Fig. S1 Illustration of the fine-mapping evolution of cmr1 Arabidopsis mutant. F 2 mutant individuals were used for mapping. Molecular markers used in this study (listed in Table S7) showed polymorphism between Col-0 and Ler accessions. Recombination rates, particularly low in the upper arm of the chromosome III, were calculated on 12 selected F 2 individuals. Fine-mapping, on up to F 2 individuals, was subsequently performed. The fine-mapping permitted to delimit the 184-kb region comprised between IN464 (4.64 Mb) and IN482 (4.83 Mb) markers where no recombinants were identified. SSLP, InDels and CAPS markers were PCR-amplified. For SSLP and InDel markers, PCR products were analyzed by 3%-agarose gel electrophoresis. For CAPS marker (SGCSNP81), PCR products were digested with BsaBI (Fermentas) according to the manufacturer’s protocol. HRM analysis of SNP markers was performed using a Light-Cycler 480 (Roche Diagnostics) at the University of Lille (France). The composition of the mixture (per 10 µl reaction) was as follows: 1 µl of 10x Dream Taq colorless buffer (Fermentas), 0.25 µl of dNTPs (10 mM each, Fermentas), 0.35 µlL of 10 µM each primer, 5.5 µl of sterile water, 0.05 µl of Dream Taq polymerase (Fermentas), 0.5 µl of ResoLight dye (Roche Diagnostics) and 2 µl of template DNA. The following conditions were used: denaturation at 96°C for 2 min, 40 cycles of amplification (30 s at 96°C, 30 s at 55°C, 45 s at 72°C), elongation at 72°C for 10 min. Fluorescence measurements were analyzed to detect variation in melting curves. Red circle indicates the location of the centromere. Stars indicate the locations of the molecular markers The position (AGI) of each marker is indicated on the left. The percentage of recombination obtained for each marker is indicated on the right. n = the number of analyzed F 2 individuals; r = the number of recombinants obtained for SNP markers. Supporting Information Figs S1-S8

Fig. S2 Root length of 15-day-old pme3 (At3g14310) seedlings exposed to high-Cu or salt conditions. Seedlings were transferred 5 days after germination on MS/2 medium supplemented with 25 µM CuSO 4 (a) or 50 mM NaCl (b). Average value (n≥30 seedlings from three different growth experimentsin two biological repeats) ±SD, no significant differences between the mutant and WT at P≤0.05. (a)(b)

Fig. S3 Detection of a mutation at the At3g14190 locus. (a) Structure of the gene and positions of primers (red) used for the PCR amplification in (b). Positions of primers are indicated with respect to the translation initiation codon (+1). Dark green boxes indicate exons and light green boxes indicate introns. The putative transcription start is deduced to be at -423 ( (b) PCR amplification of the DNA sequence delimited by the couple of primers indicated in (a), in WT, cmr1 plants and no template control NTC. At3g14190 ATG TGA WTcmr1NTC (b) (a) +882

Fig. S4 Coverage of cmr1 reads onto the Col-0 WT Arabidopsis reference genome. A read corresponds to the first 75bp of 200-bp long fragments selected after fragmentation of the genomic DNA extracted from a pool of ~300 homozygous F 2 individuals. Two regions of low coverage (black boxes) were detected in the 184-kb mapped region in the At3g14190 gene (a) and the At3g14310 gene (b). (a) (b) Reads aligned to the reference genome Coverage At3g14190 At3g14310

Fig. S5 Magnification of two low coverage regions and analysis of cmr1 pair reads. Pair reads are both 75bp extremities of 200-bp long fragments selected after the fragmentation of genomic DNA extracted from a pool of ~300 homozygous F 2 individuals. Reads # 1, 2 and 3 corresponding to At3g14190 (a) were paired with reads # 1, 2 and 3 respectively corresponding to At3g14310 (b). (a) (b) Reads aligned to the reference genome Coverage Reads aligned to the reference genome Coverage At3g14190 At3g14310

Fig. S6 Nature of the cmr1 mutation and genomic sequence of the hybrid At3g14190 gene. (a-e) FNs induced probably two breaks and a subsequent inversion of a 65-kb fragment, leading to the recombination between At3g14190 and At3g14310 (lying on the reverse strand). Red primers indicate unsuccessful amplifications in cmr1 and green primers indicate the successful ones. Positions of primers are relative to the translation initiation codon (+1). Asterisks indicate altered genes in the mutant. DNA fragments amplified by green primers were sequenced to determine the exact positions of the inversion (+160/-276, +196/-277). The sequencing enabled to detect a 35-bp deletion between positions +160 and +196 in the At3g14310 gene. (f) At3g14190 gene recombined with the At3g14310 gene (joining site indicated in grey). ATG initiation codon of the At3g14190 gene (blue) is not in frame with the ATG initiation codon of the At3g14310 gene (green). A stop codon (orange) is present between both ATG sites. (e) (f) (a) (b) (c) (d)

Fig. S7 Impact of Cu 2+ and Na + excess on cmr1-1 and WT Arabidopsis biomass in hydroponics. Plants were grown in a peat-based compost for 3 weeks at 8-h dark/16-h light regime (100 µmol m -2 s -1 ) and 20°C before transferring to hydroponics. Roots were rinsed and immediately placed on the covers of plastic containers containing a nutrient solution as described in Hermans & Verbruggen (2005). The plants were grown at 22 ± 2 °C and in relative humidity of 50 ± 5%. The nutrient solutions were replaced every 4 days. Relative fresh weight was measured the first, 7 th and 14 th day after the addition of 2.5 µM CuSO 4 or 50 mM NaCl. Average value (n=5 plants from three different growth experiments) ± SE, asterisks denote significant differences from WT within each treatment at P≤0.05; WT black closed symbols, cmr1-1 open circles.

Fig. S8 Allelism test between Arabidopsis cmr1-1 and T-DNA mutants at the At3g14190 locus. (a) Pictures were taken 10 days after germination on MS/2 or the same medium supplemented with 50 mM NaCl. (b) Quantification of root lengths in cmr1-1, cmr1-2 (SALK_070337) and their F 1 progeny grown in control and saline conditions for 7 days after transfer. Average value (n≥30 seedlings from three different growth experiments) ±SD, asterisks denote significant differences from WT within each treatment at P≤0.05. (a) (b) * WT cmr1-1 SALK_ F1:cmr1-1xSALK_ Control + NaCl WT cmr1-1 SALK_ F1:cmr1-1xSALK_ Control + NaCl WT cmr1-1 CS F1:cmr1-1xCS Control + NaCl * * * **