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1 Supplemental Data Supplemental Figure 1. Silique arrangement in the wild-type, jhs, and complemented lines. Wild-type (WT) (A), the jhs1 mutant (B,C), and the jhs1 mutant complemented with JHS1 (Com) (D) are shown. Bars=5 mm.
2 Supplemental Figure 2. Expression of ProWOX5:GFP in the roots of transgenic wild-type (WT) and jhs1 seedlings. The roots of five-day-old seedlings were examined using confocal microscopy. Green, GFP fluorescence; red, FM4-64 fluorescence. Bars=100 µm.
3 Supplemental Figure 3. Analysis of lateral root generation in wild-type and jhs1 plants. (A) Expression pattern of DR5:GUS in Col wild-type (WT) and jhs1 plants. Blue spots indicate lateral root primordia. Bars=5 mm. (B) Number of lateral roots in Col and jhs1 plants. Each point represents the average of ~30 plants. Error bars represent SD. Student s t-test; for **, P < 0.01.
4 Supplemental Figure 4. Map-based cloning and sequence analysis of JHS1. Genetic mapping of JHS1. Positions of the molecule markers used in the fine mapping of JHS1 (from left to right: LUG887, CH1-1.5, CH1-2.2, CH1-2.42, CH1-2.51, CH1-2.58, CH1-2.62, CH1-2.83, CH1-2.89, CH1-2.92, CH1-2.96, CH1-3.0, CH1-3.2, CH1-3.8, and CH1-4.3). The numbers below the markers indicate the number of recombinants at that locus. The green line represents the whole chromosome and the red dot represents the centromere.
5 Supplemental Figure 5. Genotyping and gene expression analysis of the complemented jhs1 plant. (A) Genotyping of the complemented mutant plant. The upper panel is PCR amplification of an endogenous fragment of JHS1. The lower panel is PCR amplification of an exogenous fragment from the transgene which complement the mutant. JHS1-20/JHS1-2 and BL17/JHS1-2 are PCR primers for amplification of the endogenous and exogenous fragments of JHS1 respectively. (B) Sequencing analysis of the genes analyzed in (A). Primers used for PCR and the genetic background of the template are listed on the left. Red arrows indicate the site of mutation. (C) RT-PCR analysis of the transcript level of JHS1 in WT, jhs1 and Com plants. JHS1a is a fragment (307bp-884bp) upstream of the mutation site. JHS1b is a fragment (2049bp-2325bp) downstream of the mutation site. HTA9 is a reference gene. The mrna of jhs1 seems to be degraded, probably due to nonsense-mediated mrna decay.
6 Supplemental Figure 6. Subcellular localization of JHS1 in Nicotiana benthamiana leaves. JHS1:YFP was localized to the nucleus and YFP was localized to the nucleus and cytoplasm in the epidermal cells of Nicotiana benthamiana leaves expressing JHS1:YFP and YFP, respectively. DAPI is a DNA dye used to indicate the nucleus. Images of YFP and DAPI were merged to show colocalization. Bars=50 µm.
7 Supplemental Figure 7. Subcellular localization of JHS1 in Arabidopsis leaves. The experiment was performed as in Supplemental Figure 5. Bars=50 µm.
8 Supplemental Figure 8. Homologous recombination (HR) in jhs1 mutant plants. GUS staining of WT and jhs1 plants containing the GUS reporter gene from the HR reporter line 1415 and 1406, respectively. A HR event in a cell can produce a complete and functional GUS gene, as shown in Figure 9A, and the cell will appear blue upon GUS staining. Each blue spot represents a HR event.
9 Supplemental Figure 9. qrt-pcr analysis of cell cycle-related gene expression. Real-time quantitative RT-PCR analysis of the transcript levels of cell cycle-related genes in the WT and jhs1 mutant. The level of these genes was set to 1 in the WT. Data represent the averages of four replicates. PDV2 was used as a reference gene and error bars represent SD. Student s t-test; for **, P < 0.01.
10 Supplemental Figure 10. Analysis of ploidy level distributions of wild-type (WT) and jhs1 plants. Ploidy level distributions of 10-day-old seedlings were measured by flow cytometry.
11 Supplemental Figure 11. Expression analysis of SAM-specific genes in the floral tissues of 45-day-old wild-type (WT) and jhs1 plants. (A) ProWUS:GUS; (B) ProCLV3:GUS; and (C) ProSTM:GUS. Bars = 500 µm.
12 Supplemental Figure 12. qrt-pcr analysis of TSI expression. Ten-day-old wild-type, jhs1, and complemented plants were used for the analysis. Data represent the averages of four replicates. Error bars indicate ±SD.
13 Supplemental Table 1 Molecular markers and primers used for the fine mapping of JHS1. Molecular markers LUG887 CH1-1.5 CH1-2.2 CH CH CH CH CH CH CH CH CH1-3.0 Primer sequences CH1-0.8F: 5'- CTG CCT CTG TTC TTC ATC TTA GG -3' CH1-0.8R: 5'- CCA TAA CCA GGA AGA AAC GGA AG -3' CH1-1.52F: 5'- CCT CCT CTT CCT TCT TCG TAC TC -3' CH1-1.52R: 5'- GAG ACT TAC GAG GAT GAA ACA AAC -3' CH1-2.22F: 5'- CCC AAA ACT AGA TAC CAA GTC AAA G -3' CH1-2.22R: 5'- GGT GAA TAT CGT GTC ATC CGA C -3' CH1-2.42F: 5'- GTG CTA GAT AGA TCT CTC TTT CTG C -3' CH1-2.42R: 5'- CGT GAA GGA GAA TGT ATC TTT ACT CTG -3' CH1-2.51F: 5'- GCG CGA AAG TGT GTA ATT GTC AG -3' CH1-2.51R: 5'- GAT TGC ATG GCC TGA AGA ACC -3' CH F: 5'- GAC ACG GAA AGA GAC AGC TAT C -3' CH R: 5'- GAT ATG GCT TGC TTC GGT TC -3' CH1-2.62F: 5'- GAG CCA AAT TTC TTT CAC CAA CTT G -3' CH1-2.62R: 5'- CTG CCT CAA GTT ATC AGC CTT C -3' CH1-2.83F: 5'- GCA TAG TCC TTT GCA GTG AGT ATC -3' CH1-2.83R: 5'- CCA TCA AAT CTC ATG TTG AAC GTG C -3' CH1-2.89F: 5'- CAA ACT CGC TTA TTC AAC AAG AGA C -3' CH1-2.89R: 5'- CAC AAA AGT GTT AAC GAG TGA GAG G -3' CH1-2.92F: 5'- GTG GAT CCC CAT TGT ACA GTT ACC -3' CH1-2.92R: 5'- CCG GAT GCA TTA CAA GTC AAC AGG -3' CH1-2.96F: 5'- GCT AGT TAT ATA CCA ATA GAA ATG TGC -3' CH1-2.96R: 5'- GTA GTT GTC CAA CCA AGA GTT TC -3' CH1-3.03F: 5'- CCA CTC GTT ACA TTA GAT TGC GTG -3' CH1-3.03R: 5'- GCA TTT CAA CTT TAC CAA CCA CC -3' Marker type SNP CH1-3.2 CH1-3.20F: 5'- CTC TGG TTT CTG TGC AAG C -3'
14 CH1-3.8 CH1-4.3 CH1-3.20R: 5'- CTT GTA CCT CTG ATG GCG TG -3' CH1-3.8F: 5'- GAC AAG CTA TTG GTT CTG ACT TCT GC -3' CH1-3.8R: 5'- CCT GAG TCG TTT GCC TCA TC -3' CH1-4.3F:5'- CCT TCT CCT TCT CGA TTT GGC -3' CH1-4.3R:5'- CTA GAT GAG GCC AAC CAG ATG -3' Supplemental Table 2 Primers used for the genotyping and RT-PCR analysis in Supplemental Figure 5. PCR fragments An endogenous fragment of JHS1 An exogenous fragment of JHS1 JHS1a Primer sequences JHS1-20: 5'- GTC AAT AAG GAT AGT AGC TTT GTC AC -3' JHS1-2: 5'- GTA ACA TCG GCG GCA GTT G -3' BL17: 5'- GCT TCC GGC TCG TAT GTT GTG -3' JHS1-2: 5'- GTA ACA TCG GCG GCA GTT G -3' JHS1-7: 5'- CCT TCA TCT ACA GCG CAA TCA AC -3' JHS1-22: 5'- GCT GAT AAA GCA AAG TCA AGA TGA C -3' JHS1b JHS1-25: 5'- CTT GGA AGG TAG AGA GAT GCA GG -3' JHS1-28: 5'- CAC ACG ATC TCC AGT TCT AAG TTT G -3' HTA9 HTA9-F: 5'- GGT CTC CAG TTC CCA GTT GG -3' HTA9-R: 5'- CTC CTC ATC TCC ACG AAT CGC -3'
15 Supplemental Table 3 Primers used for the qrt-pcr analysis. Gene Name WUS STM CLV3 BRCA1 RAD51 GR1 MRE11 KU70 MSH2 MSH6 CYCB1;1 CDKB1;1 CYCD4;1 CDKA;1 Primer sequences WUS-8: 5'- GCC ATA TCC CAG CTT CAA TAA CG -3' WUS-10: 5'- CTA GAC CAA ACA GAG GCT TTG C -3' STM-10: 5 - AAG CTG AGG ATA GAG AGC -3 STM-11: 5 - GGA TCG ATC AAA GCA TGG -3 CLV3-5:5'- CTT TCC AAC CGC AAG ATG ATG ATG -3' CLV3-6:5'- GTT GTT TCT TGG CTG TCT TGG TG -3' BRCA1-1: 5'- GTA ACC ATG TAT TTT GCA ATG CGT G -3' BRCA1-2: 5'- GTG ACG GAT TAT TCT GGC TAA CG -3' RAD51-1: 5'- CGA GGA AGG ATC TCT TGC AG -3' RAD51-2: 5'- CTA GAA CTT TAT CGA GCT CCC GTG -3' GR1-1:5'- CAG CAT GAG AAA TCA GCA ATC TCG -3' GR1-2:5'- GGT GAG ATG GAA GTG ATA GGT GTC -3' MRE11-1:5'- GTG ATA CAC TTC GAG TAC TTG TTG C -3' MRE11-2:5'- CTG ACT ACT TGA AAC TGC ACT GG -3' KU70-1: 5'- CGA GCT TCG TGA AAC CAG AGA TG -3' KU70-2: 5'- GTC ATA TTT TCC ATC ATC TGC GTC AC -3' MSH2-1: 5'- GTT GGT GTG GCA AAC TTC CAT G -3' MSH2-2: 5'- CAT TGT TGA TTA TCA TGG AGG AGG G -3' MSH6-1:5'- CCT GAA GAG CAT TTC ACA GAG ATT G -3' MSH6-2:5'- GAA CAC CAT CAG ATG TAG AAC CAG -3' CYCB1;1-1: 5 -CTCAAAATCCCACGCTTCTTGTGG-3 CYCB1;1-2: 5 -CACGTCTACTACCTTTGGTTTCCC-3 CDKB1;1-1: 5 - GGTGGTGACATGTGGTCTGTTGG-3 CDKB1;1-2: 5 - CGCAGTGTGGAAACACCCGG-3 CYCD4;1-1: 5 - GATGAGGGCATGATTGTTGACG-3 CYCD4;1-2: 5 - CCAAACTGGTGTACTTCACAAGC-3 CDKA;1-1: 5'- CCT GTC AGG ACA TTT ACT CAT GAG -3'
16 CDKA;1-2: 5'- GCT TTT GGC TGA TCA TCT CAG C -3' H3.1 CYCD3;3 CYCA2;1 CYCA3;1 CYCB1;2 MAD2 Histone H4 HTA9 PDV2 H3.1-1: 5'- ATG GCT CGT ACC AAG CAG ACG -3' H3.1-2: 5'- GGT TTT GAA GTC CTG AGC GAT C -3' CYCD3;3-1: 5'- CGA GAT TTC TGA GTT TTA GTC CTT CTG -3' CYCD3;3-2: 5'- CTC TTC TCC TCT TGA GCA AAG GC -3' CYCA2;1-1: 5'- CAT TTT CGG TTA TCA GTT CCC ACC -3' CYCA2;1-2: 5'- GTG TAT AGC GAT AAG AGT GCT TCC -3' CYCA3;1-1: 5'- GAT GGA AGC TGA TAT ACT TCT TGC G -3' CYCA3;1-2: 5'- GAT ACA GGC ATT GTC GCT ACA CAC -3' CYCB1;2-1: 5'- GAC AGA TTC TGG TGA TGG AGA AG -3' CYCB1;2-2: 5'- CCA TTC TCT GCC TTC GAG TAC TTC -3' MAD2-1: 5'- GAA GTT GCA GAG AGT AGT GTT AGT G -3' MAD2-2: 5'- CTC TTC TTC ATC CCA CTC GTC GTT C -3' Histone H4-1: 5'- CCT TTA GAA AAT GTC AGG TCG TGG -3' Histone H4-2: 5'- GTT TAA CAC CAC CTC TAC GAG C -3' HTA9-F: 5'- GGT CTC CAG TTC CCA GTT GG -3' HTA9-R: 5'- CTC CTC ATC TCC ACG AAT CGC -3' PDV2-12: 5'- ATG GAA GAC GAA GAA GGC ATC -3' PDV2-32: 5'- GCT TTT TCT TCC TTC TCC GG -3'
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