1. Supplemental table S1: Tri Acyl Glycerol (TAG) analysis and comparison of A3OE (A4, A10 and A17) to their isogenic negative segregants (A4N, A10N and A17N) and Tissue culture control (TCC). Data points are the average and standard error from 3 technical measurements. Results are expressed as percentage of total TAG content. A4A4NA10A10NA17A17NTCC C14:0 0.12 ± 0.020.12 ± 0.010.11 ± 0.020.13 ± 0.010.11 ± 0.000.1 ± 0.000.16 ± 0.02 C14:1 0.08 ± 0.020.12 ± 0.030.07 ± 0.000.12 ± 0.020.09 ± 0.000.1 ± 0.010 ± 0.00 C16:0 24.18 ±0.4323.17 ± 0.6020.15 ± 1.2120.01 ± 0.2220.72 ± 0.8421.42 ± 0.4323.3 ± 0.69 C16:1 0.5 ±.040.45 ± 0.040.3 ± 0.020.35 ± 0.010.32 ± 0.010.3 ± 0.010.41 ± 0.04 C18:0 2.62 ± 0.142.28 ± 0.732.21 ± 0.051.64 ± 0.162.42 ± 0.531.71 ± 0.101.88 ± 0.11 C18:1 25.72 ± 0.3823.75 ± 0.5527.97 ± 0.7821.34 ± 0.0727.21 ± 2.3624.28 ± 0.3422.97 ± 1.51 C18:2 42.21 ± 0.0644.99 ± 0.9844.51 ± 0.5050.89 ± 0.3844.23 ± 1.7147.05 ± 0.3346.7 ± 1.22 C18:3n3 1.77 ± 0.082.3 ± 0.092.07 ± 0.032.9 ± 0.012.13 ± 0.112.05 ± 0.092.29 ± 0.03 C20:0 0.29 ± 0.010.25 ± 0.060.28 ± 0.000.23 ± 0.020.32 ± 0.050.22 ± 0.000.2 ± 0.01 C20:1 0.9 ± 0.010.85 ± 0.110.91 ± 0.070.9 ± 0.070.93 ± 0.130.97 ± 0.040.81 ± 0.01 20:1iso 0.18 ± 00.17 ± 0.030.1 ± 0.010.09 ± 0.010.13 ± 0.010.11 ± 0.000.08 ± 0.12 20:2n6 0.11 ±0.010.12 ± 0.010.09 ± 0.000.12 ± 0.010.09 ± 0.000.11 ± 0.020.07 ± 0.10 C22:1 0.11 ± 00.05 ± 0.070.11 ± 0.010.1 ± 0.020.12 ± 0.020.1 ± 0.030 ±0.00 C24:0 0.14 ±0.020.15 ± 0.040.16 ± 0.000.17 ± 0.020.18 ± 0.000.15 ± 0.030 ± 0.00 C24:1 0.09 ± 00.33 ± 0.260.1 ± 0.010.13 ± 0.010.12 ± 0.030.41 ± 0.380.23 ± 0.31

2. Supplemental table S2: Protein profiling analysis and comparison of A3OE flours (A4, A10 and A17) to their isogenic negative segregants (A4N, A10N and A17N) and Tissue culture control (TCC).CF is a flour test control. Values displayed are the average and standard deviation from 3 technical measurements. Results are expressed percentage of total protein content for each class analysed. Glutenin/gliadin ratio has been measured by size exclusion chromatograpy (1) or reverse phase (2). A4A4 NA10A10 NA17A17 NTCCCF averagestdevaveragestdevaveragestdevaveragestdevaveragestdevaveragestdevaveragestdevaveragestdev Total Protein % Flour protein 15.93 0.33 16.68 0.28 15.39 0.12 14.27 0.81 16.32 1.07 15.54 0.18 15.22 0.73 14.14 0.49 Glutenins % Glu-Dy 5.36 0.23 5.53 0.17 6.04 0.13 5.24 0.01 5.51 0.23 5.48 0.35 5.41 0.31 4.78 0.22 % Glu-By 3.64 0.24 3.88 0.11 3.83 0.14 3.48 0.03 3.28 0.10 3.34 0.03 3.64 0.00 3.10 0.25 % Glu-Dx 9.96 0.06 9.94 0.18 10.51 0.04 9.01 0.29 9.08 0.31 8.83 0.14 9.20 0.38 7.50 0.23 % Glu-Bx 9.80 0.16 10.19 0.06 10.30 0.15 9.74 0.24 9.00 0.03 9.66 0.08 9.55 0.03 10.29 0.02 % Glu-Ax 3.92 0.02 4.36 0.02 4.20 0.07 3.91 0.11 3.43 0.09 4.04 0.04 3.86 0.01 5.17 0.02 % HMW 32.69 0.25 33.89 0.20 34.88 0.24 31.38 0.08 30.31 0.76 31.35 0.19 31.66 0.74 30.84 0.74 % LMW 67.31 0.25 66.11 0.20 65.12 0.24 68.62 0.08 69.69 0.76 68.65 0.19 68.34 0.74 69.16 0.74 Glu/Gli 0.49 0.01 0.51 0.00 0.54 0.01 0.46 0.00 0.44 0.02 0.46 0.00 0.46 0.02 0.45 0.02 Gliadins Omega % 24.96 0.73 22.39 1.49 22.39 0.17 23.42 0.60 23.42 1.02 22.53 2.05 24.32 0.66 22.81 0.28 Alpha % 44.99 1.00 48.78 0.91 48.78 0.77 43.45 0.85 44.44 0.46 45.52 1.25 44.50 0.58 45.49 0.13 Gamma % 30.05 0.27 28.84 0.58 28.84 0.95 33.13 1.45 32.14 0.56 31.95 0.80 31.19 0.07 31.72 0.40 %UPP Total area 6.80E+07 2.32E+06 6.75E+07 3.23E+06 6.25E+07 2.01E+06 6.07E+07 2.01E+06 6.06E+07 1.28E+06 5.45E+07 4.06E+05 6.33E+07 1.42E+06 5.88E+07 4.19E+06 %UPP s2 50.96 1.29 57.32 0.54 47.89 0.36 49.51 0.36 46.62 1.93 53.07 1.93 54.75 2.45 55.49 2.60 %UPP s4 41.71 1.20 46.97 0.50 40.41 0.11 40.75 0.11 38.60 1.25 43.52 1.32 44.99 2.27 49.06 2.55 s1s2/s3s4 2.21 0.01 1.94 0.01 2.81 0.07 2.35 0.07 2.58 0.23 2.15 0.16 2.08 0.04 3.40 0.05 Glu/Gli 1.24 0.04 1.26 0.01 1.10 0.01 1.22 0.01 1.21 0.02 1.25 0.04 1.24 0.02 1.36 0.04

3. Supplemental table S3: Ionomic analysis and comparison of A3OE flour (A4, A10 and A17) to their isogenic negative segregants (A4N, A10N and A17N) and Tissue culture control (TCC). CF is a flour test control. Table displays the average from 3 technical measurements. Results are expressed in percentage of dry mass for Calcium (Ca), Potassium (K), Magnesium (Mg), Sodium (Na), Phosphate (P) and Sulfur (S). Results are expressed in milligram per kilogram for Aluminium (Al), Boron, (B), Copper (Cu), Iron (Fe), Manganese (Mn) and Zinc (Zn) Ion A4A4NA10A10NA17A17NTCCCF Al mg/kg 6.0112.384.633.564.3344.163.57 B mg/kg 3.885.642.222.911.912.032.151.88 Ca % 0.03 0.04 Cu mg/kg 3.132.793.63.033.512.993.051.07 Fe mg/kg 19.3226.2414.8111.5416.516.4830.598.18 K % 0.19 0.170.16 0.17 Mg % 0.03 Mn mg/kg 0.881.441.040.850.831.010.896.71 Na % 000.01 P % 0.12 0.110.12 0.1 S % 0.16 0.140.150.14 Zn mg/kg 12.7515.6313.1413.6811.5714.3415.998.51

4. Supplemental table S4 Mixograph and flour parameters for A3OE flour (A4, A10 and A17), their isogenic negative segregants (A4N, A10N and A17N) and Tissue culture control (TCC). CF is a flour test control. A4A4NA10A10NA17A17NTCCCF Mixograph Mixing Time (MT) (min)3.93 ab3.6 ab3.31 ab3.16 a3.39 ab3.22 a3.46 ab3.99 b Peak Resistance (PR) (Torque %)56.56 c52.28 abc53 abc48.01 ab52.13 ac46.83 b49.67 ab50.12 ab Bandwidth at PR (Torque %)33.26 ab33.33 ab29.7 ab28.02 a34.12 ab30.72 a29.6 a37.93 b Resistance Breakdown 1 (%)40.02 a35.36 a38.31 a37.28 a38.26 a37.53 a34.89 a31.58 a Bandwidth Breakdown 1 (%)73.75 a74.28 a75.35 a75.92 a74.68 a76.46 a74.70 a53.40 b Resistance Breakdown 2 (%)32.80 abc31.89 a35.33 d34.04 cd32.74 ab33.84 bcd32.23 a29.86 e Bandwidth Breakdown 2, (%)81.74 a82.56 a81.31 a79.21 a83.55 a81.92 a80.23 a68.35 b Time to Maximum Bandwidth (min)2.25 ab2.08 ab1.75 b1.93 ab1.94 ab1.97 ab2.36 a Maximum Bandwidth (Torque %)48.11 a45.71 a44.45 a40.82 a45.52 a39.98 a36.81 a48.05 a Flour parameters Z-arm mixing properties Water Absorption (%)56.5 6056.860.258.259.260.558.2 DDT (min) 1.63.1 1.4 2.31.42.52.44.2 Stability (%)33.622.21.92.52.44.1

5. Supplemental table S5: Rapid Visco Analyser profiles of A3OE positive (A10), negative (A4N) and Tissue culture control (TCC) flours in presence or absence of the Silver Nitrate (  -amylase inhibitor). Results are expressed in RVU (Rapid Visco Unit). Table displays the average of 2 technical measurements. TestPeakTroughBreakdownFinal ViscSetback TCC113.5064.8348.67147.5882.75 TCC + AgNO 3 171.17104.1767.00227.67123.50 A1015.835.2510.585.420.17 A10 + AgNO 3 113.9262.7551.17146.3383.58 A4N124.2583.6740.58186.42102.75 A4N + AgNO 3 178.25115.3362.92253.67138.33

6. Supplemental Figure S1: Starch content and chain length distribution comparisons between A3OE positive and isogenic controls. Figure 1A displayed the relative percentage of starch present in flour. Positives lines are shown in blue, negative segregeants in purple and tissue culture control in red. Figure 1B and 1C show the Chain length distribution difference between A3OE-isogenic control in absence (B) or presence of  -amylase pre treatment (C)). Results are expressed in relative percentage for each degree of chain polymerisation. A B C