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ARS Home Northeast Area Geneva, New York Plant Genetic Resources Unit (PGRU) Research





- Fazio, Gennaro
- Plant Genetic Resources, Genomics and Genetic Improvement (301)
- Plant Diseases (303)

Research Project: **Location:
                            Plant Genetic Resources Unit (PGRU)** Project Number: 8060-21000-033-002-R
                        
                        Project Type: Reimbursable Cooperative Agreement Start Date: Oct 1, 2021
                        
                        End Date: Aug 31, 2024 **Objective:**
                        
                        Woolly apple aphids (WAA) are a major pest in apple growing regions of the world impacting international trade and survivability of the apple industry.  One of the best ways to control WAA infestation is by deploying resistant rootstocks which negate feeding shelter below ground and keep the WAA population sensitive to climate and areal treatments with parasites or insecticides. This project addresses the threat represented by the discovery of a new ecotype of WAAs in the Netherlands that can overcome the genetic resistance deployed in some Geneva rootstocks.  Organic management systems that rely on the deployment of rootstock resistance are particularly affected by this new threat.
1. Identify gene presence and expression that allow a new WAA ecotype to overcome rootstock resistance in Europe
a. ARS predicts the novel genotype overcame resistance because of overexpression or duplication of an existing effector gene. 
2. Assess presence of the emerging ecotype (Obj. 1) and overall WAA genetic diversity across North American growing regions. 
a. ARS predicts genetic variation to be highest in the eastern U.S. where wild hosts occur alongside apple orchards and that clonal populations will dominate the apple growing regions of the western U.S. ARS hopes the new ecotype has not been introduced but will find out.
3. Identify plant genes that interact with WAA genes of interest from the emerging ecotype (Obj. 1 and 2) to advance rootstock development. 
a. ARS predicts a single gene or gene cluster of effectors interact (physically bind) with known disease resistance genes to alter the apple immune response and enable colonization. **Approach:**
                        
                        The ARS lab will provide support for all objectives but will be mostly focused on collection of samples from Objective 2 and working with breeding lines from objective 3.
Approach for Objective 1: To reveal what new genes and enhanced gene expression occur in the emerging biotype (EB), we will use a combination of DNA and RNA sequencing of plants and insects across a resistance spectrum. For gene discovery and sequence analysis, EB individuals and a local genotype with reciprocal resistance will be collected in the Netherlands allowed to feed on resistant and sensitive (R5+ and R5-) plants for 10d to confirm resistance phenotypes. Two pools of each genotype will be collected (one for archival) and DNA will be extracted using a standard extraction kit. DNA will be shipped to UCR as part of the international survey of WAA genetics (Obj. 2).  
For functional gene expression profiling, EB individuals will be colonized on a R5+ rootstock (e.g, G41, G202) and a R5- rootstock (G16) alongside a local genotype that performs opposite on these genotypes. Age synchronized aphids (n=25 per plant) will be allowed to feed over time with both aphid and plant tissues collected for transcriptome profiling at 2 and 7d. 
Approach for Objective 2: To identify what gene absence or presence in the EB may underlie its resistance phenotype, DNA (Obj 1) will be compared to WAA populations across the major growing regions from both cultivated and wild hosts. This will also reveal if the emerging ecotype occurs in the US or if the genes that overcame rootstock resistance exist in similar ways among populations, To leverage a whole genome resequencing approach, insect samples will be collected from commercial and research orchards and nurseries throughout Europe (n=15), Canada (n=15), and across the major growing regions in the U.S. (n=300). Insects will also be sampled from wild hosts near orchards when present. Stakeholders and the region’s extension agents/consultants will be notified that WAA samples are needed. Regions will be chosen on the basis of density of apple production and degree of geographic isolation, with a goal have the samples be representative of the widest possible area. Samples will be both targeted and opportunistic. Field collections will occur over two years. Samples will be collected, maintained in refrigeration (on ice), and then preserved in 95% alcohol back at the lab. Samples will be stored at -20C prior to shipping to UCR and then DNA extracted and prepped for Illumina NovaSeq at the UCR core facilities. Sequencing will target >30x coverage for all samples and nucleotide polymorphisms (SNPs) will be identified using standard bioinformatics analyses.
Objective 3: We will inoculate apple rootstock breeding lines with WAAs to identify resistant material with diverse genetic origin different from Robusta 5.  To identify plant genes that interact with WAA genes of interest from the emerging ecotype we will compare gene expression profiles of resistant vs. susceptible breeding lines, identify the location of differentially expressed genes and identify those that co-locate with known QTLs for resistance.


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