Introduction

Rice heterosis, or hybrid vigor, refers to the increased yield in a hybrid offspring compared to its inbred parental lines. The rice hybrid varieties typically display a grain yield advantage of 10–30% over their parentsFull size table

Cloning and functional analysis of the QTL OsMADS1 GW3p6

In our previous study, we mapped a genomic region containing the QTL GW3p6 contributing to the high grain production of the elite hybrid rice variety Guang-Liang-You 676 (GLY-676) from F2 individuals16. To further fine clone the QTL GW3p6, we applied GPS to the F2 population derived from the elite hybrid rice variety GLY-676 (heterozygous first filial (F1)), which was generated from a cross between the varieties FH (male line) and GZ (female line).

First, we selected 1000-grain weight (TGW) as the trait for mapping heterotic genes. We ranked three categories (22.33–29.15 g/1000-grain, 29.16–31.09 g/1000-grain and 31.10–37.3 g/1000-grain) to phenotype TGW according to traits and then created simulated pools with their sequencing reads (individual sequencing reads data from European Nucleotide Archive under the accession number PRJEB13735). We implemented Ridit analysis with allelic frequencies from three bulks to calculate p values for each SNP (Fig. 2a). As numerous background noises complicated locating the QTL at a fine resolution, a noise reducing strategy followed the statistical test. After conducting all analysis, we located a 400-kb candidate interval contributing to grain weight (Fig. 2b). The mapping accuracy and resolution of GPS can reach almost the same level as that of Composite Interval Mapping. Notably, the GPS mapping results show that the method can be used as a faster and more convenient approach than conventional mapping methods in rice breeding. Considering the compatibility of the different versions of the assembled data, we remapped the TGW genes by GPS method based on Os-Nipponbare-Reference-IRGSP-1.0 (ref. 37) and MH63RS2 (ref. 38), and the genetic mapping results from the three reference genome assemblies were almost the same (Supplementary Fig. 6).

Fig. 2
figure 2

QTL mapping and map-based cloning of OsMADS1GW3p6. a, b Identification of the genomic region consisting of OsMADS1GW3p6 via GPS approach. a The p value plot is the result of Ridit analysis for 1000-grain weight before noise-reduction algorithm. The -ln (p value) plot (Y axis) is plotted against SNP positions (X axis) on each of the 12 rice chromosomes. b After the strategy of reducing background noise, the results present as ratio plot. X-axis value is set at a midpoint at each defined genomic interval and Y-axis value corresponds to ratio. c The genotype of chromosome 3 of RIL-79. Black, white and gray bars represent the heterozygous genotype, and homozygous genotypes of GZ and FH respectively. d In the fine-scale mapping (lower bar) generated from the analysis of 1,079 segregating individuals, the QTL GW3p6 falls in the heterozygous interval. The numbers below the bar indicate the number of recombinants between GW3p6 and the molecular markers shown. e Genotyping of progeny homozygous for GW3p6 delimited the locus to a ~5.9 Kb genomic region between markers MP99 and MP100. f, h 1000-grain weight and grain length of recombinant F2 lines of RIL-79 (L1-L4). Data shown as means ± SD, n = 48. Source data are provided as a Source Data file. g Schematic diagram depicting the structure of OsMADS1 and OsMADS1GW3p6, red letters stand for the nucleotides of OsMADS1 non-homozygous segment. i The sequencing electrophoresis of non-homologous segment between OsMADS1 and OsMADS1GW3p6. j The schematic illustration of OsMADS1 functional domains, M represents the MADS domain, I represents the intervening domain, K represents the keratin-like domain, and C represents the C-terminal domain. Source data of Fig. 2f, h are provided in a Source Data file

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Furthermore, we screened the recombinant inbred lines (RILs) from the self-pollination F5 generation, and RIL79, one RIL in which genomic segment of GW3p6 was heterozygous but others were homozygous, was selected as a further segregating population (Supplementary Fig. 7). We used 1,079 plants from the F1 population of RIL79 to fine-scale map GW3p6, and 36 SNP markers were used for genotyping, ultimately narrowed down the interval to a ~5.9-kb region flanked by MP99 and MP100 (Fig. 2c–h). This region contains only the second half of Os03g0215400 (RAP-DB), and further sequencing analysis indicates a 15-bp non-homologous segment at the junction of the seventh intron and eighth exon of Os03g0215400 (Fig. 2g, i), from TCCTTGGTGAAGGTA to ATGTATATATACT. The 3′ terminal bases AG of the seventh intron were altered, and we speculated that this might lead to alternative splicing. The cDNA sequencing data showed that the splice site (AG/GT) slipped to the 32nd nucleotide (AG/GC) of the last exon (Supplementary Fig. 8), directly caused a premature stop codon, and the original mature protein was truncated by 32 amino acid resides (Fig. 2j, Supplementary Fig. 9). We used the Insertion/Deletion (InDel) marker CS-92 to verify the association between grain size and alternative splicing. Totally 200 individuals of each OsMADS1 genotype were counted, and the heavier grain weight and more slender grain size were in complete agreement with OsMADS1GW3p6 (Supplementary Fig. 10). These results are also consistent with the performance of three GW3p6 genotypes in the F2 generation as previously reported16, and heterozygous OsMADS1GW3p6 showed incomplete dominance. These results indicate that this OsMADS1GW3p6 alternative splicing caused by non-homologous segment is responsible for significant grain weight as previously reported39,40.

To further examine whether the function of OsMADS1GW3p6 acted as a grain size gene, we transformed FH (male parent) with either rice Ubiquitin promoter-driven OsMADS1 cDNA from FH (pUbi::OsMADS1-FH,OE-OsMADS1) or OsMADS1GW3p6 cDNA from GZ (pUbi::OsMADS1GW3p6-GZ,OE-GW3p6). The grains of the pUbi::OsMADS1GW3p6 transgenic plants were longer and heavier than the non-transgenic control plants (Fig. 3a–c), but constitutive expression of the FH OsMADS1 cDNA driven by the rice Ubiquitin promoter led to abnormalities in lemmas and paleae, as previously described41,42 (Fig. 3a). Moreover, we designed the target primers at the last exon of OsMADS1 using the CRISPR-Cas9 system in FH (CR-FH). Sequencing analysis revealed several insertions and deletions at the last exon in transgenic plants that resulted in loss of function in the C domain of the protein encoded by OsMADS1. The transgenic panicles exhibited phenotypic alterations (Fig. 3a), including elongated leafy paleae and lemmas, as well as low fertility seriously as previously reported40,42. The result was also similar to the reported phenotype of leafy hull sterile 141. Furthermore, the C-terminal function caused changes in glume development. In addition, we transferred the same overexpression construct of OsMADS1GW3p6 and knockout construct by CRISPR/Cas9 system to the japonica variety Nipponbare (NPB) with same OsMADS1 genotype as FH. The overexpressing NPB plants (OE-GW3p6-NPB) exhibited longer and heavier grains compared with the non-transgenic plants (Supplementary Fig. 11c, d, e). The transgenic NPB plants carrying missense mutations in the C domain of OsMADS1 (CR-NPB) exhibited the same phenotype as CR-FH plants (Supplementary Fig. 11a, b, c). The above data imply that OsMADS1 plays a significant role in the development of rice flower. Typically, the C domain of OsMADS1 is closely related to the growth of glumes and the development of floral organs, and the function of OsMADS1GW3p6 is to increase grain length and weight.

Fig. 3
figure 3

The phenotype of FH transgenic plants. a The grain morphology of FH and transgenic plants. FH represents the grain morphology of non-transgenic plant FH. CR-FH represents the grain morphology of transgenic FH plants containing missense mutations in C domain of OsMADS1. OE-OsMADS1 represents the grain morphology of transgenic FH plants containing overexpression OsMADS1 construct. OE-GW3p6 represents the grain morphology of transgenic FH plants containing overexpression OsMADS1GW3p6 construct. Scale bar, 5 mm. b Grain length in transgenic overexpression plants and non-transgenic plants. OE-GW3p6-#1 and OE-GW3p6-#2 indicate the two transgenic lines containing overexpression OsMADS1GW3p6 construct. c 1000-grain weight in transgenic OsMADS1GW3p6 overexpression plants and non-transgenic plants. Data in (b, c) are given as means ± SD (n = 24) with the indicated significance by using a two-tailed Student’s t-test (**p < 0.01). Source data of Fig. 3b, c are provided as a Source Data file

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OsMADS1 encodes an MIKCc-type MADS-box transcription factor43; it is also an E-class gene involved in rice floral organ development44,45. The MIKC-type MADS-domain proteins consist of four domains: the MADS domain, Intervening domain, Keratin-like domain and C Domain respectively. The C-terminal region has been associated with transcriptional activation46, and the non-homologous segment of OsMADS1GW3p6 was located within the C domain. Therefore, we conducted transcriptional activation experiment by yeast one-hybrid system (Supplementary Fig. 12). The results showed that both full-length OsMADS1 and OsMADS1GW3p6 had no transcriptional activity, which may be associated with a full-length inhibition of its transcriptional activity46. In addition, the transcriptional activity of the OsMADS1 C-terminal region was higher than that of OsMADS1GW3p6. To further verify our experimental results, we applied a fluorescence report system for transcription activation experiments in rice protoplasts. The results were consistent with the data obtained in the yeast system, with OsMADS1 showing approximately 5 times stronger transcriptional activity than OsMADS1GW3p6 (Supplementary Fig. 13). These data were also consistent with previous reports that alternative splicing attenuates the activation of downstream genes, possibly regulating the transcriptional level of downstream auxin-related genes to change the grain size39. The analysis of rice young panicles through RT-qPCR indicated that the expression of OsMADS1GW3p6 was indeed higher than OsMADS1 (Supplementary Fig. 14). According to our analysis of the structure of OsMADS1GW3p6, a large number of SNPs and InDels are located the upstream of the 5′UTR, and we evaluated whether the observed changes in expression are associated with nucleotides differences in the promoter. To this end, we used the fluorescence report system to verify the promoter, and found no obvious difference between the promoter of OsMADS1 and OsMADS1GW3p6 (Supplementary Fig. 15). Thus, this change in expression was likely caused by a premature stop codon due to alternative splicing. Taken together, these changes in the C domain may have resulted in the change of grain size.

Improved grain yield by constructing a NIL containing GW3p6

To further investigate the genetic function of GW3p6, the near-isogenic line NIL-FH::GW3p6 was generated by introgression of GW3p6 in the FH background. Some RILs with the genetic background of FH accounting for the vast majority were selected as backcrossing materials to generate NILs and were backcrossed twice to FH. With the aid of screening using a large number of molecular markers, and ultimately through sequence-based high-throughput genotyping, we generated a NIL with the FH genetic background and a ~130-kb heterozygous segment. Meanwhile, due to the heterozygous genotype on GW3p6, we can observe phenotypic changes in the three GW3p6 genotypes among the offspring, and the phenotype of incomplete dominance could be observed (Fig. 4b). In general, the phenotypes of NIL-FH::GW3p6 were similar to that of FH (Fig. 4a), including grain width, panicle number, panicle length, seed-setting rate, grain number per panicle and plant height (Fig. 4d, g–k), though the grain length and 1000-grain weight of NIL-FH::GW3p6 were ~6–7% higher than those of the FH plants (Fig. 4c, e). In addition, the grain yield per plant of NIL-FH::GW3p6 was increased by more than 8% (Fig. 4f), while the heading date had a 1~2 days delay compared to that of FH (Fig. 4l). Thus, GW3p6 is a useful target gene in breeding. By constructing a NIL, we demonstrated that an introgression line harboring a heterosis gene from the maternal parent could achieve better performance than inbred line. It also proved rice heterosis genes’ incomplete dominance played an important role in hybrid rice (Fig. 4b).

Fig. 4
figure 4

A yield-related traits of FH and NIL-FH: GW3p6. a Plant phenotype of FH and NIL-FH::GW3p6. Scale bar, 10 cm. b Grain morphology of FH, NIL-FH:GW3p6 and FH/ NIL-FH::GW3p6. NIL and FH represent the grain morphology of NIL-FH::GW3p6 and FH, respectively. FH/NIL indicates the grain morphology of plants with heterozygous OsMADS1 (OsMADS1/OsAMDS1GW3p6) genotype. Scale bar, 5 mm. c–l A field-based comparison of the FH and NIL-FH::GW3p6 plants. c Grain length, d grain width, e 1000-grain weight, f grain yield per plant, g panicle length, h panicle number, i seed-setting rate, j grain number per panicle, k plant height, and l heading date. Data are shown as means ± SD (n = 24) with the indicated significance from a two-tailed Student’s t test. For statistical significance, *p < 0.05, **p < 0.01, NS not significant. Source data of Fig. 4c–l are provided as a Source Data file

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The heterosis effect of OsMADS1 GW3p6 in rice breeding

As shown above, the NIL carrying the heterotic gene OsMADS1GW3p6 showed significantly increased grain yield. To further explore the potential of OsMADS1GW3p6 in rice breeding, we pyramided another previously reported heterotic QTL PN3q23 underlying panicle numberFull size table