A weak allele of TGW5 enables greater seed propagation and efficient size-based seed sorting for hybrid rice production

University, Mr. He Zhang from Shenghong Planting & Seedling Co., Ltd., and Dr.SanqiangZhang from Hubei University ofTechnologyfor assisting with HSD sorting using the alveolar cylinder and Prof. Tingxu Huang (Institute of Rice Research, Fujian Academy of Agricultural Sciences) for providing the parent line Fuhui212. No conﬂict of interest is declared.

TGW5 was fine-mapped by map-based cloning to a 136.3-kb region of chromosome 5, which spanned 13 full open reading frames (ORFs).ORF4 (LOC_Os05g26890), which encodes a G protein a subunit, has previously been reported as a D1 that controls plant height and seed size (Figure 1C), although the phenotypes of the reported d1 were much more severe than those of NIL FH212 (Oki et al., 2009).Sanger sequencing of TGW5 identified 10 polymorphisms in the introns and three synonymous SNPs in the exons, including an A869T SNP located in the conserved splice site at the 5 0 end of the fifth exon (Figure 1C).RT-PCR and sequencing revealed that NIL H12-29 contains only a single transcript, whereas NIL FH212 harbors three transcript variants (Supplemental Figure 4A).Compared with the cDNA from H12-29 and Nipponbare, TGW5-FH1 (the first transcript variant from FH212) contains a 19-bp insertion and a 13-bp deletion at the splicing site, whereas TGW5-FH2 harbors only the 13-bp deletion, making the two variants indistinguishable by agarose electrophoresis.TGW5-FH3 retains the fourth intron and is thus larger in size (Supplemental Figure 4B).In terms of their amino acid sequences, both TGW5-FH2 and TGW5-FH3 show premature termination, but the TGW5-FH1 protein is only slightly modified, with four variant residues and a two-residue insertion that may impair a conserved alpha-helix structure in the protein (Supplemental Figures 4C-4E).The CRISPR-Cas9-derived TGW5 mutants crj-01 and crj-40 in the FH212 background showed a more severe dwarf phenotype and smaller grains than FH212, indicating that TGW5 FH212 is partially functional (Supplemental Figure 5).Constructs proTGW5::TGW5 H12-29 and proTGW5::TGW5 FH212(T869A) were introduced into NIL FH212 and fully rescued its plant height and seed size to NIL H12-29 levels (Figures 1D and 1E).We therefore concluded that TGW5 FH212 is a new weak allele of D1.The A869T SNP in TGW5 FH212 impairs mRNA splicing to produce a partially functional protein, TGW5-FH1, leading to a mild dwarf phenotype and a more minor seed phenotype in NIL FH212 .
Given the significant effect of recessive TGW5 FH212 on seed size, we developed two S-MSLs, S-C815S and S-WXS, by backcross

Plant Communications
Correspondence breeding in two elite temperature-sensitive MSLs, C815S and WuxiangS (WXS), respectively (Figures 1F and 1G; Supplemental Figure 6A).In terms of key MSL features, the S-MSLs showed fertility transition temperatures similar to those of their corresponding MSLs (Supplemental Table 1) but a 20% reduction in stigma exertion rate compared with the MSLs (Supplemental Figures 6B-6D).At a fertile temperature, the S-MSLs had lower GL and TGW but slightly higher GW than their corresponding MSLs (Supplemental Figure 2).Notably, despite their somewhat lower absolute yield by weight, S-C815S and S-WXS set 31.31% and 27.82% more seeds per plant than C815S and WXS, respectively, mainly owing to their increased panicle numbers and spikelet density per panicle (Figure 1H; Supplemental Figures 2B-2D).
We next grew C815S and S-C815S at a sterile temperature and crossed them with the elite RLs R143 and CH425, respectively, to produce HSDs under agricultural conditions.The seed setting rates of C815S/CH425 and S-C815S/CH425 were 45.03% ± 3.04% and 38.15% ± 2.95%, respectively.Similarly, the seed setting rate of S-C815S/R143 (36.72% ± 2.78%) was slightly lower than that of C815S/R143 (40.75% ± 3.72%), possibly because of the lower stigma exertion rate of the S-MSLs.Interestingly, S-C815S/R143 and S-C815S/CH425 produced 12.79% and 10.84% more seeds than C815S/R143 and C815S/CH425, respectively, suggesting that the S-MSLs enable significantly higher seed propagation than the corresponding MSLs (Supplemental Figure 2C).An analysis of GL distribution indicated that the significant reduction of GL conferred by TGW5 FH212 makes the S-MSL-derived HSDs separable from the RL seeds (Supplemental Figure 7).Indeed, S-C815S/CH425 HSDs (GL 6.58 ± 0.04 mm) and CH425 seeds (GL 10.72 ± 0.06 mm) could easily be sorted to reach an HSD purity of 99.87 ± 0.06% by one round of sorting with industrial alveolar cylinder equipment (Figure 1I; Supplemental Video 1).Moreover, the S-C815S/R143 HSDs (GL 6.61 ± 0.04 mm) and R143 seeds (GL 9.91 ± 0.05 mm) could also be effectively sorted to reach 96.56% ± 0.80% HSD seed purity (Figure 1I; Supplemental Video 2).By contrast, the regular MSL-derived HSDs and their corresponding RLs were not well separated, even after two rounds of sorting (Figure 1I; Supplemental Videos 3 and 4).This result clearly demonstrated that effective seed sorting of S-MSL-derived HSDs from RL seeds is highly feasible.
Finally, we evaluated the major agronomic traits of the F 1 plants of C815S/R143, S-C815S/R143, C815S/CH425, and S-C815S/ CH425 in the paddy field.The hybrid lines derived from MSLs and from their corresponding S-MSLs had almost identical phenotypes, indicating that the use of TGW5 in the S-MSLs had no negative effect on heterosis of the F 1 plants (Supplemental Figure 2).
In conclusion, we cloned a complete recessive seed size gene, TGW5 FH212, that encodes a weak allele of D1.Introduction of TGW5 FH212 converted regular MSLs into S-MSLs with increased seed numbers and reduced seed size but no penalties in terms of F 1 heterosis.Although the trade-off between seed size and seed number is unfavorable for yield in the breeding practice of inbred lines, the smaller seed size and greater seed numbers conferred by TGW5 FH212 turn out to be triple bonuses for HSD production: (1) they enable production of more HSDs per plant; (2) they facilitate effective post-harvest, size-based seed sorting that is compatible with labor-saving mechanized cultivation, which is estimated to reduce field costs by 16.3% (Tang et al., 2020); and (3) they reduce HSD storage and transport costs because of the smaller HSD seed size.Given that the currently used commercial MSLs and RLs have very similar seed sizes, TGW5 FH212 is a highly promising resource for S-MSL breeding and is expected to increase HSD propagation and cut the cost of HSD production through mechanized cultivation.

Figure 1 .
Figure 1. Cloning of TGW5, which controls grain size, and its use in mechanized production of rice hybrid seeds.(A) Flow model of HSD production using conventional alternative-row planting and S-MSL-based mixed-planting strategies.(B) Plant, panicle, and grain morphologies of the NIL lines.Plant, panicle, and grain scale bars represent 20 cm, 5 cm, and 5 mm, respectively.(C) Map-based cloning and molecular characterization of TGW5.(D and E) A genetic complementation test using proTGW5::TGW5 H12-29 and proTGW5::TGW5 FH212(T869A) constructs.Scale bars for plants and grains represent 20 cm and 5 mm, respectively.