1a. Objectives (from AD-416):
Develop Potato Virus Y (PVY) resistance in existing cultivars by: 1)testing the S. hirsutum pot-1 gene as a source of PVY resistance in potato; 2) doing a molecular characterization of eIF4E-transgenic lines; and 3) produce minitubers for proof of concept field studies.
1b. Approach (from AD-416):
Inoculation studies will be done using Potato Virus Y O strain (PVYO) Potato virus Y NTN strain (PVYNTN), and Potato virus Y N:O strain (PVYN:O) strains in a greenhouse to assay the resistance in four cultivars transformed with the pot-1 gene. Molecular characterization of the transgenic lines will be assayed for sequence insertion and copy number using a Southern Blot analysis. Minitubers of the transgenic lines will be produced in sufficient numbers to allow replicated field trials for PVY resistance in 2012.
3. Progress Report:
One of the objectives of this research is to test the tomato pot-1 (eIF4E) gene as a source of Potato Virus Y (PVY) resistance in potato. Molecular techniques allowed for the amplification of a tomato gene from Solanum hirsutum. The sequence of the cloned gene was identical to the Genbank sequence identified as pot-1 (AY723736). This sequence was subsequently cloned allowing its use in the transformation of PVY susceptible plants. Transformation experiments to introduce the S. hirsutum eIF4E gene or the potato gene into the PVY susceptible lines were completed resulting in independent clones for four different varieties and breeding lines with confirmation of the presence of the transgene. 10 lines with the S. hirsutum pot-1 gene were derived from potato cultivar Russet Norkotah, 22 from breeding clone MSE149-5Y, 30 lines from potato cultivar Classic Russet and over 45 lines from potato cultivar Silverton Russet. Select transformed lines representing all four cultivars and breeding clone were tested in the greenhouse for resistance using three PVY strains (O, N, NTN). The parent lines were highly infected by all three strains. Of the transgenic lines, 7 lines demonstrated little to no PVY accumulated in the plants across the three PVY strains. No Silverton Russet lines showed complete resistance, but the virus titre levels were reduced. In a 2012 field study at Aberdeen, Idaho, three lines were tested along with the parent line (MSE149-5Y). Based upon ELISA testing, the eIF4E lines did not get infected with PVY during the field season, while MSE149-5Y averaged 60% infection with PVY, suggesting eIF4E does confer resistance to PVY.