Recipient Organization
N Y AGRICULTURAL EXPT STATION
(N/A)
GENEVA,NY 14456
Performing Department
(N/A)
Non Technical Summary
Susceptibility of commercial apple cultivars to apple scab along with the continual emergence of new pathogenic strains have prevented widespread adoption of low-input apple production, especially in the humid Eastern and Midwestern U.S. Sources of apple scab resistance have been identified, but their use in breeding is hindered by lack of characterization of functional alleles and availability of diagnostic markers. Linkage group 1 of apple harbors two major apple scab resistance loci in close proximity to a previously well-characterized apple scab resistance gene HcrVf2. Here, we propose to fine-map, identify, and validate candidate gene function, as well as develop diagnostic markers for two major apple scab resistance loci. Availability of tightly-linked diagnostic molecular markers and characterized functional alleles for these loci will provide options to develop apple cultivars with durable disease resistance by pyramiding multiple scab resistance loci through breeding and biotechnology. To fine-map this genomic region and identify candidate genes, we will also assemble a high-quality genome of 'Antonovka', an important cultivar for apple breeding. The genome assembly will be valuable to pursue further genomics, genetics, and breeding of other commercial apple traits. This project addresses the 'Pre-breeding and germplasm enhancement' priority of the A1141 program and all USDA Roadmap for Plant Breeding priority needs, particularly (i) characterization of genetic resources; and (iv) development of novel tools to exploit genetic resources. Ultimately, the outcomes of this project will significantly reduce fungicide use, cost of sprays, and social and economic impacts of fruit quality loss from apple scab
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
0%
Goals / Objectives
Linkage group 1 of apple harbors two major apple scab resistance loci in close genomic proximity to a previously well-characterized apple scab resistance gene, HcrVf2 (historically known as the Vf locus). Whether these three loci differ with distinct mechanisms is still an outstanding question of great interest to the apple genetic and breeding community. The main goal of this project is to develop diagnostic markers, identify candidate genes, and characterize resistance mechanisms underlying two major apple scab loci in close genomic vicinity that co-localize with apple scab resistance gene HcrVf2 (Rvi6) on linkage group (LG) 1 of apples, for the development of apple cultivars with durable disease resistance. Specifically, we will perform 1) fine-mapping of the apple scab resistance loci of Rvi17 and Vhc1 on linkage group 1, and identification of candidate genes., 2) functional validation of the candidate genes underlying Rvi17 and Vhc1 scab resistance loci, and develop molecular markers linked to Rvi17 and Vhc1 scab resistance loci for apple breeding, and dissemination of data, and genetic knowledge to the apple research community.
Project Methods
Objective 1:Genetic mapping populations segregating for Rvi17, Vhc1 and Rvi6: Bi-parental populations will be used for fine- mapping of scab resistance loci Rvi17 and Vhc1 on LG1, and to identify rare recombinants between Rvi6 and Vhc1. Mature fruits will be harvested in the fall, and their seeds will be extracted and stratified for approximately three months at 4°C, and grown in the greenhouse to obtain 800-1,000 F1 progeny in each cross. Seedlings of these populations will be maintained at Cornell AgriTech, Geneva, NY. Seedlings in each cross will be evaluated in a pseudo-testcross scheme to fine-map the Rvi17 and Vhc1 loci using currently available ARD and CH-Vf1 flanking markers to identify recombinants in the region of the Rvi17 and Vhc1 loci.Chromosome-scale scaffolding and annotations of 'Antonovka' genome: Using PacBio HiFi, Omni-C, Illumina sequencing data, and RNA sequencing data, we have assembled and published a fully phased, chromosome-level genome of 'Honeycrisp' apple (Khan et al. 2022). We will use a similar approach to assemble a fully phased, chromosome-level high-quality genome of 'Antonovka' apples. We have already done PacBio long-read HiFi sequencing of 'Antonovka' and performed de novo assembly of its genome.Sequencing and genotyping to create high-resolution genetic and physical maps: Assembled genomes of 'Antonovka' (section 1.2) and 'Honeycrisp' (Khan et al. 2022) will be used to identify polymorphic markers for genotyping progeny segregating for the genomic region on LG1 harboring Rvi17 and Vhc1 resistance loci. After screening potential recombinants, we will use robust and low-cost amplicon sequencing (AmpSeq) markers for high-density genotyping and haplotyping of Rvi17 and Vhc1 apple scab resistance loci in the parents, controls, and selected recombinants. Inoculation, and phenotypic evaluation: We will use selective phenotyping of informative recombinants in these large F1 populations as a cost-effective method for fine-mapping to reduce the number of individual genotypes. Seedlings will be inoculated with V. inaequalis race 6 originating from the U.S. collection, in order to achieve segregation of LG1 Rvi genes, i.e. Rvi10 and Rvi17. Scab inoculation of plants at the seedling stage is not only convenient, but it also corresponds to major scab resistance response of mature grafted trees, as shown in previous studies (Erdin et al. 2006; Hemmat et al. 2003; Soriano et al. 2014). Raw scab susceptibility response data will be checked for outliers, mistakes and frequency distribution, standardized before performing ANOVA and to estimate heritability.Haplotype-phenotype association analysis: We will also construct marker haplotypes of resistance alleles of Rvi17 and Vhc1 to track haploblocks linked to two apple scab resistance loci by performing haplotype-phenotype association analysis. Assembled genomes of 'Antonovka' (in this project) and 'Honeycrisp' (Khan et al. 2022) and AmpSeq genotypic data will enable construction of haplotypes flanking Rvi17 and Vhc1 resistance loci. Haplotypes tightly linked to Rvi17 and Vhc1 loci will be used to genotype additional progeny to identify recombinants to narrow down Rvi17 and Vhc1 genomic regions further, if needed.Identification of candidate genes: Fine-mapped Rvi17 and Vhc1 scab resistance loci and de novo sequence of 'Honeycrisp' (Khan et al. 2022) and 'Antonovka' (in this project) will allow us to precisely localize the two loci to discreet genomic regions on LG1 and to identify a small set of underlying candidate resistance genes with high confidence. Differential gene expression analysis (qRT-PCR) will be performed in Bio-Rad CFX96 (California, USA) using gene-specific primers and a delta-delta cycle-threshold method (Silva et al. 2019).Objective 2:The functional validation of candidate genes underlying Rvi17 and Vhc1 loci will be done by 1) Over-expression of candidate genes by Agro-transformation in scab-susceptible 'Gala' and 2) knock-out of candidate genes by CRISPR-CAS9 genome-editing in 'Honeycrisp' and 'Antonovka'. 'Gala' is susceptible to apple scab, and in our experience is easy to propagate in tissue culture, transform, regenerate and root. Previously, 'Gala' was also used to functionally validate other major scab resistance genes, suggesting that its genetic background does not contain residual resistance genes for apple scab (Belfanti et al. 2004; Joshi et al. 2011; Schouten et al. 2014). Gene constructs will be designed and synthesized to transform candidate genes into a scab-susceptible 'Gala' according to the existing transformation protocols for apples (Pompili et al. 2020). To complement the overexpression studies, we will also synthesize and use Agrobacterium-based constructs to knock out candidate genes in 'Honeycrisp' and 'Antonovka' using specific gRNAs for candidate genes. Following is a detailed description of the methodology.Objective 3:Development of markers: SNP markers identified in section 1.6 through haplotype-phenotype association analysis for the Vhc1, Rvi17, Rvi6 resistance loci will be adapted for use in breeding by converting them to a low-cost, high throughput marker platform suitable for use in breeding programs, specifically Kompetitive allele specific PCR (KASP) assays which are routinely used for marker-assisted selection at UMN.Validation and utilization of markers: Seedling populations have already been developed for evaluating the markers for quality, segregation, and routine use "in-house" or with service provider(s). This includes extant field populations and seeds, suitable for field-based or greenhouse observations to confirm the marker-trait association. A subset of putative resistant and susceptible individuals, as indicated by marker alleles, will be observed for scab responses following inoculation or natural infection.Uploading data to public databases: Raw sequence data will be deposited in NCBI sequencing read archive (SRA), ensuring that data will be readily made available to the public, and using stable unique identifiers to allow for wide sharing of these data. SNP and genotype data, in vcf format, will be widely shared through the Genome Database for Rosaceae (GDR). De novo sequence assembly of 'Antonovka' will also be uploaded to NCBI and GDR.Communication and outreach. We will also present the highly reputed and relevant audiences for plant genomics, genetics, and breeding communities at ASHS, PAG, RGC, and APS meetings. Although the apple breeding and genomics community is small in the U.S., our research approach is transferable to other plant pathosystems, and highlights collaboration across disciplines and technologies to have real impact in sustainable variety development. We plan to publish at least two manuscripts in peer-reviewed journals. We will share project updates with the apple Crop Germplasm Committee and curators of the US apple germplasm collection.