Fig. 1. Mapping of red hull gene OsRH3 and its application in hybrid rice seed production. A, Mapping of OsRH3 on chromosome 3 (Chr. 3). Red ORF (Open reading frame) represents the OsRH3 gene. B, OsRH3 gene was amplified from the parental lines. M, 250 bp DNA ladder; Line 1, Shuang 1S (669 bp); Line 2, Chuan 7 (8016 bp). C, Relative expression level of OsRH3 in Shuang 1S, Chuan 7, and red-hulled sterile line. Samples were taken when panicle development was almost complete before heading date. The rice Ubiquitin gene was utilized as an internal reference. **, ***, and ****, P < 0.01, P < 0.001, and P < 0.0001, respectively, by the Student’s t-test. D, Development of functional marker OsRH3-P12. Comparison of OsRH3 gene structures from Shuang 1S (Normal/Yellow) and Chuan 7 (Red). ATG, Start codon; TAA, Stop codon. E, Selection and breeding of new red-hulled sterile lines. Shuang 1S is the sterile line; Chuan 7 is the restorer line; ‘×’ represents hybridization; ‘$\otimes$’ represents self-crossing. F-H, Plant phenotypes (F), panicles after heading (G), and grain sizes (H) of Shuang 1S and red-hulled sterile line. Scale bars in F, G, and H, 10 cm are 1 cm, and 10 mm, respectively. I-N, Plant height (I), number of tillers (J), main panicle length (K), number of grains per panicle (L), grain length (M), and grain width (N) of Shuang 1S and red-hulled sterile line. Data are Mean ± SD (n ≥ 7 in I-L) and (n = 15 in M and N). No significant differences were observed when P > 0.05.