Supplementary Figure S1 Total ureides of cultivated (C08), wild (W05) and
non-nodulating soybean (nod139). The figure shows the low level of ureides
detected on the non-nodulating soybean compared to the other two soybean cultivars
upon interactions with a fast- [S (E). fredii CCBAU45436] and a slow- (B.
diazoefficiens sp. nov. USDA110) growing rhizobium, demonstrating the potential of
this method for estimating the ureides content from biological nitrogen fixation.
Supplementary Figure S2 Electron microscopic study of the nodules. (a)
Electron microscopic images of the nodules of three wild soybean accessions, W03,
W08 and W10 upon inoculations with B. diazoefficiens sp. nov. USDA110 or S (E).
fredii CCBAU45436. Nodules were collected and fixed 28 days post-inoculation. (b)
Total nitrogen content per plant, total ureides per plant and total dry weight per plant
of 4 wild soybean accessions with reference to the cultivated soybean, C08. PHB:
poly-3-hydroxybutyrate bodies.
Supplementary Figure S3 Performance of parental lines of the RI population.
Figures showing total plant fresh weight, total nodule fresh weight, total root fresh
weight and total ureides per plant (µmol) of W05 and C08 inoculated with B.
diazoefficiens sp. nov. USDA110. Asterisks indicate significant differences using
t-test at p<0.05. N=9 per replicate.
Supplementary Figure S4. Distribution of parameters analysed on 96 RI lines
from two independent biological replicates. Nine plants per line were analysed in
each replicate.
Table S1: Seventeen wild (Glycine soja Sieb. and Zucc.) and 31 cultivated (Glycine
max (L.) Merr.) soybeans used in this work with variety names and accession codes.
Species
Variety name
Code
Origin
Glycine soja
Glycine soja
Glycine soja
Glycine soja
Glycine soja
Glycine soja
Glycine soja
Beijing 4
Donggou 16
Wuhai 4
Yanjin 3
Mengjin 1
Jidong 5
Fengcheng 20
W01
W02
W03
W04
W05
W06
W07
Beijing area, PRC
Liaoning, PRC
Inner Mongolia, PRC
Henan, PRC
Henan, PRC
Heilongjiang, PRC
Liaoning, PRC
Glycine soja
Glycine soja
Glycine soja
Glycine soja
Glycine soja
Glycine soja
Glycine soja
Glycine soja
Glycine soja
Glycine soja
Jixian 11
Kaiyuan 21
Shuangcheng 4
Taiyuan 67
Anqing 18
Yimeng 15
Yimeng 19
Yanjin 4
Hailun 19
Zhangwu 20
W08
W09
W10
W11
W12
W13
W14
W15
W16
W17
Heilongjiang, PRC
Liaoning, PRC
Heilongjiang, PRC
Shanxi, PRC
Anhui, PRC
Inner Mongolia, PRC
Inner Mongolia, PRC
Henan, PRC
Heilongjiang, PRC
Liaoning, PRC
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Wenfeng 7
Tiefeng 8
Union
Jindou 21
Brazil 10
Tainong 1
Zigongdongdou
Jilinxiaoli 1
Gandou 4
Cangdou 5
C01
C02
C08
C12
C14
C16
C17
C19
C24
C27
Shandong, PRC
Liaoning, PRC
USA
Shanxi, PRC
Brazil
Taiwan
Sichuan, PRC
Jilin, PRC
Jiangxi, PRC
Hebei, PRC
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Yudou 12
Hefeng 25
Gui 199
Guangzhoudali
Williams 82
Lee 68
Aijiaozao
Lee
C30
C33
C34
C35
C36
C37
C38
C39
Henan, PRC
Heilongjiang, PRC
Guangxi, PRC
Guangdong, PRC
USA
USA
Hebei, PRC
USA
Glycine max
Pingan dou 8
C40
Jilin, PRC
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Jilin landrace
Linshuijiasedou
Brazil 11
Bendi 1
Jack
Zhonghuang 30
Shidadou 2
Jindou 19
Beidou 26
Heihe 43
C41
C42
C43
C44
C45
C46
C47
C48
C49
C50
Jilin, PRC
Sichuan, PRC
Brazil
Guangdong, PRC
USA
Hebei, PRC
Heilongjiang, PRC
Shanxi, PRC
Heilongjiang, PRC
Heilongjiang, PRC
Glycine max
Glycine max
Huajiang 4
Jindou 23
C51
C52
Heilongjiang, PRC
Shanxi, PRC
Table S2. Averages of each parameter of each soybean accession upon interactions with fast- (S (E). fredii CCBAU45436) and slow- (B.
diazoefficiens sp. nov. USDA110) growing rhizobia. Different letters indicate a significant difference between the rhizobium-soybean pairings,
using t-test (p<0.05). N=8 to 12.
Total
Soybean accession –
Cultivated/Wild
Origin of breeding
Total
Total
Total
plant
Total N per
ureides
plant (mg)
per plant
rhizobium species
root
plant dry
fresh
number
fresh
weight
weight (g)
weight
(µmol)
per plant
weight (g)
(g)
Cultivated
Nodule
nodule
fresh
pairing
Total
(g)
C01 - USDA110
10.37
0.626
2.939
0.437
1.360
0.117 a
17.87 a
C01 - CCBAU45436
9.54
0.473
2.681
0.392
1.313
0.062 b
11.5 b
C02 - USDA110
10.46
0.843
3.517
0.442
1.621
0.110
14.00
C02 - CCBAU45436
9.85
0.630
3.320
0.382
1.565
0.086
7.17
C12 - USDA110
11.68
1.891
4.053
0.448
1.998
0.130
12.38
C12 - CCBAU45436
11.30
2.094
3.559
0.408
1.736
0.111
12.13
C16 - USDA110
13.92
4.161
4.279
0.561
1.549
0.243
27.88
C16 - CCBAU45436
11.57
1.391
3.836
0.541
1.524
0.249
21.50
C17 - USDA110
14.45
4.969
4.278
0.512
1.931
0.156
19.88
C17 - CCBAU45436
14.98
4.719
4.123
0.575
1.954
0.144
17.50
C19 - USDA110
8.71
1.113 a
2.560
0.373
1.090
0.088
11.38
C19 - CCBAU45436
7.64
0.671 b
2.341
0.351
1.079
0.070
10.50
C27 - USDA110
13.70
2.219 a
4.555
0.573
2.060
0.161
18.13
C27 - CCBAU45436
15.39
0.597 b
4.053
0.550
1.866
0.157
17.63
China
Cultivated
C30 - USDA110
13.71
3.135
3.593
0.497
1.595
0.146
20.88
C30 - CCBAU45436
15.00
1.937
3.671
0.509
1.606
0.139
22.50
C33 - USDA110
10.40
1.530 a
3.169
0.355
1.293
0.107
23.86
C33 - CCBAU45436
9.57
0.283 b
3.131
0.418
1.256
0.135
20.00
C34 - USDA110
14.18
5.385
4.107 a
0.549
1.456
0.160
23.57 a
C34 - CCBAU45436
13.48
4.320
3.655 b
0.542
1.416
0.152
28.12 b
C35 - USDA110
14.73
2.031
4.178 a
0.578 a
1.403
0.149 a
19.87 a
C35 - CCBAU45436
12.76
1.463
3.225 b
0.450 b
1.254
0.115 b
15.12 b
C38- USDA110
14.10
3.604
4.042
0.532
1.318
0.208
38.67 a
C38- CCBAU45436
14.77
7.416
3.887
0.488
1.247
0.198
27.33 b
C42- USDA110
11.51
3.948
3.147
0.404
1.005
0.155
41.16 a
C42- CCBAU45436
10.20
2.805
3.268
0.411
1.122
0.162
22.66 b
C44 - USDA110
15.36
3.974
4.183
0.535
1.388
0.191
44.87 a
C44 - CCBAU45436
14.23
5.632
4.289
0.517
1.609
0.194
22.75 b
C46 - USDA110
13.70
9.264 a
4.279
0.547
1.595
0.216
51 a
C46 - CCBAU45436
12.91
1.511 b
4.389
0.579
1.917
0.177
28.87 b
C50 - USDA110
13.33
1.357 a
4.203
0.560
1.592 a
0.162 a
40.62 a
C50 - CCBAU45436
13.26
0.494 b
4.588
0.567
1.984 b
0.124 b
20.50 b
C51 - USDA110
11.55
1.473 a
4.003
0.540
1.550
0.120 a
41.25 a
C51 - CCBAU45436
9.76
0.293 b
3.779
0.460
1.622
0.145 b
20.25 b
C24 - USDA110
14.17 a
2.143
4.548
0.621
1.710
0.184
19.63
C24 - CCBAU45436
16.99 b
1.361
4.388
0.666
1.734
0.200
21.13
China
C40 - USDA110
11.07 a
6.886 a
3.094
0.366
1.115 a
0.141 a
28.12 a
C40 - CCBAU45436
7.12 b
0.265 b
2.759
0.355
1.292 b
0.080 b
8.62 b
C41 - USDA110
6.88 a
1.344
2.370 a
0.307 a
0.916 a
0.092 a
20.00
C41 - CCBAU45436
3.60 b
0.118
1.579 b
0.217 b
0.721 b
0.056 b
14.50
C47 - USDA110
13.98 a
4.864 a
3.608
0.457
1.318
0.131
39.25
C47 - CCBAU45436
9.47 b
0.416 b
3.473
0.438
1.477
0.141
30.25
C48 - USDA110
13.80 a
2.966 a
4.508
0.537
1.969 a
0.202 a
30.62 a
C48 - CCBAU45436
11.11 b
0.577 b
4.513
0.533
2.281 b
0.133 b
13 b
C49 - USDA110
13.31 a
4.065 a
3.570
0.481
1.332
0.151
35.00
C49 - CCBAU45436
10.76 b
0.959 b
3.496
0.482
1.436
0.156
28.00
C52 - USDA110
15.79 a
2.384
5.035
0.600
2.196
0.182 a
30.00
C52 - CCBAU45436
12.93 b
1.355
4.755
0.559
2.251
0.152 b
20.75
C14 - USDA110
12.77
3.765 a
3.710 a
0.484
1.628 a
0.149
26.25 a
C14 - CCBAU45436
9.93
1.703 b
3.223 b
0.486
1.458 b
0.160
19.625 b
C36 - USDA110
14.48
3.365
4.100
0.587
1.523 a
0.157
42.25
C36 - CCBAU45436
13.81
3.795
4.093
0.591
1.689 b
0.155
36.14
C45 - USDA110
13.30
2.768 a
3.634
0.506 a
1.413 a
0.155
39.13
C45 - CCBAU45436
10.89
0.526 b
4.259
0.657 b
1.920 b
0.147
35.71
C08 - USDA110
15.43 a
4.274
4.325
0.610
1.810
0.168
25 a
C08 - CCBAU45436
11.50 b
2.337
4.121
0.628
1.729
0.189
18.5 b
C37 - USDA110
14.11 a
4.141 a
3.683
0.503 a
1.092 a
0.177
45.75 a
C37- CCBAU45436
11.83 b
1.075 b
4.135
0.641 b
1.502 b
0.185
23.5 b
China
Cultivated
Non-China
Cultivated
C39- USDA110
13.56 a
2.795 a
3.585
0.509
1.082 a
0.177 a
39 a
C39- CCBAU45436
10.51 b
0.571 b
4.124
0.584
1.542 b
0.109 b
14.87 b
C43 - USDA110
9.90 a
0.730 a
3.144 a
0.398
1.168
0.134 a
28.62 a
C43 - CCBAU45436
6.82 b
0.340 b
2.544 b
0.358
1.094
0.087 b
11.87 b
W03 - USDA110
6.39
0.961
1.771
0.240
0.790
0.091
17.12 a
W03 - CCBAU45436
5.76
1.319
1.926
0.258
0.902
0.093
9.91 b
W04 - USDA110
8.70
2.114 a
2.338
0.386
0.889
0.129
32.06 a
W04 - CCBAU45436
7.23
0.762 b
2.127
0.342
0.794
0.097
13.30 b
W05 - USDA110
6.80
1.313
1.907
0.293
0.835
0.089
19.46 a
W05 - CCBAU45436
6.46
0.839
1.859
0.268
0.834
0.079
11.60 b
W06 - USDA110
8.40
1.973 a
2.361
0.299
1.136
0.169
23.69
W06 - CCBAU45436
7.24
0.795 b
2.450
0.324
1.236
0.130
20.00
W07 - USDA110
4.04
0.790
1.107
0.160
0.451
0.058
19.85 a
W07 - CCBAU45436
3.86
0.512
1.207
0.183
0.508
0.062
10.71 b
W09 - USDA110
4.11
0.725 a
1.178
0.165
0.462
0.061
18.85 a
W09 - CCBAU45436
3.53
0.397 b
1.219
0.174
0.536
0.059
11.86 b
W10 - USDA110
6.82
1.970 a
2.112
0.274
0.898 a
0.119
29.53 a
W10 - CCBAU45436
5.85
0.759 b
2.177
0.301
1.008 b
0.129
22.71 b
W11 - USDA110
5.14
0.755
1.398
0.188
0.565
0.081
17.26 a
W11 - CCBAU45436
5.26
0.978
1.423
0.193
0.577
0.071
10.18 b
W13 - USDA110
4.46
0.684
1.526
0.204
0.722
0.084
17.71
W13 - CCBAU45436
4.38
0.998
1.583
0.207
0.779
0.074
10.27
Non-China
Wild
W17 - USDA110
5.09
0.658
1.557
0.200
0.694
0.082
25.21
W17 - CCBAU45436
4.75
0.900
1.435
0.188
0.670
0.071
19.43
W01 - USDA110
9.52 a
2.761 a
2.744
0.364
1.270 a
0.148 a
28.68 a
W01 - CCBAU45436
7.63 b
0.768 b
2.965
0.397
1.525 b
0.086 b
12.66 b
W02 - USDA110
5.34 a
0.826
1.822
0.302
0.693
0.083
13.60
W02 - CCBAU45436
4.33 b
0.542
1.634
0.281
0.639
0.070
10.64
W08 - USDA110
8.08 a
2.335 a
2.269
0.311
0.947 a
0.101 a
23.13
W08 - CCBAU45436
6.04 b
0.578 b
2.306
0.348
1.057 b
0.135 b
18.58
W12 - USDA110
2.23 a
0.330
0.799 a
0.119
0.353
0.040
17.67
W12 - CCBAU45436
2.93 b
0.266
0.916 b
0.129
0.367
0.042
15.00
W14 - USDA110
4.58 a
0.703
1.471 a
0.194 a
0.685 a
0.080
19.07
W14 - CCBAU45436
5.92 b
0.663
1.887 b
0.257 b
0.867 b
0.093
13.73
W15 - USDA110
7.20 a
0.839 a
2.207
0.336
0.948
0.120
30.19
W15 - CCBAU45436
5.61 b
0.519 b
1.919
0.307
0.829
0.095
23.73
W16 - USDA110
2.10 a
0.202
0.843 a
0.107
0.422
0.038
15.64
W16 - CCBAU45436
3.10 b
0.443
1.026 b
0.118
0.491
0.044
13.70
Wild
#
Germplasms highlighted in pale blue are cultivated germplasms bred in China which showed no difference in total nitrogen per plant between being inoculated with USDA110 and with CCBAU45436.
Germplasms highlighted in blue are cultivated germplasms bred in China which showed significant difference in total nitrogen per plant between being inoculated with USDA110 and with CCBAU45436
Germplasms highlighted in pink are non-China bred cultivated germplasms which showed no difference in total nitrogen per plant between being inoculated with USDA110 and with CCBAU45436.
Germplasms highlighted in red are non-China bred cultivated germplasms which showed significant difference in total nitrogen per plant between being inoculated with USDA110 and with CCBAU45436.
Germplasms highlighted in pale green are wild soybean germplasms showed no difference in total nitrogen per plant between being inoculated with USDA110 and with CCBAU45436.
Germplasms highlighted in green are wild soybean germplasms showed significant difference in total nitrogen per plant between being inoculated with USDA110 and with CCBAU45436.