· Web viewIndonesia-6.40 106.82 Or-I W1983 O. rufipogon India 18.50 73.15 Or-I W1993 O. rufipogon India 24.60 72.80 Or-III W2010 O. rufipogon India 19.00 73.06 Or-III W2057 O. rufipogon

Supplemental Information

Supplemental Tables

Supplemental Table 1. Summary of sequencing of DNAs of Zhu1S, SV14 and bulked F2 DNA

Sample Paired reads Coverage (%) Read depth (X)

SV14 56,937,735 86.39 36.87Zhu1S 100,062,179 89.29 53.44F2 pool 76,573,815 90.41 40.77

Coverage and Read depth were calculated based on the alignment of paired reads to the reference genome IRGSP4.0.

1

Supplemental Table 2. Information of SNPs on candidate region

SNP coordinate on Chr5

Reference base

Altered base

Information and Gene annotation

Support reads

SNPindex

25518041 C T upstream of gibberellin 2-oxidase4 ( LOC_Os05g43880)

35 0.88 25520831 A C 12 1.00 25521977 T C 21 0.88 25522540 G A 31 0.91 25525941 G T 37 0.86 25526611 T C 26 0.81 25528291 A G upstream of

hypothetical protein ( LOC_Os05g43890)

16 0.80 25528293 T C 16 0.80 25528834 C A 20 0.83

25529775 A G 27 0.87

25530146 G T upstream ofputative expressed protein( LOC_Os05g43895)

18 0.95 25530499 A G 25 0.81

25530644 T C 32 0.80

2

Supplemental Table 3. Selected reaction monitoring conditions for GA molecules [M-H]-

AnalyteIonizationMode

PrecursorIon Mass(m/z)

FragmentIon Mass(m/z)

CollisionEnergy(V)

Tube Lens(V)

GA8- 363.1 275.2 20 100- 363.1 319.2 20 100

GA29- 347.2 259.4 20 100- 347.2 303.2 20 100

GA51- 331.2 243.2 20 100- 331.2 287.2 20 100

3

Supplemental Table 4. Endogenous GAs determination using a LC-MS/MS method

Sample Compound Concentration (ng/g F.W.)

Zhu1SGA1 0.065±0.001GA8 0.028±0.002

SV14GA1 0.030±0.002GA8 0.067±0.003

CKGA1 0.024±0.001GA29 0.025±0.005

SBIPZhu1SSBIZhu1S GA1 0.005±0.001GA29 0.047±0.006

SBIPSV14SBISV14 GA1 n.d.GA29 0.199±0.014

SBIPSV14SBID308N GA1 0.007±0.001GA29 0.046±0.011

SBIPSV14SBIG338R GA1 n.d.GA29 0.176±0.011

Endogenous GAs were determined with LC-MS/MS analysis according to (Wild et al., 2012) with some modifications. GA compounds were extracted from 3 g tissue. After re-suspended in 30 μl methanol, GA compounds were determined through LC-MS/MS analysis. For quantification, calibration of standard GA compounds was set with a correlation of the MS peak area against concentration and the amount of endogenous GAs were calculated on the basis of the corresponding MS peak area. Values are means ± SD (n=3 replicates). F.W., fresh weight. n.d., not detected.

4

Supplemental Table 5. Culm phenotypes of the varieties with integration of SBISV14 allele

Rice

varieties

Integratio

n of

SBISV14

Plant height

(cm)

Length of the internodes (cm)

I II III IV V

5438 Yes 95.10±3.06* 35.77±2.59 17.47±1.11 11.70±1.13 5.83±0.46 1.97±0.30*

5438 No 114.00±2.05 40.50±3.27 19.80±2.90 11.55±1.33 6.90±1.21 4.00±0.72

5781 Yes 94.80±2.09* 34.93±3.78 17.68±3.14 11.13±1.31 7.68±0.90* 2.75±0.77*

5781 No 107.30±3.87 34.60±1.83 19.28±1.44 13.80±1.58 9.18±0.87 4.90±0.31

6204 Yes 90.90±2.41* 39.25±2.07 15.53±1.17 4.30±0.75* 2.45±0.48* 1.13±0.28*

6204 No 100.60±1.90 37.23±1.85 19.08±1.73 11.61±0.87 5.43±0.79 2.95±0.57

6389 Yes 104.89±2.14* 38.73±1.17 18.68±1.85 9.70±0.94 5.85±0.26* 2.33±0.69*

6389 No 109.70±2.11 39.80±1.79 22.30±1.37 11.78±0.86 6.90±0.36 3.28±0.19

6502 Yes 98.90±2.36* 33.80±3.21 18.15±1.47 10.75±1.13 5.40±0.92* 1.73±0.58*

6502 No 110.10±2.60 37.73±2.08 20.93±2.02 11.93±1.83 7.08±0.65 4.25±0.68

6716 Yes 96.50±2.26* 35.45±2.06 19.28±1.03 12.20±0.91 5.53±0.57 1.93±0.46*

6716 No 112.00±2.49 39.30±1.90 20.50±1.11 15.73±0.81 6.56±0.55 4.00±0.23

6878 Yes 109.00±1.81* 41.17±3.66 20.50±1.60 11.80±0.74* 7.04±0.61* 2.97±0.21*

6878 No 116.40±3.26 36.25±2.47 18.50±1.48 15.20±0.94 10.58±0.54 6.60±0.82

6990 Yes 100.60±2.26* 36.53±1.60 20.67±1.39 11.13±1.68 6.73±0.77 2.63±0.30*

6990 No 112.50±2.32 37.20±1.14 21.55±1.49 12.58±1.16 8.80±1.52 5.75±0.40

6991 Yes 97.25±2.78* 37.58±2.06 20.38±1.51 10.85±1.36* 5.68±1.03 2.18±0.19*

6991 No 114.80±3.07 39.03±1.94 22.35±1.81 15.18±1.17 7.53±0.80 4.88±0.17

7112 Yes 84.88±2.60* 33.50±1.05 18.95±1.49 8.00±0.98* 4.00±0.43* 1.5±0.30*

7112 No 109.60±2.69 36.93±1.27 19.48±1.15 12.85±1.08 7.00±0.39 4.73±0.87

7358 Yes 91.20±3.81* 35.10±1.62 19.53±1.42 10.30±0.78* 4.07±0.52* 1.57±0.44*

7358 No 109.30±1.52 35.53±2.46 21.07±1.27 16.57±1.95 8.27±1.28 4.13±1.72

7713 Yes 104.22±2.06* 36.23±2.71 18.83±1.83 14.30±0.81 8.13±0.83* 3.53±0.45*

7713 No 111.00±3.09 36.93±2.68 17.76±1.81 15.03±1.35 10.36±0.98 5.27±0.50

5

8096 Yes 100.50±1.94* 37.40±1.89 19.90±1.18 11.07±0.88 6.43±0.52 2.60±0.27*

8096 No 110.00±2.36 37.85±2.20 20.90±1.65 13.10±1.32 7.25±1.15 4.60±0.71

LYZ Yes 91.90±1.49* 37.15±1.69 18.38±1.70 8.43±1.46* 4.43±0.94* 1.65±0.91*

LYZ No 107.40±2.84 37.85±2.79 21.35±2.38 13.33±1.34 9.32±1.26 4.15±0.62

YHGZ Yes 93.33±2.93* 35.52±2.97 18.22±2.65 9.65±1.37* 4.72±1.04* 1.82±0.88*

YHGZ No 101.35±2.59 38.55±2.5 19.17±1.35 12.28±1.16 7.15±0.87 3.25±0.55

T14.13 Yes 94.70±1.29* 36.45±1.54 20.63±1.33 13.47±1.01* 4.50±0.78* 2.73±0.65*

T14.13 No 99.33±2.92 38.00±2.56 19.13±1.01 16.82±0.82 8.52±0.47 3.55±0.26

Z0041 Yes 88.87±1.76* 34.87±1.91 18.63±2.82 9.90±1.27* 3.93±0.28* 1.37±0.17*

Z0041 No 105.70±2.55 35.16±1.26 20.83±0.56 11.07±0.81 5.83±1.30 2.83±0.50

Z0108 Yes 95.80±3.38* 35.20±1.34 19.53±1.10 13.00±0.70 5.00±0.53 3.47±0.40*

Z0108 No 120.00±2.65 39.03±2.34 20.67±0.78 13.20±1.80 5.97±1.20 4.57±0.63

Z0117 Yes 105.50±2.05* 37.83±2.81 20.50±1.15 13.90±0.61 4.46±0.51* 2.13±0.26*

Z0117 No 111.40±2.23 38.92±1.91 21.55±1.48 14.92±0.92 8.43±0.68 3.63±0.15

Z0145 Yes 92.11±2.13* 33.38±2.06 17.00±1.67 12.13±1.07 5.00±0.39* 1.88±0.30*

Z0145 No 113.90±1.97 35.33±1.35 20.60±1.23 13.90±2.07 7.57±1.20 3.30±0.76

Z607 Yes 97.30±2.02* 36.47±2.14 18.87±1.70 9.65±1.67* 4.42±1.52* 1.80±0.60*

Z607 No 109.50±1.73 36.72±2.84 21.23±2.29 15.52±1.66 6.25±1.55 2.475±0.65

Culm phenotypes (height and internode length) were analyzed with/without integration of SBISV14 allele in 21

indica rice varieties. Values are means ± SD. Asterisk indicates significant difference in comparison by the

Student`s t-test at P<0.05 (n=30).

6

Supplemental Table 6. The list of 94 rice accessions sampled in the collection.

Accession ID Species

Original

producing areaLatitude Longitude

Ecotype

W0106 O. rufipogon India 20.46 85.88 Or-I

W0125 O. rufipogon India 21.00 85.10 Or-III


W0144 O. rufipogon Sri Lanka 7.07 80.30 Or-I




W0172 O. rufipogon Thailand 15.87 100.99 Or-III

W0573 O. rufipogon Malaya 3.10 101.50 Or-III

W0574 O. rufipogon Malaya 3.10 101.50 Or-I

W0623 O. rufipogon Burma 17.55 96.62 Or-III

W0626 O. rufipogon Burma 19.77 96.11 Or-I




W1161 O. rufipogon Sri Lanka 6.55 80.10 Or-III

W1214 O. rufipogon Philippines 7.86 124.86 Or-III

W1230 O. rufipogonDutch New

Guinea-4.63 138.93 Or-I

W1238 O. rufipogonNeth. New

Guinea-4.63 138.93 Or-III


W1690 O. rufipogon Thailand 19.41 99.34 Or-I

W1719 O. rufipogon China NA NA Or-I









W1970 O. rufipogon Indonesia -6.40 106.82 Or-I




W2057 O. rufipogon Bangladesh 24.48 91.78 Or-III

7

W2060 O. rufipogon Bangladesh 24.25 89.92 Or-I

W2078 O. rufipogon Australia -14.30 132.40 Or-III

W2099 O. rufipogon Australia -13.07 142.07 Or-III

W2263 O. rufipogon Cambodia 11.33 104.50 Or-I

W2265 O. rufipogon Laos 14.50 105.49 Or-I

W2267 O. rufipogon Laos 18.14 102.42 Or-III


W2319 O. rufipogon Vietnam 10.33 106.25 Or-I

W2331 O. rufipogon Vietnam 21.03 105.85 Or-I

W3022 O. rufipogon China 23.72 113.02 Or-III





GP137 O. sativa North Korea NA NA indica

GP513 O. sativa Philippine NA NA tropical japonica

GP52 O. sativa Cuba NA NA indica

GP589 O. sativa Egypt NA NA indica

GP624 O. sativa Hungary NA NA indica

GP686 O. sativa Hungary NA NA tropical japonica

GP718 O. sativa India NA NA tropical japonica

GP730 O. sativa Nigeria NA NA tropical japonica

GP76 O. sativa Madagascar NA NA indica

GP77 O. sativa IRRI NA NA tropical japonica

GP83 O. sativa India NA NA indica

GP85 O. sativa Thailand NA NA indica

HP10O. sativa

China 25.03 121.30

temperate

japonica

HP102O. sativa

China 47.35 123.18

temperate

japonica

HP11O. sativa

China 26.46 116.00

temperate

japonica

HP115O. sativa

China 26.87 104.28

temperate

japonica

HP116O. sativa

China 42.00 122.83

temperate

japonica

HP120 O. sativa China 18.25 109.50 indica

HP127O. sativa

China 44.93 127.17

temperate

japonica

HP128O. sativa

China 32.13 114.08

temperate

japonica

HP159O. sativa

China 30.40 104.04

temperate

japonica

HP171 O. sativa China 43.45 87.36 temperate

8

japonica


HP19O. sativa

China 40.13 124.37

temperate

japonica



HP29O. sativa

China 34.54 118.75

temperate

japonica



HP34O. sativa

China 29.23 113.80

temperate

japonica




HP37O. sativa

China 29.64 109.42

temperate

japonica

HP389O. sativa

China 25.58 115.79

temperate

japonica


HP54O. sativa

China 37.51 105.18

temperate

japonica

HP60O. sativa

China 45.53 131.85

temperate

japonica




HP62O. sativa

China 22.77 103.24

temperate

japonica

HP82O. sativa

China 33.00 106.93

temperate

japonica

HP9O. sativa

China 19.05 109.83

temperate

japonica

HP90O. sativa

China 38.71 115.14

temperate

japonica

9

Supplemental Table 7. Primers used in this studyPrimer Sequence(5`-3`) ExperimentsSD1F CACTCCCGCTCAACACAG

SD1 coding sequence cloningSD1R CGCCAAAAATCGGCTTCTG

SBI1F GCTTTAATTCCAGCCGAC SBI genomic DNA

overlapping cloningSBI1R GCCAACAGGCAAAAGGAC

SBI2F GGAGTGTCATGTTATC SBI genomic DNA

overlapping cloningSBI2R ACGCGAAGAACCGGACG

SBI3F ATGGTGGTGCTCGCGAAG SBI genomic DNA

overlapping cloningSBI3R TTAGGCGAGTGGGTTAG

SBI4F AACGGGAGGATGAGGAG SBI genomic DNA

overlapping cloningSBI4R GATGGATTCTAATGACTCGAG

SBI5F AGTGGCGATCCTAAAACAG SBI genomic DNA

overlapping cloningSBI5R GTTCTCTTTGTGTGATCTG

3XFLAGF GGATCCCCGGGTACCATGGACTACAAAGACCSBI promoter::SBI-3XFLAG

3XFLAGR GGGAAATTCGAGCTCCTTATCGTCATCGTCC

cSBIF GGTCTGCAGGTCGACATGGTGGTGCTCGCGAAGSBI promoter::SBI-3XFLAG

cSBIR GTAGTCCATGGTACCGGCGAGTGGGTTAGCGAC

pSBIF GCCAGTGCCAAGCTTTTTCTGGAGGGATCTGATGGSBI promoter::SBI-3XFLAG

pSBIR AGAGTCGACCTGCAGACCATGGCCAAGATCGAG

cSBIM1F TCGGCAACGGCGAGGAGGAGGAGCAGAGCCGGTACGAGGAG SBI promoter::SBID308N-

3XFLAGcSBIM1R TCCTCCTCCTCGCCGTTGCCGACGCCGGCCACCAGCAGCTG

cSBIM2FCTCAGCGACAACCGCCTGGCCCCCTTCCACCGGCAGCCACCACC

TGSBI promoter::SBIG338R-

3XFLAGcSBIM2R GGCCAGGCGGTTGTCGCTGAGGCGGGAGAGGTAGGCAGCCTTC

pMALSBIF ATCGAGGGAAGGATTTCACATATGGTGGTGCTCGCGAAG MBP-SBI construction

pMALSBIR TTAATTACCTGCAGGGAATTCTTAGGCGAGTGGGTTAG

pSBIGUS F CAGGCATGCAAGCTTTTTCTGGAGGGATCTGATGG SBI promoter::GUS

consturctionpSBIGUS R TCAGATCTACCATGGACCATGGCCAAGATCGAG

SBISNPF AACGGGAGGATGAGGAGSBIG338R SNP detection

SBISNPR AGGGTGTAAGTGGGTAAG

HYGF CTCCATACAAGCCAACCACG Screening for transgene-

positive plantsHYGR AAAAGCCTGAACTCACCGC

qUBQF TTGGCGTGGTCTTGGTTATCGGReal-time PCR of OsUBQ5

qUBQR TTACTTCGCTTCCCTGCAACAAG

qSBIF TGATGACGAACGGGAGGATGAGReal-time PCR of SBI

qSBIR ACACCCTCGACTTGAGCTTGTTG

q890F ATTCGCCTGTGTGGCCAGATTC Real-time PCR of

LOC_Os05g43890q890R ACCCGATTCATCACCTTCACACC

10

q895F CGGCCGTTGTTGCGGGAGAC Real-time PCR of

LOC_Os05g43895q895R ATCTGGCCCTCCAGGGCTAAG

Supplemental Figures

Supplemental Fig. 1. SV14 and Zhu1S displayed no significant differences in agronomic traits and yield performance.

Values are means ± SD (n=30 plants). Statistical significance of differences calculated based on two-tailed , two-sample Student`s t-test at p-value<0.01.

11

Supplemental Fig. 2. Difference in plant height between Zhu1S and SV14 accrued during culm elongation stage.

(A) 4 weeks old. (B) 8 weeks old at the basal internode elongation stage. (C) 12 weeks old at booting stage. (D) 14 weeks old plants at heading stage. (E) Height difference in (A ~ D). Values are means ± SD (n=30 plants). Scale bar, 10 cm.

12

Supplemental Fig. 3. SD1 allele and genome compare of Zhu1S and SV14.

(A) Up, mutant sd1 CDS sequence in Zhu1S and SV14. Down, truncated SD1 protein in Zhu1S and SV14. The 20G_Fell_Oxy box shows a conserved functional motif of SD1 protein. (B) Chromosomal landscape of Zhu1S and SV14 genome. The outer-ring lines represent rice 12 chromosomes. The distribution of SNPs and Indels on chromosomes is shown by line graph in 100 Kb window with 10 Kb sliding. The red graph stands for Zhu1S genome and blue graph for SV14 genome. Green graph indicates the difference between the two genomes. In the red and the blue graphs, the coordinate 0 means homozygous sites consistent with japonica rice Nipponbare genome, and 1 means homozygous sites consistent with indica rice 93-11 genome. In the green graph, the coordinate 1 means Zhu1S is closer to the indica rice 93-11 genome than SV14, and -1 means Zhu1S is closer to the japonica rice Nipponbare genome than SV14.

13

Supplemental Fig. 4. Plant height and distribution frequency in F1 and F2 progeny generated from the cross between Zhu1S and SV14.

(A) Plant height of two parents and F1 progeny. Values are means ± SD (n=30). Significant differences calculated based on two-tailed, two-sample Student`s t-test at p-value<0.01. (B) Segregation of SV14 type (<60 cm), F1 type (≥60 cm and <75 cm) and Zhu1S type phenotype (<75 cm). The actual frequency reflected a theoretical frequency of 1:2:1 based on χ2 test with a degree of freedom of 2 and significance level of 5%.

14

Supplemental Fig. 5. SNP index plots for 12 chromosomes of F2 progeny.

Red points indicate SNP position and their SNP indices. Green lines are regression lines.

15

Supplemental Fig. 6. Morphology of transgenic plants expressing different allele formats of SBI in ZH11.

(A-D) Overexpression of SBIZhu1S cDNA driven by its own promoter. (E-H) Overexpression of SBISV14 cDNA driven by Zhu1S promoter.(I-L) Overexpression of SBIZhu1S cDNA driven by SV14 promoter.Scale bar, 10 cm. Plant height (B, F, J), quantitative PCR analysis of expression of SBI (C, G, K) and protein blot detection of exogenous SBI-FLAG protein in transgenic lines (D, H, L). Rubisco (stained with coomassie brilliant blue) shows equal amount of total protein loading. CK, transgenic negative control. Plant heights are means ± SD (n=30 plants). Values are means ± SD (n=3 biological repeats). Statistical significance of differences calculated based on two-tailed , two-sample Student`s t-test at p-value<0.01.

16

Supplemental Fig.7. Overexpression of SBI in Zhu1S and SV14

(A) Morphology of SBISV14 in Zhu1S. Overexpression of SBISV14 cDNA driven by its own promoter in Zhu1S. Scale bar, 10 cm. Plant height (B), quantitative PCR analysis of expression of SBISV14 (C) . (D) Morphology of SBIZhu1S in SV14. Overexpression of SBIZhu1S cDNA driven by its own promoter in SV14. Scale bar, 10 cm. Plant height (E), quantitative PCR analysis of expression of SBISV14 (F) .CK, transgenic negative control. Plant heights are means ± s.d. (n=15 plants). Expression values are means ± s.d. (n=3 biological repeats). Statistical significance of differences calculated based on

two-tailed , two-sample Student`s t-test at p-value＜0.01.

17

Supplemental Fig. 8. Phylogenic and expression analysis of GA2ox family proteins.

(A)Multiple sequence alignment of the GA2ox family protein sequences was performed using ClustalW. The cut-off value for the condensed tree was 50%. Numbers indicate bootstrap support based on 1,000 replicates. Branch length indicates substitution per site. (B) Expression heat map of OsGA2ox family genes in different tissues based on online expression data (http://ricexpro.dna.affrc.go.jp/).

18

http://ricexpro.dna.affrc.go.jp/

Supplemental Fig. 9. GUS activity in SBI promoter-driven GUS transgenic rice plants.

Detection of GUS activity in (A) spikelets, (B) leaf blade and sheath, (C & D) stem section (the fourth internode), and (E) roots. Scale bars in (A), (B), (D), (E), 5 mm. Scale bar in (C), 1 mm. Similar results were verified in more than 3 independent transgenic lines. (F) GUS activity in various tissues of transgenic rice plants that harbor SBISV14 and SBIZhu1S promoter driven GUS construct. Mu, 4-methylumbelliferone. Values are means ± SD (n=3 biological repeats).

19

Supplemental Fig. 10 . Detection of recombinant protein

SDS-PAGE analysis (A) and protein blot detection (B) of MBP-fused recombinant SBIZhu1S and SBISV14 proteins. Recombinant proteins were produced in E. coli with (+) or without (-) 0.5 mM IPTG induction. M, Molecular weight marker.

20

Supplemental Fig. 11. Conversion of GA1 to GA8 by recombinant SBIZhu1S and SBISV14 proteins.

(A) GA8 standard, (B) Reaction product of GA1 catalyzed by SBIZhu1S, (C) Reaction product of GA1 catalyzed by SBISV14. m/z: SRM (selected-reaction monitoring) mode MS/MS spectras of one parent ion mass and two fragment ions mass, number indicates mass-to-charge ratio. Retention time of the two product ions indicated in liquid chromatograms. GA8 content was calculated by peak area of liquid chromatograms of product ion 275 (m/z) .

21


(A) GA29 standard, (B) Reaction product of GA20 catalyzed by SBIZhu1S, (C) Reaction product of GA20 catalyzed by SBISV14. m/z: SRM (selected-reaction monitoring) mode MS/MS spectras of one parent ion mass and two fragment ions mass, number indicates mass-to-charge ratio. Retention time of the two product ions indicated in liquid chromatograms. GA29 content was calculated by peak area of liquid chromatograms of product ion 303 (m/z).

22


(A) GA51 standard, (B) Reaction product of GA9 catalyzed by SBIZhu1S, (C) Reaction product of GA9 catalyzed by SBISV14. m/z: SRM (selected-reaction monitoring) mode MS/MS spectras of one parent ion mass and two fragment ions mass, number indicates mass-to-charge ratio. Retention time of the two product ions indicated in liquid chromatograms. GA51 content was calculated by peak area of liquid chromatograms of product ion 287 (m/z).

23

Supplemental Figure 14. Phylogenetic analysis of SBI gene in Zhu1S, SV14 and 94 rice accessions.

Dark gray cluster stands for wild rice, violet cluster for indica rice, yellow cluster for japonica rice and brown cluster for African rice.

24

Documents

· Web viewIndonesia-6.40 106.82 Or-I W1983 O. rufipogon India 18.50 73.15 Or-I W1993 O. rufipogon India 24.60 72.80 Or-III W2010 O. rufipogon India 19.00 73.06 Or-III W2057 O. rufipogon