Duplication of an upstream silencer of FZP increases grain yield in rice

Transcription

1 SUPPLEMENTARY INFORMATION Articles In the format provided by the authors and unedited. Duplication of an upstream silencer of FZP increases grain yield in rice Xufeng Bai 1,2, Yong Huang 1, Yong Hu 1, Haiyang Liu 1, Bo Zhang 1, Cezary Smaczniak 3, Gang Hu 1, Zhongmin Han 1 and Yongzhong Xing 1 * 1 National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan , China. 2 Hubei Collaborative Innovation Center for Grain Industry, Yangtze University, Jingzhou , China. 3 Plant Cell and Molecular Biology Institute for Biology Humboldt-Universität zu Berlin, Berlin 10115, Germany. * yzxing@mail.hzau.edu.cn Nature Plants Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

2 Supplementary Table 1 The recombinants from the large NIL-F 2 population selfing by RI76 for SGDP7 fine mapping Plant No. RM6389 RM22115 SNP2 SNP4 SNP3 SNP8 SNP1 InDel76 InDel710 InDel711 Progeny Test 360 C H H H H H H H H H segregated 551 C C C C C C C H H H Small grain & more SPP 747 C C C C C C C H H H Small grain& more SPP 112 C C C C C C C N N N Small grain& more SPP 688 H C C C C C C C C C Small grain& more SPP 79 H C C C C C C C C C Small grain& more SPP 1812 H H C C C C C C C C Small grain& more SPP 4881 H H H C C C C C C C Small grain& more SPP 6810 H H H H H H C C C C segregated

3 Supplementary Table 2 The phenotypes of the complementary transgenic plants in NIL-CC background (T 0 ) Lines PL (cm) NPB NSB SPP GL (mm) Empty Vector 26.0 ± ± ± ± ± 0.1 C ± ± ± ± ± 0.08 C ± ± ± ± ± 0.04 C ± ± ± 2.9** 138 ± 5** 7.2 ± 0.06** C ± ± ± 4.5** 124 ± 12** 7.4 ± 0.07** C ± ± ± 0.7** 137 ± 3** 7.2 ± 0.02** C ± ± ± 4.0** 137 ± 22** 7.3 ± 0.02** C ± ± ± 5.1** 151 ± 26** 7.1 ± 0.02** C ± ± ± 1.6** 122 ± 6** 7.3 ± 0.1** PL, panicle length; NPB, number of primary branches; NSB, number of secondary branches; SPP, spikelets per panicle; GL, grain length; C0, the positive transgenic plant of the vector with the fragment DF1 containing the whole gene LOC_Os07g47340; C4, the positive transgenic plant of the vector with the fragment DF2 containing the polymorphism sites (SNP4 and InDel712); the others (C1-C3 and C5-C7) were the different positive transgenic plants with the fragment DF3 of 8232bp containing the CDS and the 5.3-kb upstream sequence of FZP. All data are presented as mean±sd. **, P < P value was calculated using the Student s t-test (n = 5 panicles).

4 Supplementary Table 3 The phenotypes of the complementary transgenic plants in NIL-CC background (T 1 ) Lines Genotype NPB NSB NSB/NPB SPP GL TGW C1-T 1 (-) 8.2± ± ± ± ± ±0.7 (+) 9.3± ± ± ± ± ±0.9 P value 3.0E E E E E E-06 C2-T 1 (-) 8.3± ± ± ± ± ±0.7 (+) 9.5± ± ± ± ± ±0.9 P value 2.9E E E E E E-06 C4-T 1 (-) 8.0± ± ± ± ± ±0.8 (+) 7.9± ± ± ± ± ±0.5 P value C7-T 1 (-) 8.9± ± ± ± ± ±0.4 (+) 8.6± ± ± ± ± ±0.9 P value E E E E E-04 NPB, number of primary branches; NSB, number of secondary branches; NSB/NPB, the ratio of NSB to NPB; SPP, spikelets per panicle; GL, grain length; TGW, 1000-grain weight; C1-T 1, C2-T 1, C4-T 1 and C7-T 1 were respectively the T 1 generation of the corresponding T 0 lines (C1, C2, C4 and C7) in Supplementary Table 2. - and + were the transgenic negative plants and positive plants, respectively. All data are presented as mean ± SD. P value were calculated using the Student s t-test (n = 10 plants).

5 Supplementary Table 4 The phenotypes of the NILs in Chuan 7 genetic backgrounds Traits Chuan7-NN Chuan7-CC P value PN 8.9± ± NPB 11.0± ± NSB 28.3± ± E-13 NSB/NPB 2.6± ± E-07 SPP 125.5± ± E-09 GL (mm) 6.9± ± E-15 TGW (g) 14.2± ± E-17 YD (g) 14.1± ± E-03 PN, panicle number per plant; NPB, number of primary branches; NSB, number of secondary branches; NSB/NPB, the ratio of NSB to NPB; SPP, spikelets per panicle; GL, grain length; TGW, 1000-grain weight; YD, grain yield per plant; All data are presented as mean ± SD. P value were calculated using the Student s t-test (n = 20 plants).

6 Supplementary Table 5 The phenotypes of the heterozygous inbred family (HIF) derived from the cross between Haoboka and Chuan 7 Trait HIF-Haoboka HIF-Chuan7 P value PN 9.4± ± NPB 8.1± ± NSB 20.6± ± E-14 NSB/NPB 2.5± ± E-15 SPP 119± ± E-13 GL (mm) 6.8± ± E-11 TWG (g) 20.6± ± E-10 YD (g) 15.0± ± E-03 PN, panicle number per plant; NPB, number of primary branches; NSB, number of secondary branches; NSB/NPB, the ratio of NSB to NPB; SPP, spikelets per panicle; GL, grain length; TGW, 1000-grain weight; YD, yield; All data are presented as mean ±SD. P value was calculated using the Student s t-test (n = 12 plants).

7 Supplementary Table 6 The phenotypes of grain quality in the near isogenic lines with the genetic background of RI76 Alkali Rice length Rice width Ratio length Chalkiness Chalkiness Amylose Gel consistency Genotype spreading (mm) (mm) to width Ratio (%) degree (%) content (%) (mm) value NIL-NN 4.3±0.04** 2.2± ±0.01** 54.9±2.7** 18.9±0.8** 26.8± ±4.5** 1.0±0.1 NIL-CC 4.2± ± ± ± ± ± ± ±0.2 All data are presented as mean ± SD. **, P < 0.01 (n = 10 plants, Student s t-test).

8 Supplementary Table 7 The phenotypes of RNAi-3 and negative plants (T 3 ) in NIL- NN genetic background Trait Negative (25) RNAi-FZP (63) P value PN 8.8± ± NPB 7.7± ± NSB 20.0± ± E-16 NSB/NPB 2.6± ± E-15 SPP 105±10 165± E-09 GL (mm) 8.0± ± E-23 TWG (g) 19.8± ± E-25 YD (g) 14.4± ± PN, panicle number per plant; NPB, number of primary branches; NSB, number of secondary branches; NSB/NPB, the ratio of NSB to NPB; SPP, spikelets per panicle; GL, grain length; TGW, 1000-grain weight; YD, yield; All data are presented as mean ±SD. P value was calculated using the Student s t-test (n 25 plants).

9 Supplementary Table 8 The information of the 60 wild rice accessions used for examining the CNV-18bp No. of wild rice IRGC accession Species Name Source Country W O. glaberrima TANZANIA W O. rufipogon INDIA W O. rufipogon INDIA W O. rufipogon INDIA W O. rufipogon PAPUA NEW GUINEA W O. rufipogon MYANMAR W O. rufipogon INDIA W O. nivara NEPAL W O. rufipogon NEPAL W O. rufipogon NEPAL W O. alta SURINAME W O. nivara INDIA W O. rufipogon CHINA W O. rufipogon SRI LANKA W O. rufipogon THAILAND W O. rufipogon INDIA W O. nivara CHINA W O. rufipogon INDIA W O. rufipogon INDIA W O. rufipogon THAILAND W O. rufipogon THAILAND W O. nivara SRI LANKA W O. nivara INDIA W O. nivara INDIA W O. rufipogon THAILAND W O. rufipogon THAILAND W O. rufipogon MALAYSIA W O. rufipogon INDONESIA W29 O. rufipogon W O. rufipogon VIETNAM W31 O. punctata W32 GUANGDONG, CHINA W33 HAINAN, CHINA W O.rufipogon KAMPUCHEA W35 O.rufipogon W36 O. rufipogon W37 GUANGDONG, CHINA W38 O.rufipogon W O.rufipogon INDIA W O.rufipogon LAOS W O. punctata KENYA

10 W42 O.rufipogon W43 O.rufipogon W44 O.rufipogon W45 O. rufipogon GUANGDONG, CHINA W46 O. rufipogon GUANGDONG, CHINA W47 O. rufipogon GUANGDONG, CHINA W48 O. rufipogon GUANGDONG, CHINA W49 O. rufipogon GUANGDONG, CHINA W O. nivara CHINA W O. longistaminata TANZANIA W O.rufipogon CHINA W O. nivara INDIA W O. nivara THAILAND W O. nivara SRI LANKA W O. nivara SRI LANKA W57 O. rufipogon HUNAN, CHINA W58 O. rufipogon HUNAN, CHINA W59 O. rufipogon HUNAN, CHINA W60 O. rufipogon CHINA

11 Supplementary Table 9 The primers were used for mapping, vector construction and qrt-pcrs Markers Primers Forward(5'-3') Reverse(5'-3') InDel712 GCTTCCAGCGCCTACTGC TGCTCTGTTCTCCTCCGTTT InDel711 GCACATGCATGCTAGGACAT AGCCGGTAAATTTCTTGCAC InDel710 CATGTTATCGTGTGGGCTTT TCTGTTGCTGCAGCTGAACT InDel76 CACCGAAGACTGATCAGCAA TCACATTCGAGTGGAGCAA SNP1/SNP8 GGTTATTTCACATTGCCAAGC CGCTCAAAAGACAAGCTCCT SNP2 GCCTTAAGCTTGCATGGAGT GGTGGCCGGTGAAAAGTAGT SNP3 AGCTTTCCCCACAAGTGACA GTTGCCGAGTTACCATGTGA SNP4 CGAGCTTTGCCTACAAGGAT CGGGGAACAATGTAACACGA SNP5 GCATCCCAGAATCGTTCATC GGAATGCATGTATTGCGTGT SNP6 CAATGGGCGCAAACATAAAT ATGCAAAGTTCAGCCTGCTT SNP7 CAGCAACGGAAGAAGTGTCA CGGGTTAGCTGATTTGGTTT X4 CACCAGAACTTGAACCATCG GTCCAAGAGCCCAACTGAAC X3 GCATTAGCTGGTGGACAAAA AACGCAGGGGAATAACAATG U1 GTATCTCGGCAGCGTACCTC CGAAGGAAAGCAAAGCCATA U3 GCCGGTGTTAGGTTCAAATG CTTGTGTGCTGTGCTCGTTT U4 TGATGACCTTGACTTCCCCTA CAGTTGCGTAATTGCTTGTCA U5 CCTTAACAATTGTCAGTGCTTGC ACTACCGAACGGGGCTTTAT U7 AAGGGCAACGAGAGATGGT ATGCTCAGTTGGCCGATAAT U8 GACTTGTACACCGGCTACGG TATCCTTCTACCCCCGTTCC U9 GAGCGAGTGAAAGGCATGA TTTCCCGAGGAGTCAACATT RiFZP GGACTAGTGGTACCGTACCTG- CGCGAGCTCGGATCCCAATG- AGCAGCGTGGTG GGAGAGGAAGCTGAA FZP-In Situ ATGAACACTCGAGGCAGC CTCGCTCATCGGCGACGA FZP-qRT CAGGGCTCCGACTCCTACTCT CAATGCCCGGTGCAACTC OsBZR1-qRT CTGCCTCCTCCCGTTCCT GCCGTCCCTTCTATCTCCAT CDC20-qRT TCGAATCACCTGTTTGTTGGC TGGAGACAATCCAACGCAAAG

12 CDKA-2-qRT CGAGATTTGAAGCCCCAGAA TCCGCGAGCTTCAATGAGTT CYCA2-1-qRT AGGTTGTCAAGATGGAGAGCGA CGCTTTTTGTCTTCCTGGCA CYCD3-qRT CCTTCCACACTGACGGTACAGTT TGCCGCTGCCAAATAGACA CYCT1-qRT GCATTTGTTGCAGCTCAAG TCACCACTTCGCTGACTTATTG E2Fa-qRT TGTTGGTGGCTGCCGATAT CGCCAGGTGCACCCTTT H1-qRT GCAAGGCACCTGCAGCTT AGGCAGCCTTTGTACAGATCCT MAD2-qRT GAGCCATGCATATTCGACGTG GGTGTCGAAGGAATGCAGCTT MCM3-qRT TTCATGCGTCACTAAATGCGAG TGAATCTGGAAGCCCAATGTTC Ubiquitin-qRT AACCAGCTGAGGCCCAAGA ACGATTGATTTAACCAGTCCATGA OsBZR1-1 AACTGCAGATGACGTCCGGG CGGAATTCTCATTTCGCGCC CC-Bait-probe GCGCGTGCGCGTCCGTGCGTGCGTGCG- GTCACGCACGCACGCACGGACGCGCACG- CGTCCGTGCGTGCGTGCGTGAC CACGCACGGACGCGCACGCGC CC-Bait-Mtprobe GCGCGGTCGCGTCCGGACGCTCGATCG- CGTCCGGTCGGACGCTCGATAC GTATCGAGCGTCCGACCGGACGCGATCGA- GCGTCCGGACGCGACCGCGC NN-Bait-probe GCGCGTGCGCGTCCGTGCGTGCGTGCGTGAC GTCACGCACGCACGCACGGACGCGCACGCGC NN-Bait-Mtprobe GCGCGGTCGCGTCCGGACGCTCGATCGATAC GTATCGATCGAGCGTCCGGACGCGACCGCGC ChIP-qRT- OsBZR1 TACTATAGCGCGCGCTCTG AACGATCGATCGATCGGC

13 Supplementary Fig. 1 The panicle architecture and grain shape of Nanyangzhan and Chuan 7 Panicle architecture of Nanyangzhan (left) and Chuan 7 (right) ; (b) and (c) grains of Nanyangzhan and Chuan 7, respectively. Scale bar, 4 cm for a, 1 cm for b & c.

14 Supplementary Fig. 2 The genome constitution of the HIF (RI76) NYZ, Nanyangzhan; C7, Chuan 7; H, heterozygous.

15 Supplementary Fig. 3 The panicle of the transgenetic plants (T 0 ) of the genetic complementary of SGDP7 in NIL-CC background Vector, represents the empty of pcambia1301; C4, the positive transgenic plant of the vector with a fragment (DF2) only containing the polymorphism sites (SNP4 and InDel712); the others (C1-C3 and C5-C7) were the different positive transgenic plants with a whole fragment (DF3) of 8232bp containing the CDS and the 5.3-kb upstream sequence. Scale bar = 4 cm.

16 Supplementary Fig. 4 Comparison of yield related traits between the NILs of SGDP7 All data are presented as mean ± SD. P value were calculated using the Student s t- test (n = 20 plants).

17 Supplementary Fig. 5 The genome constitution of the HIF derived from the cross between Haoboka and Chuan 7 HBK, Haoboka; C7, Chuan 7; H, heterozygous.

18 Supplementary Fig. 6 The panicle architecture and grain shape of the heterozygous inbred line derived from the cross between Haoboka and Chuan 7 (a) panicle architecture, Haoboka (left) and Chuan 7 (right) ; (b) grains of haoboka (up) and Chuan 7 (down). (c), (d) and (e) were panicle architecture, primary branch and secondary branch of HIF-Haoboka (left) and HIF-Chuan7 (right), respectively; (f) and (e) were grains and brown rice of HIF-Haoboka (up) and HIF-Chuan7 (down). Scale bar, 4 cm for a & c, 1 cm for b & d-g.

19 Supplementary Fig. 7 The grains of the near isogenic lines of SGDP7 in Chuan 7 genetic backgrounds (a) the grains and (b) brown rice of Chuan7-NN (right) and Chuan7-CC (left). Scale bar, 2.5 mm for a & b.

20 Supplementary Fig. 8 Characterization of grain filling and transverse section of the grain in NIL-NN and NIL-CC (a) Time-course of the increase in endosperm fresh weight. (b) Time-course of the increase in endosperm dry weight. The data are presented as the means ± SD (n = 12 plants) in a and b. (c) Loose starch granule in a cross section of NIL-NN. (d) The starch grains were compactly arranged with no gap in a cross section of NIL-CC. Scale bars, 20 μm for c & d.

21 Supplementary Fig. 9 The observation of young panicles and the expression of floral identity genes in young panicles The observation of the young panicle by scanning electron microscopy; (a) NIL-NN and (b) NIL-CC, in the secondary branching stage; (c) NIL-NN and (d) NIL-CC in the floral meristem stage; (e) the expression pattern of the floral identity genes in the 2- mm-long and 5-mm-long young panicle from the data of RNA-Seq for NIL-NN and NIL-CC; BM, branch meristem; SM, spikelet meristem; FM, floral meristem; FM (F), the former stage of FM; FM (L), the later stage of FM; Scale bar, 100 μm for a-d.

22 Supplementary Fig. 10 The panicle length in different developmental stage (n = 10 plants)

23 Supplementary Fig. 11 Expression of FZP in the 2-mm-long young panicle (a) The relative expression levels of FZP of NILs. Ubiquitin served as the control. The bars indicate standard deviations. ** P < 0.01 (n = 3, Student s t-test). (b) RNA in situ hybridization analysis of FZP. The Spikelet meristems (SM) and floral meristem (FM) marked with black arrows; blue arrows indicate areas of expression in FM, sense probe control is also showed. Scale bar = 100 μm.

24 Supplementary Fig. 12 Comparison grain length between CK and RNAi of FZP Primary branch (PB); Secondary branch (SB); The bars indicate standard deviations. ** P < 0.01 (n = 10 plants, Student s t-test).

25 Supplementary Fig. 13 The relative expression of 9 cell cycle related genes (n = 3).

26 Supplementary Fig. 14 Interaction analysis of OsBZR1 and CNV-18bp by electrophoretic mobility shift assay

27 Supplementary Fig. 15 The electrophoresis gel map of the maker InDel712 (a) the polymorphism of InDel712 was displayed in polyacrylamide gel (4%), blue arrows indicated the bind of the aus varieties with a 18-bp insertion; (b) the polymorphism of InDel712 was displayed in agarose gel (3%); (c) and (d) the genotypes of the 60 wild rice at InDel712. NYZ, Nanyangzhan; C7, Chuan 7; H, Heterozygote.