Plant Materials. Seeds were surface-sterilized and grown on Murashige and Skoog medium supplemented with 50 mg/liter kanamycin. Resistant seedlings were transplanted to soil and grown to maturity in growth chambers with 70% humidity and daily cycles of 16 h light and 8 h darkness at 21°C.

Isolation of Ds Flanks. For inverted PCR (iPCR), genomic DNA was prepared with Nucleon Phytopure (Amersham Pharmacia Life Science), 400 ng was digested by ApoI in 20 m l at 50°C for 1.5 h, and the enzyme was heat-inactivated at 75°C for 20 min. Digested products were religated overnight using 4 units of T4-ligase (NEB) in a 4-fold volume of the digests. One-tenth of the ligation reaction served as template in PCR using GUSs1 (5'-AACATACGGCGTGACATCGG-3') and GUSas1 (5'-CAACTGGACAAGGCACTAGCG-3'), and subsequently 2 m l of a 100-fold dilution of the primary reaction served as template for nested PCR with GUSs2 (5'-ATATCTGCATCGGCGAACTGATCG-3' and GUSas1. PCR conditions were the same for both reactions: 1× PCR buffer (Amersham Pharmacia Life Science), 1.5 mM MgCl₂, 0.2 mM of each dNTP, 10 pmol of each primer, and 2 units of TaqDNA polymerase, using 40 cycles at an annealing temperature of 57°C and an extension time of 90 sec.

Southern Blot Analysis and Long-Range PCR. FMI32361 (5'- ATCCCGTACCGACCGTTATCG-3') and FMI32360 (5'-CGTGTGAATGTGTGATGC-3') were used for the digoxigenin (DIG)-labeling reaction (Roche Applied Science) of the Ds5' probe. The primer sequences used for the respective mutants and indicated in Fig. 2C are: ana: DP216 (5'-CTTGCTAGTGTTCTAGGTTTGAACG-3') and DP217 (5'-AATCTTATTCATTGCCGAGCAACTAC-3') in the primary reaction, DP25 (5'-GAACGTTTCAATTGAGGTCCG-3') and Ds3-1as (5'-CGAACGGGATAAATACGGTAATCG-3') for the secondary reaction; hma, DP218 (5'-ATTGTTGTGCAAGCAGGTAGCTCG-3') and DP219 (5'-AGACACAGTACCGTGCCTGGAGC-3'); tms, DP188 (5'-TACAGATGAAGACAGTGGCTGCG-3') and CK164 (5'-CCTGGGAAGACAAATTCTAT-3') in the primary reaction, Ds3-1 (35) and CK147 (5'-AGCATGTGCTCACATTTTGTG-3') in the secondary reaction. One microliter of a 100-fold dilution of the primary reactions served as template for secondary reactions. Except for the annealing temperature, the cycling conditions were the same for the respective mutant, in both the primary and nested PCR. After initial denaturation at 95°C for 4 min, 10 cycles with 95°C for 15 sec, 55°C (ana), 56°C (hma), 54°C (tms) for 20 sec, 68°C for 12 min were performed, followed by 20 cycles with 20 sec per cycle increasing extension time. One-quarter of the PCR products were digested with BamHI and EcoRI.

PCR Genotyping. The annealing temperature was 55°C, the extension time was 30 sec, and 30 cycles were performed. We used Ds3-1/DP25, Ds5-1/DP25, and TA3550/TA2482 to specifically amplify Ds in the starter line and in ana, as well as Ac. Ds3-1 and Ds5-1 (35); TA3550 (5'-AGAGTCTAGCACCTCGAGATC-3'); TA2482 (5'-GATGGAATTCAACCTATTTGATGTTGAG-3').