New Phytologist Supporting Information for the Article
Relaxation of Tyrosine Pathway Regulation Underlies the Evolution of Betalain Pigmentation in Caryophyllales
Figure S1-S10 and Table S1-S4 Samuel Lopez-Nieves, Ya Yang, Alfonso Timoneda, Minmin Wang, Tao Feng, Stephen A. Smith, Samuel F. Brockington, and Hiroshi A. Maeda
Article acceptance date: August 25, 2017 a 25.3 kbp
BvADHα BvADHβ predicted gene (2781bp) (1160bp) (6845bp) b
BvADHα BvADHβ AtADH1 AtADH2 GmPDH1 AaPDH EcPDH SyADH BvADHα 100 66 66 61 52 18 28 24 BvADHβ 66 100 72 59 54 24 26 25 AtADH1 66 72 100 61 56 22 25 34 AtADH2 61 59 61 100 52 23 23 32 GmPDH1 52 54 56 52 100 23 23 29 AaPDH 18 24 22 23 23 100 21 28 EcPDH 28 26 25 23 23 21 100 23 SyADH 24 25 34 32 29 28 23 100
Figure S1a and b. Physical location, homology, and phylogeny of BvADHα and BvADHβ. (a) The location and physical distance of BvADHα and BvADHβ on the chromosome 8 of the B. vulgaris genome. A nearby gene is indicated in gray. (b) Amino acid identity of ADH and PDH proteins from different plants and bacteria. AaPDH, Aquifex aeolicus; AtADH1 and AtADH2, Arabidopsis thaliana; GmPDH1, Glycine max; EcPDH, Escherichia coli; and SyADH, Synechocystis sp. PCC6803. (c) See the next page. c
Figure S1c. (c) Maximum likelihood phylogeny of BvADHs together with other plant ADHs and PDHs. Previously characterized Arabidopsis ADH and legume PDH enzymes are indicated in green and blue, respectively. Red indicates ADH candidates from Caryophyllales, which are derived from a Caryophyllales-specific duplication event (98%). These Caryophyllales genes fall within in the clade containing Arabidopsis ADH genes. Numbers at branches indicate bootstrap support values. Scale bar indicates inferred number of amino acid substitution per site. d
Figure S1d. (d) Maximum likelihood phylogeny of BvADHs together with other plant ADHs only. Previously characterized Arabidopsis ADH enzymes are indicated in green. Red indicates ADH candidates from Caryophyllales, which are derived from a recent duplication event shown in a red circle. These analyses still allow us to claim that ADHα and ADHβ lineages fall within the ADH clade as they are nested with the eudicot clade. Numbers at branches indicate bootstrap support values. Scale bar indicates inferred number of amino acid substitution per site. 3 1 Chenopodiaceae_Chenopodium_giganteum=Chenopodium_amaranticolor_GREEN@c36044_g1_i1_252_710_minu s 1 3 Chenopodiaceae_Salicornia_europaea_GREEN@GAIA01070440_1_2_460_plus 1 23 0Chenopodiaceae_Atriplex_hortensis_GREEN@64913 5 Chenopodiaceae_Atriplex_rosea_GREEN@9658 Chenopodiaceae_Atriplex_prostrata_GREEN@54973 e 3 8 2 Amaranthaceae_Amaranthus_cruentus_GREEN@10395 Amaranthaceae_Amaranthus_retroflexus_GREEN@65691 2 3 5 Chenopodiaceae_Chenopodium_giganteum=Chenopodium_amaranticolor_GREEN@c28658_g1_i1_2_448_minus Chenopodiaceae_Chenopodium_quinoa_GREEN@25077 100 5 9 9 BvADH_alpha Chenopodiaceae_Beta_vulgaris_GREEN@8_197560_ojzz_t1 BvADHα 2 Chenopodiaceae_Beta_maritima_GREEN@8815 SoADH_alpha 5 5 5 Chenopodiaceae_Salicornia_europaea_GREEN@SRAc14078_g1_i1_2_598_minus SoADHα Chenopodiaceae_Bassia_scoparia_GREEN@4394 Amaranthaceae_Alternanthera_brasiliana_GREEN@20089 2 0 1 8 Amaranthaceae_Blutaparon_vermiculare_GREEN@60141 Amaranthaceae_Froelichia_latifolia_GREEN@c44777_g1_i1_191_1384_plus 0 5 Amaranthaceae_Amaranthus_cruentus_GREEN@13383 74 0 Amaranthaceae_Alternanthera_brasiliana_GREEN@36054 1 Amaranthaceae_Alternanthera_sessilis_GREEN@18746 4 Amaranthaceae_Alternanthera_tenella_GREEN@12702 Amaranthaceae_Alternanthera_brasiliana_GREEN@13635 1 9 6 4 Amaranthaceae_Aerva_lanata_GREEN@6582 2 Amaranthaceae_Alternanthera_caracasana_GREEN@27692 Caryophyllaceae_Polycarpaea_repens_GREEN@10601 2 8Amaranthaceae_Aerva_lanata_GREEN@57613 1 2 Amaranthaceae_Aerva_javanica_GREEN@21266 Amaranthaceae_Alternanthera_tenella_GREEN@90591 1 9 2 8 Achatocarpaceae_Phaulothamnus_spinescens_GREEN@c40402_g1_i1_2_601_plus 1 2 Caryophyllaceae_Spergularia_media_GREEN@61777 1 1 Caryophyllaceae_Polycarpaea_repens_GREEN@68899 TiADH_alpha
ClADH_alpha A 3 5 5 4 Portulacaceae_Portulaca_grandiflora_BLUE@4264 5 4 Portulacaceae_Portulaca_pilosa_BLUE@4346 1 3 5 2 Portulacaceae_Portulaca_suffrutescens_BLUE@43495 Portulacaceae_Portulaca_amilis_BLUE@9163 D H α o r t h l g s 1 0 Portulacaceae_Portulaca_suffrutescens_BLUE@41741 1 1 3 2 Cactaceae_Lophophora_williamsii_BLUE@98034 3 0 Molluginaceae_Mollugo_verticillata_BLUE@163776 Portulacaceae_Portulaca_cryptopetala_BLUE@12629 2 0 7 7 Portulacaceae_Portulaca_molokiniensis_BLUE@25184 Portulacaceae_Portulaca_oleracea_BLUE@3234 6 3 0 Basellaceae_Basella_alba_BLUE@5746 1 Talinaceae_Talinum_sp_BLUE@16801 2 8 2 9 Aizoaceae_Trianthema_portulacastrum_RED@2352 Aizoaceae_Zaleya_pentandra_RED@52470 7 9 4 Aizoaceae_Cypselea_humifusa_RED@2955 6 5 5 4 Aizoaceae_Sesuvium_verrucosum_RED@71095 Aizoaceae_Zaleya_pentandra_RED@2368 Aizoaceae_Sesuvium_portulacastrum_RED@16776 Aizoaceae_Zaleya_pentandra_RED@10923 Microteaceae_Microtea_debilis@175537 7 0 1 4 7 6 Portulacaceae_Portulaca_pilosa_BLUE@44514 9 5 Portulacaceae_Portulaca_suffrutescens_BLUE@44168 Portulacaceae_Portulaca_grandiflora_BLUE@4265 2 3 Portulacaceae_Portulaca_amilis_BLUE@59704 9 9 Basellaceae_Basella_alba_BLUE@26752 5 2 BaADH_alpha 9 9 9 6 Portulacaceae_Portulaca_oleracea_BLUE@8581 7 8 1 6 PoADH_alpha PoADHα Portulacaceae_Portulaca_molokiniensis_BLUE@178 Portulacaceae_Portulaca_molokiniensis_BLUE@18408 Talinaceae_Talinum_sp_BLUE@3485 5 8 5 9 Nyctaginaceae_Boerhavia_burbidgeana_RED@18081 1 3 Nyctaginaceae_Boerhavia_coccinea_RED@27163 Nyctaginaceae_Anulocaulis_leiosolenus_var_gypsogenus_RED@c96785_g4_i1_244_1023_minus 6 Nyctaginaceae_Cyphomeris_gypsophiloides_RED@c1683_g1_i1_3_785_plus 1 1 8 9 MjADH_alpha Nyctaginaceae_Mirabilis_jalapa_RED@8420 MjADHα 1 5 9 Nyctaginaceae_Acleisanthes_lanceolata_RED@c30211_g1_i1_1_327_plus Nyctaginaceae_Acleisanthes_obtusa_RED@c40761_g1_i1_1_1410_plus 4 Nyctaginaceae_Acleisanthes_lanceolata_RED@c99516_g1_i1_3_1316_plus 5 0 Nyctaginaceae_Anulocaulis_leiosolenus_var_gypsogenus_RED@c96785_g1_i1_59_373_plus 1 2 Nyctaginaceae_Cyphomeris_gypsophiloides_RED@c28221_g1_i1_106_417_plus 2 0 3 5 Nyctaginaceae_Acleisanthes_obtusa_RED@c18031_g2_i1_2_346_plus 1 1 Nyctaginaceae_Acleisanthes_obtusa_RED@c18031_g1_i1_1_543_minus Nyctaginaceae_Mirabilis_multiflora_RED@c14082_g1_i1_1_402_plus 5 3 2 0 1 6 Nyctaginaceae_Boerhavia_coccinea_RED@16100 1 5 Nyctaginaceae_Boerhavia_burbidgeana_RED@24463 Nyctaginaceae_Acleisanthes_obtusa_RED@c64669_g1_i1_1_324_plus 3 Nyctaginaceae_Boerhavia_coccinea_RED@11273 3 0 3 4 Nyctaginaceae_Mirabilis_jalapa_RED@117470 7 Nyctaginaceae_Mirabilis_multiflora_RED@c62462_g1_i1_154_1344_plus 9 9 Nyctaginaceae_Anulocaulis_leiosolenus_var_gypsogenus_RED@c96785_g2_i1_221_1183_minus 2 8 2 7 Nyctaginaceae_Abronia_nealleyi_RED@c19062_g1_i2_83_673_plus 1 6 Nyctaginaceae_Abronia_nealleyi_RED@c17054_g1_i1_466_1698_plus Nyctaginaceae_Bougainvillea_spectabilis_RED@144651 1 5 Nyctaginaceae_Bougainvillea_stipitata_RED@c10529_g1_i1_2_958_minus 2 0 Nyctaginaceae_Pisonia_umbellifera_RED@c82751_g1_i1_167_640_plus 4 8 Nyctaginaceae_Pisonia_umbellifera_RED@c52568_g1_i1_2_742_plus Nyctaginaceae_Pisonia_aculeata_RED@c38458_g1_i1_34_1275_minus 2 8 Nyctaginaceae_Guapira_obtusata_RED@c34036_g1_i1_46_1479_minus 6 2 100 2 5 Petiveriaceae_Rivina_humilis_RED@c109306_g1_i1_104_1381_minus 4 1 RhADH2_alpha Petiveriaceae_Petiveria_alliacea_RED@19271 RhADHα Petiveriaceae_Seguieria_aculeata_RED@c69105_g4_i1_1_1473_plus 4 1 Phytolaccaceae_Phytolacca_dioica_RED@c95021_g1_i1_1_345_plus 2 8 Phytolaccaceae_Phytolacca_bogotensis_RED@12214 5 8 5 8 6 9 Phytolaccaceae_Phytolacca_bogotensis_RED@12213 Phytolaccaceae_Ercilla_volubilis_RED@c4424_g2_i1_3_437_plus 2 1 Phytolaccaceae_Ercilla_volubilis_RED@c69688_g1_i1_1_414_plus 9 6 5 5 2 4 5 9 Phytolaccaceae_Ercilla_volubilis_RED@c4424_g1_i1_148_504_plus Sarcobataceae_Sarcobatus_vermiculatus_RED@17241 Phytolaccaceae_Phytolacca_dioica_RED@c81159_g1_i1_1_438_plus 1 6 Phytolaccaceae_Phytolacca_dioica_RED@c27761_g1_i1_2_532_plus 2 8 3 2 Phytolaccaceae_Phytolacca_americana_RED@17976 4 4 Phytolaccaceae_Phytolacca_bogotensis_RED@12215 Phytolaccaceae_Phytolacca_bogotensis_RED@88377 Phytolaccaceae_Ercilla_volubilis_RED@c80895_g1_i1_236_1447_plus Nyctaginaceae_Guapira_obtusata_RED@c9168_g1_i2_366_1616_minus Simmondsiaceae_Simmondsia_chinensis@91744 5 6 4 6 Nyctaginaceae_Abronia_nealleyi_RED@c13801_g1_i2_3_968_plus 9 Nyctaginaceae_Anulocaulis_leiosolenus_var_gypsogenus_RED@c87255_g1_i1_2_403_plus Nyctaginaceae_Boerhavia_coccinea_RED@10808 5 Nyctaginaceae_Boerhavia_burbidgeana_RED@14891 08 8 Nyctaginaceae_Mirabilis_multiflora_RED@c19939_g1_i2_1_1203_plus Nyctaginaceae_Mirabilis_jalapa_RED@21565 5 5 Petiveriaceae_Petiveria_alliacea_RED@24748 3 4 1 7 9 Nyctaginaceae_Cyphomeris_gypsophiloides_RED@c60800_g1_i1_63_1220_plus Nyctaginaceae_Acleisanthes_obtusa_RED@c20024_g1_i1_3_1265_plus 3 Nyctaginaceae_Acleisanthes_lanceolata_RED@c7211_g1_i1_64_1224_plus Microteaceae_Microtea_debilis@31447 9 3 4 Petiveriaceae_Rivina_humilis_RED@c111225_g1_i1_597_1820_minus Petiveriaceae_Hilleria_latifolia_RED@89186 4 6 8 3 Nyctaginaceae_Bougainvillea_stipitata_RED@c52563_g1_i1_645_1808_minus 3 Nyctaginaceae_Bougainvillea_spectabilis_RED@10404 Nyctaginaceae_Bougainvillea_spectabilis_RED@37955 6 8 0 5 3 Nyctaginaceae_Pisonia_aculeata_RED@c42565_g1_i1_1_1260_plus Nyctaginaceae_Guapira_obtusata_RED@c31473_g1_i1_88_1269_plus 1 Nyctaginaceae_Pisonia_umbellifera_RED@c24985_g1_i1_3_1322_plus Nyctaginaceae_Guapira_obtusata_RED@c25947_g1_i1_2_856_plus Petiveriaceae_Seguieria_aculeata_RED@c70681_g1_i1_172_1344_plus 2 3 5 3 7 Phytolaccaceae_Ercilla_volubilis_RED@c7718_g2_i1_1_309_plus 6 5 Phytolaccaceae_Phytolacca_americana_RED@20770 6 3 Phytolaccaceae_Phytolacca_bogotensis_RED@15293 5 Phytolaccaceae_Phytolacca_dioica_RED@c40123_g1_i1_1_1029_plus Phytolaccaceae_Ercilla_volubilis_RED@c7718_g1_i1_3_914_plus 8 8 4 0 Phytolaccaceae_Phytolacca_americana_RED@17706 Phytolaccaceae_Phytolacca_bogotensis_RED@17620 1 6 A D H β o r t h l 5 Aizoaceae_Delosperma_echinatum_RED@12040 1 0 Phytolaccaceae_Phytolacca_americana_RED@91914 4 5 Phytolaccaceae_Phytolacca_dioica_RED@c35873_g1_i1_2_337_plus Phytolaccaceae_Ercilla_volubilis_RED@c8264_g1_i1_2_337_plus Achatocarpaceae_Phaulothamnus_spinescens_GREEN@c76345_g1_i1_1_756_plus 2 8 2 4 Portulacaceae_Portulaca_grandiflora_BLUE@47715 5 9 Portulacaceae_Portulaca_amilis_BLUE@61715 1 7 4 Portulacaceae_Portulaca_suffrutescens_BLUE@48134 Portulacaceae_Portulaca_pilosa_BLUE@47723 8 7 Portulacaceae_Portulaca_cryptopetala_BLUE@16132 3 4 4 8 Portulacaceae_Portulaca_molokiniensis_BLUE@11363 5 9 Portulacaceae_Portulaca_oleracea_BLUE@24479 8 3 Cactaceae_Lophophora_williamsii_BLUE@21424 3 4 Talinaceae_Talinum_sp_BLUE@70056 4 Basellaceae_Basella_alba_BLUE@26057 1 9 7 4 8 0 Aizoaceae_Sesuvium_portulacastrum_RED@3428 6 3 Aizoaceae_Sesuvium_verrucosum_RED@12145 9 1 Aizoaceae_Cypselea_humifusa_RED@11733 Aizoaceae_Zaleya_pentandra_RED@65980 Aizoaceae_Trianthema_portulacastrum_RED@2351 5 6 Petiveriaceae_Petiveria_alliacea_RED@130218 Aizoaceae_Delosperma_echinatum_RED@53817 Sarcobataceae_Sarcobatus_vermiculatus_RED@12976 5 4 8 1 Chenopodiaceae_Atriplex_hortensis_GREEN@65725 100 Chenopodiaceae_Atriplex_prostrata_GREEN@55909 6 1 Chenopodiaceae_Atriplex_rosea_GREEN@13810 1 7 Chenopodiaceae_Chenopodium_quinoa_GREEN@32868 7 SoADH_beta SoADHβ 3 9 Chenopodiaceae_Salicornia_europaea_GREEN@GAIA01012201_1_1_1023_plus 6 3Chenopodiaceae_Beta_maritima_GREEN@50340 3 3 100 Chenopodiaceae_Beta_vulgaris_GREEN@8_197570_yaar_t1 BvADH_beta Chenopodiaceae_Bassia_scoparia_GREEN@3431 BvADHβ g s 7 0 Amaranthaceae_Alternanthera_tenella_GREEN@23415 3 2 5 5 4 1 Amaranthaceae_Alternanthera_caracasana_GREEN@23275 9 9 Amaranthaceae_Alternanthera_caracasana_GREEN@23276 Amaranthaceae_Alternanthera_sessilis_GREEN@19308 9 0 Amaranthaceae_Alternanthera_brasiliana_GREEN@21098 4 9 7 9 3 9 8 Amaranthaceae_Aerva_javanica_GREEN@17317 Amaranthaceae_Aerva_lanata_GREEN@11408 7 6 Amaranthaceae_Froelichia_latifolia_GREEN@c56613_g1_i1_188_1366_plus 4 1 Amaranthaceae_Blutaparon_vermiculare_GREEN@12398 100 Amaranthaceae_Amaranthus_cruentus_GREEN@83498 Amaranthaceae_Amaranthus_retroflexus_GREEN@65503 Achatocarpaceae_Phaulothamnus_spinescens_GREEN@c5714_g1_i1_2_478_plus 4 0 1 3 7 1 4 6 Caryophyllaceae_Saponaria_officinalis_GREEN@66006 4 7 Caryophyllaceae_Schiedea_membranacea_GREEN@33817 Caryophyllaceae_Spergularia_media_GREEN@67827 8 2 Caryophyllaceae_Saponaria_officinalis_GREEN@10051 100 4 4 Caryophyllaceae_Silene_latifolia_SRA_GREEN@c51675_g1_i5_64_1206_plus 3 3 Caryophyllaceae_Silene_vulgaris_GREEN@c55632_g1_i1_1236_2330_minus Caryophyllaceae_Cerastium_arvense_GREEN@c15955_g1_i1_58_1179_plus 100 3 9 3 7 Caryophyllaceae_Drymaria_cordata_GREEN@c8964_g2_i1_1_1302_plus Caryophyllaceae_Polycarpaea_repens_GREEN@19691 5 1 Molluginaceae_Mollugo_cerviana_BLUE@5060 Molluginaceae_Mollugo_nudicaulis_BLUE@1572 3 5 Molluginaceae_Mollugo_verticillata_BLUE@14008 Physenaceae_Physena_madagascariensis@84536 Simmondsiaceae_Simmondsia_chinensis@103214 9 6 9 6 Polygonaceae_Rumex_acetosa@c16827_g1_i1_165_1328_minus 7 3 Polygonaceae_Rumex_palustris@c21408_g1_i1_2_1327_plus O u t g r o p s Polygonaceae_Rheum_rhabarbarum@c65804_g3_i2_1_1302_plus 4 5 3 Polygonaceae_Fagopyrum_esculentum@c12355_g1_i1_8_715_minus 5 0 4 7 Polygonaceae_Antigonon_leptopus@c29837_g1_i1_2_1243_plus 3 4 9 6 Polygonaceae_Fallopia_convolvulus@15527 Polygonaceae_Reynoutria_japonica@c33073_g1_i1_139_945_minus 4 2 Plumbaginaceae_Limonium_spectabile@2563 9 1 2 5 Polygonaceae_Fagopyrum_esculentum@c59579_g1_i1_1_420_plus 2 0 Polygonaceae_Antigonon_leptopus@c19405_g1_i1_2_370_plus Polygonaceae_Rheum_rhabarbarum@c56685_g3_i1_2_478_minus 100 Droseraceae_Dionaea_muscipula@c34918_g1_i6_2_1345_plus 4 0 Droseraceae_Aldrovanda_vesiculosa@c34697_g1_i1_1_933_plus 4 8 2 8 3 1 Tamaricaceae_Tamarix_hispida@c73590_g1_i1_307_1485_minus 8 9 Tamaricaceae_Tamarix_hispida@c61173_g1_i1_3_548_plus Frankeniaceae_Frankenia_laevis@7591 6 3 Tamaricaceae_Reaumuria_trigyna@c24036_g1_i2_1_1170_minus 100 NaADH Nepenthaceae_Nepenthes_alata@1277 Arab@AT5G34930_1__PACid_19672874 NaADHβ Soly@c07g007590_1_1__PACid_27294624 100 6 7 Vitis_vinifera@GSVIVT01017443001 5 6 Vitis_vinifera@GSVIVT01017439001 Vitis_vinifera@GSVIVT01017448001 Arab@AT1G15710_1__PACid_19657213 100 4 8 Oryza_sativa@LOC_Os06g49505_1 Oryza_sativa@LOC_Os06g49520_1 Oryza_sativa@LOC_Os06g35050_1
0.2 Figure S1e. (e) Maximum-likelihood phylogeny of ADH genes in Caryophyllales. The cartoon version of this tree is shown in Figure 4a. Numbers above branches indicate bootstrap support values. Scale bar indicates inferred number of amino acids substitution per site. ADH enzymes characterized in this study are indicated by arrows. Clades containing BvADHα and BvADHβ orthologs are indicated by orange and blue, respectevely. a ADH activity b 200 PDH activity 120 110 Active enzyme 180 Active enzyme No substrate No substrate 100 160 Heated enzyme Heated enzyme 90 140 80 120 70 Tyr std 100 60 soybean 50 80 HPLA std soybean
40 red beet L 60 Absorbance nm) (270Absorbance 30 red beet L 40 20 red beet R/S red beet R/S 20
10 Fluorescence (Ex = 360 nm, Em455 = 360 nm) nm, = (ExFluorescence 0 0 8.5 9 9.5 10 10.5 11 11.5 3 4 5 6 7 Retention time (min) Retention time (min)
PDH activity Figure S2. ADH but not PDH activity 250 c Active enzyme was detected from B. vulgaris tissues No substrate (a,b) or recombinant enzyme (c). Heated enzyme Arogenate (a) or prephenate (b,c) 200 substrates were incubated with NADP+ cofactor and desalted protein crude extract (a,b) of beet leaf (L), root/stem (R/S) tissues or recombinant enzyme of 150 BvADHα or BvADHβ together with HPLA std NADP+ cofactor (c). The production of Tyr (a) or HPP (which was converted to 100 MtPDH 4-hydroxyphyenyllactic acid, HPLA) (b,c) were analyzed by HPLC. The HPLC
traces were offset for presentation. Absorbance (270nm)Absorbance BvADHα Arabidopsis thaliana ADH2 (AtADH2) 50 and Medicago truncatula PDH (MtPDH) BvADHβ were used as a control for the ADH and PDH assay, respectively. 0 3 4 5 6 7 Retention time (min) 30 NADP+NADP+ NAD+
25 NAD+
protein) 1 1
- 20
mg
1 1
- s
15 nmol 10
5
ADH activity ( activity ADH 0 AtADH2 BvADHβ BvADHα
Figure S3. BvADHs prefer NADP+ over NAD+ as cofactor. ADH activity was analyzed using NADP+ or NADP+ cofactor, which is expressed as the mean of three independent experiments ± s.e.m. in nmol s-1 mg-1 of protein. a) Nucleotide sequence alignment of BvADHα Redbeet1_BvADHα 1 ATGATTTCACTCTCTTCTTTTCATCCTTCCTCCACCACCGCCACCGCCAC Yellowbeet_BvADHα 1 ATGATTTCACTCTCTTCTTTTCATCCTTCCTCCACCACCGCCACCGCCAC Whitebeet_BvADHα 1 ATGATTTCACTCTCTTCTTTTCATCCTTCCTCCACCACCGCCACCGCCAC Sugarbeet_BvADHα 1 ATGATTTCACTCTCTTCTTTTCATCCTTCCTCCACCACCGCCACCGCCAC Seabeet_BvADHα 1 ATGATTTCACTCTCTTCTTTTCATCCTTCCTCCACCACCGCCACCGCCAC Redbeet2_BvADHα 1 ATGATTTCACTCTCTTCTTTTCATCCTTCCTCCACCACCGCCACCGCCAC
Redbeet11_BvADHα 51 CGCCGCCGCCGCCACC------ACCCACC Yellowbeet_BvADHα 51 CGCCGCCGCCGCCACC------ACCCACC Whitebeet_BvADHα 51 CGCCGCCGCCGCCACC------ACCCACC Sugarbeet_BvADHα 51 CGCCGCCACCGCCACC------ACCCACC Seabeet_BvADHα 51 CGCCGCCACCGCCACCGCCACCGCCGCCACCGCCACCGCCACCACCCACC Redbeet2_BvADHα 51 CGCCGCCGCCGCCACC------ACCCACC
Redbeet1_BvADHα 74 CACCTCAACAATGTCCCGCTTTTTCCTCTCCTCCGTCGCATCTCTCGCTT Yellowbeet_BvADHα 74 CACCTCAACAATGTCCCGCTTTTTCCTCTCCTCCGTCGCATCTCTCGCTT Whitebeet_BvADHα 74 CACCACAACAATGTCCCGCTTTTTCCTCTCCTCCGTCGCATCTCTCGCTT Sugarbeet_BvADHα 74 CACCACAACAATGTCCCGCTTTTTCCTCTCCTCCATCGCATCTCTCGCTT Seabeet_BvADHα 101 CACCACAACAATGTCCCGCTTTTTCCTCTCCTCCATCGCATCTCTCGCTT Redbeet2_BvADHα 74 CACCTCAACAATGTCCCGCTTTTTCCTCTCCTCCGTCGCATCTCTCGCTT
Redbeet1_BvADHα 124 CCTTTACGCCACCCTCGCCAACACCTTGTAGTTCGGTGCGGTGGAGGTGG Yellowbeet_BvADHα 124 CCTTTACGCCACCCTCGCCAACACCTTGTAGTTCGGTGCGGTGGAGGTGG Whitebeet_BvADHα 124 CCTTTACGCCACCCTCGCCAACACCTTGTAGTTCGGTGCGGTGGAGGTGG Sugarbeet_BvADHα 124 CCTTTACGCCACCCTCGCCAACACCTTGTAGTTCGGTGCGGTGGAGGTGG Seabeet_BvADHα 151 CCTTTACGCCACCCTCGCCAACACCTTGTAGTTCGGTGCGGTGGAGGTGG Redbeet2_BvADHα 124 CCTTTACGCCACCCTCGCCAACACCTTGTAGTTCGGTGCGGTGGAGGTGG
Redbeet1_BvADHα 174 TTCGGCCTCCGAATCGGTATTTAACCGTGATAGTGCTGCTACTCGTGTTT Yellowbeet_BvADHα 174 TTCGGCCTCCGAATCGGTATTTAACCGTGATAGTGCTGCTACTCGTGTTT Whitebeet_BvADHα 174 TTCGGCCTCCGAATCGGTATTTAACCGTGATAGTGCTGCTACTCGTGTTT Sugarbeet_BvADHα 174 TTCGGCCTCCGAATCGGTATTTAACCGTGATAGTGCTGCTACTCGTGTTT Seabeet_BvADHα 201 TTCGGCCTCCGAATCGGTATTTAACCGTGATAGTGCTGCTACTCGTGTTT Redbeet2_BvADHα 174 TTCGGCCTCCGAATCGGTATTTAACCGTGATAGTGCTGCTACTCGTGTTT
Redbeet1_BvADHα 224 CTAATGATCATCTTGACGTTAGTAAAAGAGATGTTAAGCTTAAGATTGCT Yellowbeet_BvADHα 224 CTAATGATCATCTTGACGTTAGTAAAAGAGATGTTAAGCTTAAGATTGCT Whitebeet_BvADHα 224 CTAATGATCATCTTGACGTTAGTAAAAGAGATGTTAAGCTTAAGATTGCT Sugarbeet_BvADHα 224 CTAATGATCATCTTGACGTTAGTAAAAGAGATGTTAAGCTTAAGATTGCT Seabeet_BvADHα 251 CTAATGATCATCTTGACGTTAGTAAAAGAGATGTTAAGCTTAAGATTGCT Redbeet2_BvADHα 224 CTAATGATCATCTTGACGTTAGTAAAAGAGATGTTAAGCTTAAGATTGCT
Redbeet1_BvADHα 274 ATTATTGGGTTTGGTAACTTTGGCCAGTTTTTGGCTAAGACAATGGCTAA Yellowbeet_BvADHα 274 ATTATTGGGTTTGGTAACTTTGGCCAGTTTTTGGCTAAGACAATGGCTAA Whitebeet_BvADHα 274 ATTATTGGGTTTGGTAACTTTGGCCAGTTTTTGGCTAAGACAATGGCTAA Sugarbeet_BvADHα 274 ATTATTGGGTTTGGTAACTTTGGCCAGTTTTTGGCTAAGACAATGGCTAA Seabeet_BvADHα 301 ATTATTGGGTTTGGTAACTTTGGCCAGTTTTTGGCTAAGACAATGGCTAA Redbeet2_BvADHα 274 ATTATTGGGTTTGGTAACTTTGGCCAGTTTTTGGCTAAGACAATGGCTAA
Redbeet1_BvADHα 324 GCAAGGTCATAGAGTGTTGGCTTACTCACGCTCGGACTACTCCCGCGCTG Yellowbeet_BvADHα 324 GCAAGGTCATAGAGTGTTGGCTTACTCACGCTCGGACTACTCCCGCGCTG Whitebeet_BvADHα 324 GCAAGGTCATAGAGTGTTGGCTTACTCACGCTCGGACTACTCCCGCGCTG Sugarbeet_BvADHα 324 GCAAGGTCATAGAGTGTTGGCTTACTCACGCTCGGACTACTCCCGCGCTG Seabeet_BvADHα 351 GCAAGGTCATAGAGTGTTGGCTTACTCACGCTCGGACTACTCCCGCGCTG Redbeet2_BvADHα 324 GCAAGGTCATAGAGTGTTGGCTTACTCACGCTCGGACTACTCCCGCGCTG
Redbeet1_BvADHα 374 CTAAGGAGATCGGCGTCGAGTATTTTACTGACGCCGATGACCTCTGCGAG Yellowbeet_BvADHα 374 CTAAGGAGATCGGCGTCGAGTATTTTACTGACGCCGATGACCTCTGCGAG Whitebeet_BvADHα 374 CTAAGGAGATCGGCGTCGAGTATTTTACTGACGCCGATGACCTCTGCGAG Sugarbeet_BvADHα 374 CTAAGGAGATCGGCGTCGAGTATTTTACTGACGCCGATGACCTCTGCGAG Seabeet_BvADHα 401 CTAAGGAGATCGGCGTCGAGTATTTTACTGACGCCGATGACCTCTGCGAG Redbeet2_BvADHα 374 CTAAGGAGATCGGCGTCGAGTATTTTACTGACGCCGATGACCTCTGCGAG
Redbeet1_BvADHα 424 GAGCACCCTGAGGTTATTCTGTTGTGCACATCCATCCTCTCAACGGAGAA Yellowbeet_BvADHα 424 GAGCACCCTGAGGTTATTCTGTTGTGCACATCCATCCTCTCAACGGAGAA Whitebeet_BvADHα 424 GAGCACCCTGAGGTTATTCTGTTGTGCACGTCCATCCTCTCAACGGAGAA Sugarbeet_BvADHα 424 GAGCACCCTGAGGTTATTCTTTTGTGCACGTCCATCCTCTCAACGGAGAA Seabeet_BvADHα 451 GAGCACCCTGAGGTTATTCTTTTGTGCACGTCCATCCTCTCAACGGAGAA Redbeet2_BvADHα 424 GAGCACCCTGAGGTTATTCTGTTGTGCACATCCATCCTCTCAACGGAGAA
Redbeet1_BvADHα 474 GGTCCTCCGATCACTCCCCCTCCACCGGCTCCGTCGTTCAACCCTCTTTG Yellowbeet_BvADHα 474 GGTCCTCCGATCACTCCCCCTCCACCGGCTCCGTCGTTCAACCCTCTTTG Whitebeet_BvADHα 474 GGTCCTCCGATCACTCCCCCTCCACCGGCTCCGTCGTTCAACCCTCTTTG Sugarbeet_BvADHα 474 GGTCCTCCGATCACTCCCCCTCCACCGGCTCCGTCGTTCAACCCTCTTTG Seabeet_BvADHα 501 GGTCCTCCGATCACTCCCCCTCCACCGGCTCCGTCGTTCAACCCTCTTTG Redbeet2_BvADHα 474 GGTCCTCCGATCACTCCCCCTCCACCGGCTCCGTCGTTCAACCCTCTTTG
Redbeet1_BvADHα 524 CGGATGTTCTCTCGGTCAAGGAATTTCCTCGATCGCTCTTCCTTCAACTA Yellowbeet_BvADHα 524 CGGATGTTCTCTCGGTCAAGGAATTTCCTCGATCGCTCTTCCTTCAACTA Whitebeet_BvADHα 524 CGGATGTTCTCTCGGTCAAGGAATTTCCTCGATCGCTCTTCCTTCAACTA Sugarbeet_BvADHα 524 CGGATGTTCTCTCGGTCAAGGAATTTCCTCGATCGCTCTTCCTTCAACTA Seabeet_BvADHα 551 CGGATGTTCTCTCGGTCAAGGAATTTCCTCGATCGCTCTTCCTTCAACTA Redbeet2_BvADHα 524 CGGATGTTCTCTCGGTCAAGGAATTTCCTCGATCGCTCTTCCTTCAACTA
Redbeet1_BvADHα 574 CTTCCTAAGGACTTTGATATCCTATGCACCCACCCTATGTTTGGCCCAGA Yellowbeet_BvADHα 574 CTTCCTAAGGACTTTGATATCCTATGCACCCACCCTATGTTTGGCCCAGA Whitebeet_BvADHα 574 CTTCCTAAGGACTTTGATATCCTATGCACCCACCCTATGTTTGGCCCAGA Sugarbeet_BvADHα 574 CTTCCTAAGGACTTTGATATCCTATGCACCCACCCTATGTTTGGCCCAGA Seabeet_BvADHα 601 CTTCCTAAGGACTTTGATATCCTATGCACCCACCCTATGTTTGGCCCAGA Redbeet2_BvADHα 574 CTTCCTAAGGACTTTGATATCCTATGCACCCACCCTATGTTTGGCCCAGA
Redbeet1_BvADHα 624 CTCGGGCAAAGACGGGTGGGGTGGACTACCTTTTGTGTTCGATAAAGTTA Yellowbeet_BvADHα 624 CTCGGGCAAAGACGGGTGGGGTGGACTACCTTTTGTGTTCGATAAAGTTA Whitebeet_BvADHα 624 CTCGGGCAAAGACGGGTGGGGTGGACTACCTTTTGTGTTCGATAAAGTTA Sugarbeet_BvADHα 624 CTCGGGCAAAGACGGGTGGGGTGGACTACCCTTTGTGTTTGATAAAGTTA Seabeet_BvADHα 651 CTCGGGCAAAGACGGGTGGGGTGGACTACCCTTTGTGTTTGATAAAGTTA Redbeet2_BvADHα 624 CTCGGGCAAAGACGGGTGGGGTGGACTACCTTTTGTGTTCGATAAAGTTA
Redbeet1_BvADHα 674 GAGTCGGATCAGATCAGAGTCGGACATCTCGTGCTGAGGCATTCCTAGAC Yellowbeet_BvADHα 674 GAGTCGGATCAGATCAGAGTCGGACATCTCGTGCTGAGGCATTCCTAGAC Whitebeet_BvADHα 674 GAGTCGGATCAGATCAGAGTCGGACATCTCGTGCTGAGGCATTCCTAGAC Sugarbeet_BvADHα 674 GAGTCGGATCAGATCAGAGTCGGACGTCTCGTGCTGAGGCATTCCTAGAC Seabeet_BvADHα 701 GAGTCGGATCAGATCAGAGTCGGACGTCTCGTGCTGAGGCATTCCTAGAC Redbeet2_BvADHα 674 GAGTCGGATCAGATCAGAGTCGGACATCTCGTGCTGAGGCATTCCTAGAC
Redbeet1_BvADHα 724 GTGTTTAGGAATGCCGGGTGTAGGATGGTGGAAATGAGTTGTGTTGATCA Yellowbeet_BvADHα 724 GTGTTTAGGAATGCCGGGTGTAGGATGGTGGAAATGAGTTGTGTTGATCA Whitebeet_BvADHα 724 GTGTTTAGGAATGCCGGGTGTAGGATGGTGGAAATGAGTTGTGTTGATCA Sugarbeet_BvADHα 724 GTGTTTAGGAATGCCGGGTGTAGGATGGTGGAAATGAGTTGTGTTGATCA Seabeet_BvADHα 751 GTGTTTAGGAATGCCGGGTGTAGGATGGTGGAAATGAGTTGTGTTGATCA Redbeet2_BvADHα 724 GTGTTTAGGAATGCCGGGTGTAGGATGGTGGAAATGAGTTGTGTTGATCA
Redbeet1_BvADHα 774 TGACAAGCATGCAGCCGGGTCTCAATTTATTACACATATGATGGGACGAG Yellowbeet_BvADHα 774 TGACAAGCATGCAGCCGGGTCTCAATTTATTACACATATGATGGGACGAG Whitebeet_BvADHα 774 TGACAAGCATGCAGCCGGGTCTCAATTTATTACACATATGATGGGACGAG Sugarbeet_BvADHα 774 TGACAAGCATGCAGCCGGGTCTCAATTTATTACACATATGATGGGACGAG Seabeet_BvADHα 801 TGACAAGCATGCAGCCGGGTCTCAATTTATTACACATATGATGGGACGAG Redbeet2_BvADHα 774 TGACAAGCATGCAGCCGGATCTCAATTTATTACACATATGATGGGACGAG
Redbeet1_BvADHα 824 TTTTGGAGAAATTGGCCTTGGAAAATACACCAATTAATACAAAAGGGTAC Yellowbeet_BvADHα 824 TTTTGGAGAAATTGGCCTTGGAAAATACACCAATTAATACAAAAGGGTAC Whitebeet_BvADHα 824 TTTTGGAGAAATTGGCCTTGGAAAATACACCAATTAATACAAAAGGGTAC Sugarbeet_BvADHα 824 TTTTGGAGAAATTGGCCTTGGAAAATACACCAATTAATACAAAAGGGTAC Seabeet_BvADHα 851 TTTTGGAGAAATTGGCCTTGGAAAATACACCAATTAATACAAAAGGGTAC Redbeet2_BvADHα 824 TTTTGGAGAAATTGGCCTTGGAAAATACACCAATTAATACAAAAGGGTAC
Redbeet1_BvADHα 874 GAAAGTTTGTTAAATTTGGTGGATAATACTGCAAGGGATAGTTTTGAGTT Yellowbeet_BvADHα 874 GAAAGTTTGTTAAATTTGGTGGATAATACTGCAAGGGATAGTTTTGAGTT Whitebeet_BvADHα 874 GAAAGTTTGTTAAATTTGGTGGATAATACTGCAAGGGATAGTTTTGAGTT Sugarbeet_BvADHα 874 GAAAGTTTGTTAAATTTGGTGGATAATACTGCAAGGGATAGTTTTGAGTT Seabeet_BvADHα 901 GAAAGTTTGTTAAATTTGGTGGATAATACTGCAAGGGATAGTTTTGAGTT Redbeet2_BvADHα 874 GAAAGTTTGTTAAATTTGGTGGATAATACTGCAAGGGATAGTTTTGAGTT
Redbeet1_BvADHα 924 GTTTTACGGGTTGTTTTTGTACAATAAAAATGCAATGGAGCAATTGGATA Yellowbeet_BvADHα 924 GTTTTACGGGTTGTTTTTGTACAATAAAAATGCAATGGAGCAATTGGATA Whitebeet_BvADHα 924 GTTTTACGGGTTGTTTTTGTACAATAAAAATGCAATGGAGCAATTGGATA Sugarbeet_BvADHα 924 GTTTTATGGGTTGTTTTTGTACAATAAAAATGCAATGGAGCAATTGGATA Seabeet_BvADHα 951 GTTTTATGGGTTGTTTTTGTACAATAAAAATGCAATGGAGCAATTGGATA Redbeet2_BvADHα 924 GTTTTACGGGTTGTTTTTGTACAATAAAAATGCAATGGAGCAATTGGATA
Redbeet1_BvADHα 974 GAATGGATTGGGCTTTCGAGATGGTAAAAAAGCAACTTTCGGGATATTTG Yellowbeet_BvADHα 974 GAATGGATTGGGCTTTCGAGATGGTAAAAAAGCAACTTTCGGGATATTTG Whitebeet_BvADHα 974 GAATGGATTGGGCTTTCGAGATGGTAAAAAAGCAACTTTCGGGATATTTG Sugarbeet_BvADHα 974 GAATGGATTGGGCTTTCGAGATGGTAAAAAAGCAACTTTCGGGATATTTG Seabeet_BvADHα 1001 GAATGGATTGGGCTTTCGAGATGGTAAAAAAGCAACTTTCGGGATATTTG Redbeet2_BvADHα 974 GAATGGATTGGGCTTTCGAGATGGTAAAAAAGCAACTTTCGGGATATTTG
Redbeet1_BvADHα 1024 CATGATCTTGTTAGAAAACAATTGATGTTGGAGGGTAATAATGATCAAGC Yellowbeet_BvADHα 1024 CATGATCTTGTTAGAAAACAATTGATGTTGGAGGGTAATAATGATCAAGC Whitebeet_BvADHα 1024 CATGATCTTGTTAGAAAACAATTGATGTTGGAGGGTAATAATGATCAAGC Sugarbeet_BvADHα 1024 CATGATCTTGTTAGAAAACAATTGATGTTGGAGGGTAATAATGATCAAGC Seabeet_BvADHα 1051 CATGATCTTGTTAGAAAACAATTGATGTTGGAGGGTAATAATGATCAAGC Redbeet2_BvADHα 1024 CATGATCTTGTTAGAAAACAATTGATGTTGGAGGGTAATAATGATCAAGC
Redbeet1_BvADHα 1074 TGAGGTTACTTTTGACAAACCATTGATGCTTCCTTCTCCTACTATTAATC Yellowbeet_BvADHα 1074 TGAGGTTACTTTTGACAAACCATTGATGCTTCCTTCTCCTACTATTAATC Whitebeet_BvADHα 1074 TGAGGTTACTTTTGACAAACCATTGATGCTTCCTTCTCCTACTATTAATC Sugarbeet_BvADHα 1074 TGAGGTTACTTTTGACAAACCATTAATGCTTCCTTCTCCTACTATTAATC Seabeet_BvADHα 1101 TGAGGTTACTTTTGACAAACCATTAATGCTTCCTTCTCCTACTATTAATC Redbeet2_BvADHα 1074 TGAGGTTACTTTTGACAAACCATTGATGCTTCCTTCTCCTACTATTAATC
Redbeet1_BvADHα 1124 CTCCACAAATAGTTCCCTCTGCTGATATGGCTGAGAAGAAGCATGATTTA Yellowbeet_BvADHα 1124 CTCCACAAATAGTTCCCTCTGCTGATATGGCTGAGAAGAAGCATGATTTA Whitebeet_BvADHα 1124 CTCCACAAATAGTTCCCTCTGCTGATATGGCTGAGAAGAAGCATGATTTA Sugarbeet_BvADHα 1124 CTCCACAAATAGTTCCTTCTGCTGATATGGCTGAGAAGAAGCATGATTTA Seabeet_BvADHα 1151 CTCCACAAATAGTTCCTTCTGCTGATATGGCTGAGAAGAAGCATGATTTA Redbeet2_BvADHα 1124 CTCCACAAATAGTTCCCTCTGCTGATATGGCTGAGAAGAAGCATGATTTA
Redbeet1_BvADHα 1174 GTGGTGGTTAATGGTACTAGATAG Yellowbeet_BvADHα 1174 GTGGTGGTTAATGGTACTAGATAG Whitebeet_BvADHα 1174 GTGGTGGTTAATGGTACTAGATAG Sugarbeet_BvADHα 1174 GTGGTGGTTAATGGTACTAGATAG Seabeet_BvADHα 1201 GTGGTGGTTAATGGTACTAGATAG Redbeet2_BvADHα 1174 GTGGTGGTTAATGGTACTAGATAG
b) Nucleotide sequence alignment of BvADHβ Sugarbeet_BvADHβ 1 ATGCTTTCTCTCTCCTCCACAACCACCGCAAAACCCTCGCCGTCGCCATC Yellowbeet_BvADHβ 1 ATGCTTTCTCTCTCCTCCACAACCACCGCAAAACCCTCGCCGTCGCCATC Redbeet1_BvADHβ 1 ATGCTTTCTCTCTCCTCCACAACCACCGCAAAACCCTCGCCGTCGCCATC Whitebeet_BvADHβ 1 ATGCTTTCTCTCTCCTCCACAACCACCGCAAAACCCTCGCCGTCGCCATC Seabeet_BvADHβ 1 ATGCTTTCTCTCTCCTCCACAACCACCGCAAAACCCTCGCCGTCGCCATC Redbeet2_BvADHβ 1 ATGCTTTCTCTCTCCTCCACAACCACCGCAAAACCCTCGCCGTCGCCATC
Sugarbeet_BvADHβ 51 TCCGGCGAATTTTCCGGCGAAACTTTCTTCTCTCTCCACCATCACCACCA Yellowbeet_BvADHβ 51 TCCGGCGAATTTTCCGGCGAAACTTTCTTCTCTCTCCACCATCACCACCA Redbeet1_BvADHβ 51 TCCGGCGAATTTTCCGGCGAAACTTTCTTCTCTCTCCACCATCACCACCA Whitebeet_BvADHβ 51 TCCGGCGAATTTTCCGGCGAAACTTTCTTCTCTCTCCACCATCACCACCA Seabeet_BvADHβ 51 TCCGGCGAATTTTCCGGCAAAACTTTCTTCTCTCTCCACCATCACCACCA Redbeet2_BvADHβ 51 TCCGGCGAATTTTCCGGCAAAACTTTCTTCTCTCTCCACCATCACCACCA
Sugarbeet_BvADHβ 101 CTCTCTCTTTCTCTCCTCGCCGGAGATATTTTCATGGCGTCAAAACCCTA Yellowbeet_BvADHβ 101 CTCTCTCTTTCTCTCCTCGCCGGAGATATTTTCATGGCGTCAAAACCCTA Redbeet1_BvADHβ 101 CTCTCTCTTTCTCTCCTCGCCGGAGATATTTTCATGGCGTCAAAACCCTA Whitebeet_BvADHβ 101 CTCTCTCTTTCTCTCCTCGCCGGAGATATTTTCATGGCGTCAAAACCCTA Seabeet_BvADHβ 101 CTATCTCCTTCTCTCCTCGCCGGAGATATTTTCATGGCGTCAAAACCCTA Redbeet2_BvADHβ 101 CTCTCTCCTTCTCTCCTCGCCGGAGATATTTTCATGGCGTCAAAACCCTA
Sugarbeet_BvADHβ 151 ACAATTCGCAGCATCGACGCCGCACAATTCTTCGATTACGAATCAAAACT Yellowbeet_BvADHβ 151 ACAATTCGCAGCATCGACGCCGCACAATTCTTCGATTACGAATCAAAACT Redbeet1_BvADHβ 151 ACAATTCGCAGCATCGACGCCGCACAATTCTTCGATTACGAATCAAAACT Whitebeet_BvADHβ 151 ACAATTCGCAGCATCGACGCCGCACAATTCTTCGATTACGAATCAAAACT Seabeet_BvADHβ 151 ACAATTCGCAGCATCGACGCTGCACAATTCTTCGATTACGAATCAAAACT Redbeet2_BvADHβ 151 ACAATTCGCAGCATCGACGCTGCACAATTCTTCGATTACGAATCAAAACT
Sugarbeet_BvADHβ 201 TGCCGCCATTAACACAACCTCTTCGTCTTCATCTTCATCTTATTCGAAGC Yellowbeet_BvADHβ 201 TGCCGCCATTAACACAACCTCTTCGTCTTCATCTTCATCTTATTCGAAGC Redbeet1_BvADHβ 201 TGCCGCCATTAACACAACCTCTTCGTCTTCATCTTCATCTTATTCGAAGC Whitebeet_BvADHβ 201 TGCCGCCATTAACACAACCTCTTCGTCTTCATCTTCATCTTATTCGAAGC Seabeet_BvADHβ 201 CGCCGCCATTAACACAACCTCTTCATCTACATCGTCATCTTATTCGAAAC Redbeet2_BvADHβ 201 CGCCGCCATTAACACAACCTCTTCATCTACATCTTCATCTTATTCGAAAC
Sugarbeet_BvADHβ 251 TCAAAATCGCAATCGTAGGGTTCGGAAATTACGGACAATTTCTCGCGAAA Yellowbeet_BvADHβ 251 TCAAAATCGCAATCGTAGGGTTCGGAAATTACGGACAATTTCTCGCGAAA Redbeet1_BvADHβ 251 TCAAAATCGCAATCGTAGGGTTCGGAAATTACGGACAATTTCTCGCGAAA Whitebeet_BvADHβ 251 TCAAAATCGCAATCGTAGGGTTCGGAAATTACGGACAATTTCTCGCGAAA Seabeet_BvADHβ 251 TCAAAATCGCAATCGTAGGTTTCGGAAATTACGGACAATTTCTCGCGAAA Redbeet2_BvADHβ 251 TCAAAATCGCAATCGTAGGTTTCGGAAATTACGGACAATTTCTGGCGAAA
Sugarbeet_BvADHβ 301 ACCCTAGTTTCTCAAGGTCATACTGTTCTCGCTTATTCTCGCTCTGATTA Yellowbeet_BvADHβ 301 ACCCTAGTTTCTCAAGGTCATACTGTTCTCGCTTATTCTCGCTCTGATTA Redbeet1_BvADHβ 301 ACCCTAGTTTCTCAAGGTCATACTGTTCTCGCTTATTCTCGCTCTGATTA Whitebeet_BvADHβ 301 ACCCTAGTTTCTCAAGGTCATACTGTTCTCGCTTATTCTCGCTCTGATTA Seabeet_BvADHβ 301 ACCCTAGTTTCTCAAGGTCATACTGTTCTCGCTTATTCTCGCTCTGATTA Redbeet2_BvADHβ 301 ACCCTAGTTTCTCAAGGTCATACTGTTCTCGCTTATTCTCGCTCTGATTA
Sugarbeet_BvADHβ 351 CTCTAAAATCGCTGCGAATCTCGGCGTTTCTTACTTTTCTGATCCTGATG Yellowbeet_BvADHβ 351 CTCTAAAATCGCTGCGAATCTCGGCGTTTCTTACTTTTCTGATCCTGATG Redbeet1_BvADHβ 351 CTCTAAAATCGCTGCGAATCTCGGCGTTTCTTACTTTTCTGATCCTGATG Whitebeet_BvADHβ 351 CTCTAAAATCGCTGCGAATCTCGGCGTTTCTTACTTTTCTGATCCTGATG Seabeet_BvADHβ 351 CTCTAAAATCGCTGCGAATCTCGGCGTTTCTTACTTTTCTGATCCTGATG Redbeet2_BvADHβ 351 CTCTAAAATCGCTGCGAATCTCGGTGTTTCTTACTTTTCTGATCCTGATG
Sugarbeet_BvADHβ 401 ATCTTTGCGAAGAACATCCTGAGGTAATTATGTTGTGTACTTCGATTTTA Yellowbeet_BvADHβ 401 ATCTTTGCGAAGAACATCCAGAGGTAATTATGTTGTGTACTTCGATTTTA Redbeet1_BvADHβ 401 ATCTTTGCGAAGAACATCCTGAGGTAATTATGTTGTGTACTTCGATTTTA Whitebeet_BvADHβ 401 ATCTTTGCGAAGAACATCCTGAGGTAATTATGTTGTGTACTTCGATTTTA Seabeet_BvADHβ 401 ATCTTTGCGAAGAACATCCTGAGGTAATTATGTTGTGTACTTCGATTTTA Redbeet2_BvADHβ 401 ATCTTTGCGAAGAACATCCCGAGGTAATTATGTTGTGTACTTCGATTTTA
Sugarbeet_BvADHβ 451 TCAACTGAAGTTATGTTGAATTCGTTACCATTGCAGCGACTTAAACGATC Yellowbeet_BvADHβ 451 TCAACTGAAGTTATGTTGAATTCGTTACCATTGCAGCGACTTAAACGATC Redbeet1_BvADHβ 451 TCAACTGAAGTTATGTTGAATTCGTTACCATTGCAGCGACTTAAACGATC Whitebeet_BvADHβ 451 TCAACTGAAGTTATGTTGAATTCGTTACCATTGCAGCGACTTAAACGATC Seabeet_BvADHβ 451 TCAACTGAAGTTATGTTGAATTCGTTACCATTGCAGCGACTTAAACGATC Redbeet2_BvADHβ 451 TCAACTGAAGTTATGTTGAATTCGTTACCATTGCAGCGACTTAAACGATC
Sugarbeet_BvADHβ 501 GACGCTTTTTGTTGATGTTTTATCGGTGAAAGAATTTCCGCGTAATTTGT Yellowbeet_BvADHβ 501 GACGCTTTTTGTTGATGTTTTATCGGTGAAAGAATTTCCGCGTAATTTGT Redbeet1_BvADHβ 501 GACGCTTTTTGTTGATGTTTTATCGGTGAAAGAATTTCCGCGTAATTTGT Whitebeet_BvADHβ 501 GACGCTTTTTGTTGATGTTTTATCGGTGAAAGAATTTCCGCGTAATTTGT Seabeet_BvADHβ 501 GACGCTTTTTGTTGATGTTTTATCGGTGAAAGAATTTCCGCGTAATTTGT Redbeet2_BvADHβ 501 GACGCTTTTTGTTGATGTTTTATCGGTGAAAGAATTTCCGCGTAATTTGT
Sugarbeet_BvADHβ 551 TTCTTCAAACTTTACCGTCTGATTTTGATATATTATGTACTCATCCTATG Yellowbeet_BvADHβ 551 TTCTTCAAACTTTACCGTCTGATTTTGATATATTATGTACTCATCCTATG Redbeet1_BvADHβ 551 TTCTTCAAACTTTACCGTCTGATTTTGATATATTATGTACTCATCCTATG Whitebeet_BvADHβ 551 TTCTTCAAACTTTACCGTCTGATTTTGATATATTATGTACTCATCCTATG Seabeet_BvADHβ 551 TTCTTCAAACTTTACCGTCTGATTTTGATATATTATGTACTCATCCTATG Redbeet2_BvADHβ 551 TTCTTCAGACTTTACCGTCTGATTTTGATATATTATGTACTCATCCTATG
Sugarbeet_BvADHβ 601 TTTGGGCCTGAATCTGGGAAAAATGGTTGGGGAAGTTTGCCTTTTGTTTA Yellowbeet_BvADHβ 601 TTTGGGCCTGAATCTGGGAAAAATGGTTGGGGAAGTTTGCCTTTTGTTTA Redbeet1_BvADHβ 601 TTTGGGCCTGAATCTGGGAAAAATGGTTGGGGAAGTTTGCCTTTTGTTTA Whitebeet_BvADHβ 601 TTTGGGCCTGAATCTGGGAAAAATGGTTGGGGAAGTTTGCCTTTTGTTTA Seabeet_BvADHβ 601 TTTGGGCCTGAATCTGGGAAAAATGGTTGGGGAAGTTTGCCTTTTGTTTA Redbeet2_BvADHβ 601 TTTGGGCCTGAATCTGGGAAAAATGGTTGGGGAAGTTTGCCGTTTGTTTA
Sugarbeet_BvADHβ 651 TGATAAGGTTAGGATTGGGAAAGATGAGGGTAGAATTAAGAGATGTGAGA Yellowbeet_BvADHβ 651 TGATAAGGTTAGGATTGGGAAAGATGAGGGTAGAATTAAGAGATGTGAGA Redbeet1_BvADHβ 651 TGATAAGGTTAGGATTGGGAAAGATGAGGGTAGAATTAAGAGATGTGAGA Whitebeet_BvADHβ 651 TGATAAGGTTAGGATTGGGAAAGATGAGGGTAGAATTAAGAGATGTGAGA Seabeet_BvADHβ 651 TGATAAGGTTAGGATTGGGAAAGATGAGGGTAGAATTAAGAGATGTGAGA Redbeet2_BvADHβ 651 TGATAAAGTTAGGATTGGGAAAGATGAGGGTAGAATTAAGAGATGTGAGA
Sugarbeet_BvADHβ 701 GTTTTTTGGATGTTTTTAGGAGAGAAGGTTGTAGGGTTGAGGAAATGACT Yellowbeet_BvADHβ 701 GTTTTTTGGATGTTTTTAGGAGAGAAGGTTGTAGGGTTGAGGAAATGACT Redbeet1_BvADHβ 701 GTTTTTTGGATGTTTTTAGGAGAGAAGGTTGTAGGGTTGAGGAAATGACT Whitebeet_BvADHβ 701 GTTTTTTGGATGTTTTTAGGAGAGAAGGTTGTAGGGTTGAGGAAATGACT Seabeet_BvADHβ 701 GTTTTTTGGATGTTTTTAGGAGAGAAGGTTGTAGGGTTGAGGAAATGACT Redbeet2_BvADHβ 701 GTTTTTTGGATGTTTTTAGGAGAGAAGGTTGTAGGGTTGAGGAAATGACT
Sugarbeet_BvADHβ 751 TGTGCTGAGCATGATAAGTTTGCAGCAGGGTCTCAGTTTATAACACATTT Yellowbeet_BvADHβ 751 TGTGCTGAGCATGATAAGTTTGCAGCAGGGTCTCAGTTTATAACACATTT Redbeet1_BvADHβ 751 TGTGCTGAGCATGATAAGTTTGCAGCAGGGTCTCAGTTTATAACACATTT Whitebeet_BvADHβ 751 TGTGCTGAGCATGATAAGTTTGCAGCAGGGTCTCAGTTTATAACACATTT Seabeet_BvADHβ 751 TGTGCTGAGCATGATAAGTTTGCAGCAGGGTCTCAGTTTATTACACATTT Redbeet2_BvADHβ 751 TGTGCTGAGCATGATAAGTTTGCAGCAGGATCTCAGTTTATAACACATTT
Sugarbeet_BvADHβ 801 CTTAGGGAGGGTTTTGGAGAAGCTTGATTTGGAGGATACGCCGATTAATA Yellowbeet_BvADHβ 801 CTTAGGGAGGGTTTTGGAGAAGCTTGATTTGGAGGATACGCCGATTAATA Redbeet1_BvADHβ 801 CTTAGGGAGGGTTTTGGAGAAGCTTGATTTGGAGGATACGCCGATTAATA Whitebeet_BvADHβ 801 CTTAGGGAGGGTTTTGGAGAAGCTTGATTTGGAGGATACGCCGATTAATA Seabeet_BvADHβ 801 CTTAGGGAGGGTTTTGGAGAAGCTTGATTTGGAGGATACGCCGATTAATA Redbeet2_BvADHβ 801 CTTAGGGAGGGTTTTGGAGAAGCTTGATTTGGAGGATACGCCGATTAATA
Sugarbeet_BvADHβ 851 CGAAAGGGTATGAGAGTTTGTTGAATTTGGTGGATAATACGTCGAAGGAT Yellowbeet_BvADHβ 851 CGAAAGGGTATGAGAGTTTGTTGAATTTGGTGGATAATACGTCGAAGGAT Redbeet1_BvADHβ 851 CGAAAGGGTATGAGAGTTTGTTGAATTTGGTGGATAATACGTCGAAGGAT Whitebeet_BvADHβ 851 CGAAAGGGTATGAGAGTTTGTTGAATTTGGTGGATAATACGTCGAAGGAT Seabeet_BvADHβ 851 CGAAAGGGTATGAGAGTTTGTTGAATTTGGTGGATAATACGTCGAAGGAT Redbeet2_BvADHβ 851 CGAAAGGGTATGAGAGTTTGTTGAATTTGGTGGATAATACGTCGAAGGAT
Sugarbeet_BvADHβ 901 AGTTTCGAGTTGTTTTATGGGTTGTTTTTGTATAATCAGAATGCTATGGA Yellowbeet_BvADHβ 901 AGTTTCGAGTTGTTTTATGGGTTGTTTTTGTATAATCAGAATGCTATGGA Redbeet1_BvADHβ 901 AGTTTCGAGTTGTTTTATGGGTTGTTTTTGTATAATCAGAATGCTATGGA Whitebeet_BvADHβ 901 AGTTTCGAGTTGTTTTATGGGTTGTTTTTGTATAATCAGAATGCTATGGA Seabeet_BvADHβ 901 AGTTTCGAGTTGTTTTATGGGTTGTTTTTGTATAATCAGAATGCTATGGA Redbeet2_BvADHβ 901 AGTTTCGAGTTGTTTTATGGGTTGTTTTTGTATAATCAGAATGCTATGGA
Sugarbeet_BvADHβ 951 GCAGTTAGAGAGGTTAGATTGGGCGTTTGAGTTGGTTAAGAAGCAATTGT Yellowbeet_BvADHβ 951 GCAGTTAGAGAGGTTAGATTGGGCGTTTGAGTTGGTTAAGAAGCAATTGT Redbeet1_BvADHβ 951 GCAGTTAGAGAGGTTAGATTGGGCGTTTGAGTTGGTTAAGAAGCAATTGT Whitebeet_BvADHβ 951 GCAGTTAGAGAGGTTAGATTGGGCGTTTGAGTTGGTTAAGAAGCAATTGT Seabeet_BvADHβ 951 GCAGTTAGAGAGGTTAGATTGGGCATTTGAGTTGGTTAAGAAGCAATTGT Redbeet2_BvADHβ 951 GCAGTTAGAGAGGTTAGATTGGGCGTTTGAGTTGGTTAAGAAGCAATTGT
Sugarbeet_BvADHβ 1001 TTGGACACTTGCATGGGTTGCTAAGGAAACAGTTGTTTGGGTTTTCTGAG Yellowbeet_BvADHβ 1001 TTGGACACTTGCATGGGTTGCTAAGGAAACAGTTGTTTGGGTTTTCTGAG Redbeet1_BvADHβ 1001 TTGGACACTTGCATGGGTTGCTAAGGAAACAGTTGTTTGGGTTTTCTGAG Whitebeet_BvADHβ 1001 TTGGACACTTGCATGGGTTGCTAAGGAAACAGTTGTTTGGGTTTTCTGAG Seabeet_BvADHβ 1001 TTGGACACTTGCATGGGTTGCTAAGGAAACAGTTGTTTGGGTTTTCTGAG Redbeet2_BvADHβ 1001 TTGGACACTTGCATGGGTTGCTAAGGAAACAGTTGTTTGGGTTTTCTGAG
Sugarbeet_BvADHβ 1051 ATAGATGAACGTATTGGGAAGGCGAAGGAGATCAAATTTCTCTCTGATGC Yellowbeet_BvADHβ 1051 ATAGATGAACGTATTGGGAAGGCGAAGGAGATCAAATTTCTCTCTGATGC Redbeet1_BvADHβ 1051 ATAGATGAACGTATTGGGAAGGCGAAGGAGATCAAATTTCTCTCTGATGC Whitebeet_BvADHβ 1051 ATAGATGAACGTATTGGGAAGGCGAAGGAGATCAAATTTCTCTCTGATGC Seabeet_BvADHβ 1051 ATAGATGAACGTATTGGGAAGGCGAAGGAGATCAAATTTCTCTCTGATGC Redbeet2_BvADHβ 1051 ATAGATGAACGTATTGGGAAGGCGAAGGAGATCAAATTTCTCTCTGATGC
Sugarbeet_BvADHβ 1101 TGCAGAACAGAATGGCTCTGCCTTGTCTGCTAGGGAGAATGCAAATTCGG Yellowbeet_BvADHβ 1101 TGCAGAACAGAATGGCTCTGCCTTGTCTGCTAGGGAGAATGCAAATTCGG Redbeet1_BvADHβ 1101 TGCAGAACAGAATGGCTCTGCCTTGTCTGCTAGGGAGAATGCAAATTCGG Whitebeet_BvADHβ 1101 TGCAGAACAGAATGGCTCTGCCTTGTCTGCTAGGGAGAATGCAAATTCGG Seabeet_BvADHβ 1101 TGCAGAACAGAATGGCTCTGCCTTGTCTGCTAGGGAGAATGCAAATTCGG Redbeet2_BvADHβ 1101 TGCAGAACAGAATGGCTCTGCCTTGTCTGCTAGGGAGAATGCAAATTCGG
Sugarbeet_BvADHβ 1151 AGACAAATTGA Yellowbeet_BvADHβ 1151 AGACAAATTGA Redbeet1_BvADHβ 1151 AGACAAATTGA Whitebeet_BvADHβ 1151 AGACAAATTGA Seabeet_BvADHβ 1151 AGACAAATTGA Redbeet2_BvADHβ 1151 AGACAAATTGA
c) Amino acid sequence alignment of BvADHα Redbeet1_BvADHα 1 MISLSSFHPSSTTATATAAAAT------THPPQQCPAFSSPPSHLSL Whitebeet_BvADHα 1 MISLSSFHPSSTTATATAAAAT------THPPQQCPAFSSPPSHLSL Yellowbeet_BvADHα 1 MISLSSFDPSSTTATATAAAAT------THPPQQCPAFSSPPSHLSL Sugarbeet_BvADHα 1 MISLSSFHPSSTTATATAATAT------THPPQQCPAFSSPPSHLSL Seabeet_BvADHα 1 MISLSSFHPSSTTATATAATATATAATATATTHPPQQCPAFSSPPSHLSL Redbeet2_BvADHα 1 MISLSSFHPSSTTATATAAAAT------THPPQQCPAFSSPPSHLSL
Redbeet1_BvADHα 42 PLRHPRQHLVVRCGGGGSASESVFNRDSAATRVSNDHLDVSKRDVKLKIA Whitebeet_BvADHα 42 PLRHPRQHLVVRCGGGGSASESVFNRDSAATRVSNDHLDVSKRDVKLKIA Yellowbeet_BvADHα 42 PLRHPRQHLVVRCGGGGSASESVFNRDSAATRVSNDHLDVSKRDVKLKIA Sugarbeet_BvADHα 42 PLRHPRQHLVVRCGGGGSASESVFNRDSAATRVSNDHLDVSKRDVKLKIA Seabeet_BvADHα 51 PLRHPRQHLVVRCGGGGSASESVFNRDSAATRVSNDHLDVSKRDVKLKIA Redbeet2_BvADHα 42 PLRHPRQHLVVRCGGGGSASESVFNRDSAATRVSNDHLDVSKRDVKLKIA
Redbeet1_BvADHα 92 IIGFGNFGQFLAKTMAKQGHRVLAYSRSDYSRAAKEIGVEYFTDADDLCE Whitebeet_BvADHα 92 IIGFGNFGQFLAKTMAKQGHRVLAYSRSDYSRAAKEIGVEYFTDADDLCE Yellowbeet_BvADHα 92 IIGFGNFGQFLAKTMAKQGHRVLAYSRSDYSRAAKEIGVEYFTDADDLCE Sugarbeet_BvADHα 92 IIGFGNFGQFLAKTMAKQGHRVLAYSRSDYSRAAKEIGVEYFTDADDLCE Seabeet_BvADHα 101 IIGFGNFGQFLAKTMAKQGHRVLAYSRSDYSRAAKEIGVEYFTDADDLCE Redbeet2_BvADHα 92 IIGFGNFGQFLAKTMAKQGHRVLAYSRSDYSRAAKEIGVEYFTDADDLCE
Redbeet1_BvADHα 142 EHPEVILLCTSILSTEKVLRSLPLHRLRRSTLFADVLSVKEFPRSLFLQL Whitebeet_BvADHα 142 EHPEVILLCTSILSTEKVLRSLPLHRLRRSTLFADVLSVKEFPRSLFLQL Yellowbeet_BvADHα 142 EHPEVILLCTSILSTEKVLRSLPLHRLRRSTLFADVLSVKEFPRSLFLQL Sugarbeet_BvADHα 142 EHPEVILLCTSILSTEKVLRSLPLHRLRRSTLFADVLSVKEFPRSLFLQL Seabeet_BvADHα 151 EHPEVILLCTSILSTEKVLRSLPLHRLRRSTLFADVLSVKEFPRSLFLQL Redbeet2_BvADHα 142 EHPEVILLCTSILSTEKVLRSLPLHRLRRSTLFADVLSVKEFPRSLFLQL
Redbeet1_BvADHα 192 LPKDFDILCTHPMFGPDSGKDGWGGLPFVFDKVRVGSDQSRTSRAEAFLD Whitebeet_BvADHα 192 LPKDFDILCTHPMFGPDSGKDGWGGLPFVFDKVRVGSDQSRTSRAEAFLD Yellowbeet_BvADHα 192 LPKDFDILCTHPMFGPDSGKDGWGGLPFVFDKVRVGSDQSRTSRAEAFLD Sugarbeet_BvADHα 192 LPKDFDILCTHPMFGPDSGKDGWGGLPFVFDKVRVGSDQSRTSRAEAFLD Seabeet_BvADHα 201 LPKDFDILCTHPMFGPDSGKDGWGGLPFVFDKVRVGSDQSRTSRAEAFLD Redbeet2_BvADHα 192 LPKDFDILCTHPMFGPDSGKDGWGGLPFVFDKVRVGSDQSRTSRAEAFLD
Redbeet1_BvADHα 242 VFRNAGCRMVEMSCVDHDKHAAGSQFITHMMGRVLEKLALENTPINTKGY Whitebeet_BvADHα 242 VFRNAGCRMVEMSCVDHDKHAAGSQFITHMMGRVLEKLALENTPINTKGY Yellowbeet_BvADHα 242 VFRNAGCRMVEMSCVDHDKHAAGSQFITHMMGRVLEKLALENTPINTKGY Sugarbeet_BvADHα 242 VFRNAGCRMVEMSCVDHDKHAAGSQFITHMMGRVLEKLALENTPINTKGY Seabeet_BvADHα 251 VFRNAGCRMVEMSCVDHDKHAAGSQFITHMMGRVLEKLALENTPINTKGY Redbeet2_BvADHα 242 VFRNAGCRMVEMSCVDHDKHAAGSQFITHMMGRVLEKLALENTPINTKGY
Redbeet1_BvADHα 292 ESLLNLVDNTARDSFELFYGLFLYNKNAMEQLDRMDWAFEMVKKQLSGYL Whitebeet_BvADHα 292 ESLLNLVDNTARDSFELFYGLFLYNKNAMEQLDRMDWAFEMVKKQLSGYL Yellowbeet_BvADHα 292 ESLLNLVDNTARDSFELFYGLFLYNKNAMEQLDRMDWAFEMVKKQLSGYL Sugarbeet_BvADHα 292 ESLLNLVDNTARDSFELFYGLFLYNKNAMEQLDRMDWAFEMVKKQLSGYL Seabeet_BvADHα 301 ESLLNLVDNTARDSFELFYGLFLYNKNAMEQLDRMDWAFEMVKKQLSGYL Redbeet2_BvADHα 292 ESLLNLVDNTARDSFELFYGLFLYNKNAMEQLDRMDWAFEMVKKQLSGYL
Redbeet1_BvADHα 342 HDLVRKQLMLEGNNDQAEVTFDKPLMLPSPTINPPQIVPSADMAEKKHDL Whitebeet_BvADHα 342 HDLVRKQLMLEGNNDQAEVTFDKPLMLPSPTINPPQIVPSADMAEKKHDL Yellowbeet_BvADHα 342 HDLVRKQLMLEGNNDQAEVTFDKPLMLPSPTINPPQIVPSADMAEKKHDL Sugarbeet_BvADHα 342 HDLVRKQLMLEGNNDQAEVTFDKPLMLPSPTINPPQIVPSADMAEKKHDL Seabeet_BvADHα 351 HDLVRKQLMLEGNNDQAEVTFDKPLMLPSPTINPPQIVPSADMAEKKHDL Redbeet2_BvADHα 342 HDLVRKQLMLEGNNDQAEVTFDKPLMLPSPTINPPQIVPSADMAEKKHDL
Redbeet1_BvADHα 392 VVVNGTR Whitebeet_BvADHα 392 VVVNGTR Yellowbeet_BvADHα 392 VVVNGTR Sugarbeet_BvADHα 392 VVVNGTR Seabeet_BvADHα 401 VVVNGTR Redbeet2_BvADHα 392 VVVNGTR
d) Amino acid sequence alignment of BvADHβ Sugarbeet_BvADHβ 1 MLSLSSTTTAKPSPSPSPANFPAKLSSLSTITTTLSFSPRRRYFHGVKTL Yellowbeet_BvADHβ 1 MLSLSSTTTAKPSPSPSPANFPAKLSSLSTITTTLSFSPRRRYFHGVKTL Redbeet1_BvADHβ 1 MLSLSSTTTAKPSPSPSPANFPAKLSSLSTITTTLSFSPRRRYFHGVKTL Whitebeet_BvADHβ 1 MLSLSSTTTAKPSPSPSPANFPAKLSSLSTITTTLSFSPRRRYFHGVKTL Seabeet_BvADHβ 1 MLSLSSTTTAKPSPSPSPANFPAKLSSLSTITTTISFSPRRRYFHGVKTL Redbeet2_BvADHβ 1 MLSLSSTTTAKPSPSPSPANFPAKLSSLSTITTTLSFSPRRRYFHGVKTL
Sugarbeet_BvADHβ 51 TIRSIDAAQFFDYESKLAAINTTSSSSSSSYSKLKIAIVGFGNYGQFLAK Yellowbeet_BvADHβ 51 TIRSIDAAQFFDYESKLAAINTTSSSSSSSYSKLKIAIVGFGNYGQFLAK Redbeet1_BvADHβ 51 TIRSIDAAQFFDYESKLAAINTTSSSSSSSYSKLKIAIVGFGNYGQFLAK Whitebeet_BvADHβ 51 TIRSIDAAQFFDYESKLAAINTTSSSSSSSYSKLKIAIVGFGNYGQFLAK Seabeet_BvADHβ 51 TIRSIDAAQFFDYESKLAAINTTSSSTSSSYSKLKIAIVGFGNYGQFLAK Redbeet2_BvADHβ 51 TIRSIDAAQFFDYESKLAAINTTSSSTSSSYSKLKIAIVGFGNYGQFLAK
Sugarbeet_BvADHβ 101 TLVSQGHTVLAYSRSDYSKIAANLGVSYFSDPDDLCEEHPEVIMLCTSIL Yellowbeet_BvADHβ 101 TLVSQGHTVLAYSRSDYSKIAANLGVSYFSDPDDLCEEHPEVIMLCTSIL Redbeet1_BvADHβ 101 TLVSQGHTVLAYSRSDYSKIAANLGVSYFSDPDDLCEEHPEVIMLCTSIL Whitebeet_BvADHβ 101 TLVSQGHTVLAYSRSDYSKIAANLGVSYFSDPDDLCEEHPEVIMLCTSIL Seabeet_BvADHβ 101 TLVSQGHTVLAYSRSDYSKIAANLGVSYFSDPDDLCEEHPEVIMLCTSIL Redbeet2_BvADHβ 101 TLVSQGHTVLAYSRSDYSKIAANLGVSYFSDPDDLCEEHPEVIMLCTSIL
Sugarbeet_BvADHβ 151 STEVMLNSLPLQRLKRSTLFVDVLSVKEFPRNLFLQTLPSDFDILCTHPM Yellowbeet_BvADHβ 151 STEVMLNSLPLQRLKRSTLFVDVLSVKEFPRNLFLQTLPSDFDILCTHPM Redbeet1_BvADHβ 151 STEVMLNSLPLQRLKRSTLFVDVLSVKEFPRNLFLQTLPSDFDILCTHPM Whitebeet_BvADHβ 151 STEVMLNSLPLQRLKRSTLFVDVLSVKEFPRNLFLQTLPSDFDILCTHPM Seabeet_BvADHβ 151 STEVMLNSLPLQRLKRSTLFVDVLSVKEFPRNLFLQTLPSDFDILCTHPM Redbeet2_BvADHβ 151 STEVMLNSLPLQRLKRSTLFVDVLSVKEFPRNLFLQTLPSDFDILCTHPM
Sugarbeet_BvADHβ 201 FGPESGKNGWGSLPFVYDKVRIGKDEGRIKRCESFLDVFRREGCRVEEMT Yellowbeet_BvADHβ 201 FGPESGKNGWGSLPFVYDKVRIGKDEGRIKRCESFLDVFRREGCRVEEMT Redbeet1_BvADHβ 201 FGPESGKNGWGSLPFVYDKVRIGKDEGRIKRCESFLDVFRREGCRVEEMT Whitebeet_BvADHβ 201 FGPESGKNGWGSLPFVYDKVRIGKDEGRIKRCESFLDVFRREGCRVEEMT Seabeet_BvADHβ 201 FGPESGKNGWGSLPFVYDKVRIGKDEGRIKRCESFLDVFRREGCRVEEMT Redbeet2_BvADHβ 201 FGPESGKNGWGSLPFVYDKVRIGKDEGRIKRCESFLDVFRREGCRVEEMT
Sugarbeet_BvADHβ 251 CAEHDKFAAGSQFITHFLGRVLEKLDLEDTPINTKGYESLLNLVDNTSKD Yellowbeet_BvADHβ 251 CAEHDKFAAGSQFITHFLGRVLEKLDLEDTPINTKGYESLLNLVDNTSKD Redbeet1_BvADHβ 251 CAEHDKFAAGSQFITHFLGRVLEKLDLEDTPINTKGYESLLNLVDNTSKD Whitebeet_BvADHβ 251 CAEHDKFAAGSQFITHFLGRVLEKLDLEDTPINTKGYESLLNLVDNTSKD Seabeet_BvADHβ 251 CAEHDKFAAGSQFITHFLGRVLEKLDLEDTPINTKGYESLLNLVDNTSKD Redbeet2_BvADHβ 251 CAEHDKFAAGSQFITHFLGRVLEKLDLEDTPINTKGYESLLNLVDNTSKD
Sugarbeet_BvADHβ 301 SFELFYGLFLYNQNAMEQLERLDWAFELVKKQLFGHLHGLLRKQLFGFSE Yellowbeet_BvADHβ 301 SFELFYGLFLYNQNAMEQLERLDWAFELVKKQLFGHLHGLLRKQLFGFSE Redbeet1_BvADHβ 301 SFELFYGLFLYNQNAMEQLERLDWAFELVKKQLFGHLHGLLRKQLFGFSE Whitebeet_BvADHβ 301 SFELFYGLFLYNQNAMEQLERLDWAFELVKKQLFGHLHGLLRKQLFGFSE Seabeet_BvADHβ 301 SFELFYGLFLYNQNAMEQLERLDWAFELVKKQLFGHLHGLLRKQLFGFSE Redbeet2_BvADHβ 301 SFELFYGLFLYNQNAMEQLERLDWAFELVKKQLFGHLHGLLRKQLFGFSE
Sugarbeet_BvADHβ 351 IDERIGKAKEIKFLSDAAEQNGSALSARENANSETN Yellowbeet_BvADHβ 351 IDERIGKAKEIKFLSDAAEQNGSALSARENANSETN Redbeet1_BvADHβ 351 IDERIGKAKEIKFLSDAAEQNGSALSARENANSETN Whitebeet_BvADHβ 351 IDERIGKAKEIKFLSDAAEQNGSALSARENANSETN Seabeet_BvADHβ 351 IDERIGKAKEIKFLSDAAEQNGSALSARENANSETN Redbeet2_BvADHβ 351 IDERIGKAKEIKFLSDAAEQNGSALSARENANSETN
Figure S4 . No amino acid changes were found in the mature protein coding region of BvADHα among different B. vulgaris varieties. The BvADHα and BvADHβ genes were sequenced from five different varieties of domesticated (red 1 [W357B], red 2 [Boltardy], sugar, yellow, and white) and a wild beet (sea beet ascension number PI562585). In nucleotide sequence comparisons of BvADHα (a) and BvADHβ (b), several single nucleotide polymorphisms (SNPs) were found among varieties. Amino acid sequence alignments of BvADHα (c) and BvADHβ (d), however, showed that these SNPs were mostly synonymous (no changes in amino acid), with two exceptions found in the N-terminal predicted chloroplast transit peptide, which was eliminated for recombinant enzyme expression. The predicted chloroplast transit peptide cleavage sites are denoted by green triangles. 120
M AtADH2 μ 100 BvADHα
80
60
40
20
% ADH activity 0 to relative activity ADH %
N.D.
N.D.
N.T. N.T. 0 N.T. 0 10 100 200 400 800 1000 2000 4000 8000 L-Tyrosine concentrations (μM)
Figure S5. Recombinant His-tagged BvADHα also exhibits reduced sensitivity to Tyr relative to AtADH2. BvADHα and AtADH2 recombinant enzymes were also generated as 6xHis-tag proteins to determine if GST-tag affects Tyr sensitivity of BvADHα. The His-BvADHα recombinant enzyme still exhibited relaxed sensitive to Tyr inhibition. Data are expressed as the percentage of respective control activity without Tyr (0 μM) and the means of three independent experiments ± s.e.m. N.D., not detectable; N.T., not tested. 120
μM 100
80 No effector 60 L-Phe L-Trp 40 Betanin
20 % ADH activity 0 to relative activity ADH % 0 AtADH2 BvADHβ BvADHα
Figure S6. BvADHs are not inhibited by phenylalanine, tryptophan, and betanin. ADH activity of BvADHα, BvADHβ and AtADH2 was measured in the presence and absence of 1 mM final concentration of L-phenylalanine (L-Phe), L-tryptophan (L-Trp), and betanin as an effector. Data are expressed as the percentage of respective control activity without effector and the mean of three independent experiments ± s.m.e. No significant reduction was observed by any effector treatment relative to respective no effector control (P < 0.05, student-t test).
a
GFP
α
BvADH
β BvADH
b 200
150 )
100
gFW /
50
Tyrosine Tyrosine
nmol (
0
2a 3a 4a 5a
5b 2b 3b 4b
2G 1G 3G 4G 5G
1a-1 1a-2
1b-1 1b-2 GFP BvADHα BvADHβ Figure S7. Transgene expression and tyrosine levels of individual leaf samples of infiltrated Nicotiana benthamiana. Agrobacterium tumefaciens carrying the construct of 35S::GFP, 35S::BvADHα, or 35S::BvADHβ was infiltrated to Nicotiana benthamiana leaves (sample names ending with G, a, and b, respectively). 1a-1 and 1a-2 are technical replicates of the same leaf infiltrated with 35S::BvADHα, so do 1b-1 and 1b-2 for 35S::BvADHβ. (a) Expression of respective transgenes shown by RT-PCR. (+) denotes a positive control using the original plasmid as a template, while (-) indicates a negative control cDNA from a leaf area without infiltration. (-RT) is an additional negative control without reverse transcriptase to detect genomic DNA contamination. (b) Tyrosine contents of individual samples. Two technical replicates showed very similar results. Means ± s.e.m. of Tyr and other amino acids analysis are shown in Figure 3 and Table S2. protein protein + 3-fluoro-Tyr boiled protein 140 Spinacia oleracea 120
100
80
60 /mg protein) /mg
ADH Activity Activity ADH 40
nmol ( 20
0 0 1 2 3 25 Dianthus barbatus
20
15
/mg protein) /mg 10
ADH Activity Activity ADH nmol ( 5
0 0 1 2 3 120 Arabidopsis thaliana 100
80
60 /mg protein) /mg
40
ADH Activity Activity ADH nmol ( 20
0 0 1 2 3 Reaction time (hours) Figure S8. Tyr sensitivity of ADH activity from plant tissues. The plastid extracts of spinach (Spinacia oleracea), and the crude extracts of Dianthus barbatus and Arabidopsis thaliana were incubated with 1 mM arogenate substrate and 1 mM NADP+ cofactor for indicated times. Plastids were isolated for spinach ADH assays to eliminate strong polyphenol oxidase activity present in the crude extracts. Data are means ± s.e.m. (n = 4). Activity increased linearly during the first two hours, which were used to calculate ADH activity presented in Table 1. a) ADHα CDS b) ADHα Peptide
100 Mirabilis jalapa ADH-α 9 9 Mirabilis jalapa ADH-α 100 Rivina humilis ADH2-α 8 3 Rivina humilis ADH2-α Basella alba ADH-α 100 9 5 Basella alba ADH-α Portulaca oleracea ADH-α Portulaca oleracea ADH-α Beta vulgaris ADH-α 9 6 8 3 Beta vulgaris ADH-α Spinacia oleracea ADH-α Spinacia oleracea ADH-α 100 7 7 Telephium imperatii ADH-α 8 3 Corrigiola litoralis ADH-α Corrigiola litoralis ADH-α Telephium imperatii ADH-α 9 5 7 7 Paronychia polygonifolia ADH-α Paronychia polygonifolia ADH-α 5 7 Spergularia media@join2061778_2046855 100 100 Spergularia media@join2061778_2046855 3 7 6 0 Spergularia marina ADH-α 8 9 Spergularia marina ADH-α Polycarpaea repens@2010602 Polycarpaea repens@2010602 7 5 6 5 5 6 Herniaria latifolia ADH-α 5 5 Herniaria latifolia ADH-α Polycarpaea repens@2068900 6 0 Polycarpaea repens@2068900
0.05 0.04
Figure S9. ADHα sequences used for testing relax selection. (a and b) ADHα orthologs of Caryophyllace- ae (blue, designated as test branches in RELAX analysis, Table S4), as compared to those betalain-producing Caryophyllales species (pink, designated as reference branches in RELAX analysis, Table S4). Blue branches showed no obvious acceleration of substitution in their coding sequences (CDS, a), whereas there was appar- ent acceleration in their peptide sequences (b). Tips marked with '@' are from assembled transcriptomes (see Supporting Information Methods S1). The rest of the sequences are from PCR and Sanger sequencing from DNA (H. latifolia, S. marina, and P. polygonifolia) or RNA. AtADH2 1 ------MLLHFSPAKPLISPP------NLRRNSPTFLISPPR------SLR BvADHβ 1 ----MLSLSSTTTAKPSPSPSPANFPAKLSSLSTITTTLSFSPRRRYFHGVKTLT BvADHα 1 MISLSSFHPSSTTATATAAAATTHPPQQCPAFSSPPSHLSLPLRHPRQHLVVRCG AaPDH 1 ------MAILSSMFNPSPPQ------SyADH 1 ------
AtADH2 34 IRAIDAAQIFDYETQLKSEYRKSS------ALKIAVLGFGNFGQFLSKTLIRHG BvADHβ 52 IRSIDAAQFFDYESKLAAINTTSSSSSSSYSKLKIAIVGFGNYGQFLAKTLVSQG BvADHα 56 GGGSASESVFNRDSAATRVSNDHLDVSKRDVKLKIAIIGFGNFGQFLAKTMAKQG AaPDH 15 --GFCKKNIIKILKSLSMQN------VLIVGVGFMGGSFAKSLRRSG SyADH 1 ------MKIGVVGLGLIGASLAGDLRRRG
AtADH2 82 HDLITHSRSD----YSDAANSIGARFFDNPHDLCEQHPDVVLLCTSILSTESVLR BvADHβ 107 HTVLAYSRSD----YSKIAANLGVSYFSDPDDLCEEHPEVIMLCTSILSTEVMLN BvADHα 111 HRVLAYSRSD----YSRAAKEIGVEYFTDADDLCEEHPEVILLCTSILSTEKVLR AaPDH 54 FKGKIYGYDINPESISKAVDLGIIDEGTTSIAKVEDFSPDFVMLSSPVRTFREIA SyADH 24 HYLIGVSRQQ----STCEKAVERQLVDEAGQDLSLLQTAKIIFLCTPIQLILPTL
AtADH2 133 SFPFQRLRRSTLFVDVLSVKEFPKALFIKYLPKEFDILCTHPMFGPESGKHSWSG BvADHβ 158 SLPLQRLKRSTLFVDVLSVKEFPRNLFLQTLPSDFDILCTHPMFGPESGKNGWGS BvADHα 162 SLPLHRLRRSTLFADVLSVKEFPRSLFLQLLPKDFDILCTHPMFGPDSGKDGWGG AaPDH 109 KKLSYILSEDATVTDQGSVKGKLVYDLENILGKRFVGG--HPIAGTEKSGVEYSL SyADH 75 EKLIPHLSPTAIVTDVASVKTAIAEPASQLWSG-FIGG--HPMAGTAAQGIDGAE
AtADH2 188 LPFVYDKVRIGDAASRQ--ERCEKFLRIFENEGCKMVEMSCEKHDYYAAGSQFVT BvADHβ 213 LPFVYDKVRIGKDEGRI--KRCESFLDVFRREGCRVEEMTCAEHDKFAAGSQFIT BvADH2 217 LPFVFDKVRVGSDQSRT--SRAEAFLDVFRNAGCRMVEMSCVDHDKHAAGSQFIT AaPDH 162 DNLYEGKKVILTPTKKTDKKRLKLVKRVWEDVGGVVEYMSPELHDYVFGVVSHLP SyADH 127 ENLFVNAPYVLTPTEYTDPEQLACLRSVLEPLGVKIYLCTPADHDQAVAWISHLP
AtADH2 241 HTMGRVLEKYGVESSPINTKGYETLLDLVENTSSDSFELFYGLFMYNPNALEQLE BvADHβ 266 HFLGRVLEKMDLEDTPINTKGYESLLNLVDNTSKDSFELFYGLFLYNQNAMEQLE BvADHα 270 HMMGRVLEKLALENTPINTKGYESLLNLVDNTARDSFELFYGLFLYNKNAMEQLD AaPDH 217 HAVAFALVDTLIHMS------TPEVDLFKYPGGGFKDFTRIAKSDP SyADH 182 VMVSAALIQACAGEKDG------DILKLAQNLASSGFRDTSRVGGGNP
AtADH2 296 RLDMAFESVKKELFGRLHQQYRKQMFGG---EVQSPKKTEQKLLNDGGVVPMNDI BvADHβ 321 RLDWAFELVKKQLFGHLHGLLRKQLFGFSEIDERIGKAKEIKFLSDAAEQNGSAL BvADHα 325 RMDWAFEMVKKQLSGYLHDLVRKQLMLEGNNDQAEVTFDKPLMLPSPTINPPQIV AaPDH 257 IMWRDIFLENKENVMKAIEGFEKSLNHLKELIVREAEEELVEYLKEVKIKRMEID SyADH 224 ELGTMMATYNQRALLKSLQDYRQHLDQLITLISNQQWPELHRLLQQTNGDRDKYV
AtADH2 348 SSSSSSSSSSS------BvADHβ 376 SARENANSETN------BvADHα 380 PSADMAEKKHDLVVVNGTR AaPDH 312 ------SyADH 279 E------Figure S10. Histidine 217 residue responsible for Tyr sensitivity of Aquifex aeolicus PDH (AaPDH) is still present in BvADHα. Previous studies showed that the H217 residue of AaPDH (denoted by red triangles) is absent in Tyr-insensitive ADH of Synechocystis sp. PCC6803 (SyADH) and confers Tyr sensitivity of AaPDH (Sun et al., 2009, Legrand, P. et al. 2008). The amino acid alignment of AaPDH, SyADH together with BvADHα, BvADHβ, and Arabidopsis ADH (AtADH2) showed that corresponding His residues are present in all plant ADHs. This result suggests that yet to be identified novel residues and mechanism are involved in the relaxed Tyr sensitivity of BvADHα Table S1. Primers used as indicated in the text and methods
Species (gene) Purpose Primer name Primer sequence 5’ to 3’ Beta vulgaris (BvADHβ) RT-PCR pHM0290SLNBvADHβF GGTTCCGCGTGGATCCCTAACAATTCGCAGCAT Beta vulgaris (BvADHβ) RT-PCR pHM0291SLNRBvADHβR AATTCGGAGACAAATTGAGAATTCATCGTGACTG Beta vulgaris (BvADHα) RT-PCR pHM0372SLNBvADHαF CTGGTTCCGCGTGGATCCTGCGGTGGAGGTGGTTCG Beta vulgaris (BvADHα) RT-PCR pHM0373SLNBvADHαR GTTAATGGTACTAGATAGGAATTCATCGTGACTGA Arabidopsis thaliana (AtADH2) Cloning pHM0384SLNAtADHαF CTGGTTCCGCGTGGATCCGCAATCGACGCCGCCCAA Arabidopsis thaliana (AtADH2) Cloning pHM0385SLNAtADHαR TCATCATCATCATCTTAAGAATTCATCGTGACTGA Spinacea oleracea (SoADHβ) Cloning pHM0582SoADHβF CTGGTTCCGCGTGGATCCGCCGCTACCAATACCTCC Spinacea oleracea (SoADHβ) Cloning pHM0583SoADHβR AATTCAGAGATCAATTGAGAATTCATCGTGACTGA Spinacea oleracea (SoADHα) Cloning pHM0584SoADHαF CTGGTTCCGCGTGGATCCTGCGCCGCCTCTGACTCC Spinacea oleracea (SoADHα) Cloning pHM0585SoADHαR TGGTAATAATTCTAGATAGGAATTCATCGTGACTGA Nepenthes alata (NaADHβ) Cloning pHM0603SLNNaADHF CTGGTTCCGCGTGGATCCGCCGCGCTGCCAAACGACT Nepenthes alata (NaADHβ) Cloning pHM0604SLNNaADHR AAATGTTGAGAGAAATTGAGAATTCATCGTGACTGA Portulaca oleracea (PoADHα) RT-PCR pHM0609SLNPoADHαAF CTGGTTCCGCGTGGATCCTGCTCATCATCATCATCAT Portulaca oleracea (PoADHα) RT-PCR pHM0610SLNPoADHαAR CGTCAACGATAGATCATAGGAATTCATCGTGACTGA Mirabilis jalapa (MjADHα) Cloning pHM0624SLNMjADHαAF CTGGTTCCGCGTGGATCCATAGCGATAGTTGGGTTTG Mirabilis jalapa (MjADHα) Cloning pHM0625SLNMjADHαAR TATCAATGGTCGTCGATAGGAATTCATCGTGACTGA Rivina humilis (RhADHα) Cloning pHM0647SLNRhADHαF CTGGTTCCGCGTGGATCCTGCACGGCCTTCACTAAAAC Rivina humilis (RhADHα) Cloning pHM0648SLNRhADHαR TCAATGGATCAAAGCGGTAGGAATTCATCGTGACTGA Beta vulgaris (BvADHα) RT-PCR BvADHα_q_F TCAAGCTGAGGTTACTTTTGACA Beta vulgaris (BvADHα) RT-PCR BvADHα_q_R AAGAAGCATGATTTAGTGGTGGT Beta vulgaris (BvADHβ) RT-PCR BvADHβ_q_F TGCAGCGACTTAAACGATCG Beta vulgaris (BvADHβ) RT-PCR BvADHβ_q_R TTGGGGAAGTTTGCCGTTTG Beta vulgaris (BvADHα) RT-PCR pHM0793SLNBvADHαF AGTTCCCTCTGCTGATATG Beta vulgaris (BvADHα) RT-PCR pHM0794SLNBvADHαR GTGGTTAATGGTACTAGATAG Beta vulgaris (BvADHβ) qPCR pHM0791SLNBvADHβF GCGAAGGAGATCAAATTTCT Beta vulgaris (BvADHβ) qPCR pHM0792SLNBvADHβR TCAATTTGTCTCCGAATTTGC Beta vulgaris (BvADHα) qPCR BvADHα_F ATGATTTCACTCTCTTCTTTTCATCC Beta vulgaris (BvADHα) qPCR BvADHα_R GATTTAGTGGTGGTTAATGGTACTAGATAG Beta vulgaris (BvADHβ) qPCR BvADHβ_F ATGCTTTCTCTCTCCTCCAC Beta vulgaris (BvADHβ) qPCR BvADHβ_R CAAATTCGGAGACAAATTGA Beta vulgaris (BvActin) qPCR pHM0001HMBvACT TCTATCCTTGCATCTCTCAG Beta vulgaris (BvActin) qPCR pHM0002HMBvACT TCTCCAAGGGCGAGTATGAT Beta vulgaris (BvDODA) qPCR pHM0003HMBvDODA CATTGGTTCAGGAAGTGCAA Beta vulgaris (BvDODA) qPCR pHM0004HMBvDODA CCTTTGATTCATGGCTTCGT Beta vulgaris (BvMYB1) qPCR pHM0576BvMYB1F TATCAAACGAGGGCACTTC Beta vulgaris (BvMYB1) qPCR pHM0577BvMYB1R GATGGTCTTTGATAGCAGC Beta vulgaris (BvCYP76AD1) qPCR pHM0005HMBvCYP76AD1 CTTTTCAGTGGAATTAGCCCACC Beta vulgaris (BvCYP76AD1) qPCR pHM0006HMBvCYP76AD1 TGGAACATTATGGAAGATATTGGG GFP qPCR tGFP_q_F GGCTGGAAGAGTGATCGGAG GFP qPCR tGFP_q_R ACGCTACTGTTGAGCATCTTCA Gene Racer oligoT RT-PCR GeneRacer OligoT GCTGTCAACGATACGCTACGTAACGGCATGACAGTG(T)20 Eukaryotic translational elongation factor 1α qPCR EF1α_q_F AGCTTTACCTCCCAAGTCATC Eukaryotic translational elongation factor 1α qPCR EF1α_q_R CCAAGATTGACAGGCGTTCT Table S2. Sequences of Caryophyllales (ingroups) and non-Caryophyllales (outgroups) used in this study. Taxon Source Accession code Citation Ingroups Achatocarpaceae_Phaulothamnus_spinescens Smith Lab MJM1677 (Brockington et al., 2015) Aizoaceae_Cypselea_humifusum 1KP GJNX (Matasci et al., 2014) Aizoaceae_Delosperma_echinatum 1KP BJKT (Matasci et al., 2014) Aizoaceae_Sesuvium_portulacastrum 1KP HZTS (Matasci et al., 2014) Aizoaceae_Sesuvium_verrucosum 1KP EDIT (Matasci et al., 2014) Aizoaceae_Trianthemum_portulacastrum 1KP OMYK (Matasci et al., 2014) Aizoaceae_Zaleya_pentandra 1KP BERS (Matasci et al., 2014) Amaranthaceae_Aerva_javanica 1KP HDSY (Matasci et al., 2014) Amaranthaceae_Aerva_lanata 1KP PDQH (Matasci et al., 2014) Amaranthaceae_Alternanthera_brasiliana 1KP ZBPY (Matasci et al., 2014) Amaranthaceae_Alternanthera_caracasana 1KP OHKC (Matasci et al., 2014) Amaranthaceae_Alternanthera_sessilis 1KP BWRK (Matasci et al., 2014) Amaranthaceae_Alternanthera_tenella 1KP EYRD (Matasci et al., 2014) Amaranthaceae_Amaranthus_cruentus 1KP XSSD (Matasci et al., 2014) Amaranthaceae_Amaranthus_retroflexus 1KP WMLW (Matasci et al., 2014) Amaranthaceae_Atriplex_hortensis 1KP ONLQ (Matasci et al., 2014) Amaranthaceae_Atriplex_prostrata 1KP AAXJ (Matasci et al., 2014) Amaranthaceae_Atriplex_rosea 1KP CBJR (Matasci et al., 2014) Amaranthaceae_Bassia_scoparia 1KP WGET (Matasci et al., 2014) Amaranthaceae_Beta_maritima 1KP FVXD (Matasci et al., 2014) Amaranthaceae_Beta_vulgaris Genome v1.1 (Dohm et al., 2014) Amaranthaceae_Blutaparon_vermiculare 1KP CUTE (Matasci et al., 2014) Amaranthaceae_Chenopodium_amaranticolor SRA SRX151423 (Zhang et al., 2012) Amaranthaceae_Chenopodium_quinoa 1KP SMMC (Matasci et al., 2014) Amaranthaceae_Froelichia_floridana Smith Lab MJM1665 (Brockington et al., 2015) Amaranthaceae_Salicornia_europaea SRA SRX302090 (Fan et al., 2013) Basellaceae_Basella_alba 1KP CTYH (Matasci et al., 2014) Cactaceae_Lophophora_williamsii 1KP CPKP (Matasci et al., 2014) Cactaceae_Pereskia_aculeata 1KP JLOV (Matasci et al., 2014) Caryophyllaceae_Cerastium_arvense Smith Lab MJM1767 (Brockington et al., 2015) Caryophyllaceae_Dianthus_caryophyllus Genome v1.0 (Yagi et al., 2014) Caryophyllaceae_Drymaria_cordata Smith Lab LCMsn (Brockington et al., 2015) Caryophyllaceae_Polycarpaea_repens 1KP RXEN (Matasci et al., 2014) Caryophyllaceae_Saponaria_officinalis 1KP SKNL (Matasci et al., 2014) Caryophyllaceae_Schiedea_membranacea 1KP OLES (Matasci et al., 2014) Caryophyllaceae_Silene_latifolia 1KP FZQN (Matasci et al., 2014) SRX118777– Caryophyllaceae_Silene_latifoliaSRA SRA (Muyle et al., 2012) SRX118782 Caryophyllaceae_Silene_vulgaris SRA SRX096120 N/A Caryophyllaceae_Spergularia_media 1KP TJES (Matasci et al., 2014) Droseraceae_Aldrovanda_vesiculosa Smith Lab MJM1652 (Brockington et al., 2015) Droseraceae_Dionaea_muscipula SRA SRX312294 (Jensen et al., 2015) Frankeniaceae_Frankenia_laevis 1KP WPYJ (Matasci et al., 2014) Microteaceae_Microtea_debilis 1KP YNFJ (Matasci et al., 2014) Molluginaceae_Mollugo_cerviana 1KP RNBN (Matasci et al., 2014) Molluginaceae_Mollugo_nudicaulis 1KP SCAO (Matasci et al., 2014) Molluginaceae_Mollugo_verticillata 1KP NXTS (Matasci et al., 2014) Nepenthaceae_Nepenthes_alata 1KP WQUF (Matasci et al., 2014) Nyctaginaceae_Abronia_carletonii Smith Lab MJM1751 (Brockington et al., 2015) Nyctaginaceae_Acleisanthes_lanceolata Smith Lab MJM1741 (Brockington et al., 2015) Nyctaginaceae_Acleisanthes_obtusa Smith Lab MJM1697 (Brockington et al., 2015) Nyctaginaceae_Anulocaulis_leiosolenus Smith Lab SRX717838 (Yang et al., 2015) Nyctaginaceae_Boerhavia_burbidgeana 1KP VJPU (Matasci et al., 2014) Nyctaginaceae_Boerhavia_coccinea 1KP ZBTA (Matasci et al., 2014) Nyctaginaceae_Bougainvillea_spectabilis 1KP JAFJ (Matasci et al., 2014) Nyctaginaceae_Bougainvillea_stipitata Smith Lab SRX718672 (Yang et al., 2015) Nyctaginaceae_Cyphomeris_gypsophiloides Smith Lab MJM1714 (Brockington et al., 2015) Nyctaginaceae_Guapira_obtusata Smith Lab SRX718384 (Yang et al., 2015) Nyctaginaceae_Mirabilis_jalapa 1KP JGAB (Matasci et al., 2014) Nyctaginaceae_Mirabilis_multiflora Smith Lab MJM1771 (Brockington et al., 2015) Nyctaginaceae_Pisonia_aculeata Smith Lab SRX718389 (Yang et al., 2015) Nyctaginaceae_Pisonia_umbellifera Smith Lab SFB29 (Brockington et al., 2015) Physenaceae_Physena_madagascariensis 1KP RUUB (Matasci et al., 2014) Phytolaccaceae_Ercilla_volubilis Smith Lab MJM1649 (Brockington et al., 2015) Phytolaccaceae_Hilleria_latifolia 1KP SFKQ (Matasci et al., 2014) Phytolaccaceae_Petiveria_alliacea 1KP AZBL (Matasci et al., 2014) Phytolaccaceae_Phytolacca_americana 1KP BKQU (Matasci et al., 2014) Phytolaccaceae_Phytolacca_bogotensis 1KP MRKX (Matasci et al., 2014) Phytolaccaceae_Phytolacca_dioica Smith Lab SFB31 (Brockington et al., 2015) Phytolaccaceae_Rivina_humilis Smith Lab SRX718277 (Yang et al., 2015) Phytolaccaceae_Seguieria_aculeata Smith Lab SRX718486 (Yang et al., 2015) Plumbaginaceae_Limonium_spectabile 1KP WOBD (Matasci et al., 2014) Polygonaceae_Antigonon_leptopus Smith Lab MJM1811 (Brockington et al., 2015) Polygonaceae_Fagopyrum_esculentum SRA SRX112838 N/A Polygonaceae_Polygonum_convolvulus 1KP FYSJ (Matasci et al., 2014) Polygonaceae_Polygonum_cuspidatum SRA SRX079484 (Hao et al., 2012) Polygonaceae_Rheum_nobile SRA SRX621187 N/A Polygonaceae_Rheum_rhabarbarum SRA SRX286365 N/A Polygonaceae_Rumex_acetosa SRA ERX190940 N/A ERX190941, Polygonaceae_Rumex_palustris SRA N/A ERX190942 Portulacaceae_Portulaca_amilis 1KP LDEL (Matasci et al., 2014) Portulacaceae_Portulaca_cryptopetala 1KP LLQV (Matasci et al., 2014) Portulacaceae_Portulaca_grandiflora 1KP CPLT (Matasci et al., 2014) Portulacaceae_Portulaca_molokiniensis 1KP UQCB (Matasci et al., 2014) Portulacaceae_Portulaca_oleracea 1KP EZGR (Matasci et al., 2014) Portulacaceae_Portulaca_pilosa 1KP IWIS (Matasci et al., 2014) Portulacaceae_Portulaca_suffruticosa 1KP GCYL (Matasci et al., 2014) Sarcobataceae_Sarcobatus_vermiculatus 1KP GIWN (Matasci et al., 2014) Simmondsiaceae_Simmondsia_chinensis 1KP CVDF (Matasci et al., 2014) Talinaceae_Talinum_sp 1KP LKKX (Matasci et al., 2014) SRX099851, Tamaricaceae_Reaumuria_trigyna SRA N/A SRX105466 All 8 runs in Tamaricaceae_Tamarix_hispida SRA (Wang et al., 2014) PRJNA170420 Outgroups Accessed May 28, Arabidopsis_thaliana Genome (Goodstein et al., 2012) 2014 Accessed Apr 21, Oryza_sativa Genome (Goodstein et al., 2012) 2015 Accessed May 28, Solanum_lycopersicum Genome (Goodstein et al., 2012) 2014 Accessed Apr 21, Vitis_vinifera Genome (Goodstein et al., 2012) 2015
Table S3. Amino acid levels of Nicotiana benthamiana leaves expressing GFP, BvADHα, or BvADHβ. Agrobacteria carrying the 35S::GFP, 35S::BvADHα, or 35S::BvADHβ construct were infiltrated to Nicotiana benthamiana leaves and the levels of amino acids were analyzed after three days post-infiltration. Data are mean ± s.e.m. (nmol/gFW, n = 5 biological replications). Asterisks denote values significantly different from the control 35S::GFP sample (Student t-test, p<0.01). Tryptophan, lysine, cysteine, and histidine levels were below quantification threshold.
Amino Acids 35S::GFP 35S::BvADHα 35S::BvADHβ alanine 99.8 ± 15.5 93.0 ± 14.8 88.1 ± 20.0 glycine 15.5 ± 1 17.5 ± 2.1 13.6 ± 0.2 valine 23.9 ± 9.7 23.8 ± 8.3 22.1 ± 8.4 leucine 21.3 ± 10.4 21.8 ± 9.2 18.8 ± 8.3 isoleucine 13.8 ± 7 13.3 ± 5.7 13.3 ± 6.7 proline 154.8 ± 67.4 126.7 ± 56.3 137.3 ± 75.4 methionine 2.8 ± 0.4 3.1 ± 0.4 2.6 ± 0.2 serine 57.4 ± 8 58.6 ± 11.7 43.9 ± 3.9 threonine 69.4 ± 7.5 67.8 ± 8.6 58.1 ± 6.5 phenylalanine 10.8 ± 0.7 5.9 ± 1.2* 9.7 ± 0.7 aspartic acid 173.5 ± 45.5 176.8 ± 40.6 132.7 ± 41.5 glutamic acid 941.6 ± 45.8 968.1 ± 91.6 746.4 ± 111.4 ornithinea 54.9 ± 1.6 56.2 ± 2.4 48.4 ± 2.9 asparagine 6.8 ± 1.2 6.9 ± 1.5 4.9 ± 1.0 glutamine 345.2 ± 116.1 348.7 ± 138.4 291.3 ± 107.7 tyrosine 11.2 ± 2.8 116.8 ± 15.1* 17.2 ± 3.2 aArginine was quantified as its non-enzymatic degradation product ornithine. Table S4. RELAX analysis support the acceleration in amino acid substitution in Caryophyllales is due to relaxed purifying selection, instead of intensified positive selection.
Model log L # par. AICc Ltree Branch ω1 (purifying ω2 (nearly ω3 (positive set selection) neutral) selection)
Partitioned -5484.8 38 11046.5 2.23 Reference 0.0743 (100%) MG94xREV Test 0.166 (100%)
Null -5374.3 41 10831.7 11.9 Reference 0.00 (83%) 0.550 (15%) 30.9 (1.4%)
Test 0.00 (83%) 0.550 (15%) 30.9 (1.4%)
Alternative -5359.6 42 10804.2 84.5 Reference 0.00598 (91%) 0.650 540 (1.5%) (7.9%) Test 0.0646 (91%) 0.794 29.0 (1.5%) (7.9%)
K = 0.54. Test for selection relaxation (K < 1) was significant (p = 5.6e-8, LR = 29.48)
Supplemental References for Table S2
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