Heading date in parent plants
The varieties DH and HS showed significant differences regarding heading date in 2020 and 2021 (Fig. 1a, Fig. S2, Table S2); in 2020, the mean DTH value of DH was 92.3 days, and that of HS was 98.8 days (Table S2). The range of DTH was 90–97 in DH and 96–102 in HS (Fig. 1a). This trend in heading date variation between the parents was also observed in 2021 (Fig. S2).
The frequency distribution of DTH in the F2 population is shown in Fig. 1b. DTH ranged from 90 to 104 in the parents. The continuous distribution of heading date indicated that qDTH3 altered the earlier heading date within the observed range under local environmental conditions.
Fine mapping of qDTH3
To map qDTH3 on the physical map of IRGSP 1.0, plants showing the recombination genotype between DNA markers 3SHSDC01 and InDel05018 were selected from 592 plants of the F2 population. In addition, the F3 progeny test for qDTH3 was performed on the recombinants. In total, 116 plants were identified as recombinants (Table S3). Because of phenotypic redundancy in the parents, seven recombinants were excluded from fine mapping. In addition, ten recombinants with unexpected genotypes were excluded from fine mapping. Ninety-nine recombinants were used for fine mapping of qDTH3 (Table S3). The qDTH3 locus was limited to the region between the two markers, HR7 and HR13, corresponding to the 158-kb region for the rice reference genome of IRGSP 1.0 (Fig. 2, Table S4). In this region, 19 protein-coding genes were predicted using the Rice Annotation Project Database (RAP DB; https://rapdb.dna.affrc.go.jp; Table S5). Only two genes (Os03g0122500 and Os03g0122600) were known to affect heading date.
Genotyping of the qDTH3 locus among Oryza species
To elucidate genetic differences in the qDTH3 locus between the parent varieties, the genotype of the 656-kb chromosomal region including the qDTH3 locus, denoted as GRID, was compared among four Oryza populations: OR, WRC, JRC, and 20V. Many nucleotide polymorphisms were observed in OR population. Whereas small were observed in the JRC population and 20V (Table 1).
The GRID genotype was compared across the four Oryza populations. Five polymorphic sites (EGM01–EGM05) were used to determine the GRID genotype (Tables S6, S7, Fig. 3). Eleven GRID genotypes were identified across all varieties of the four Oryza populations (Table 2). The OR population was divided into four genotypes (GG01, GG02, GGr01, and GGr02; Table S8), the WRC population also comprised four genotypes (GG01, GG02, GG03, and GG05; Table S9), the JRC population included nine genotypes (GGDH, GGHS, and GG01–GG07; Table S10), and the 20V population comprised three genotypes (GGDH, GGHS, and GG04; Table S11).
Four genotypes (GG01, GG02, GG03, and GG05) were identified in the Japonica group of WRC population, and eight genotypes were found in the Japonica group of JRC population. The GGDH and GGHS genotypes were found in five and four varieties, respectively in japonica in JRC population. However, no varieties with GGDH and GGHS occurred in WRC population and OR population. In 20V population, ten and five varieties showed the GGDH and GGHS genotypes, respectively.
The relationships between GRID and qDTH3 are shown in Table 4. Recombination genotype in qDTH3 within the GRID, were four genotypes, GG01, GG04, GG06, and GGr01. GGDH, GG02, GG03, and GGr02 genotypes of GRID carried the DH genotype in qDTH3, and GGHS, GG05 and GG07 genotypes of GRID carried the HS genotype in qDTH3. The qDTH3 DH genotype was found in the four OR varieties and 14 WRC varieties. The qDTH3 HS genotype in WRC population was found only in one varieties (Nipponbare), which is breeding line derived from rice breeding programs. Both qDTH3 DH and HS were found in JRC population and 20V.