Location: Vegetable Research
Project Number: 6080-22000-031-032-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jan 1, 2024
End Date: Jun 30, 2025
Objective:
Screen a core set of Citrulus amarus Plant Introductions (PIs) for resistance to Reniform nematode (RN) and use this data to conduct Genome Wide Association Study (GWAS) that will identify the genes that underly this resistance.
Approach:
We plan to evaluate a core set of 108 citron melon lines (Citurlus amarus) from the USDA, ARS, genebank (table 1) and 2 watermelon cultivars as reference controls. We have used these same PI lines to conduct a successful Genome Wide Association Study (GWAS) identifying the genomic loci associated with root-knot nematode resistance in C. amarus. Reniform nematode (Rotylenchulus reniformis, RN) cultures are maintained in a greenhouse at cooperator's institution and will be increased on suitable hosts for 8-12 weeks before each greenhouse screen. Each of three temporally independent greenhouse phenotyping screens will be performed over the course of 10-11 weeks at the cooperator's institution according to well established protocols used to screen for RN resistance . Watermelon lines will be seeded in Deepot™ containers filled with a heat sterilized sand:soil medium, and arranged into a randomized complete block design with two biological replications in each of the three temporally independent greenhouse screens. Once roots are established (2-3 weeks), each pot will be inoculated with 10,000 RN eggs. Inoculated plants will be maintained for an additional 8 weeks post inoculation to allow nematodes to infect and reproduce. At the end of the experiment, nematode eggs will be extracted by agitating each root system for 4 minutes in a 10% bleach solution, and then rinsing through stacked 8” sieves (#60, #200, #500) to collect eggs . Each extracted egg sample will be counted by hand using specialized counting dishes under a dissecting microscope. Nematode reproduction will be measured in terms of number of RN eggs per root system. The relative reproductive index will be estimated and is calculated from the number of nematode eggs per gram of root weight for each plant divided by the value for the susceptible control root-stock ‘Carnivor’. The relative reproductive index data will be used as input for the Genome Wide Association Study pipeline as previously described.
