Location: Southern Horticultural Research Unit
2020 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 researchers at 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, interspecific hybrids from crosses between different Hibiscus species were obtained by ARS researchers at Poplarville, Mississippi, without a need for embryo rescue. Further, two interspecific hybrids were obtained from crosses between Vaccinum tenellum and Vaccinium darrowii, between Vaccinium darrowii and Vaccinium arboretum and between rabbiteye and southern high bush cultivars. The new hybrids were propagated and transplanted by ARS researchers into a research plot at Poplarville, Mississippi.
Ojective 2, ARS researchers at Poplarville, Mississippi, made significant progress toward characterizing the blueberry diploid mapping populations. Further, a tetraploid mapping population was transplanted into a research plot established at Stone County research farm and characterized for phenological traits. The two populations were genotyped with 2000 single nucleotide polymorphisms (SNP) using SeqSNP platform. The most valuable outcomes of this objective will be a genotyping platform that blueberry breeders can use in marker-assisted breeding to improve breeding efficiency and estimate genetic diversity of blueberry germplasm collection.
Objective 3, ARS researchers at Poplarville, Mississippi, selected four interspecific hybrid grape cultivars and one muscadine cultivar to evaluate for the effect of crop load stress on the ability of Xyllela fastidiosa (Xf) to infect vines and cause the Pierce’s Disease (PD) symptoms in the canopy. At this point, ARS researchers at Poplarville, Mississippi, established that OK392 and Blanc du Bois had higher yield under the full load compared to the reduced and minimal loads. In contrast, Miss Blanc and Mid-South performed better under reduced crop load. Furthermore, population levels of Xf in OK392 and Villard Blanc were much higher than in Miss Blanc, while Mid-South and Blanc du Bois were Xf-free despite of crop load treatment.
Accomplishments
1. Determination of nuclear DNA content, ploidy, and Fluorescence In Situ Hybridization (FISH) location of ribosomal DNA (rDNA) in Hibiscus hamabo. Genome size is a distinguishing character of living organisms and estimates of genome size have been useful in systematic and evolutionary studies as well as in planning sequencing work. ARS researchers in Poplarville, Mississippi, in collaboration with scientists of USDA-Forest Service, determined genome size, ploidy, and rDNA location in Hibiscus hamabo. H. hamabo is salt-tolerant, so it is considered one of the best afforestation plants used in reclamation areas and shelterbelt tree species along the coasts as well as urban landscaping. H. hamabo is a good plant material to study the response mechanisms of salt-stressed woody plants. A practical application of these results was the hybridization of H. hamabo with an ornamentally superior species, H. paramutabilis, which has the same chromosome number as H. hamabo. Seedlings of these crosses are being evaluated.
2. Comparative analysis of rhizosphere microbiomes of southern highbush, Darrow’s, and rabbiteye blueberry. Plants are inhabited by millions of microorganisms that coexist in complex ecological communities, and profoundly affects the plant’s productivity and capacity to cope with abiotic stress. An ARS researcher in Poplarville, Mississippi, in collaboration with university collaborators, employed molecular approaches to characterize and compare microbial communities inhabiting the roots of three Vaccinium species, Darrow’s blueberry, rabbiteye, and southern highbush. Results from this study revealed that the Vaccinium species share a common core rhizobiome, but at the same time differ significantly in the diversity, relative abundance, richness, and evenness of multiple groups of prokaryotic and eukaryotic microorganisms. Further, they differ in the abundance of beneficial rhizobacteria and ericoid mycorrhizal fungi, which play a vital role in their adaptation to soils with low pH and slow turnover of organic matter. Results of the study will contribute to the selection of cultivars that maximally benefit from the mycorrhizal associations and exhibit better adaptability to prevalent field conditions.
3. Draft whole genome sequencing of Xylella fastidiosa strains OK3, VB11, and NOB1. Pierce’s disease, caused by the bacterium X. fastidiosa, is an economically important grapevine disease that endangers the grape industry worldwide. Analysis of whole genome sequences of X. fastidiosa strains from bunch and muscadine grapes is important to better understand the pathogenic potential and to breed new resistant cultivars. ARS researchers in Poplarville, Mississippi, in collaboration with universities scientists generated high-quality draft whole-genome assemblies of X. fastidiosa subsp. fastidiosa strains OK3, VB11, and NOB1 isolated from symptomatic bunch and muscadine grape plants grown in southern Mississippi. Findings from this study will benefit grape growers by providing knowledge required to screen germplasm for resistance and to develop novel methods of disease control.
4. Genomic insight into the developmental history of southern highbush blueberry populations. Southern highbush blueberry (SHB) is a cultivar group that has been intensively bred by multiple interspecific crosses between northern highbush blueberry (NHB) and low-chill Vaccinium species to expand the geographic limits of highbush blueberry production. Despite years of research, little is known about the genetic diversity and population structure of SHB. An ARS researcher in Poplarville, Mississippi, working with collaborators from Japan, used DNA sequencing technique to genotype a diverse set of SHB, NHB, and rabbiteye accessions. Sequence data indicated that rabbiteye accessions were genetically distinct from SHB and NHB cultivars, whereas NHB and SHB were genetically indistinguishable. The population structure results appeared to reflect the differences in the allele selection strategies breeders used for developing germplasm adapted to local climates. The genotype data implied there are no or very few genomic segments that were commonly introgressed from low-chill Vaccinium species to the SHB genome. These findings will enable genetic variability to be associated with specific phenotype and enhance germplasm selection based on DNA sequences.
Review Publications
Li, J., Mavrodi, O., Hou, J., Blackman, C., Babiker, E.M., Mavrodi, D. 2020. Comparative analysis of rhizosphere microbiomes of southern highbush blueberry (Vaccinium corymbosum L.), Darrow’s blueberry (V. darrowii Camp.), and rabbiteye blueberry (V. virgatum Aiton). Frontiers in Microbiology. 11:370. https://www.frontiersin.org/articles/10.3389/fmicb.2020.00370/full.
Mavrodi, O.V., Mavrodi, D.V., Stafine, E.T., Adamczyk Jr, J.J., Babiker, E.M. 2020. Draft genome sequences of Xylella fastidiosa subsp. fastidiosa strains OK3, VB11, and NOB1 isolated from bunch and muscadine grapes grown in southern Mississippi. Microbiology Resource Announcements. 9(25):1-2. https://doi.org/10.1128/MRA.00562-20.
