Phenotypic analysis
The two parents G1025 and K1561 showed highly significant differences in TGW under five environments including two locations in Nan-Ning (NN), and Wu-Han (WH), in China with an average of 16.01 g and 32.07 g (Table 1), respectively. TGW values of the 201 individuals were mostly distributed between 20–30 g with an average of 24.60 g, 25.69 g, 22.80 g, 24.97 g, and 25.69 g in 2013NN (2013 in Nan-Ning), 2014NN, 2015NN, 2016NN, 2016WH (in 2016 in Wu-Han)(Table 1, Figure 1, Table S1), respectively. TGW values of 104, 106, 104, 107, and 107 out of the 201 individuals were smaller than the average in 2013NN, 2014NN, 2015NN, 2016NN, 2016WH, and TGW of the remaining individuals was either equal to or larger than the average (Table S1).
QTL mapping of TGW by simple sequence repeats (SSR)
TGW QTLs were preliminarily detected by 300 SSR markers evenly distributed on the 12 chromosomes. The population were F6, F7 RILs derived from the cross of G1025 and K1561 planted in NN in 2013, 2014. Four QTLs TGW3, TGW7, TGW9.2, and TGW12 were stably detected on the chromosomes 3, 7, 9 and 12 in the two environments (Table 2). TGW12 had the greatest effect, which located on the region of RM247 and RM7003 (Table 2), so it was selected for further analysis. There were other 166 SSR markers (Table S2) in the region based on the genome sequencing data of Nipponbare [29]. The polymorphism of the 166 SSRs was firstly detected between the parental lines G1025 and K1561. As a result, nine SSRs showed polymorphism but only five displayed clear bands. The five SSRs were further used to detect F6, F7 RILs population in 2013 and 2014. Finally, TGW12 was mapped to the 5.1 cM region between RM27638 and RM27748 (Figure 2).
QTL mapping of TGW by SLAF markers
We have developed 5521 SLAF markers by SLAF sequencing [30]. To further map TGW QTLs, those SLAF markers were used to screen F8 RILs in NN in 2015 and F9 RILs in NN and WH in 2016, respectively. A total of eight QTLs were detected in three environments, namely, TGW7, TGW9.1, and TGW12 in 2015NN; TGW7, TGW9.2, and TGW12 in 2016NN; TGW7 and TGW12 in 2016WH (Table 2, Figure 3). TGW7 and TGW12 were both detected in three environments, and TGW9.1 and TGW9.2 were each detected once. TGW7 explained the phenotypes for 8.01%, 10.76%, and 10.43% inheritance with an LOD of 6.94, 7.69, and 7.48 in 2015NN, 2016NN, and 2016WH, respectively, whereas TGW12 showed 22.36%, 17.48%, and 17.95% inheritance explaining for the phenotypes with an LOD of 15.42, 11.99, and 11.96 in the three environments, respectively (Table 2). TGW12 had a greater effect than TGW7, which was consistent with the results analyzed by SSR mapping (mentioned above). Further analysis for TGW12 was conducted by comparing the linkage map constructed by SSR and SLAF markers. Consequentially, nine SLAF markers fell into the region of RM27638-RM27748, and TGW12 was further narrowed to 204.12 kb region between SLAFs Marker 2768345 and Marker 2853491 (Figure 2).
Evaluation of TGW12 phenotype and genetic origin identification of TGW12 segment
In order to evaluate whether the phenotypes were determined by TGW12, 16 out of the 201 RILs containing the TGW12 region were identified by means of the markers nearby the region (Figure 4). Then, the phenotypes and genotypes of the 16 RILs were compared. All the 16 RILs with one or two segments of K1561 showed TGW increase than the recurrent parent G1025, suggesting TGW12 control TGW (Figure 4). To clarify whether the increasing effect of TGW12 was originated from O. minuta, the genotypes of G1025, K1561, IR24, and O. minuta were examined using markers nearby or within TGW12. The genotype of K1561 was the same as that of IR24 but different from that of G1025 and O. minuta on the sites of RM27638 and RM27748, which are nearby TGW12 (Figure 2, Figure 5). However, the genotype of K1561 was the same as those of IR24 and O. minuta, but it was different from that of G1025 at the site of Marker 2768345, which is linked with TGW12. It was hard to draw a conclusion whether TGW12 originated from IR24 or O. minuta based on the above results. It has been suggested that translocation through centric break-fusion occurred more frequently than recombination in the introgression lines with interspecific cross, which didn’t always result in an O. minuta chromosome arm onto a complete or incomplete O. sativa chromosome [31][30]. Thus, TGW12 origination remains to be determined in the near future. It is feasible to compare sequence of TGW12 candidate among O. minuta, IR24, and K1561 once it was fine mapped.
Preliminary prediction of candidate genes for TGW12
Analysis of annotated genes indicated that 32 ORFs located in the 241.47 kb region based on Nipponbare genomeannotation (http://rice.plantbiology.msu.edu)(Table 3). Among them, 13 ORFs encoded functional proteins and 19 ORFs were annotated as transposon/retrotransposon proteins, hypothetical proteins, or expressed protein. It is worth noting that there were four transcription factors (TFs) among the functional proteins: two MADS-box proteins (ORF12, ORF14),, one ZF-HD protein (ORF24),, and one B-box zinc finger protein (ORF27).. Because TFs play crucial roles in regulation of plant growth and development [32], the four TFs were considered preferentially as putative candidate genes of TGW12. Reverse transcript (RT)-PCR were conducted to amplify the CDS (coding domain sequence) of the four transcription factors from the parents G1025 and K1561. Sequence comparison indicated that the amplified sequence of ORF12 in K1561 was 56 bp shorter than that of G1025, which resulted in premature transcription termination, consequently leading to a peptide with 45 amino acid residues only in K1561, whereas ORF12 in G1025 encoded a protein with 202 amino acid residues (Figure S1). Further analysis revealed that lack of 56 bp of ORF12 in K1561 was due to alternative splicing (AS) in the first intron (Figure 6). There were no difference in the CDS of ORF14, ORF24, and ORF27 between K1561 and G1025 (data not shown). Thus, the MADS-box (ORF12) was likely one putative candidate of TGW12. However, possibility of other nine functional proteins and the hypothetical proteins, or expressed protein independently or collectively affecting TGW could not be excluded. Further investigation is required.