Comnaturegenetics letter fig 2 segregating phenotypes

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Unformatted text preview: ishing Group http://www.nature.com/naturegenetics letter Fig. 2 Segregating phenotypes in T2 progeny. Wild-type segregants (MET1+/+; +/+) were identical to wild-type Columbia plants, heterozygous (MET1+/–; +/–) plants showed high variation in flowering time and homozygous met1 mutants (MET1–/–; –/–) were consistently delayed in flowering. Scale bar = 2 cm. obtained from the Syngenta Arabidopsis Insertion Library. The TDNA inserts in the met1-3 and met1-4 loci are of 7.1 kb and 4.7 kb and disrupt the conserved motif region of DNA methyltransferases8–10 and the first exon, respectively (Fig. 1a). RT–PCR analysis did not detect MET1 transcripts spanning the catalytic domain in strains homozygous with respect to either mutation (Fig. 1b), suggesting that each mutated allele is null. The insertions created new restriction sites in the met1 loci, allowing for genotyping by Southern-blot analysis (Fig. 1c). A. thaliana mutants deficient in DNA methylation usually have reduced levels of cytosine methylation at centromeric repeats, transposonrelated sequences and silent transgenic loci11–15. Bisulfite analysis of cytosine methylation at a 180-bp centromeric repeat showed that CpG methylation at the locus was almost completely erased in homozygous met1-3 plants. Notably, we also observed hypomethylation in heterozygous met1 plants in the T2 population (Fig. 1d). Once the A. thaliana genome has been exposed to mutations that reduce DNA methylation, remethylation is extremely slow despite the presence of wild-type alleles2,11,12,14. But our T2 plants that were heterozygous with respect to the met1 mutated alleles were derived from mutagenized plants that were never homozygous with respect to the mutation, excluding the possibility that hypomethylation in heterozygotes was carried over from homozygous ancestors. To examine whether demethylation reflects release of transcriptional suppression in heterozygotes, we hybridized RNA from these plants with a probe for transcriptionally silent information (TSI) that was derived from pericentromeric repeat sequences. These sequences are transcriptionally silent in wildtype A. thaliana strains but reactivated in TGS mutants16, including strains compromised in MET1 gene expression owing to transgenic production of MET1 antisense transcripts (AsMET1; refs. 12,16). TSI was reactivated in both homozygous and heterozygous met1-3 and met1-4 plants (Fig. 1e). Hypomethylation is often accompanied by characteristic developmental abnormalities17. In particular, it correlates well with late-flowering phenotypes caused by ectopic expression of the gene FWA, a regulator of flowering time18. Indeed, lateflowering plants were frequently observed among plants heterozygous with respect to both met1 alleles (Fig. 2). Therefore, the appearance of this particular set of phenotypes (hypomethylation of repetitive DNA, TSI reactivation and 66 delayed flowering) in T2 heterozygotes, without passage through a homozygous geno...
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