Volume 8, Issue 2, Pages 303-314 (February 2015) Isolation of a Novel Lodging Resistance QTL Gene Involved in Strigolactone Signaling and Its Pyramiding with a QTL Gene Involved in Another Mechanism  Kenji Yano, Taiichiro Ookawa, Koichiro Aya, Yusuke Ochiai, Tadashi Hirasawa, Takeshi Ebitani, Takeshi Takarada, Masahiro Yano, Toshio Yamamoto, Shuichi Fukuoka, Jianzhong Wu, Tsuyu Ando, Reynante Lacsamana Ordonio, Ko Hirano, Makoto Matsuoka  Molecular Plant  Volume 8, Issue 2, Pages 303-314 (February 2015) DOI: 10.1016/j.molp.2014.10.009 Copyright © 2015 The Author Terms and Conditions

Figure 1 QTL Analysis and Molecular Cloning of SCM3. (A and B) Gross morphology in (A) and cross-sections of the fourth internode in (B) of Koshihikari (left), Habataki (center), and Chugoku117 (right). Scale bars in (A) represent 15 cm; scale bars in (B) represent 2 mm (left) and 100 μm (right). (C) Comparison of BMB, SM, and BS values of the fourth internode at 20 days after heading (n = 3). The letters (a, b, and c) indicate significant differences by Tukey's significant difference test. Numbers in parentheses indicate corresponding values for Koshihikari. (D) QTL map for SM (red) and BMB (blue). Chugoku117 alleles enhanced BMB and SM in the Koshihikari background. (E) Stepwise positional cloning of SCM3, and comparison of the gene structure of OsTB1 between Koshihikari and Chugoku117. The number of recombinants between markers is indicated below the maps. (F) Gross morphology of Nipponbare (NP) plants independently transformed with the OsTB1 genome from Chugoku117 (NP-Chugoku117) and Koshihikari (NP-Koshihikari), and the empty vector (NP-Vector). Scale bar represents 15 cm. (G) Cross-sections of the first (I, panicle-neck internode) down to the fifth (V, basal internode) of the transgenic lines. (H) SM of the transgenic lines at 20 days after heading. Each column represents the mean ± SD (n = 5). Molecular Plant 2015 8, 303-314DOI: (10.1016/j.molp.2014.10.009) Copyright © 2015 The Author Terms and Conditions

Figure 2 Effect of SCM3, SCM2, and SCM2+SCM3 on Culm Morphology and OsTB1 Expression Level. (A) NIL-SCM3, NIL-SCM2, and NIL-SCM2+SCM3 were respectively introgressed with the Chugoku117 genomic segment carrying SCM3 (shown in red), Habataki genomic segment carrying SCM2 (blue), and both. (B) Expression analysis of OsTB1 in various organs of Koshihikari, NIL-SCM3, NIL-SCM2, NIL-SCM2+SCM3, and Chugoku117 by qRT-PCR. SAM at 30 days after germination. IM at 30 days before heading. Each column represents the mean ± SD (n = 3). (C) Cross-section of the first to fifth internodes of Koshihikari, NIL-SCM3, NIL-SCM2, NIL-SCM2+SCM3, and Chugoku117. (D and E) Comparison of culm diameter (D) and culm thickness (E). Each column represents the mean ± SD (n = 3). (F) Comparison of culm physical strength among Koshihikari, NIL-SCM3, NIL-SCM2, NIL-SCM2+SCM3, Chugoku117, and Habataki. Curved lines indicate BMB (n ≥ 8). Molecular Plant 2015 8, 303-314DOI: (10.1016/j.molp.2014.10.009) Copyright © 2015 The Author Terms and Conditions

Figure 3 Histological Analyses of IM and Internode. (A) Cross-section of the fourth internodes of Koshihikari, NIL-SCM3, NIL-SCM2, NIL-SCM2+SCM3, and Chugoku117 at 20 days after heading. Scale bar represents 100 μm. (B) Longitudinal section of the IMs of the plants at 30 days before heading. Red and blue bidirectional arrows indicate the width and height of the meristem, respectively. Scale bar represents 100 μm. (C and D) Quantitative comparison of the number of parenchyma cells (C) and cell area per parenchyma cell (D). (E and F) Quantitative comparison of the width (E) and height (F) of the meristems, respectively. Each column represents the mean ± SD (n = 3). Molecular Plant 2015 8, 303-314DOI: (10.1016/j.molp.2014.10.009) Copyright © 2015 The Author Terms and Conditions

Figure 4 Effect of SCM3, SCM2, and SCM2+SCM3 on Crop Productivity. (A) Panicle morphology of Koshihikari, NIL-SCM3, NIL-SCM2, NIL-SCM2+SCM3, and Chugoku117. Scale bar represents 5 cm. (B–D) Comparison of spikelet number (B), primary branch number (C), and secondary branch number (D) per panicle observed from the main culm of each plant. (E and F) Panicle number (E) and spikelet number (F) of each plant. (G and H) 1000-grain weight (G) and grain yield (H) of each plant. Each column represents the mean ± SD (n = 3). Molecular Plant 2015 8, 303-314DOI: (10.1016/j.molp.2014.10.009) Copyright © 2015 The Author Terms and Conditions

Figure 5 Agromorphological Characteristics of SL-Related Mutants, fc1-2 and d10-2. (A) Plant morphology of fc1-2, d10-2, and their original strain, Nipponbare (NP), at 20 days after heading. Scale bar represents 15 cm. (B and G) Cross-section of their fourth internodes at 20 days after heading. Scale bar represents 2 mm in (B) and 100 mm in (G). (C) The plant height at 20 days after heading. (D) The panicle number per plant. (E) BMB of the fourth internodes at 20 days after heading. (F) Panicle structure. Scale bar represents 5 cm. (H) Longitudinal sections of their IMs. Scale bar represents 100 mm. (I and J) Number of parenchyma cells in (I) and cell area per parenchyma cell in (J), with respect to the cross-section of their fourth internodes. Each column represents the mean ± SD (n = 3). Molecular Plant 2015 8, 303-314DOI: (10.1016/j.molp.2014.10.009) Copyright © 2015 The Author Terms and Conditions

Figure 6 Effect of OsTB1 and APO1 Overproduction. (A) Gross morphology of Nipponbare (NP) plants independently transformed with OsTB1 cDNA (FC1ox) and APO1 cDNA (APO1ox) under the control of the UBIQUITIN promoter, and the empty vector (Vector). Yellow arrows indicate the top positions of panicle. (B) Cross-sections of their fourth internodes at 20 days after heading. Scale bar represents 2 mm. (C and D) Expression of OsTB1 (C) and APO1 (D) in these transgenic plants. RNAs were extracted from their inflorescences at the primary branch initiation stage and quantified by qRT-PCR. Each column represents the mean ± SD (n ≥ 5). (E) Leaf blade morphology. Scale bar represents 5 cm. (F) Panicle number per plant. Each column represents the mean ± SD (n = 5). (G) Heading date. Each column represents the mean ± SD (n = 5). (H) Panicle morphology. Scale bar represents 5 cm. Molecular Plant 2015 8, 303-314DOI: (10.1016/j.molp.2014.10.009) Copyright © 2015 The Author Terms and Conditions

Figure 7 Comparison of the Gene Expression Patterns in IMs of OsTB1 and APO1 Overexpressors. (A) RNAs were independently extracted from wild-type, OsTB1, and APO1 overexpressors and used for microarray. The y axis represents log2 ratios of the signal intensities of probes in the APO1 overexpressor and control plants, whereas the x axis shows those of the OsTB1 overexpressor. The red line represents the regression line (y = 0.6887x + 0.0193), which shows a moderate coefficient of determination (R2 = 0.5432). (B and C) Venn diagrams of probes up- (B) or downregulated (C) in OsTB1 (shown in blue) or APO1 (shown in yellow) overexpressors. Previously reported genes involved in panicle and flower development are shown in the corresponding areas. Genes in black characters had ≥10-fold change in expression, whereas genes in red had ≥3-fold change. Molecular Plant 2015 8, 303-314DOI: (10.1016/j.molp.2014.10.009) Copyright © 2015 The Author Terms and Conditions

Figure 8 Model for the Overlapping Function of SCM3/OsTB1 and SCM2/APO1 in Regulating Culm Diameter, Grain Number per Panicle, and Panicle Number per Plant. Molecular Plant 2015 8, 303-314DOI: (10.1016/j.molp.2014.10.009) Copyright © 2015 The Author Terms and Conditions