A F28A21A(3) F28A21B(1) F15J5A(20) F28J12A(6) F28A21C(1) T16H5(25) G4539 AG(4) CWI7 F28A21 Centromere T5K18 Telomere F28J12 F15J5 F13C5 F24J7 T18B16 T9A21 T16H5 AT4G18710 AT4G18720 AT4G18730 AT4G18740 AT4G18750 AT4G18760 AT4G18770 AT4G18790 AT4G18800 AT4G18810 AT4G18820 AT4G18780 CESA8 N D683 W114 * ern1/irx1-6/lew2-1 irx1-1 B CESA4 (AT5G44030) T-DNA T-DNA nws2-1 irx5-4 /irx5-5 Supplemental Figure 1. Cloning of ERN1/IRX1 and NWS2/IRX5 genes. (A)Map-based cloning of ERN1/IRX1 gene. CAPS and SSLP marker analysis located ern1/irx1-6 in a 1.9 Mpb interval bordered by markers G4539 and CWI7 on chromosome 4. To fine-map ERN1 430 F2 plants from a cross between ern1-1 (Col-0 background) and Ler, were screened for individuals with recombination break points between these markers. Figures in brackets represnt the number of plants with recombination between ERN1 and the indicated markers. Use of additional CAPS and SSLP-like markers allowed delimitation to an interval of approximately 41 kb (bordered by SNP markers F28A21B and F28A21C) comprising 12 predicted ORFs (AT4G18710 to AT4G18820). Sequencing of genomics ORFs in ern1-1irx1-6 mutants revealed a single stop codon (*) mutation (W114 to *) in the fourth exon of the coding region of AT4G18780 gene, which encodes the CESA8/IRX1 protein (Taylor et al., 2003; Brown et al., 2005). The ern1-1 mutant was identical to the lew2 (leaf wilting 2) mutant (Chen et al., 2005), and allelic to the previously described irx1-1-irx1-5 (irregular xylem) mutants (Taylor et al., 2003; Brown et al., 2005), and consequently we designated the ern1 allele as irx1-6. The previously described irx1-1 (Taylor et al., 2003; Brown et al., 2005) is a weak allele, in which the mutation located in the tenth exon causes a single amino acid substitution (D683N) in the CESA8 catalytic domain. (B)Schematic representation of the CESA4 / IRX5 gene. The position of the T-DNA insertion in the nws2 mutant is indicated. nws2 mutant was renamed irx5-5 as it was allelic to irx5-1-irx5-4 mutants, which are loss of function mutants in the CESA4 (Taylor et al., 2003; Brown et al., 2005). The position of the T-DNA insertion in the irx5-4 allele used in this study is indicated.