Cloning of resistance gene to rice tungro disease: confirmation mapping and genomic library construction

Confirmation mapping of resistance to rice tungro spherical virus (RTSV) and to the vector green leafhopper (GLH) was performed. Twenty-five DNA markers were evaluated for polymorphism between tungro-susceptible TNI and resistant ARC 11554 and the near-isogenic line T1-11. Thirteen polymorphic markers were found and used to estimate site and size of introgression of ARC 11554 DNA into T1-11. Genotyping this marker indicated that ARC 11554 DNA is present at two places - a ~1000 -kb segment at the middle portion of chromosome 4 short arm and about 80 _kb piece toward the centromere. Markers 4800, 5291, 5600 and 6100 within the 1000bp introgression were converted into CAP DNA markers. Based on the ELISA of 153 F3 families from the T1- 11 x TNI cross, 73 F2 plants were selected representing definite homozygous and heterozygous genotypes for marker genotyping and linkage analysis. Only markers 5600 and 6100, showing normal segregation, were used in linkage analysis. Unexpectedly, Mapmaker analysis showed no linkage between RTSV resistance and the two markers. Based on the GLH antibiosis tests of 114 F3 families, 84 F2 plants were selected for marker genotyping and linkage analysis. Again, no linkage between GLH resistance to the 2 markers was found. With this, a new QTL analysis for RTSV and GLH resistance was initiated with the production of new F2 population (n=264) from ARC 11554 x T1. DNA was isolated from 147 of these plants. ELISA and antibiosis test will commence after adequate F3 seeds are produced. Furthermore, 181 of the 332 PCR- based markers around the rice genome were screened, of which 51 were found to be polymorphic. A genomic library of ARC11554 was constructed in the Fosmid vector with 3.4 x genome coverage giving 99.9% probability of containing any gene of interest. This library will facilitate physical mapping and positional cloning of QTL for RTSV and GLH resistance.