CHARACTERIZATION OF GAMMA-IRRADIATION-INDUCED DELETION MUTATIONS AT ASELECTABLE LOCUS IN ARABIDOPSIS

Seeds of transgenic Arabidopsis, containing a negatively selectable su icide marker, a 35S :: tms2 construct introduced as a transgene, were gamma-irradiated at a range of doses from 20-120 krad. Batches of M2 s eeds, from M1 plants irradiated at doses of 40, 45 and 60 krad, were s creened by germinating them on medium containing NAM under conditions that selectively inhibited growth of plants expressing the tms2 gene p roduct. Nine candidate loss-of-transgene mutants were isolated. The fr equency of such mutations (0.0125 to 0.025%) did not vary significantl y with irradiation dose or M1 pool size. DNA from the mutants and the parent was hybridized in Southern blots, using probes complementary to various regions of the transgene, All nine mutants were null for both the tms2 coding sequence and the 35S promoter. Six of the nine mutant s were null for the entire transgene construct of 9 kbp. DNA from one mutant contained one of the T-DNA borders and gave a hybridization pat tern consistent with a deletion at least 5 kbp. The two remaining muta nt lines gave identical patterns of hybridization, consistent with a 5 .6-kbp internal deletion within the transgene, From the Southern blots , and on the basis of lineage, the nine lines represent the progeny of either seven or eight independent mutations. We have established cond itions capable of producing deletion mutations of at least 5 kbp, but without apparently introducing small deletions or rearrangements. Such deletion mutations are ideally suited for cloning by subtractive hybr idization, and should also be readily detectable by RFLP analysis, fac ilitating map-based cloning procedures. (C) 1998 Elsevier Science B.V. All rights reserved.