A TRANSGENIC MUTANT OF LACTUCA-SATIVA (LETTUCE) WITH A T-DNA TIGHTLY LINKED TO LOSS OF DOWNY MILDEW RESISTANCE

One hundred and ninety-two independent primary transformants of lettuc e cv. Diana were obtained by co-cultivation with Agrobacterium tumefac iens carrying constructs containing maize Ac transposase and Ds. R-2 f amilies were screened for mutations at four genes (Dm) for resistance to downy mildew. One family, designated dm3t524, had lost resistance t o an isolate of Bremia lactucae expressing the avirulence gene Avr3. L oss of resistance segregated as a single recessive allele of Dm3. The mutation was not due to a large deletion as all molecular markers flan king Dm3 were present. Loss of Dm3 activity co-segregated with a T-DNA from which Ds had excised. Genomic DNA flanking the right border of t his T-DNA was isolated by inverse polymerase chain reaction. This geno mic sequence was present in four to five copies in wild-type cv. Diana . One copy was missing in all eight deletion mutants of Dm3 and altere d in dm3t524, indicating tight physical linkage to Dm3. Three open rea ding frames (ORFs) occurred in a 6.6-kb region flanking the insertion site; however, expression of these ORFs was not detected. No similarit ies were detected between these ORFs and resistance genes cloned from other species. Transgenic complementation with 11- to 27-kb genomic fr agments of Diana spanning the insertion site failed to restore Dm3 fun ction to two ethyl methanesulfonate (EMS)-induced mutants of Dm3 or to cv. Cobham Green, which naturally lacks Dm3 activity. Therefore, eith er the T-DNA inserted extremely close to, but not within, Dm3 and the mutation may have been caused by secondary movement of Ds, or Dm3 acti vity is encoded by a gene extending beyond the fragments used for comp lementation.