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Supporting Information Figs S1-S8
Fig. S1 Illustration of the fine-mapping evolution of cmr1 Arabidopsis mutant. F2 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
F2 individuals. Fine-mapping, on up to 1.500 F2 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 F2 individuals; r = the
number of recombinants obtained for SNP markers.
25 μM CuSO4
(b)
60.0
60
50.0
50
Root length (mm)
Root length (mm)
(a)
40.0
30.0
20.0
10.0
50 mM NaCl
40
30
20
10
0.0
0
WT
pme3
WT
pme3
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 CuSO4 (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)
At3g14190
+1
-487
WT
+314
cmr1
+882
NTC
(b)
1000
800
600
400
200
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
(www.arabidopsis.org). (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.
(a)
Coverage
Reads
aligned to the
reference
genome
At3g14190
(b)
Coverage
Reads
aligned to the
reference
genome
At3g14310
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)
Coverage
231
Reads aligned
to the
reference
genome
At3g14190
(b)
Coverage
3
1
2
Reads aligned
to the
reference
genome
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)
(c)
(d)
(e)
(f)
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.
Fig. S7 Impact of Cu2+ 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, 7th and 14th day after the
addition of 2.5 µM CuSO4 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.
WT
cmr1-1
SALK_035661
F1:cmr1-1xSALK_035661
WT
cmr1-1
SALK_070337
F1:cmr1-1xSALK_070337
WT
cmr1-1
CS811152
F1:cmr1-1xCS811152
+ NaCl
Control
+ NaCl
Control
+ NaCl
Control
(a)
80
Root length [mm]
(b)
*
60
*
*
40
*
*
*
20
0
Control
WT
cmr1-1
50 mM NaCl
cmr1-2
F1 cmr1-1 x cmr1-2
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 F1 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.