Three key factors determine yield in rice (Oryza sativa): panicle number, grain number, and grain weight. Panicle number is strongly associated with tiller number. Although many genes regulating tillering have been identified, whether Dof proteins are involved in controlling plant architecture remains unknown. The dwarf and less tillers on chromosome 3 (dlt3) rice mutant produces fewer tillers than the wild type. We cloned DLT3, which encodes a Dof protein that interacts with MONOCULM 3 (MOC3) in vivo and in vitro and recruits MOC1, forming a DLT3-MOC3-MOC1 complex. DLT3 binds to the promoter of FLORAL ORGAN NUMBER 1 (FON1) to activate its transcription and positively regulate tiller number. The overexpression of MOC1, MOC3, or FON1 in the dlt3 mutant increased tiller number. Collectively, these results suggest a model in which DLT3 regulates tiller number by maintaining the expression of MOC1, MOC3, and FON1. We discovered that DLT3 underwent directional selection in the Xian/indica and Geng/japonica populations during rice domestication. To provide genetic resources for breeding varieties with optimal panicle numbers, we performed large-scale diversity sequencing of the 1,080-bp DLT3 coding region of 531 accessions from different countries and regions. Haplotype analysis showed that the superior haplotype, DLT3^H1, produced the most tillers, while haplotype DLT3^H6 produced the fewest tillers. Our study provides important germplasm resources for breeding super high-yielding rice varieties with combinations of superior haplotypes in different target genes, which will help overcome the challenge of food and nutritional security in the future.

Tiller number (TN) is an important trait that contributes to yield in rice (Oryza sativa L.). A set of 76 chromosome segment substitution lines (CSSLs) derived from the super-hybrid cross between Zhonghui 9308 (ZH9308) and Xieqingzao B (XQZB) was used to map quantitative trait loci (QTL) controlling tiller number (TN). A total of four QTLs were detected in Fuyang, Zhejiang Province (30.15° N, 120° E). Two QTLs were detected in Lingshui, Hainan Province (18.5° N, 110° E) in our previous study. To further map the QTL on chromosome 4, namely qTN4, the line CSSL29 with a lower tiller number was selected to cross with ZH9308 to develop the secondary F₂ population. In the F_2:3 population, the qTN4 was validated and subsequently narrowed down to a 4.08 Mb region. What is more, combined phenotype with genotype, qTN4 was dissected into two QTLs, qTN4.1 and qTN4.2, in the F_4:5 population. The qTN4.1 and qTN4.2 explained 34.31% and 32.05% of the phenotypic variance, with an additive effect of 1.47 and 1.38, respectively. Finally, the qTN4.1 and qTN4.2 were fine-mapped into a 193.55 Kb and 175.12 Kb intervals on chromosome 4, respectively. Based on genotype and phenotype, four near-isogenic lines (NILs) were selected in the mapping populations. Compared with NIL^CSSL29, tiller number (TN), grain setting rate, grain length (GL), the ratio of grain length to width (LWR) and grain yield per plant of NIL^ZH9308, NIL-qTN4.1^ZH9308 and NIL-qTN4.2^ZH9308 were increased, and the heading date of these three lines were earlier than that of NIL^CSSL29. Interestingly, among the candidate genes of qTN4.1 and qTN4.2, except for LOC_Os04g23550, none of the other genes has been cloned, indicating the existence of a novel gene-controlling tiller number. These results lay a foundation for the analysis of QTL controlling tiller number in ZH9308 and provide a theoretical basis for the application of ZH9308 in super-hybrid breeding.

Size is a critical determinant of rice grain appearance quality and yield. In this study, we isolated a dominant mutant of rice grain size, RE10498, from an EMS-induced mutant library of ZH8015 characterized by long grains. Using the MutMap method, we cloned the causal gene, OsGLW3, which encodes an expressed protein of unknown function. Targeted editing of position 3,119 in the OsGLW3 coding region to “A” in the wild-type ZH8015 produced positive transgenic lines whose grain size matched that of RE10498. Knocking out OsGLW3 in the wild-type ZH8015 did not alter the grain size of positive plants (KO-OsGLW3ZH), whereas knocking out OsGLW3 in the mutant RE10498 resulted in decreased grain length and increased grain width in its positive plants (KO-OsGLW3RE), reverting to a phenotype consistent with the wild type. Overexpression of the OsGLW3RE allele in ZH8015 resulted in a highly significant increase in grain length. These findings indicate that RE10498 is a gain-of-function mutant. OsGLW3RE positively regulated the level of brassinolide, which affected grain size by regulating the proliferation and extension of glume cells. Crossing ZH8015 and RE10498 with the 3-line sterile lines Fu387A (F387A), Nei5A (N5A), and Zhongxiang131A (ZX131A) showed that the hybrids F387A/RE10498, N5A/RE10498, and ZX131A/RE10498, respectively, exhibited improved rice appearance quality: longer grains with reduced chalkiness. The OsGLW3RE allele identified in this study, along with its functional marker dCAPs-G, holds significant reference value for breeding high-quality hybrid rice varieties.