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Additional file 1: Fig. S1. Pedigree of #1203, #1206, #1203 (BC3), and #1206 (BC3) lines. Fig. S2. Elution profiles of debranched endosperm starch separated by gel filtration chromatography. Fig. S3. Comparison of amylopectin structure before and after backcrossing. Fig. S4. Differences in amylopectin structure between #1203 (BC3) lines and Akita 63 to determine the effect of BEIIb loss in addition to the effects of SSIIa and/or GBSSI. Fig. S5. Differences in amylopectin structure showing the effect of active SSIIa. Fig. S6. Differences in amylopectin structure between #1206 (BC3) lines and Akita 63 to determine the effect of the loss of SSIIIa and BEIIb in addition to the effects of SSIIa and/or GBSSI. Fig. S7. Differences in amylopectin structure showing the effect of high expression level of GBSSI. Fig. S8. Typical differential scanning calorimetry profiles of rice lines showing peak gelatinization temperature.

Additional file 2: Table S1. Genotypes and grain weight of rice lines before backcrossing. Table S2. Flowering dates of rice lines and cumulative temperature before and after backcrossing. Table S3. Apparent amylose, long amylopectin chain, and short amylopectin contents and short-to-long amylopectin chain ratio in the endosperm starch of rice lines after backcrossing measured by gel filtration chromatography using debranched starch. Table S4. Apparent amylose, long amylopectin chain, and short amylopectin contents and short-to-long amylopectin chain ratio in the endosperm starch of rice lines before backcrossing measured by gel filtration chromatography using debranched starch. Table S5. Differential scanning calorimetry analysis of the thermal properties of starch in rice lines before backcrossing. Table S6. RS contents of raw and cooked rice flour and un-mashed and mashed cooked rice grains.

Additional file 1: Table S1. Percentages and average chain length of amylopectin branches analyzed by capillary electrophoresis.

Additional file 2: Fig. S1. Elution profiles of debranched starch and amylopectin analyzed by gel filtration chromatography. Fraction I contains amylose or extra-long amylopectin chains. Fraction II contains long amylopectin chains. Fraction III contains short amylopectin chains. Red lines indicate patterns obtained from starch, and blue lines indicate patterns obtained from the purified amylopectin. Differences in retention time (RT) in Kinmaze were due to difference of the lot of the column.

Analyses of protein-protein interactions between rice starch biosynthetic isozymes by co-immunoprecipitation. Immuno-precipitation experiments were performed using the isozyme specific antibodies indicated above and the soluble protein extract from SS1/ss2aL/SS3a (WT), ss1L/ss2aL/SS3a, SS1/ss2aL/ss3a and ss1L/ss2aL/ss3a. Immuno-blotting was performed using the antibodies indicated on the right. (PDF 903Â kb)

Molecular weight distributions of BE isozymes from developing rice endosperm determined by gel filtration chromatography. Soluble proteins were separated by gel filtration chromatography. Fractions were denatured and separated by SDS-PAGE, and immuno-blotting was performed using the indicated antibodies. (PDF 903Â kb)

SS activities were visualized by non-denaturing zymograms using rice starch biosynthetic enzymes separated by gel filtration chromatography from SS1/ss2aL/ss3a. Gel was incubated in the presence of 1 mM ADP-glucose (A) and in the absence of ADP-glucose (B). Black arrowhead shows the SSI activity. Gray arrowheads represent glycosyl hydrolase or glucan transferase activities. White arrowheads are SS activity bands found only in ss3a. (PDF 903Â kb)

Size separation of debranched endosperm starch and purified amylopectin by gel filtration showing the amylose content in fraction I.Gel filtration chromatography was performed for debranched endosperm starch and purified amylopectin using the SS1/ss2aL/SS3a (Nipponbare; A), ss1L/ss2aL/SS3a (B), SS1/ss2aL/ss3a (C), and ss1L/ss2aL/ss3a (D) harvested in 2013. Each graph shows typical elution profiles of isoamylase-debranched starch (blue lines) and purified amylopectin (red lines). Each fraction (Fr. I, II, and III) is separated according to the carbohydrate content curve determined by refractive index detectors (left Y-axis). The panels show one typical data set (of at least three replicates prepared from starch and purified amylopectin). (PDF 901Â kb)

Immuno-blotting of SS isozymes resolved by non-denaturing PAGE using the fractions obtained from gel filtration chromatography. Antibodies used for immuno-blotting are indicated. Black arrowheads indicate the same migration distances as the main stained activity bands shown in Fig. 4, and white arrowheads correspond to the activity bands found in the zymogram of SS1/ss2aL/ss3a in Fig. 4. (PDF 902Â kb)