1. Fig. S1 Figure S1. Multiple alignment of the selected OSC sequences from Barbarea vulgaris and Arabidopsis thaliana, using the software Muscle; this alignment was used to construct the phylogenetic tree in Fig 2.

2. Fig. S1, continued

3. Bv1460 Bv14724 Bv14843 Bv18 69 13 Bv1520 Bv6614 Bv4315 Bv1621 Bv2426 Bv15334 14 1.4 0.1 15.0 15.8 12.8 17.0 16.9 16.5 5.8 Mb chromosome 4 A. thaliana Bv1540 Bv4110 Bv15515 p10m50-43318 p11m48-8124 Bv15629 p40m49-22039 p41m49-30258 p41m60-49266 p41m60-13085 p15m60-7597 p15m60-76101 p15m60-292107 15 CYP716A 0 Bv15918 Bv16126 16 Bv1650 Bv16714 Bv626 17 23.4 26.4 3.5 6.4 3.5 4.0 16.3 16.5 Mb 14.2 chromosome 5 A. thaliana CYP72A8 Bv70Bv740 Bv5611 Bv3213 Bv7818 Bv8321 Bv2924 Bv3426 28 Bv1733 Bv12442 Bv6453 10 Bv1330 Bv2012 p35m62-10919 p14m59-24425 Bv11327 p14m59-37235 p41m60-12639 Bv10752 p41m49-11566 11 Bv1100 p10m50-1078 p40m49-46617 p40m49-47529 12 9.8 15.5 23.4 1.3 0.8 1.4 1.8 3.5 2.8 4.7 4.4 5.3 Mb 4.9 chromosome 3 A. thaliana AB CD 1 2 3 4 dominance p41m60-2150 Bv1239 p38m49-13521 p35m62-13926 p14m48-22729 p16m49-34132 Bv6533 Bv3640 p14m59-38343 p14m48-39450 Bv15 52 Bv11658 p41m60-19464 570 p38m49-27589 p40m49-178114 1 p40m49-1370 p41m49-1382 Bv12114 p10m50-11720 Bv3528 Bv12635 2 Bv120 Bv51-212 Bv87-221 Bv12835 Bv87-148 Bv8650 Bv89-164 3 LUP5 0 Bv89-214 Bv9633 Bv31 47 4 5 Bv600 Bv63-15 p40m49-26611 p11m59-25112 LUP2 17 p40m49-26527 p16m49-13834 p38m49-19143 9.0 7.5 4.8 4.1 0.6 18.9 20.7 20.1 22.9 22.8 23.9 23.3 23.9 22.8 25.1 25.0 25.1 25.4 29.7 Mb 28.3 25.7 chromosome 1 A. thaliana p10m50-4210 Bv13521 6 p15m60-1330 1220 p14m47-20140 7 8 Bv1370 Ra1229 Bv13936 UGT73C 45 Bv14550 Bv14354 Bv14161 Bv2365 9 7.8 16.1 15.3 19.6 18.2 15.4 12.0 13.5 9.5 9.8 Mb 7.1 chromosome 2 A. thaliana Bv270 20 31 Bv51-142 Bv136 2S dominance A BCD 2R dominance QTL for P-type saponins QTL for glucosinolates QTL for G-type saponins A. Hederagenin cellobioside B. Oleanolic acid cellobioside C. Gypsogenin cellobioside D. 4-Epihederagenin cellobioside 1-4 QTL for flea beetle resistance Fig. S2 Figure S2. B. vulgaris QTL map showing 17 linkage groups by vertical bars, aligned to the five A. thaliana chromosomes. QTLs for flea beetle resistance, saponins of the G- and P-type, and glucosinolates are indicated by vertical colored bars, and positions of the six mapped genes for saponin biosynthesis by circles. Genetic distances (cM) are listed at the left of each linkage group and genetic markers (AFLP, SSR) at the right; corresponding marker positions on the A. thaliana chromosomes are indicated in millions of base pairs (Mb). QTLs for the four unknown saponins from the P-type are marked 1 to 4; see Kuzina et al. (2011) for more details; two of these were dominant, as were two QTLs for glucosinolates. Confidence intervals, explained variation and position of the maximal LOD score are in Table S2. AFLP markers are designated with the MseI (m) and PstI (p) primer combinations followed by molecular weight (bp).

4. 55.1 73.1 95.1 129.1 147.1 175.1 189.2 203.2 218.2 257.2 279.2408.4 498.5 0 20 40 60 80 100 [%] 100200300400500600700 m/z Relative Abundance RT: 22.50 min 55.1 75.1 95.1 129.1 147.1 175.1 189.2 218.2 231.2 279.2 361.2 408.4 498.5 0 20 40 60 80 100 [%] 100200300400500600700 m/z Relative Abundance RT: 22.69 min a b β-amyrin α-amyrin Fig. S3 Figure S3. EI-MS fragmentation patterns and retention times (RT) of trimethylsilyl (TMS) derivatives of 13 authentic triterpene standards and five tentatively identified triterpenoids (unknowns 1-5). Tentatively identified triterpenoids correspond to peak numbers as illustrated in Fig. 3. EI-MS patterns, RT and structures of (a) β-amyrin, (b) α-amyrin, (c) lupeol, (d) erithrodiol, (e) uvaol, (f) betulin, (g) oleanolic acid, (h) oleanolic aldehyde, (i) betulinic acid, (j) betulinic aldehyde, (k) ursolic acid, (l) ursolic aldehyde, (m) hederagenin were compared against authentic standards. Tentative identification of triterpenoids (unknowns 1-5 (n-u)) were based on common EI-MS fragmentation patterns observed for most trimethylsilylated triterpenes and mass spectral comparison with authentic standards. Characteristic fragmentation patterns represented by loss of methyl (-15 m/z), followed by loos of dimethylsilyl oxonium (-75 m/z) and loss of the second methyl (-15 m/z) group from the final trimethylsilylated products (v) and allowed estimation of molecular masses of unknowns 1-5 (Table S8).

5. 55.1 75.1 95.1 109.1 121.1 135.2 175.2 189.2 203.2 218.2 231.2 245.2 257.2 279.2 299.3 325.3 369.4 393.4 483.4 498.5 0 20 40 60 80 100 [%] 100200300400500600700 m/z Relative Abundance RT: 22.73 min 55.1 75.1 91.1 147.1 216.2 281.1 361.1 496.4 567.5 RT: 23.3 min 0 20 40 60 80 100 [%] 100200300400500600700 erythrodiol 55.1 75.1 91.1 147.1 203.2 281.1 361.1 429.2 496.5 567.5 RT: 23.5min 0 20 40 60 80 100 [%] 100200300400500600700 uvaol 55.1 73.1 91.1 147.1 204.1 281.0 361.2 393.3483.4 567.5 RT: 23.6 min 0 20 40 60 80 100 [%] 100200300400500600700 betulin c d lupeol m/z e f Relative Abundance Fig. S3, continued

6. 55.1 73.1 105.1 129.1 189.2 203.2 219.2 241.2 279.2 306.2 320.2 393.4 482.4 585.5 0 20 40 60 80 100 [%] 100200300400500600700 RT: 23.76 min 75.1 105.1 129.1 203.2 279.2 RT: 23.8min 0 20 40 60 80 100 [%] 100200300400500600700 oleanolic aldehyde H 73.1 95.1 129.1 189.2 257.2 292.2 353.3393.4 483.4 585.5 RT: 23.9 min 0 20 40 60 80 100 [%] 100200300400500600700 betulinic acid 55.1 75.1 91.1 129.1 189.2 279.2 383.3 484.5 512.5 RT: 23.9min 0 20 40 60 80 100 [%] 100200300400500600700 betulinic aldehyde H Relative Abundance oleanolic acid m/z Relative Abundance g h i j Fig. S3, continued

7. 55.1 73.1 133.1 203.2 256.2 279.3 320.2 393.4 482.5 585.5 RT: 24.0 min 0 20 40 60 80 100 [%] 100200300400500600700 55.1 75.1 91.1 133.1 203.2 232.2 279.3 RT: 24.1 min 0 20 40 60 80 100 [%] 100200300400500600700 H ursolic aldehyde 55.1 73.1 105.1 147.1 203.2 278.2 320.2 391.3 570.5 24.5 min 0 20 40 60 80 100 [%] 100200300400500600700 hederagenin ursolic acid m/z Relative Abundance k l m Fig. S3, continued

8. 55.1 75.1 129.1 147.1 159.1 190.2 216.2 243.1 279.2 291.2 306.2 331.1 361.2 406.3 496.4 586.6 0 20 40 60 80 100 [%] 100200300400500600700 m/z Relative Abundance RT: 23.09 min 55.1 73.1 91.1 103.1 129.1 147.1 201.2 217.1 243.2 255.2 271.1 291.2 331.2 361.2 379.3 451.4 494.5 505.4 541.5 584.5 0 20 40 60 80 100 [%] 100200300400500600700 m/z Relative Abundance RT: 23.67 min 55.1 73.1 103.1 129.1 147.1 189.2 201.2 214.2 253.2 291.2 361.1 389.4 481.4 494.4 584.5 0 20 40 60 80 100 [%] 100200300400500600 m/z Relative Abundance RT: 23.15 min RT: 23.70 min 73.0 103.0 129.0 201.1 253.1 291.2 389.3 494.4 584.5 0 20 40 60 80 100 [%] 100200300400500 m/z Relative Abundance 600 unknown-1unknown-4 unknown-2 unknown-2 identified in the GC-MS analysis of saponified leaf extracts of the G-type B. vulgaris n o p q Fig. S3, continued,

9. 55.1 73.1 103.1 129.1 147.1 189.1 201.2 214.2 253.2 304.2 361.2389.3 481.4 494.4 584.5 0 20 40 60 80 100 [%] 100200300400500600 m/z Relative Abundance RT: 23.29 min RT: 23.90 min 73.0 103.0 129.0 201.1 253.1 304.2 389.3 494.4 584.5 0 20 40 60 80 100 [%] 100200300400500 m/z Relative Abundance 600 481.4 55.1 73.1 91.1 129.1 147.1 171.1 187.2 238.1 255.1 271.1 304.2 347.2 361.2 391.4 413.4 481.4 508.4 583.4 598.5 0 20 40 60 80 100 [%] 100200300400500600 m/z Relative Abundance RT: 24.20 min RT: 24.90 min 73.0 95.0 129.0 147.0 187.1 238.1 304.1 368.2 413.2 481.4 508.4 557.1 598.4 0 20 40 60 80 100 [%] 100200300400500 m/z Relative Abundance 600 unknown-3 unknown-5 unknown-3 identified in the GC-MS analysis of saponified leaf extracts of the G-type B. vulgaris unknown-5 identified in the GC-MS analysis of saponified leaf extracts of the G-type B. vulgaris r s t u Fig. S3, continued

10. CH 3 - - 15 m/z 499 m/z.. + 484 m/z + - 75 m/z. + 409 m/z CH 3 - 15 m/z + 394 m/z - 360380400420440460480500 0 1 2 3 499.0 393.9 408.9 483.9 365.6 377.3 455.5 425.4 469.6441.3 m/z [%] 55.1 75.1 95.1 147.1 218.2 281.1 361.2 499.0 0 20 40 60 80 100 [%] 100200300400500600700 m/z Zoomed in v Characteristic EI-MS of β-amyrin-tms Relative Abundance Fig. S3, continued

11. Peak 1Peak 2 Peak 3Peak 4 Fig. S4 (a) Figure S4. LC-MS/MS spectra of tentatively identified saponins produced by expressing (a) LUP5G + CYP716A80 + UGT73C11 and (b) LUP2P + CYP716A80 + UGT73C11 in N. benthamiana, as illustrated in Fig. 4 left (LUP5G) and right (LUP2P) panels, respectively. Mass spectra numbers correspond to peak numbers in Fig. 5. Mass spectra were recorded in negative mode, thus proposed aglycone masses correspond to [M – 1] -.

12. Peak 5 Peak 7 Peak 6 Peak 8 Fig. S4 (a)

13. Peak 9 Peak 10 Peak 11 Peak 12 Fig. S4 (a)

14. Peak 13Peak 14 Peak 15 Peak 16 Fig. S4 (a)

15. Peak 17 Peak 18 Peak 19Peak 20 Fig. S4 (a)

16. Peak 21 Peak 22 Fig. S4 (a)

17. Peak 1Peak 2 Peak 3Peak 4 Fig. S4 (b)

18. Peak 7 Peak 8 Peak 5Peak 6 Fig. S4 (b)

19. Peak 11 Peak 12 Peak 9 Peak 10 Fig. S4 (b)

20. Peak 15 Peak 16 Peak 13 Peak 14 Fig. S4 (b)

21. Fig. S5 Saponin aglycone molecular mass of 456(––); 458(––); 472(––) 14161820222426281230 14* 15* 7 2 6 4* 5 8* 1 3 9*9* 6* 12 13* 11 16 10* 14161820222426281230 2* 4* 5 6 7* 8 9 10 11 12 13 14 15 1 3 14161820222426281230 2A (EIC) 1 (TIC) 2 (TIC) 2B (EIC) 2C (EIC) 2D (EIC) 1 3* 4 6* 7*7* 9 10* 11* 8 12 14 15* 16 17* 18* 19 20* 21* 5 2 RT (min) a LUP2G + CYP716A80 + UGT73C11 b LUP2P + CYP716A81 + UGT73C13 c LUP2P + CYP716A80 + UGT73C11 Figure S5. Saponins produced by expression of different combinations of B. vulgaris genes coding for OSCs, P450s and UGTs in N. benthamiana plant leaves. Panels (a-c) show LC-MS profiles for (1) (a) LUP2G, (b) and (c) LUP2P; (2) LUPs in combination with (a) CYP716A80, (b) CYP716A81, and (c) BvCYP716A80; (3) LUPs and CYPs in combination with (a) UGT73C11, (b) UGT73C13, and (c) UGT73C11. (2A) – (3D) EICs of (2) representing saponins with (2A) five sugar moieties (m/z 1266-1268, 1308-1310, 1380-1382, 1418-1420); (2B) four moieties (m/z 1088-1091, 1105-1110, 1145-1147, 1190-1192) (2C) two moieties (m/z 829-831, 901-903, 1564-1566) and (2D) one moiety (m/z 617-619, 666-668, 1236-1238, 1413-1415, 1408-1411). The y axis (ion count) of each chromatogram is scaled to the highest peak. Peaks highlighted with asterisk (*) correspond to saponins that significantly decreased or disappeared after sodium hydroxide-based saponification, indicating presence of sugar moieties at the C28 position of the triterpene aglycones. See Tables S5-S7 for more detailed information on detected saponins from these enzyme combinations.