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 Beijing 4 W01 Beijing area, PRCGlycine soja Donggou 16 W02 Liaoning, PRCGlycine soja Wuhai 4 W03 Inner Mongolia, PRCGlycine soja Yanjin 3 W04 Henan, PRCGlycine soja Mengjin 1 W05 Henan, PRCGlycine soja Jidong 5 W06 Heilongjiang, PRCGlycine soja Fengcheng 20 W07 Liaoning, PRCGlycine soja Jixian 11 W08 Heilongjiang, PRCGlycine soja Kaiyuan 21 W09 Liaoning, PRCGlycine soja Shuangcheng 4 W10 Heilongjiang, PRCGlycine soja Taiyuan 67 W11 Shanxi, PRCGlycine soja Anqing 18 W12 Anhui, PRCGlycine soja Yimeng 15 W13 Inner Mongolia, PRCGlycine soja Yimeng 19 W14 Inner Mongolia, PRCGlycine soja Yanjin 4 W15 Henan, PRCGlycine soja Hailun 19 W16 Heilongjiang, PRCGlycine soja Zhangwu 20 W17 Liaoning, PRCGlycine max Wenfeng 7 C01 Shandong, PRCGlycine max Tiefeng 8 C02 Liaoning, PRCGlycine max Union C08 USAGlycine max Jindou 21 C12 Shanxi, PRCGlycine max Brazil 10 C14 BrazilGlycine max Tainong 1 C16 TaiwanGlycine max Zigongdongdou C17 Sichuan, PRCGlycine max Jilinxiaoli 1 C19 Jilin, PRCGlycine max Gandou 4 C24 Jiangxi, PRCGlycine max Cangdou 5 C27 Hebei, PRCGlycine max Yudou 12 C30 Henan, PRCGlycine max Hefeng 25 C33 Heilongjiang, PRCGlycine max Gui 199 C34 Guangxi, PRCGlycine max Guangzhoudali C35 Guangdong, PRCGlycine max Williams 82 C36 USAGlycine max Lee 68 C37 USAGlycine max Aijiaozao C38 Hebei, PRC

Glycine max Lee C39 USAGlycine max Pingan dou 8 C40 Jilin, PRCGlycine max Jilin landrace C41 Jilin, PRCGlycine max Linshuijiasedou C42 Sichuan, PRCGlycine max Brazil 11 C43 BrazilGlycine max Bendi 1 C44 Guangdong, PRCGlycine max Jack C45 USAGlycine max Zhonghuang 30 C46 Hebei, PRCGlycine max Shidadou 2 C47 Heilongjiang, PRCGlycine max Jindou 19 C48 Shanxi, PRCGlycine max Beidou 26 C49 Heilongjiang, PRCGlycine max Heihe 43 C50 Heilongjiang, PRCGlycine max Huajiang 4 C51 Heilongjiang, PRCGlycine max Jindou 23 C52 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.

Cultivated/Wild Origin of breeding

Soybean accession –

rhizobium species

pairing

Total N per

plant (mg)

Total

ureides

per plant

(µmol)

Total

plant

fresh

weight

(g)

Total

plant dry

weight (g)

Total

root

fresh

weight

(g)

Total

nodule

fresh

weight (g)

Nodule

number

per plant

Cultivated China

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

Cultivated China

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

Cultivated

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

Non-China

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

Cultivated Non-China

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

Wild

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

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# 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.