Map-Based Cloning

Fresh rice leaves of plants at the grain filling stage were obtained from the two parents, the F2 population, and its family groups. Thirty normal embryo plants and 30 giant embryo plants were randomly selected from the F2 population; the same amount of leaves were mixed to form a normal embryo pool and a giant embryo pool. The genomic DNA of each pool was extracted using the Plant Genomic DNA kit (Tiangen, Beijing, China) and detected by single nucleotide polymorphism (SNP) chip array (RICE6K) to initially locate the target gene. HiScan scanner (Illumina Inc., San Diego, CA, United States) was used for chip scanning, and GenomeStudio software was used for raw data analysis. R platform was used for further analysis, such as genotype identification, comparison and map drawing (R Development Core Team, 2011). Simple sequence repeat (SSR) markers were identified from the Gramene database for grasses¹. Molecular markers containing polymorphism between two parents were screened, and the genotypes of all markers in the target region were determined for recombinant plants in the F2 population and its family groups. Mapmaker/Exp v3.0 was used for linkage analysis and the Kosambi function was used to calculate the genetic distance (Lincoln et al., 1992). The candidate gene (OsZS_07T0416900) in the final positioning interval was amplified separately from the two parents, sequenced, and compared using GENtle software (v1.9.4, Magnus Manske, Cologne, NRW). All the primers used in the current study are listed in Supplementary Table 1.