... pathway determines grain size in rice. Authors: Shengyuan Sun, Lei Wang, Hailiang Mao, Lin Shao, Xianghua Li, Jinghua Xiao,. Yidan Ouyang. *. , Qifa Zhang.
Title: A G-protein pathway determines grain size in rice
Authors: Shengyuan Sun, Lei Wang, Hailiang Mao, Lin Shao, Xianghua Li, Jinghua Xiao, Yidan Ouyang*, Qifa Zhang*
5 4 3 2 1 1
2 WT
1
2
1
2
1
2
DEP1OE dep1OE DEP1Ri
6 5 4 3 2 1 0
d
1
2 WT
1
2
1
2
1
2
GS3-1OE GS3-4OE GS3-1Ri
1.4
Relative expression of GGC2
Relative expression of GS3
Relative expression of DEP1/dep1
7
6
0
c Relative expression of endo-DEP1
b
a
1.2 1.0 0.8 0.6 0.4 0.2 0
1
2 WT
1
2
dep1OE
25 20 15 10 5 0
1
2 WT
1
2
GGC2OE
0.25
Relative expression (/ubi)
e
DEP1 GGC2 GS3
0.20
0.15
0.10
0.05
0.00
t oo Sh
af Le a Le
e
2 4 4 10 40 -2 -3 -4 G G G AG AG A A A -D -D -D -D -D SA SA SA SA SA
d la fb
Vegetative stage
m m cm cm 5cm cm 8cm cm 9cm 1c 2c . .5 .5 -1 -2 1 1 2 3 6 6 e e e e e e cl cl ee- nicl -1 cl cl cl ni ni cl cl le ni ni ni ni ni ic a a a Pa Pa Pa a a n p P P P P Pa
m
t oo
Pa ni cl e35m
R
Reproductive stage
Supplementary Figure 1 Relative expression levels of three Gγ proteins. (a) Relative expression level of DEP1 or dep1 in WT (wild type ZH11), DEP1OE, dep1OE, and DEP1Ri transgenic plants. Transformants from two independent T2 homozygous lines were assayed for each transgene. Transcriptional level is determined by quantitative Real-time PCR based on cDNA template prepared from 0.5 cm young panicles. Rice ubiquitin is used as the reference gene. (b) Relative expression level of GS3 in WT, GS3-1OE, GS3-4OE, and GS3-1Ri transgenic plants. (c) Relative expression level of endogenous DEP1 in dep1OE transgenic plants. (d) Relative expression level of GGC2 in GGC2OE transgenic plants. (e) Relative expression levels of DEP1, GGC2, and GS3 in root, shoot, flag leaf, leaf blade, shoot apex at 10, 22, 34, 40, and 44 days after germination, and developing panicles in ZH11. SA, shoot apex; DAG, days after germination. Rice ubiquitin is used as the reference gene. Data for all the assays are shown as mean ± SEM (n = 3).
10 0
G S3
dep1OE GS3-1Ri /dep1OE
DG Gd
6
5
Actin GS3-1
TGA
W1 W2
M1
M2
2715R+SR17 H W M W M W M H W M H M W W H H W H
P = 4.02E-11
P = 4.39E-13
P = 2.99E-08
P = 6.56E-14
P = 1.18E-06
P = 1.05E-12
P = 1.07E-12
DEP1
b
GS3-1OE GS3-1OE/DEP1ko
bc c d
GS3-4OE/DEP1
d
e 5
SF28+SR17
8 a
ko
RB 3’
Grain length (mm) 6 7
WT
GS3-4OE
5’ ATG
1 -2 -1 -2 d-1 d-2 d-1 d-2 WTgs3GG-G G DG DG G G D D
d
2715R SR17
5
ko
b LB SF28
7 6
4
Dd 5
G W S3 T -1 de -1R R p1 i i/d O ep E 1O E
WT GS3-1Ri
GG
c c
8
P = 9.67E-12
Grain length (mm)
GS3-1Ri dep1OE /dep1OE
a 7
G W S3 T -1 de 1R R p1 i i/d O ep E 1O E
GS3-1Ri
b b
gs3
b
G S3
Panicle length (cm)
a a
20
WT
WT
8
30
Grain length (mm)
c
P = 3.04E-08
a
Grain length (mm) 6 7 8 9 10
GS3-1OE P = 0.21
GS3-1OE/DEP1OE GS3-1Ri
P = 5.44E-07
GS3-1Ri/DEP1OE
Supplementary Figure 2 Genetic analysis of DEP1 and GS3. (a) Panicles, panicle length, grains, and grain length in WT (wild type ZH11, n = 7), GS3-1Ri (n = 14), dep1OE (n = 10), and GS3-1Ri/dep1OE (n = 28) plants. Values are given as mean ± SEM. Different letters indicate significant differences ranked by the LSD test (P < 0.05). Panicle, bar = 5 cm; grains, bar = 5 mm. (b) Description for the T-DNA insertion mutant of GS3 (gs3). The genotype of the mutant is confirmed using PCR. T-DNA is inserted into the second intron. SF28, SR17, and 2715R indicate primers for genotyping. RB and LB indicate the right border and left border of the T-DNA. The transcription level of GS3 is investigated in 2 cm panicle. W, the wild type Dongjing; H, plants heterozygous for T-DNA insertion; M, plants homozygous for T-DNA insertion. (c) Grains and grain length of WT (wild type Dongjing) (n = 8), gs3 (n = 7), GG-1 (n = 10), GG-2 (n = 10), DG-1 (n = 14), DG-2 (n = 11), Gd-1 (n = 7), Gd-2 (n = 12), Dd-1 (n = 18), and Dd-2 (n = 8). Values are given as mean ± SEM. P values are given based on two-tailed t-tests. GG and DG indicate gs3 plants transformed with GS3-1 under GS3 and DEP1 promoters, respectively. Gd and Dd indicate gs3 plants transformed with dep1 under GS3 and DEP1 promoters, respectively. Bar = 5 mm. (d) Grains and grain length of WT (wild type ZH11) (n = 6), DEP1ko (n = 7), GS3-1OE (n = 10), GS3-1OE/DEP1ko (n = 8), GS3-4OE (n = 6), and GS3-4OE/DEP1ko (n = 8). Values are given as mean ± SEM. Different letters indicate significant differences ranked by the LSD test (P < 0.05). Bar = 5 mm. (e) Grains and grain length of GS3-1OE (n = 5), GS3-1OE/DEP1OE (n = 13), GS3-1Ri (n = 17), and GS3-1Ri/DEP1OE (n = 26). Values are given as mean ± SEM. P values are given based on two-tailed t-tests. Bar = 5 mm.
a
G1: GACGCGCTCCACCGCGAGAT G2: GCCCCGGCCCAAGTCGCCGC GS3-1 D1: GGAGGAGGCGGTGGTGATGG D2: AAAGAGATAAATGAGTTTGT DEP1 DO1: GGGTACCTCGGCGGCGACTT DO2: GGTACCCGGACCTGTGCGGC 500bp
GGC2 O1: AACGGATTCAGCCGGTCTCG O2: GGGACTTGAACGGATTCAGC
b c d
e f
Pubi
Cas9
OsU6a G1
OsU6c G2
GS3-1 WT CGGCGGCGCCCCGGCCCAAGTCGC ― CGCCGG^GTCGACGCGCTCCACCGCGA― GATCGGA ko GS3-1 -1 CGGCGGCGCCCCGGCCCAAGTCGCGCGCCGG^GTCGACGCGCTCCACCGCGAAGATCGGA GS3-1ko-2 CGGCGGCGCCCCGGCCCAAGTCGC ACGCCGG^GTCGACGCGCTCCACCGCGA ― GATCGGA Pubi
Cas9
OsU3
T3 D1
OsU6b D2
DEP1 WT AGATGGGGGAGGAGGCGGTGGTGATGG AGG^CCTGAG ― CCGCGAGATCACGTTCCTCAA DEP1ko-1 AGATGGGGGAGGAGGCGGTGGTGATGG AGG^CCTGAGACCGCGAGATCACGTTCCTCAA DEP1ko-2 AGATGGGGGAGGAGGCGGTGGTGATGG AGG^CCTGAG ― ― CGCGAGATCACGTTCCTCAA Pubi
Cas9
OsU3
G1
OsU6a D1
OsU6c G2
OsU6b
Cas9
OsU3
T3 O1
Cas9
OsU6a DO1
+2 bp +1 bp
+1 bp +1 bp
OsU6a O2
GGC2 WT AGCTACAGGGACTTGAACGGATTC ― AGCCGGTCTCGAGGTGTTG GGC2 ko-1 AGCTACAGGGACTTGAACGG ― ― ― ― ― AGCCGGTCTCGAGGTGTTG ko GGC2 -2 AGCTACAGGGACTTGAACGGATTCAAGCCGGTCTCGAGGTGTTG Pubi
+1 bp -1 bp
D2
GS3-1 WT CGGCGGCGCCCCGGCCCAAGTCGC ― CGCCGG^GTCGACGCGCTCCACCGCGA― GATCGGA ko GS3-1koDEP1 -1 CGGCGGCGCCCCGGCCCAAGTCGCGCGCCGG^GTCGACGCGCTCCACCGCGAAGATCGGA ko ko GS3-1 DEP1 -2 CGGCGGCGCCCCGGCCCAAGTCGCGCGCCGG^GTCGACGCGCTCCACCGCGA ― GATCGGA DEP1 WT AGATGGGGGAGGAGGCGGTGGTGATGG AGG^CCTGAG ― CCGCGAGATCACGTTCC TCAA ko GS3-1ko DEP1 -1 AGATGGGGGAGGAGGCGGTGGTGATGG AGG^CCTGAGACCGCGAGATCACGTTCC TCAA ko ko GS3-1 DEP1 -2 AGATGGGGGAGGAGGCGGTGGTGATGG AGG^CCTGAGTCCGCGAGATCACGTTCC TCAA Pubi
+2 bp +1 bp
-4 bp +1 bp
OsU6b DO2
DEP1 WT GAGGCCCAAG - TCGCCGCCGAGGTACCCGGACCTGTGC - GGCCGGCGGCGGAT DEP1koGGC2 ko-1 GAGGCCCAAG TTCGCCGCCGAGGTACCC GGACCTGTGC - GGC CGGCGGCGGAT DEP1 koGGC2 ko-2 GAGGCCCAAG ATCGCCGCCGAGGTACCCGGACCTGTGCC GGCCGGCGGCGGAT DEP1koGGC2 ko-3 GAGGCCCAAG - - - GCCGCCGAGGTACCCGGACCTGTGC T GGC CGGCGGCGGAT
+1 bp +2 bp -2+1 bp
GGC2 WT GAGGCCCAAG - TCGCCGCCGAGGTACCCGGACCTGTGC - GGC CGGCGGCGGCT DEP1koGGC2 ko-1 GAGGCCCAAG ATCGCCGCCGAGGTACCCGGACCTGTGC T GGCCGGCGGCGGCT +2 bp DEP1koGGC2 ko-2 GAGGCCC - - - - - - - - - - - - GAGGTACCCGGACCTGTGCC GGCCGGCGGCGGCT -11+1 bp ko ko DEP1 GGC2 -3 GAGGCCCAAG CTCGCCGCCGAGGTACCCGGACCTGTGC - GGCCGGCGGCGGCT +1 bp
g
Pubi
Cas9
OsU3
DO1
OsU6a DO2
OsU6c G2
DEP1 WT GAGGCCCAAG - TCGCCGCCGAGGTACCC GGACCTGTGC - GGCCGGCGGCGGAT ko ko DEP1 GGC2 GS3-1ko-1 GAGGCCCAAGGTCGCCGCCGAGGTACCC GGACCTGTGCC GGC CGGCGGCGGAT ko ko DEP1 GGC2 GS3-1ko-2 GAGGCCCAAG ATCGCCGCCGAGGTACCC GGACCTGTGC T GGCCGGCGGCGGAT ko ko DEP1 GGC2 GS3-1ko-3 GAGGCCCAAG TTCGCCGCCGAGGTACCC GGACCTGTGC - GGCCGGCGGCGGAT GGC2 WT ko ko ko DEP1 GGC2 GS3-1 -1 ko ko ko DEP1 GGC2 GS3-1 -2 ko ko ko DEP1 GGC2 GS3-1 -3
GAGGCCCAAG - TCGCCGCCGAGGTACCCGGACCTGTGC - GGCCGGCGGCGGCT GAGGCCCAAGGTCGCCGCCGAGGTACCCGGACCTGTGC - GGCCGGCGGCGGCT GAGGCCCAAG ATCGCCGCCGAGGTACCCGGACCTGTGCC GGCCGGCGGCGGCT GAGGCCCAAG ATCGCCGCCGAGGTACCCGGACCTGTGC - GGCCGGCGGCGGCT
GS3-1 WT CGGCGGCGCCCCGGCCCAAGTCGC - CGCCGGCGCC ko ko ko DEP1 GGC2 GS3-1 -1 CGGCGGCGCCCCGGCCCAAGTCGCA CGCCGGCGCC ko ko ko DEP1 GGC2 GS3-1 -2 CGGCGGCGCCCCGGCCCAAGTCGC T CGCCGGCGCC ko ko ko DEP1 GGC2 GS3-1 -3 CGGCGGCGCCCCGGCCCAAGTCGC T CGCCGGCGCC
+2 bp +2 bp +1 bp
+1 bp +2 bp +1 bp
+1 bp +1 bp +1 bp
Supplementary Figure 3 Knock-out mutants of three Gγ proteins created by CRISPR/Cas9. (a) Schematic map of the sgRNA target sites in GS3, DEP1, and GGC2. (b) Vector construction and sequence alignment for GS3-1ko single mutant. (c) Vector construction and sequence alignment for DEP1ko single mutant. (d) Vector construction and sequence alignment for GS3-1koDEP1 ko double mutant. (e) Vector construction and sequence alignment for GGC2 kosingle mutant. (f) Vector construction and sequence alignment for DEP1koGGC2 ko double mutant. (g) Vector construction and sequence alignment for DEP1koGGC2 ko GS3-1ko triple mutant.
a
c
A1: ATCCACAAGCTCTTACTTCT A2: GCTTTGATGAGGCAGAACTT A3: GAAAAACTATCAGATATTGA 500 bp RGA1
B1: GCCGGGCGTCCGTGGCGGAGCTCA B2: GGCAAGAGGTACTCGAGGACGCAG 500 bp
RGB1 B3: GTTGGCAGCATCTGCCGCCTCTGA Pubi
Cas9
OsU3 B1
OsU6a B2
OsU6b B3
Pubi
Cas9
OsU3 A1
OsU6a A2
(bp) 1465 1024 660
(bp) 445 308 206 5
6 7 8 9 10 11 12 13 14 15 16 17 CR-RGB1-13 T1 445 206 CR-RGB1-17 T1
1465
445 206
196
RGB1Ri
WT RGB1Ri-1
P35S
RGB1
RGB1 Nos
Relative expression of RGB1
CK
b
CR-RGA1-8 T1
1.5 1 0.5 0
RGB1Ri-2
196
M 1 2 3 4 5 6 7 8 9 10 111213 14
2 WT Ri-1 1RiB1 GB G R R
Grain length (mm)
M 1 2 3 4
OsU6b A3
WT
8 7
-09
9E
7.6
1 P=
P=
11
ko
RGA1
Brown lamina joint
E-
5 2.0
6 5
2 WT1Ri-1 1RiB GB G R R
WT
RGB1Ri-1
Supplementary Figure 4 Generation of knock-out and knock-down transgenic plants of RGB1 and RGA1. (a) Generation of RGB1ko mutant by CRISPR/Cas9 technology in ZH11 background. The vector and schematic map of the sgRNA target sites in RGB1 are shown. The genotypes of 17 T0 transgenic plants and two T1 families from RGB1 ko-13 and RGB1 ko-17 were investigated, and no homozygous mutation was identified. (b) Generation of transgenic plants with suppression of RGB1 in ZH11 background. The vector construction for RGB1Ri is shown. Relative expression level of RGB1 in RGB1Ri transgenic plants is given as mean ± SEM (n = 3). The grain length of WT (wild type ZH11), RGB1Ri-1, and RGB1Ri-2 is given as mean ± SEM (n = 10). P values are given based on two-tailed t-tests. Bar = 5 mm. Brown lamina joint was observed in RGB1Ri transgenic plants. (c) Generation of RGA1ko mutant by CRISPR/Cas9 technology in ZH11 background. The vector and schematic map of the sgRNA target sites in RGA1 are shown. The genotypes of 14 T0 transgenic plants and one T1 family from RGA1ko -8 were investigated. Whole plants and grains of WT (wild type ZH11) and RGA1ko mutant are shown. Plant, bar = 20 cm; grains, bar = 5 mm.
04
16
.1
GG
AC
AC
C2
54
T7
I25
5.1
po
di
44
.1
86
44
um
Tr
91
di
itic
.1
st
ac
C2
XP
XP
_00
_0
02
44
DA
A4
88
80 67
lg ar is x su run 06 XP_ 96. bs 4 ca 65 1R 012 p. tul 90 icin 068 2.1 99 a vu us 866 Cit lg com .1 J ar ru atro is pha munis s sin en curc XP_ sis 0100 as 4934 85 5.1 E 73 ucaly 85 ptus gran dis 96
ot
ma
ag
_0
dic
XP _0
155
839
XP
Me .2
27 73
62 03 _0
99
vu
ta
ja
yc
Gl
22 76
H4 KR
acea
. oler
a var
XP
ine
Be
.1
ine
58
.1
22 14
N0
1 5.
3
ica ole race
KH
67
ts re ina x b ent s m ru le sis 84 Py s c en 37 5.1 Citru sin 3 s 1 as 08 72 .1 itru urc s _0 933 814 .1 C ha c uni P X 00 452 099 trop omm tica a _ 6 5 c J hra 0 7 XP _0 064 60.1 icinus eup s 2 u 6 arpa 8 6 XPP_0 770 .1 R pul hoc X 120 2906 6.1 Po ulus tric atica 99 0 7 9 op _ 57 uphr XP_015 11039 614.1 P opulus e a 0 P 1 P _ 8 .1 o 3 h carp X XP 006 7771 lus tric 92 50 XP_ P_01100 141.2 Popu dii on im 90 X 4 ra 2 3 um P_002 2866.1 Gossypi 100 X XP_01246 .1 Theobroma cacao 2624 96 XP_00701 99 XP_008459850.1 Cucumis melo XP_011648578.1 Cuc umis sativus XP_00450 81 KYP57 8000.1 Cicer arietin 89 um 52 NP_0 831.1 Caja nus ca 0 XP_ jan 00711242228.1 99 XP 54574.1 Glycine m 88 ax KH _00353 Phase 52 olu N1 49 XP 2284 47.1 G s vulgar is .1 G _0 lyci 10 0 0 ne m XP X 4488 lycine ax P 0 _ soj a XP XP_ 008 _015 92.1 C _0 01 450 954 74 icer a 10 166 92 9.1 rie 53 00 8.1 tinu Ar 58 20 Cu m ac 11 .1 cu h is Cu mi .1 du sm cu Ta ran m elo re en is na sis sa ya tiv ha us ss le ria na us
al
M
G
XP
_0
.1
10
Gl
66
yc
68
Zm
so
3
GS
56
d
i
er
eid
n ch
96
83
ta ra ly p. bs su ta ra ly s a na si ea ia fer ac op al a lei ler id th ap .o r is r. o ab sp va ica sub ps Ar s o ea id .1 as apa rac Br r 08 rab ole 40 A 1.1 ica ca s s ssi 87 .2 46 a 5 us Bra 02 17 126 Br nap _0 680 09 87.1 69.1 sica iva sat ras XP _ _0 14 187 lina NP XP D3 36 .1 B me CC _01 076 1 Ca a 97 in XP Y45 70. lp 543 a 97 is a CD 4 b a 0 apus lerace 1 Ar ica n _01 var. o XP 26289. .1 Brass racea 52 ica ole KFK 16550 Brass CDY 372.1 apus 13607 XP_0 447.1 Brassica n a 59 CDX92 .1 Brassica rap XP_009120789 AIS73136.1 Brassica nigra AIO11838.1 Brassica rapa XP_0091318 05.1 Brassi CDX ca rapa 7 0 XP_0 936.1 Brassic 1 a napu 3 6 2707 s 6.1 B rass
GS3
55
om
AG
u Ho Triti m a hyo rd cum es n e t 44 357 05 um ur ivu 69 XP_ 226 . .1 a m 015 8.1 So 1 Ze vulg rtu 649 Bra r a a 592 chy ghum ma re 8 94 8 .1 O 3 pod 6 bic ys ryza i u X o 99 P_01 md sativ l XP_0 5695 ista or a Ja 09400 90 630. c pon 109.1 1 Or ica hyon Musa yza acum brac Group XP_009 in a 9 h ta 8 y 391231 subsp a .1 Musa . mala ntha 96 54 acumin ccens ata sub is s p . malacc XP_0109324 e n 06.1 Elaeis guineenssisis 78 85 XP_008784313.1 Phoenix dactylifera fera XP_008782613.1 Phoenix dactyli 99 ineensis 32.1 Elaeis gu alaccensis 91 XP_0109373 m . sp b is ccens minata su la cu a a m sa sp. .1 Mu ta sub up 94 414819 XP_009 umina Gro sa ac a u ic M n po 811.1 98 tha a Ja 09408 yan 79 sativ XP_0 rach ryza b O a 1 n . z a 2 Ory 5 hyo 3007 od tac a9 33.1 0156 op dis vum italic a 899 XP_ 82 i h m 6 t 5 u s c i c 01 ae tri ria ali pod XP_ um eta ia it ays 91 lla chy ritic Bra 1 S etar r ore m . T 1 o 1 0 . l a 7 7 72. 1 S Ze bico mb 04 195 553 A K9 061. 0.1 475 HC m .2 KQ _01 4 0 A u P 8 6 4 X h 49 02 org 971 0 Z _0 AC .1 S 684 XP 2 00 15 65 XP 4 02 _0 XP
GG
a
tic
es
hy
I2
58 85 78
Zm
Br
ac
AC
90
J9
XP_004243703.1 Solanum lycopersicum XP_015080497.1 Solanum pennellii XP_00634 98 2388.1 S olanum tu gAIL52 berosum XP_0 190.1 Capsic 0959 um an 6092 C nuum .1 Nic DP0 88 otian XP XP_0 3079.1 a tom 52 _01 128 C entos 107 4806 offea ca iform XP is nep 4.1 809 XP XP _01 hora E 5 _ r X y . 0 1 S _0 01 2 thra P_ 6 nth esa XP 158 065 183 010 eg utta _0 89 611 2.1 2738 mum ta ind 47 N 11 04 5. icu 46 5.1 1 V elum .1 N m elu itis 08 Z bo mb 19 izip nu vin on cif h .1 if uc era Fr us j era ife ra uju ag ba ar ia ve sc a su bs p. ve sc a
55 s 92 54 may s y Zea r ma o l 61.1 o ea 616 1 Z m bic lica 96 DAA 67. a u t 1 p i h ou ha A40 Sorg aria t DA Gr ant 99 e .1 S 75 ica hy .1 02 on rac 39 46 b ap 69 02 a 95 aJ _0 z v y 04 XP ati _0 Or n as XP .1 z yo 8 y 36 tach Or 6 s 2.1 69 di 89 15 10 um _0 56 di XP 01 po P_ hy ac Br
a EP1 ZmD EP1b D Zm
X P1 DE
.1 84 78 75 14 _0
XP
KQ
Monocots
Dicots 0.1
Supplementary Figure 5 The maximum-likelihood tree based on protein sequences of 96 Gγ proteins from 55 species showing significant similarity to three rice Gγ proteins. The blue color indicates proteins from monocots, and the purple color indicates ones from dicots. One protein from a relict plant is shown in red. The accession number and species for each of the protein is labeled in the tree. Scale bar represents 0.1 substitutions per site.
a
Anti-Flag
Rubisco
100 70 55 40 35 25 15 1#
b
2#
1#
2#
1#
2#
1#
2#
WT
GS3-1FOE GS3-4FOE DEP1FOE dep1FOE
100 70 55 40 35 25
CK
6
12 20 CHX
c
6
12 20 MG132
100 70 55 40 35 25 15
CK
6
12 CHX
20
6
12
20
MG132
Supplementary Figure 6 Immunoblot analysis of the proteins in transgenic plants with Flag tag. (a) Immunoblot analysis of the transgenic plants. Total proteins are extracted from the shoots of 15-day-old seeding, and 20 μg amount was used for western blotting. (b-c) Immunoblot analysis of Flag fused GS3-1(b) and GS3-4 (c) proteins after the treatments with 30 μM CHX and 50 μM MG132. CK, the GS3-1F and GS3-4F transgenic plants without treatment. CHX, Actidione. The two bands of GS3-1 protein are likely due to protein modification by ubiquitination. The numbers (6, 12, 20) indicate hours after the treatments.
Supplementary Table 1 Phenotypes of transgene positive and negative plants in the T 1 or T2 generation. Genotype DEP1OE-1 (+) DEP1OE-1 (-) Variation P -value DEP1OE-2 (+) DEP1OE-2 (-) Variation P -value DEP1OE-3 (+) DEP1OE-3 (-) Variation P -value dep1OE-1 (+) dep1OE-1 (-) Variation P -value dep1OE-2 (+) dep1OE-2 (-) Variation P -value DEP1Ri-1 (+) DEP1Ri-1 (-) Variation P -value DEP1Ri-2 (+) DEP1Ri-2 (-) Variation P -value GS3-1OE-1 (+) GS3-1OE-1 (-) Variation P -value GS3-1OE-2 (+) GS3-1OE-2 (-) Variation P -value GS3-1OE-3 (+) GS3-1OE-3 (-) Variation P -value GS3-4OE-1 (+) GS3-4OE-1 (-)
Number of plants 14 12
18 8
20 10
16 4
17 11
23 7
12 5
12 7
9 6
9 8
13 6
Grain length 7.95 ± 0.04 7.44 ± 0.03 6.85% 1.32E-10 7.89 ± 0.04 7.20 ± 0.05 9.58% 1.10E-08 7.93 ± 0.04 7.30 ± 0.03 8.63% 4.60E-12 6.92 ± 0.03 7.26 ± 0.01 -4.68% 7.12E-10 6.75 ± 0.04 7.07 ± 0.03 -4.53% 1.47E-07 6.69 ± 0.03 7.10 ± 0.05 -5.77% 3.35E-05 6.65 ± 0.05 7.19 ± 0.03 -7.51% 1.04E-07 6.73 ± 0.05 7.33 ± 0.03 -8.19% 6.56E-09 6.41 ± 0.08 7.19 ± 0.06 -10.85% 2.50E-06 6.97 ± 0.03 7.59 ± 0.06 -8.17% 2.11E-06 5.91 ± 0.06 7.21 ± 0.05
Number of plants Grains per panicle 10 183.80 ± 2.52 9 200.33 ± 5.80 -8.25% 2.42E-02 9 173.67 ± 4.63 8 213.63 ± 12.31 -18.71% 1.41E-02 13 192.69 ± 5.39 10 218.40 ± 9.11 -11.77% 0.0281 14 233.79 ± 9.05 12 225.17 ± 10.03 3.83% 0.53 11 190.30 ± 10.78 11 183.55 ± 9.23 3.68% 0.64 10 211.20 ± 3.47 10 221.70 ± 5.19 -4.74% 0.11 10 215.00 ± 3.46 10 215.6 ± 4.61 -0.28% 0.92 15 179.93 ± 8.41 14 210.71 ± 6.97 -14.61% 9.03E-03 15 189.80 ± 4.24 13 202.15 ± 6.60 -6.11% 0.13 15 188.40 ± 6.43 13 198.69 ± 5.90 -5.18% 0.25 11 251.55 ± 11.35 8 233.13 ± 11.83
Variation Variation P -value GS3-4OE-2 (+) GS3-4OE-2 (-) Variation P -value GS3-4OE-3 (+) GS3-4OE-3 (-) Variation P -value GS3Ri-1 (+) GS3Ri-1 (-) Variation P -value GS3Ri-2 (+) GS3Ri-2 (-) Variation P -value GS3Ri-3 (+) GS3Ri-3 (-) Variation P -value GGC2OE-1 (+) GGC2OE-1 (-) Variation P -value GGC2OE-2 (+) GGC2OE-2 (-) Variation P -value
12 5
10 6
18 12
19 11
26 4
5 5
5 5
-18.03% 2.57E-11 5.91 ± 0.05 7.21 ± 0.08 -18.03% 2.82E-06 5.67 ± 0.08 7.20 ± 0.07 -21.25% 1.63E-09 7.83 ± 0.02 7.33 ± 0.04 6.82% 6.16E-10 7.46 ± 0.03 7.09 ± 0.03 5.22% 2.51E-09 7.76 ± 0.02 7.37 ± 0.04 5.29% 2.16E-04 8.13 ± 0.08 7.26 ± 0.01 11.98% 9.87E-05 8.07 ± 0.12 7.14 ± 0.03 13.03% 1.11E-03
9 8
8 8
14 11
11 14
13 10
12 12
12 12
7.90% 0.28 253.67 ± 10.27 215.00 ± 11.55 17.99% 0.02 245.63 ± 8.80 233.75 ± 10.00 5.08% 0.12 181.93 ± 2.99 205.64 ± 2.22 -11.53% 1.92E-06 208.73 ± 6.22 245.17 ± 8.06 -14.86% 1.15E-03 204.77 ± 1.78 230.30 ± 2.04 -11.09% 1.11E-08 148.83 ± 1.80 187.50 ± 1.87 -20.62% 9.34E-14 116.67 ± 1.32 197.92 ± 2.17 -41.05% 1.94E-17
All data are given as mean ± SEM. P values are obtained by Student’s t tests between the positive and negative plants in the same family.
Supplementary
Table
2
Grain
length
of
transgene-positive
and
transgene-negative segregants in T1 families in DHX background. Grain size Constructs
Positive (mm)
Negative (mm)
P
DEP1OE
9.01 ± 0.13 (n = 9)
8.11 ± 0.05 (n = 6)
9.19E-05
11.09%
AGG3OE
6.40 ± 0.06 (n = 10)
7.94 ± 0.23 (n = 6)
7.93E-04
-19.31%
AGG3OSROE
5.22 ± 0.06 (n = 25)
8.42 ± 0.06 (n = 20)
1.34E-33
-38.03%
ZmGS3OE
6.51 ± 0.10 (n = 10)
8.10 ± 0.04 (n = 6)
8.62E-09
-19.61%
ZmGS3OSROE
5.07 ± 0.05 (n = 8)
8.34 ± 0.09 (n = 6)
1.24E-09
-39.20%
GmDEP1OE
5.61 ± 0.05 (n = 26)
8.61 ± 0.09 (n = 8)
1.59E-12
-34.84%
GS3-1OE
7.32 ± 0.06 (n = 10)
8.24 ± 0.05 (n = 12)
1.83E-10
-11.15%
GS3-4OE
5.44 ± 0.03 (n = 10)
8.05 ± 0.08 (n = 12)
1.71E-14
-32.43%
variationa
a
Extent of grain length variation of the transgene positive plants relative to the
negative plants. b
All data are given as mean ± SEM. P values are obtained by Student’s t tests between
the positive and negative plants in the same family.
