Location: Southern Horticultural Research Unit
2021 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 by ARS scientists in Poplarville, Mississippi on all three objectives and their subobjectives, all of which fall under the National Program 301, Plant Genetic Resources, Genomics and Genetic improvement. Objective 1, an efficient protocol for micropropagation of Hibiscus moscheutos has been developed by ARS scientists in Poplarville, Mississippi. This protocol can be used for plant transformation, in vitro selection, and industrial mass micropropagation of improved H. moscheutos selections or cultivars. The true-to-type regenerants generated through the protocol were transplanted into a research plot by ARS scientists in Poplarville, Mississippi, for field evaluation. Also, the confusion about the ploidy of H. moscheutos was removed as its chromosome number (2n = 2x = 38) was unequivocally established. Finally, the genome size of H. moscheutos was determined by ARS scientists. Objective 2, a diverse panel of southern highbush blueberry and two tetraploid mapping populations were characterized for phenological and fruit quality traits. A diverse panel of 105 southern highbush blueberry accessions were genotyped using double-digest restriction site-associated DNA sequencing. Genome wide association analyses of phenology-related traits were performed and one QTL for chilling requirement was detected on chromosome 4. Further, a chromosomal-level genome of Vaccinium darrowii, a progenitor species for the commercially grown southern highbush blueberry, has been assembled. For this high-quality reference genome, a combination of PacBio sequencing and high throughput chromatin conformation capture scaffolding technologies have been used. As result, over 97.8% of the genome is anchored to chromosomes and 34,809 protein-coding genes were annotated. V. darrowii possesses good adaptation to the soil and climate of the southeast. Therefore, the genome sequence will accelerate the development of improved blueberry cultivars adapted to fluctuating climatic conditions. Objective 3, one rabbiteye blueberry (MS 794) has been advanced for release as an early rabbiteye cultivar for growers in the southeast of the United States. MS 794 normally ripe by May 15th. The market period of May 15th is still profitable for growers in this region.
Accomplishments
1. Development of an efficient protocol for micropropagation of Hibiscus moscheutos L. ARS scientists in Poplarville, Mississippi, suggests Hibiscus moscheutos L., also known as hardy hibiscus, is a perennial shrub native to wetland areas of North America and desired for its showy white to pink flowers. Propagation by stem cuttings is the most used method to propagate new cultivars. ARS researchers in Poplarville, Mississippi, in collaboration with the United States forest service collaborator in College Station, Texas, developed an efficient protocol for micropropagation of H. moscheutos. The protocol can be used for rapid and mass production of new cultivars and eliminate viruses and other pathogens from valuable clones, thus facilitating the risk-free interchange of plant breeding materials. Further, the protocol will reduce the production costs and allow United States nursery industry to remain competitive in the market.
2. The genome sequence of Darrow's blueberry (Vaccinium darrowii). ARS scientists in Poplarville, Mississippi suggests Marker-assisted selection at the seedling stage can reduce the costs of breeding by identifying the best parents for crosses and the best resulting progeny. A crop genome sequence is fundamental to accelerate the development of a genotyping platform for marker-assisted selection. In collaboration with university collaborator, ARS researchers in Poplarville, Mississippi, and Stoneville, Mississippi, assembled the first chromosomal-level reference genome for V. darrowii, a progenitor species for the commercially grown southern highbush blueberry. The genome sequence will facilitate transfer of genes for adaption traits from V. darrowii into cultivated blueberry.
Review Publications
Nishiyama, S., Fujikawa, M., Yamane, H., Shirasawa, K., Babiker, E.M., Tao, R. 2020. Genomic insight into the developmental history of southern highbush blueberry populations. Heredity. https://doi.org/10.1038/s41437-020-00362-0.
Sakhanokho, H.F., Islam-Faridi, N., Babiker, E.M., Nelson, C., Stringer, S.J., Adamczyk Jr, J.J. 2020. Determination of nuclear DNA content, ploidy, and FISH location of ribosomal DNA in Hibiscus hamabo. Scientia Horticulturae. 264:109167. https://doi.org/10.1016/j.scienta.2019.109167.
Cao, S., Stringer, S.J., Gunawan, G., Conner, P.J., Mcgregor, C. 2020. Genetic diversity and pedigree analysis of muscadine grapes (Vitis rotundifolia) using SSR markers. HortScience. 14(3):143-151. https://doi.org/10.21273/JASHS04856-20.
