Unformatted text preview: of the met1 mutant. We extracted genomic DNA from the TSIexpressing T-DNA line using Nucleon Phytopure Plant and Fungal DNA
Extraction Kits according to the manufacturer’s instruction (Amersham
Pharmacia Biotech). We cloned a genomic fragment ﬂanking the left border
of the inserted T-DNA by thermal asymmetric interlaced PCR. We cloned
PCR products into the T/A vector pCR2.1 (Invitrogen) and sequenced them.
We compared the ﬂanking genomic DNA with the A. thaliana genome database using The Arabidopsis Information Resource website. We carried out
Southern hybridization analysis and DNA sequencing as previously
described16. A second insertion in MET1 was identiﬁed in and supplied by the
Syngenta Arabidopsis Insertion Library. We determined the T-DNA insertions
in MET1 and heterozygous or homozygous MET1 mutant genotypes either
by Southern blotting or by PCR with primers differentiating between wildtype and mutated loci. We obtained RT–PCR products for MET1 transcripts
68 with poly-T primer for cDNA synthesis, MEF-1 and MER-1 primers (Fig. 1a)
for PCR. We used the gene encoding actin 2 (ACT2) as an RT–PCR control.
Primer sequences used for genotyping MET1 mutants and for RT–PCR are
available on request.
Segregation tests using the dominant marker gene residing on the TDNA showed that the frequency of plants with the mutated allele was signiﬁcantly lower than the expected 75% (see Supplementary Table 2
online). Frequent seed abortion observed in siliques of heterozygous plants
corroborates gametophytic or early embryonic lethality of strains carrying
the met1 mutated allele. The transmission frequency of the met1 mutated
allele varied signiﬁcantly between individual lines (see Supplementary
Table 2 online), suggesting secondary effects of DNA demethylation in
met1 strains. The loss of seeds was not restricted to homozygous mutant
plants, as T-DNA transmission in most cases was signiﬁcantly lower than
the 66% expected for survival of all heterozygotes. Indeed, genotyping in
T2 segregating populations identiﬁed 46 wild-type, 63 heterozygous and 3
homozygous mutant plants. Considering the expectation based on the
number of wild-types, the number of heterozygotes was 32% lower and of
homozygotes was 94% lower. To determine whether the distortion in heterozygotes occurred during maternal or paternal transmission, we did reciprocal outcrosses of plants heterozygous with respect to the mutations.
Maternal transmission of the mutated allele, which resulted in viable seeds,
was roughly equal to the wild-type but paternal transmission was 20%
lower (see Supplementary Table 2 online). This may reﬂect a role for MET1
in transcriptional inactivation of the paternal genome during gametogenesis or early embryogenesis.
DNA methylation analysis. To analyze the methylation status at 180-bp
pericentromeric repeats, we carried out genomic bisulﬁte sequencing. We
chose a sequence from BAC clone F9M8 on chromosome I (nucleotides
61,536–62,127) because a unique sequence ﬂanks...
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This document was uploaded on 03/17/2014.
- Fall '09