A Transgenic Mutant of Lactuca sativa (Lettuce) with a T-DNA Tightly Linked to Loss of Downy Mildew Resistance

摘要

One hundred and ninety-two independent primary transformants of lettuce cv. Diana were obtained by co-cultivation with Agrobacterium tumefaciens carrying constructs containing maize Ac transposase and Ds . R2 families were screened for mutations at four genes ( Dm ) for resistance to downy mildew. One family, designated dm3t524, had lost resistance to an isolate of Bremia lactucae expressing the avirulence gene Avr3 . Loss of resistance segregated as a single recessive allele of Dm3 . The mutation was not due to a large deletion as all molecular markers flanking 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 this T-DNA was isolated by inverse polymerase chain reaction. This genomic 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 altered in dm3t524, indicating tight physical linkage to Dm3 . Three open reading frames (ORFs) occurred in a 6.6-kb region flanking the insertion site; however, expression of these ORFs was not detected. No similarities were detected between these ORFs and resistance genes cloned from other species. Transgenic complementation with 11-to 27-kb genomic fragments of Diana spanning the insertion site failed to restore Dm3 function to two ethyl methanesulfonate (EMS)-induced mutants of Dm3 or to cv. Cobham Green, which naturally lacks Dm3 activity. Therefore, either 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 activity is encoded by a gene extending beyond the fragments used for complementation.