SupplementaryMaterials
Reduced weed seed shattering by silencing a cultivated rice gene:strategic mitigation for escaped transgenes
Huanxin Yan, Lei Li, Ping Liu, Xiaoqi Jiang, Lei Wang, Jia Fang, Zhimin Lin, Feng Wang, Jun Su, Bao-Rong Lu
Supplementary Figure 1.Structure of anti-SH4 vectors. (a) Artificial microRNA vector MR1. (b) Artificial microRNA vector MR2. (c) Antisense RNA vector AS. Tnos: terminator of nopaline synthetase; HYG(R): hygromycin-resistance gene hpt; 35S: cauliflower mosaic virus (CaMV) 35S promoter; ShMR: artificial microRNA-based specific silencing fragment on SH4; Ubi: maize Ubiquitin gene promoter; pSH4: rice SH4 gene promoter; SH4: rice seed shattering gene SH4; LB: left border; RB: right border.
Supplementary Table 1.The design of the anti-SH4 genes, including their position in SH4 mRNA and PCR primers for amplification
Anti-SH4 gene / Position in SH4 mRNA / Primers for amplifying anti-SH4 genesShMR / -71 ~ -51 / mir-s 5’-agtactggcgagagccaaggcaacaggagattcagtttga
mir-a 5’-tgttgccttggctctcgccagtactgctgctgctacagcc
mir*s 5’-ctttgccatgggtctcgccagtattcctgctgctaggctg
mir*a 5’-aatactggcgagacccatggcaaagagaggcaaaagtgaa
oligoA 5’-ctgcaaggcgattaagttgggtaac
oligoB 5’-gcggataacaatttcacacaggaaacag
ShAS / 6 ~ 1175 / sh4mr-a 5’-gggctcctctgccgaccc
sh4mr-b 5’-gatcatcggccggaggagtc
Supplementary Table 2.Primers for identifying anti-SH4 genes
Primer / SequencemiR5-F / 5’-agcagcagcagccacagcaaa
miR5-R / 5’-gctgctgatgctgatgccat
sh4-F / 5’-tctgccgacccctcgccatc
sh4-R / 5’-gaggcagccgttcttccagc
Supplementary Table 3.Hygromycinresistance analyses of the anti-SH4 (MR1, MR2 and AS) rice lines
Transgenes / Hygromycinresistance / 2 value† / P valueResistant / Susceptible
MR1 / 134 / 43 / 0.047 / 0.828
MR2 / 134 / 42 / 0.121 / 0.728
AS / 81 / 22 / 0.728 / 0.393
†2 analysis was performed to test whether the segregation ratios of the anti-SH4 genes (T1 generation)were close to the expected ratio 3:1 as one-copy of the transgene, and P values were not significant.
Supplementary Table 4.One-way ANOVA to determine the effect of anti-SH4 transgenes (MR1, MR2 and AS) on seed shattering in F1 and F2 crop-weed hybrid lineages with two weedy rice accessions (Wd-1292 and Wd-7125). N = 6 plots (transgene-present and -absent) for F1 and10 plots(transgene-present and -absent) for F2 treatments
Seed shattering / Transgenes*MR1 / MR2 / AS
df / F / P / df / F / P / df / F / P
F1(Wd-1292) / 1 / 54.693 / 0.002 / 1 / 22.313 / 0.009 / 1 / 11.284 / 0.025
F2(Wd-1292) / 1 / 5.404 / 0.049 / 1 / 40.988 / <0.001 / 1 / 13.775 / 0.006
F1(Wd-7125) / 1 / 18.517 / 0.013 / 1 / 74.260 / 0.001 / 1 / 38.089 / 0.004
F2(Wd-7125) / 1 / 12.481 / 0.008 / 1 / 14.109 / 0.005 / 1 / 23.091 / 0.001
* df, degrees of freedom; F, F value; P, P value.
Supplementary Table 5.The breaking tensile strength (BTS) and seed shattering index in the transgene-present (TM) and -absent (NT) F1 and F2 hybrid lineages derived from crosses between the anti-SH4 rice (MR1, MR2 and AS) lines and two weedy rice accessions (Wd-1292 and Wd-7125)
Combination / Transgene / BTS (gf) / Seed shattering index (%) / Relative seed shattering index(%)†Crossingparents
MR1 / TM / 150.29.5 a / - / -
MR2 / TM / 151.612.2 a / - / -
AS / TM / 153.93.0 a / - / -
MH86 / NT / 145.67.1 a / - / -
F1 hybrids
Crop × Wd-1292
MR1 / TM / 117.2 2.0 b / 5.36 0.26 a / 41%
MR2 / TM / 116.4 5.9 b / 5.57 0.83 a / 43%
AS / TM / 120.5 10.6 b / 5.30 1.52 a / 41%
MH86 / NT / 82.0 4.3 a / 13.02 1.39 b / 100%
Crop × Wd-7125
MR1 / TM / 107.4 1.7 a / 6.83 0.30 b / 68%
MR2 / TM / 131.1 3.3 b / 3.80 0.32 a / 38%
AS / TM / 133.4 5.9 b / 3.63 0.50 a / 36%
MH86 / NT / 92.1 3.1 a / 10.07 0.80 c / 100%
F2 progeny
Crop × Wd-1292
MR1 / TM / 62.5 4.6 * / 21.512.32 * / 70%
NT / 73.9 3.1 / 15.97 1.22 / 100%
MR2 / TM / 57.0 2.1 *** / 24.16 1.35 *** / 57%
NT / 48.0 4.1 / 30.742.88 / 100%
AS / TM / 51.3 1.7 ** / 27.811.14 ** / 77%
NT / 46.5 1.9 / 31.431.47 / 100%
Crop × Wd-7125
MR1 / TM / 79.9 2.7 ** / 13.70 0.97 ** / 74%
NT / 87.8 3.8 / 11.33 1.07 / 100%
MR2 / TM / 75.2 1.6 ** / 15.310.61 ** / 62%
NT / 67.9 2.1 / 18.43 0.98 / 100%
AS / TM / 68.4 3.5 ** / 18.35 1.53** / 65%
NT / 58.5 3.1 / 23.45 1.78 / 100%
†The relative index of seed shattering was calculated by the seed shattering index of the TM plants relative to that of their corresponding NT ones, respectively.The means standard error between TM and NT plants were compared (N = 3 plots for parents and F1, and N = 5 plots for F2). Different letters indicate significant differences at P < 0.05 based on ANOVA; *, **, *** indicate significant differences at P < 0.05, P< 0.01, and P< 0.001, respectively, based on independent ttest.
Supplementary Table 6.The number of seeds in the soil seed-bank per m2 in the field plots grown transgene-present (TM) and transgene-absent (NT) F1 and F2 hybrid lineages of anti-SH4 cultivated rice (MR1, MR2 and AS) crossed with weedy rice Wd-1292
Combination / Transgene / Number of seeds in the soil seed-bank/m2 / Relative number of seeds in the soil seed bank(%)†MR1 / TM / 266.7 51.8 * / 48%
NT / 554.4 111.8 / 100%
MR2 / TM / 241.7 44.2 * / 44%
NT / 543.3 92.1 / 100%
AS / TM / 256.7 39.9 * / 50%
NT / 518.3 72.5 / 100%
†The relative number of seeds in the soil seed bankwas calculated by the number of seeds in the soil seed bank of field plots grownTM plants relative to that of their corresponding NT ones, respectively. The means standard error from TM and NT plants were compared (N = 5 plots), * indicates significant differences at P < 0.05 based on independent ttest.
Supplementary Table 7.One-way ANOVA to determine the effect of anti-SH4 transgenes (MR1, MR2 and AS) on the productivity-related traits in cultivated rice, F1 and F2 crop-weed hybrid lineages. Only hybrid lineages with Wd-1292 were included for analysis. N = 10 plots(transgene-present and -absent) for cultivated rice, F1 and F2 treatments
Traits / Transgenes*MR1 / MR2 / AS
df / F / P / df / F / P / df / F / P
Cultivated rice
Number of seeds / 1 / 0.309 / 0.608 / 1 / 0.701 / 0.450 / 1 / 1.096 / 0.354
1,000 grain weight / 1 / 0.728 / 0.442 / 1 / 0.132 / 0.735 / 1 / 2.351 / 0.200
Seed germination / 1 / 0.199 / 0.668 / 1 / 1.900 / 0.205 / 1 / 0.281 / 0.610
Seedling survival / 1 / 0.449 / 0.522 / 1 / 1.000 / 0.347 / 1 / 1.704 / 0.228
F1
Number of seeds / 1 / 0.040 / 0.851 / 1 / 0.387 / 0.567 / 1 / 0.671 / 0.459
1,000 grain weight / 1 / 0.055 / 0.826 / 1 / 0.002 / 0.968 / 1 / 5.896 / 0.072
Seed germination / 1 / 0.449 / 0.522 / 1 / 1.000 / 0.347 / 1 / 1.704 / 0.228
Seedling survival / 1 / 0.288 / 0.606 / 1 / 0.015 / 0.906 / 1 / 0.044 / 0.838
F2
Number of seeds / 1 / 0.175 / 0.686 / 1 / 0.445 / 0.524 / 1 / 0.008 / 0.524
1,000 grain weight / 1 / 0.004 / 0.949 / 1 / 4.379 / 0.070 / 1 / 1.021 / 0.070
Seed germination / 1 / 4.231 / 0.074 / 1 / 2.316 / 0.055 / 1 / 4.023 / 0.167
Seedling survival / 1 / 5.161 / 0.053 / 1 / 2.176 / 0.053 / 1 / 4.080 / 0.178
* df, degrees of freedom; F, F value; P, P value.
Supplementary Table 8.The relative expression of SH4 in the transgene-present (TM) and transgene-absent (NT) of cultivate/weedy rice, F1 and F2 hybrid lineages of anti-SH4 cultivated rice (MR1, MR2 and AS)crossed with weedy rice
Combination / Transgene / Relative expression of SH4†Cultivated rice
MR1 / TM / 0.15 0.03
MR2 / TM / 0.16 0.02
AS / TM / 0.13 0.06
MH86 / NT / 1.00 0.04
Weedy rice
Wd-1292 / - / 9.99 0.96
Wd-7125 / - / 4.29 0.42
F1 hybrids
Crop × Wd-1292
MR1 / TM / 1.46 0.13
MR2 / TM / 1.23 0.16
AS / TM / 0.99 0.11
MH86 / NT / 2.29 0.20
Crop × Wd-7125
MR1 / TM / 0.93 0.18
MR2 / TM / 0.56 0.07
AS / TM / 0.71 0.16
MH86 / NT / 1.72 0.39
F2 progeny
Crop × Wd-1292
MR1 / TM / 0.97 0.40
NT / 5.92 1.27
MR2 / TM / 0.74 0.14
NT / 4.86 0.23
AS / TM / 1.57 0.27
NT / 6.26 0.89
Crop × Wd-7125
MR1 / TM / 0.57 0.12
NT / 3.28 1.30
MR2 / TM / 0.69 0.26
NT / 1.97 0.56
AS / TM / 1.30 0.40
NT / 2.96 1.53
†The means standard error of relative expression of SH4 (N = 3 replicates) wherethe relative expression of MH86 was set as control.