Location: Southern Horticultural Research Unit
2019 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 was made on all three objectives and their subobjectives, all of which fall under the National Program 301, Plant Genetic Resources, Genomics and Genetic improvement. Under Objective 1, interspecific hybrids from crosses between Hibiscus moscheutos and Hibiscus syriacus were obtained without a need for embryo rescue. The new hybrids will be propagated and tested under field conditions in two locations. To expand the geography where blueberries are grown, accessions from five Vaccinium species were used in crosses. Two interspecific hybrids were obtained from crosses between Vaccinum tenellum and Vaccinium darrowii and between Vaccinium darrowii and Vaccinium arboreum. Two pentaploid hybrids were developed from crosses between rabbiteye and southern highbush cultivars. Under Objective 2, we made significant progress toward characterizing a diverse panel of 216 southern highbush blueberry genotypes collected from different breeding programs. Knowledge obtained from this objective can be used to select genotypes for crosses to generate hybrids with blended traits from the parental genotypes. Further, the diversity panel was used to organize a field day and allow growers to have new information on performance of different southern highbush cultivars in the U.S. gulf-coast region. To investigate the role of the host microbial community structure and composition and the plant anatomy in tolerance to Pierce’s disease, two muscadine grape cultivars were micro propagated to obtain disease-free plants to be tested against Xylella fastidiosa (plant pathogen). Results from this objective are essential to develop grape cultivars with resistance to Pierce’s disease and broader adaption to prevalent environmental conditions.
Accomplishments
1. Development and identification of two pentaploid hybrids. The native rabbiteye blueberry has been grown commercially in the Southeastern U.S. for well over a century. However, the southern highbush blueberry is continually gaining popularity in much of this geography due to earlier ripening periods and quality attributes. Several growers in the region have established trial plantings of southern highbush cultivars but have reported declines in plant vigor, growth, and survival in the field following several years of productivity. Two fertile pentaploid hybrids with low chill requirements were developed by ARS scientists in Poplarville, Mississippi, from crosses between two rabbiteye cultivars and two southern highbush cultivars. These hybrids will be utilized by breeders to develop new early-ripening blueberry cultivars possessing greater adaptation and vigor, coupled with the ability to produce high quality, earlier ripening berries.
2. Development of a tissue culture protocol for mass-production of milkweed species. Monarch butterfly populations have declined by more than 80 percent since the 1990s due in large part to the widespread eradication of milkweed plants. A tissue culture method capable of mass-producing plants in three milkweed species, namely Asclepias latifolia (broadleaf milkweed), Asclepias speciosa (showy milkweed), and Asclepias subverticillata (horsetail milkweed) was developed by ARS scientists in Poplarville, Mississippi. The in-vitro grown plants were characterized using the flow cytometry to verify that the regeneration system produces genetically stable plants. The new protocol can be used by the nursery industry to propagate milkweed plants and conduct interspecific hybridization using embryo rescue.
Review Publications
Babiker, E.M., Stringer, S.J., Smith, B.J., Sakhanokho, H.F. 2018. Reaction of different vaccinium species to the blueberry leaf rust pathogen Thekopsora minima. HortScience. 53(10):1447-1452. https://doi.org/10.21273/HORTSCI13319-18.
Babiker, E.M., Stringer, S.J., Adamczyk Jr, J.J., Sakhanokho, H.F., Draper, A. 2018. ‘Muffin Man’ an edible ornamental rabbiteye blueberry. HortScience. 53(10):1523-1524. https://doi.org/10.21273/HORTSCI13372-18.
Sakhanokho, H.F., Babiker, E.M., Smith, B.J., Drackett, P.R. 2019. High-frequency somatic embryogenesis, nuclear DNA estimation, and genome size stability of micropropagated milkweed (Asclepias spp.) plants. Plant Cell Tissue and Organ Culture. 137:149-156.
Kambiranda, D., Basha, S.M., Stringer, S.J., Obuya, J.O., Snowden, J. 2019. Multi-year evaluation of stilbene levels among selected muscadine grape cultivars. Molecules. 24(5):981. https://doi.org/10.3390/molecules24050981.
Babiker, E.M., Stringer, S.J., Sakhanokho, H.F., Smith, B.J., Polashock, J.J. 2019. Characterization and pathogenicity of stem blight complex isolates associated with stem blight disease on Vaccinium species. HortScience. 54:1199-1203. https://doi.org/10.21273/HORTSCI14033-19.
