Performing Department
(N/A)
Non Technical Summary
Soilborne diseases of cultivated strawberry are an increasingly critical economic burden in both nursery and fruit production. The two most damaging and widespread soilborne diseases in the US are Phytophthora crown rot (PhCR) and charcoal rot (CR).The leading cultivars are susceptible to one or both diseases, and chemical controls are either expensive or ineffective. Cultivars with improved resistance are an urgent need and offer the most sustainable form of control. Our goal is to identify sequence variations and genes associated with the resistance and employ conventional strategies alongwith marker-assisted selection breeding efforts to more effectively introgress resistance into strawberry cultivars.To accelerate the resistant breeding, we will also develop new molecular breedingtools and resources aimed at enhancing strawberry varieties with desirable traits such as sweetness, flavorful, and high level of disease resistance. Throughout this research project, improved germplasm will be available to the US strawberry industry and breeding communitiesas a critical component of an integrated disease control strategy.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Fresh strawberries are a vital component of the U.S. fruit and vegetable industry, with an annual farm gate value of approximately $2.5 billion. Economic losses due to root and crown rots caused by soilborne pathogens are estimated at $150 million annually in the U.S. In the Florida strawberry industry alone, yield losses due to soilborne diseases are estimated tocost growers $15 million in revenue each year, despite annual pre-plant soil fumigation. Phytophthora crown rot (PhCR), caused by Phytophthora cactorum, and charcoal rot (CR), caused by Macrophomina phaseolina, are currently the most damaging and widespread soilborne diseases of strawberry in the U.S. and the world, causing up to 40% production losses. Crop mortality can surpass 80% in CR-infected fields of susceptible cultivars. Limitations on fumigants such as methyl bromide have led to deterioration in the control of P. cactorum and M. phaseolina in recent years. Registration of new fumigants has been difficult, and alternatives to methyl bromide have had lower and inconsistent efficacy for a cultivar of pathogens. Commercial cultivars vary widely in their genetic resistance to these soilborne pathogens. The cultivars most resistant to PhCR provide sufficient levels of disease control, but currently, the cultivars most resistant to CR provide only moderate disease reduction. Thus, the development of resistant cultivars is becoming a top priority for strawberry breeding programs, and a critical strategy for combatting these diseases in an era of alternative fumigants. Breeding efforts and genomics research at the University of Florida have identified large-effect QTL for resistance to both PhCR and CR under field conditions. For PhCR, the resistance locus is located on chromosome 7B and is referred to as FaRPc2. Within the FaRPc2 locus, two significant haplotypes, FaRPc2-H2 and FaRPc2-H3, confer resistance against P. cactorum. Each resistant haplotype explained about 40% of the phenotypic variance in the tested populations. The resistant haplotype, FaRPc2-H3, is more prevalent in commercial strawberry cultivars in the United States. The two haplotypes originated from different sources, suggesting the presence of two distinct functional alleles associated with the FaRPc2-mediated resistance. Cultivars with FaRPc2-H2 display strong resistance to PhCR; however, genomic tools to investigate FaRPc2-H2 are missing, which is a bottleneck for deploying genomics-assisted breeding strategies. For CR resistance, our group recently discovered three large-effect resistance QTL on chromosomes 2B (FaRMp1), 4A (FaRMp2), and 4C (FaRMp3). Each locus explained 20% to 40% of the phenotypic variance in the tested populations. While FaRMp1 and FaRMp2 are prevalent in breeding germplasm, FaRMp3 was identified in FVC 11-58, a reconstituted F. ×ananassa of diverse parentage that was previously identified as resistant to CR (Zurn et al. 2020). The resistance effect of FaRMp3 is stronger than FaRMp1 and FaRMp2 combined. The potential effects of pyramiding the three loci are currently undetermined. Moreover, FaRMp2 and FaRMp3 appear to reside in homoeologous regions that originate from two different ancestral diploids. Thus, marker-assisted pyramiding of FaRMp1, FaRMp2 and FaRMp3 may lead to stronger and more durable resistance against CR in new cultivars. Previous research in our group has shown that enhanced disease resistance can be effectively achieved through the combination of conventional and genomics-assisted breeding approaches. Developing subgenome- or allele-specific DNA markers in allo-octoploid strawberry is very challenging due to high levels of heterogenicity and complexity of homoeologous sequences in the subgenome. Our group recently developed a telomere-to-telomere (T2T) haplotype-phased reference genome of 'Florida Brilliance'. However, the high level of allelic and structural diversity in cultivated strawberry means that a single reference genome can only capture a fraction of relevant genes of interest. Without additional genomes, the search for candidate genes at all relevant loci will not be successful. In this project, new genomic tools and resources will be developed to enhance breeding for resistance to PhCR and CR.ObjectivesObjective 1. Characterizing Genomic Regions Conferring Resistance to Phytophthora Crown Rot and Charcoal Rot. Define genomic regions and fine-map QTL to discover subgenome-specific sequence variants underlying resistance to PhCR and CR.Objective 2. Developing Chromosome-Scale Haplotype-Phased Genomes Containing FaRPc2-H2 and FaRMp3. Generate high-quality haplotype-phased genome assemblies for elite breeding selections harboring loci conferring resistance to PhCR and CR.Objective 3. Designing Subgenome-Specific DNA Markers for Marker-Assisted Breeding. Develop high-throughput subgenome-specific DNA markers tightly linked to resistant alleles.?Objective 4. Accelerating Introgression of Multiple QTL Through Marker-Assisted Pyramiding. Combine multiple QTL for the resistance to PhCR and CR via marker-assisted pyramiding for increased resistance in elite breeding germplasm.
Project Methods
Objective #1 - Characterizing Genomic Regions Conferring Resistance to Phytophthora Crown Rot and Charcoal RotSub-objective 1.1. Defining the QTL Region for FaRPc2-H2:Approximately 400 accessions with multiple seasons of field phenotypic data (2013 to 2020) will be used for fine-mapping analysis. We will develop sub-genome-specific SNP or Indel-based DNA markers from FanaSNP array markers located in the FaRPc2 region and use them to examine marker-phenotype associations. We will also develop additional fine-mapping population for FaRPc2-H2 (N=500). The highly susceptible cultivar 'Florida Medallion' (homozygous of farpc2-h2) will be crossed with a resistant advanced selection 'FL10-92' (heterozygous of FaRPc2-H2). Inoculation and phenotyping will be conducted in field conditions. All individual genotypes will be collected by 50K FanaSNP array, and genome-wide association study (GWAS) will be performed using a multi-locus mixed-model approach with SNP genotypes to determine the precise genomic region associated with FaRPc2-H2-mediated resistance.Sub-objective 1.2. Develop Genomic Tools to Investigate QTL Structure and Deploy Genome-Assisted Breeding Strategies for CR Resistance:To narrow the QTL region for three Mp loci, marker probe sequence from FanaSNP 50k array will be used to develop subgenome-specific markers across each locus. The SNP or InDel-based subgenome-specific markers will be designed at 50 kb intervals and tested with populationsdeveloped in our previous studies.All marker testing sets of accessions (approximately 500 in the association mapping panels) will be genotyped with SNP or Indel-based high-throughput markers such as KASP, PACE and HRM. After evaluating the marker segregation with field CR phenotype, the fine-mapped regions of FaRMp1, FaRMp2, and FaRMp3 will be selected for further sequencing analysis (target captured and amplicon sequencing). To further narrow the FaRMp3 region and facilitate pyramiding with other Mp loci, we will develop at least five backcross populations from FVC 11-58 progeny. Each crossing family will contain about 150 individuals, and the population size will be about 650. Distributions of seedling response to infection will be plotted for each of the F1 introgression populations and compared against the susceptible and resistant parents' response.Objective #2: Developing Chromosome-Scale Haplotype-Phased Genomes Containing FaRPc2-H2 and FaRMp3.Sub-objective 2.1 - Assemble and Annotate Reference Genomes Representing FaRPc2-H2 and FaRMp3:The trio-binning assembly pipeline will be built on Hifiasm.The phased contigs will be scaffolded with the reference genome 'Florida Brilliance'.Protein-coding genes will be annotated using publicly available F. × ananassa Iso-Seq transcript data and an expansive RNA-seq atlas.The assemblies will be evaluated in terms of contiguity, completeness, and accuracy. The completeness of the assembly and gene annotation will also be assessed using BUSCO. The quality of scaffolding will be evaluated in terms of grouping confidence, location confidence, orientation confidence using outputs from Ragtag. All the above-mentioned matrices will be compared to the gold-standard 'Florida Brilliance' and 'Royal Royce' assemblies.Sub-objective 2.2 - Validation of Candidate Genes Using Agrobacterium Transient Gene Expression Assay:A comparative genomic analysis between the resistant and susceptible FaRPc2-H2 and FaRMp3 alleles will be performed to determine if meaningful polymorphisms or structural variations exist within or nearby candidate genes identified by the RNA-seq study.Susceptible and resistant alleles for potential candidate genes will be compared to detect functional polymorphisms. 'Florida Brilliance' and 'Royal Royce' Iso-Seq data will be used to confirm the expression of transcript sequences and validate the start and stop codons.For RNAi-mediated knockdown assay, hairpin structures will designed for target candidate genes.For overexpression study, coding region of candidate gene (CDS) will be obtained based on the 'Florida Brilliance' genome annotation.Objective #3: Designing Subgenome-Specific DNA Markers for Marker-Assisted Breeding.Sub-objective 3.1. Develop and Validate Predictive DNA markers for FaRPc-H2:PCR primers will be designed to amplify SNP or indel polymorphisms that distinguish haplotypes carrying resistant and susceptible alleles. Both HRM and KASP assays will be developed.Markers that show three distinct genotyping clusters denoting each allelic state for the polymorphism (co-dominant markers) will be tested in the larger populations for marker-traits association. The resulting validated biallelic subgenome-specific markers showing at least 95% correspondence between marker and phenotype will be publicly available and used for the marker assisted seedling selection pipeline.Sub-objective 3.2. Transcriptome, eQTL, Candidate Gene Identification and Marker Development for Mp Loci:To identify functional variants associated with CR resistance, we will analyze the transcriptome for differentially expressed genes (DEGs) in the fine-mapped QTL regions of three loci.RNA-seq analysis will be conducted to identify changes in gene expression that correlate with genomic variants. Plants from three resistant and susceptible accession for each Mp locus will be inoculated with M. phaseolina, and crown tissue samples will be collected 72 hr after infection. For expression quantitative trait locus (eQTL) analysis, about 150 individuals will be used for RNA sequencing. RNAseq data will be quality controlled and standardized before being used for eQTL and TWAS analyses.Genes associated with CR resistance through both TWAS and eQTL will be considered as candidate genes. Candidate gene sequences that exhibit significant association with resistance to CR will be selected for designing subgenome-specific functional markers.Based upon the candidate genes identified in the previous steps, subgenome-specific markers will be developed according to the methods above for PhCR and further validated with diverse breeding germplasm and populations used in the QTL discovery study (a discovery population and two validation populations - approximately 300 individuals).Objective #4: Accelerating Introgression of Multiple QTL Through Marker-Assisted Pyramiding.Sub-objective 4.1. Marker-Assisted Introgression of FaRPc2-H2 and FaRPc2-H3:All crossingparentswill be screened with FaRPc2-H2 and FaRPc2-H3 markers in order to maximize the number of crosses segregating for resistance or producing 100% of offspring with at least one resistance allele.Inoculation and field screening will be conducted. Using marker-assisted backcrossing approach, both resistant haplotypes present in the locus of FaRPc2 will be pyramided to maximize the resistance to PhCR. All resulting breeding selections will be evaluated in field conditions for enhanced disease resistance in clonally replicated trials.Sub-objective 4.2. Marker-Assisted Pyrimiding of FaRMp1, FaRMp2, and FaRMp3: In previous years, crossing population were developed for pyramiding thes three Mp loci. In this project, selections from these seedling populations will be screened via inoculated field trials in order to validate the presence of the resistance locus or loci and determine the phenotypic effects of pyramiding. Inoculation and field trials will be conducted according to the established methods in our previous study. Assuming the validation shows that pyramiding further decreases plant mortality in inoculated trials, a modified backcross approach for FaRMp3 will be conducted with crosses to different elite parents in each cycle (to avoid inbreeding depression), with marker selection conducted for each cross to ensure the transmission of the resistant allele.