winged sharpshooter was predictably abundant from May through July, other species also were prominent including the invasive Sophonia orientalis in August. Results obtained from this study represent the initial efforts toward vector and disease management and will facilitate future research quantifying Xylella fastidiosa in these species.

Review Publications
Yu, J., Hulse-Kemp, A.M., Babiker, E.M., Staton, M. 2021. High-quality reference genome and annotation aids understanding of berry development for evergreen blueberry (Vaccinium darrowii). Horticulture Research. 8:228. https://doi.org/10.1038/s41438-021-00641-9.
Sakhanokho, H.F., Islam-Faridi, N., Smith, B.J. 2022. Determination of genome size and chromosome number of a Ziziphus species (Z. mauritiana Lam.) from eastern Senegal. HortScience. 57(3):349-352. https://doi.org/10.21273/HORTSCI16267-21.
Sakhanokho, H.F., Nurual, I., Smith, B.J. 2022. Nuclear DNA content and chromosome number determination in a Sahel medicinal plant, Combretum micranthum G. Don. Journal of Crop Improvement. https://doi.org/10.1080/15427528.2022.2030447.
Nagasaka, K., Nishiyama, S., Fujikawa, M., Yamane, H., Shirasawa, K., Babiker, E.M., Tao, R. 2022. Genome-wide identification of loci associated with phenology-related traits and their adaptive variations in a highbush blueberry collection. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2021.793679.
Edger, P.P., Iorizzo, M., Bassil, N.V., Benevenuto, J., Ferrao, L.F., Giongo, L., Hummer, K.E., Lawas, L.F., Leisner, C.P., Li, C., Munoz, P., Ashrafi, H., Atucha, A., Babiker, E.M., Canales, E., Chagne, D., DeVetter, L., Ehlenfeldt, M.K., Espley, R.V., Gallardo, K., Gunther, C.S., Hardigan, M.A., Hulse-Kemp, A.M., Jacobs, M.L., Lila, M., Luby, C.H., Main, D., Mengist, M.F., Owens, G.L., Perkins-Veazie, P., Polashock, J.J., Pottorff, M., Rowland, L.J., Sims, C.A., Song, G., Spencer, J., Vorsa, N., Yocca, A.E., Zalapa, J.E. 2022. There and back again; historical perspective and future directions for Vaccinium breeding and research studies. Horticulture Research. 9. Article uhac083. https://doi.org/10.1093/hr/uhac083.
Smith, B.J., Rezazadeh, A., Stafne, E., Sakhanokho, H.F. 2022. Effect of LED, UV-B, and fluorescent supplemental greenhouse lights on strawberry plant growth and response to infection by the anthracnose pathogen, Colletotrichum gloeosporioides. HortScience. 57(8):856–863. https://doi.org/10.21273/HORTSCI16591-22.
Hamm, T., Boggess, S., Sthapit Kandel, J., Staton, M., Huff, M., Hadziabdic, D., Shoemaker, D., Adamczyk Jr, J.J., Nowicki, M., Trigiano, R. 2022. Development and characterization of 20 genomic SSR markers for ornamental cultivars of weigela. Plants. 11(11):1444. https://doi.org/10.3390/plants11111444.
Islam-Faridi, N., Sakhanokho, H.F., Nelson, C. 2020. New chromosome number and cytomolecular characterization of the African Baobab (Adansonia digitata L.) – "The Tree of Life". Scientific Reports. 10:13174. https://doi.org/10.1038/s41598-020-68697-6.