Figure 6.—

Preferential DNA methylation and siRNA accumulation in A. thaliana centromeres over A. arenosa centromeres in allotetraploids. (A) Diagram of probes used for DNA and RNA blot analyses. Full-length repeats of A. thaliana (At, 1) and A. arenosa (Aa, 8) were used for DNA blot analysis. Three pairs of antisense and sense DNA oligo probes (∼20 bp) are shown, and the numbers with an asterisk indicate the sequences derived from A. arenosa. (B) A. thaliana centromeric DNA is heavily methylated in met1-RNAi A. suecica lines. Low hybridization signals detected in lane “Aa” suggest the DNA probe is specific to A. thaliana centromeres. The lines are A. thaliana 2x (At2), A. thaliana 4x (At4), A. arenosa (Aa), A. suecica-GUS (AsG), and met1-1 and met1-12-RNAi A. suecica lines. (C) DNA blot analysis showing hypomethylation of A. arenosa centromeres in met1-RNAi A. suecica lines. Low hybridization signals in lanes “At2” and “At4” suggest the DNA probe is specific to the A. arenosa centromere. (D) High accumulation levels of small RNAs derived from A. thaliana centromeres in met1-RNAi A. suecica lines. The small RNA blot was hybridized with an antisense A. thaliana centromeric probe (3). The RNA materials are At2 (lane 1), At4 (lane 2), Aa (lane 3), AsG (lane 4), allotetraploid F₁ (lane 5), allotetraploid (F₂) (lane 6), allotetraploid F₅ (lane 7), met1-1-RNAi A. suecica (lane 8), and met1-12-RNAi A. suecica (lane 9). RNA size markers are shown in lane “M,” and the sizes of small RNAs are indicated in the right. (E) Low accumulation levels of small RNAs derived from A. arenosa centromeres in met1-RNAi A. suecica. The same blot was stripped off the probe and rehybridized with an antisense A. arenosa centromeric probe (3*). (F) Accumulation of antisense small RNAs derived from the A. thaliana centromere in met1-RNAi A. suecica. The same blot was used except that a sense A. thaliana centromeric probe (6) was used for hybridization. (G) The small RNA blot was hybridized with an antisense U6 probe.