Supplementary Information for Unleashing floret fertility by a mutated homeobox gene improved grain yield during wheat evolution under domestication Shun Sakuma, Guy Golan, Zifeng Guo, Taiichi Ogawa, Akemi Tagiri, Kazuhiko Sugimoto, Nadine Bernhardt, Jonathan Brassac, Martin Mascher, Goetz Hensel, Shizen Ohnishi, Hironobu Jinno, Yoko Yamashita, Idan Ayalon, Zvi Peleg, Thorsten Schnurbusch, Takao Komatsuda Corresponding authors: Shun Sakuma; [email protected] Thorsten Schnurbusch; [email protected] Takao Komatsuda; [email protected] This PDF file includes: Figs. S1 to S9 Tables S1 to S5 Other supplementary materials for this manuscript include the following: Datasets S1 to S4 1 16 14 14 13 12 12 11 10 10 8 9 Plant height (cm) Spike length (cm) 6 8 4 7 Bobwhite Bobwhite PKVB40PKVB40 (–) PKVB48 (+) PKVB48 (–) PKVB59 (+) PKVB59 (–) PKVB78 (+) PKVB78 (–) (+) PKVB40PKVB40 (–) PKVB48 (+) PKVB48 (–) PKVB59 (+) PKVB59 (–) PKVB78 (+) PKVB78 (–) (+) 16 24 14 22 12 10 20 8 18 6 Spike number per plant Spikelet number per spike 16 4 Bobwhite Bobwhite PKVB40PKVB40 (–) PKVB48 (+) PKVB48 (–) PKVB59 (+) PKVB59 (–) PKVB78 (+) PKVB78 (–) (+) PKVB40PKVB40 (–) PKVB48 (+) PKVB48 (–) PKVB59 (+) PKVB59 (–) PKVB78 (+) PKVB78 (–) (+) 16 4.0 ) 2 15 3.5 14 3.0 13 2.5 12 2.0 11 10 Grain width (mm) 1.5 Grain area size (mm 9 1.0 Bobwhite Bobwhite PKVB40PKVB40 (–) PKVB48 (+) PKVB48 (–) PKVB59 (+) PKVB59 (–) PKVB78 (+) PKVB78 (–) (+) PKVB40PKVB40 (–) PKVB48 (+) PKVB48 (–) PKVB59 (+) PKVB59 (–) PKVB78 (+) PKVB78 (–) (+) 18 8.0 7.5 17 7.0 16 6.5 15 6.0 14 Grain length (mm) 5.5 Grain perimeter length (mm) 13 5.0 Bobwhite Bobwhite PKVB40PKVB40 (–) PKVB48 (+) PKVB48 (–) PKVB59 (+) PKVB59 (–) PKVB78 (+) PKVB78 (–) (+) PKVB40PKVB40 (–) PKVB48 (+) PKVB48 (–) PKVB59 (+) PKVB59 (–) PKVB78 (+) PKVB78 (–) (+) Fig.Supplementary S1. Phenotype Figure of GNI1 1.- PhenotypeA RNAi transgenic of GNI1-RNAi Bobwhite transgenic wheat inwheat T1 generation. in T2 generation. White boxes White and green boxes indicate non-transgenic and transgenic plants, respectively. indicate construct-negative plants and blue boxes construct-positive plants. 2 A Kitami35 Hokushin Chihokukomugi Kun383-46 (F3) Kitamoe Wichita Kitakei1354 Takunekomugi Kitami19 Kitahonami Chihokukomugi Kitakei1463 Kitami53 Norman PI 173438 Kitakei1660 Horoshirikomugi Kun1051-166-8 105Y (F4) Chihokukomugi 105N Chihokukomugi 105K Kitami33 Kitakei1409 Not available Kitakei1033 B 7 P = 0.0990 5 P = 0.0008 P = 0.0034 P = 0.0757 6 4 5 3 Grains per spikelet Florets per spikelet 4 2 3 105K 105N 105Y 105K 105N 105Y P = 0.0933 P = 0.0015 P = 0.2372 80 60 P = 0.0416 70 50 60 40 Grains per spike Florets per spike 50 30 40 20 105K 105N 105Y 105K 105N 105Y Supplementary Fig. 2 Pedigree analysis of Kitahonami. (a) Genealogy of Japanese cultivar Kitahonami. (b) Phenotype of floret and grain in Kitahonami pedigrees. Fig. S2. Pedigree of Kitahonami and their yield performance. (A) Genealogy of Japanese winter wheat cultivar Kitahonami. (B) Phenotype of florets per spikelet and grains per spikelet in the pedigree. 3 90 ns 60 ns + 23 ns 85 50 + 105N 21 + ns 105Y 80 40 + + 19 ns 75 30 + + 70 20 17 + ns Plant height (cm) 65 Spike number per plant 10 15 + + P = 0.039 60 0 + -5 13 + P = 3.1x10 KH KH + 105N 105Y 105N 105Y 11 -6 + P = 1.8x10 25 140 + -7 ns spikelet position 9 + P = 1.8x10 24 130 + 7 = 4.1x10-4 23 a + P 120 b b + -5 22 5 P = 1.4x10 110 + 21 + 3 = 0.009 100 P 20 + Spike length (mm) 1 + 19 90 P = 0.001 Spikelet number per spike + 18 80 0 2 4 6 8 KH KH Grains per spikelet 105N 105Y 105N 105Y Fig. S3. Phenotype of TILLING mutant. Homozygous plants were compared in the M3 family. 105N indicates revertant mutant (functional) allele and 105Y wildtype (reduced-function) allele as Kitahonami (KH). Box edges represent the 25% quantile and 75% quantile with the median values shown by bold lines. Whiskers extend to data less than 1.5 times the interquartile range, and remaining data are indicated by circles. + indicate mean value. Differences between the haplotypes were analyzed by Tukey HSD or Student’s t-test (P ≤ 0.05). ns; not significant. 4 P = 0.0889 1000 50 45 P = 0.0510 P = 0.9658 900 40 P = 0.3091 800 40 P = 0.3158 700 P = 0.0004 30 600 35 20 Grain yield (kg/10a) 500 Grain number per spike Thousand grain weight (g) 400 10 30 Naganuma Kitami Naganuma Kitami Naganuma Kitami P = 0.2568 P = 0.7434 1200 2400 45 P = 0.0001 2 2200 P = 0.0509 1000 2000 40 800 P = 0.0051 P = 0.6579 1800 600 1600 35 1400 Biomass (kg/10a) Harvest index (%) Spike number per m 400 1200 200 1000 30 Naganuma Kitami Naganuma Kitami Naganuma Kitami Fig. S4.Fig. Yield S4. test Yield in the testtwo field of mutantconditions. in the two field conditions. Kitahonami revertant mutant family at Kitahonami revertant mutant family at M4 generation was compared. Blue box and white box indicate 105Y allele and 105N allele, respectively. DifferencesM4 generation were determined was by compared. Student’ s t-test Bluebox (N=4 plots indicates in Kitami and105Y N=3 alleleplots in andNaganuma). white box 105N allele. Differences were determined by Student’s t-test (n = 4 plots in Kitami and n = 3 plots in Naganuma). 5 0.07 Triticum monococcum (KT3-5) 0.06 0.05 0.04 0.03 GNI1-A / Actin / GNI1-A 0.02 0.01 0.00 GP LP FP TS WA GA YA Spike developmental stage 0.07 Triticum durum (cv. Langdon) 0.06 0.05 0.04 0.03 GNI1-A / Actin / GNI1-A 0.02 0.01 0.00 LP WA GA TP TP (central) (apical) Spike developmental stage Fig. S5.Fig. ExpressionS5 Expression pattern pattern ofof GNI1-AGNI1- Ain indiploid immature and tetraploid spikes wheat. of diploid and tetraploid wheat. GP: glumeGP: primordium glume primordium stage; stage; LP: lemmaLP: lemma primordium primordium stage;stage; FP:FP: floret floret primordium primordium stage; stage; TS: terminalTS: terminalspikelet spikelet stage; stage; WA: WA:white white anther anther stage; stage; GA: GA: green anther anther stage; stage; YA: YA: yellow yellow anther antherstage. stage; TP: tipping stage. Means ± SE of three biological replicates are shown. 6 Fig. S6. Structure of GNI1 homoeologs in Triticum aestivum. Boxes indicate exons and lines introns. White boxes indicate UTRs and grey boxes coding regions. Translucent boxes in GNI1-B and GNI1-D indicate un-transcribed sequences. 7 Traes_7AS_EBD5F1F54.1 Traes_7BS_581AA844D.1 Traes_7DS_12C14942B.1 study High levelTissue tissue High levelAge age High levelStress-disease stress-diseaseHigh levelVariety variety roots, roots, veg, 24_d, no_stress, no_stress, CS, CS (n=3) roots, roots, veg, 24_d, abiotic, P_starvation_10d, CS, CS (n=3) FT-A1 FT-B1 FT-D1 lf_shoot, shoots, veg, 24_d, no_stress, no_stress, CS, CS (n=3) lf_shoot, shoots, veg, 24_d, abiotic, P_starvation_10d, CS, CS (n=3) spike, spikelet, repr, anthesis, disease, Fus_50h, other, CM-3 (n=4) spike, spikelet, repr, anthesis, no_stress, mock_30h, other, CM-82036 (n=3) spike, spikelet, repr, anthesis, no_stress, mock_50h, other, CM-3 (n=2) spike, spikelet, repr, anthesis, disease, Fus_30h, other, CM-4 (n=3) spike, spikelet, repr, anthesis, disease, Fus_30h, other, CM-82036 (n=3) spike, spikelet, repr, anthesis, disease, Fus_50h, other, CM-2 (n=3) spike, spikelet, repr, anthesis, no_stress, mock_50h, other, CM-2 (n=3) spike, spikelet, repr, anthesis, no_stress, mock_50h, other, CM-1 (n=4) spike, spikelet, repr, anthesis, no_stress, mock_50h, other, CM-4 (n=3) spike, spikelet, repr, anthesis, no_stress, mock_30h, other, CM-4 (n=3) spike, spikelet, repr, anthesis, disease, Fus_30h, other, CM-3 (n=4) spike, spikelet, repr, anthesis, no_stress, mock_30h, other, CM-1 (n=3) spike, spikelet, repr, anthesis, disease, Fus_30h, other, CM-2 (n=2) spike, spikelet, repr, anthesis, disease, Fus_50h, other, CM-82036 (n=3) spike, spikelet, repr, anthesis, no_stress, mock_30h, other, CM-2 (n=4) spike, spikelet, repr, anthesis, disease, Fus_50h, other, CM-4 (n=3) spike, spikelet, repr, anthesis, disease, Fus_50h, other, CM-1 (n=2) spike, spikelet, repr, anthesis, no_stress, mock_30h, other, CM-3 (n=2) spike, spikelet, repr, anthesis, disease, Fus_30h, other, CM-1 (n=3) spike, spikelet, repr, anthesis, no_stress, mock_50h, other, CM-82036 (n=3) grain, endosp, repr, 10dpa, no_stress, no_stress, CS, CS (n=4) grain, transfer, repr, 20dpa, no_stress, no_stress, CS, CS (n=4) grain, aleurone, repr, 20dpa, no_stress, no_stress, CS, CS (n=6) grain, aleu_endo, repr, 30dpa, no_stress, no_stress, CS, CS (n=4) grain, sta_endosp, repr, 30dpa, no_stress, no_stress, CS, CS (n=4) grain, sta_endosp, repr, 20dpa, no_stress, no_stress, CS, CS (n=4) grain, endosp, repr, 20dpa, no_stress, no_stress, CS, CS (n=4) lf_shoot, flag_leaf, repr, 12dpa, transg, GPC_RNAi, other, GPC_RNAi (n=4) lf_shoot, flag_leaf, repr, 12dpa, no_stress, no_stress, other, Bobw (n=3) grain, aleurone, repr, 6dpa, no_stress, no_stress, other, Banks (n=1) grain, sta_endosp, repr, 6dpa, no_stress, no_stress, other, Banks (n=1) grain, aleurone, repr, 9dpa, no_stress, no_stress, other, Banks (n=1) grain, sta_endosp, repr, 9dpa, no_stress, no_stress, other, Banks (n=1) grain, aleurone, repr, 14dpa, no_stress, no_stress, other, Banks (n=1) grain, sta_endosp, repr, 14dpa, no_stress, no_stress, other, Banks (n=1) lf_shoot, leaves, seedling, seedling, disease, Stripe_6+, other, AvocetS (n=1) lf_shoot, 2_leaf, seedling, 14_d, no_stress, no_stress, other, Sevin (n=4) lf_shoot, 2_leaf, seedling, 14_d, disease, Sept_4d, other, Sevin (n=2) lf_shoot, 2_leaf, seedling, 14_d, disease, Sept_10d, other, Sevin (n=1) lf_shoot, 2_leaf, seedling, 14_d, disease, Sept_13d, other, Sevin (n=1) lf_shoot, shoots, veg,