Most agronomical traits of crop species are complex traits controlled by multiple genes and affected by environmental factors. While considerable efforts have been made to fine-map and clone major quantitative trait loci (QTLs) for yield-related traits in rice, it is not until recently that the attention has been paid to minor QTLs. Following previous dissection of QTLs for grain weight and grain size in a 12-Mb interval on the long arm of chromosome 1 in rice, this study targeted at one putative QTL region for a more precise mapping and for analyzing the genotype-by-environment interaction of minor QTLs. Four BC2F10 plants of the indica rice cross ZS97///ZS97//ZS97/MY46 were selected. They carried overlapped heterozygous segments that jointly covered the entire putative region for qTGW1.1 detected previously. Four sets of near isogenic lines (NILs) were developed from selfing progenies of the four plants. Each NIL set consisted of 32 ZS97 homozygous lines and 32 MY46 homozygous lines that differed in the corresponding heterozygous region. They were grown in two locations having distinct ecological conditions and measured for 1000-grain weight, grain length and grain width. Two QTLs were separated in an 835.2-kb interval flanked by DNA markers Wn28447 and RM11569. They both showed consistent effects across the two environments. The qTGW1.1a located within the 120.4-kb interval Wn28447 − RM11543 significantly affect all the three traits with the enhancing allele derived from ZS97, showing a stronger influence on grain weight than on grain length and width. The qTGW1.1b located in the 521.8-kb interval RM11554 − RM11569 significantly affect grain weight and length with the enhancing allele derived from MY46, having a stronger influence on grain length than on grain weight. Consistent performance of the two QTLs was confirmed in a validation experiment using five NIL-F2 populations segregated for either qTGW1.1a or qTGW1.1b. Separation of closely-linked QTLs having small effects is achievable in the absence of major-QTL segregation. Minor QTLs for complex traits could act consistently in diverse environments, offering the potential of pyramiding beneficial alleles of multiple minor QTLs through marker-assisted selection.

Important role of flowering genes in enhancing grain productivity in rice has become well recognized for a number of key genes regulating the florigen production, but little has been known for the two florigen genes themselves. In this study, pleiotropism ofRice Flowering Locus T 1(RFT1), one of the two florigen genes in rice, was firstly evaluated using near isogenic lines (NILs) carryingRFT1alleles from theindicarice cultivars Zhenshan 97 (ZS97) and Milyang 46, respectively, and then determined by transformation of theRFT1^ZS97allele into ajaponicarice variety, Zhonghua 11. TheRFT1^ZS97allele was shown to delay heading and increase plant height, grain weight, grain number and grain yield, indicating thatRFT1plays an important role in the growth and development of rice. This study has also validated the potential of using a new type of genetic resource, sequential residual heterozygotes (SeqRHs), for QTL fine-mapping. A step-by-step approach was employed for SeqRHs identification, NIL development and QTL fine-mapping. The heterozygous segments and candidate QTL regions were gradually narrowed down. Eventually, the QTL region was delimited to a 1.7 kb region containing a single gene.

Grain weight and size are important traits determining grain yield and influencing grain quality in rice. In a previous study, a quantitative trait locus controlling thousand-grain weight (TGW) in rice, qTGW10-20.8, was mapped in a 70.7 kb region on chromosome 10. Validation of the candidate gene for qTGW10-20.8, OsMADS56 encoding a MADS-box transcription factor, was performed in this study. In a near-isogenic line (NIL) population segregated only at the OsMADS56 locus, NILs carrying the OsMADS56 allele of IRBB52 were 1.9% and 2.9% lower in TGW than NILs carrying the OsMADS56 allele of Teqing in 2018 and 2020, respectively. Using OsMADS56 knock-out mutants and overexpression transgenic plants, OsMADS56 was validated as the causal gene for qTGW10-20.8. Compared with the recipients, the TGW of the knock-out mutants was reduced by 6.0–15.0%. In these populations, decreased grain weight and size were associated with a reduction in the expression of OsMADS56. In transgenic populations of OsMADS56 driven by a strong constitutive promoter, grain weight and size of the positive plants were significantly higher than those of the negative plants. Haplotype analysis showed that the Teqing-type allele of OsMADS56 is the major type presented in cultivated rice and used in variety improvement. Cloning of OsMADS56 provides a new gene resource to improve grain weight and size through molecular design breeding.

Key message: A minor QTL for grain weight in rice, qTGW1.2b, was fine-mapped. Its casual gene OsVQ4 was confirmed through CRISPR/Cas9-targeted mutagenesis, exhibiting an effect that was larger than the original QTL effect. The CRISPR/Cas system exhibits a great potential for rice improvement, but the application was severely hindered due to insufficient target genes, especial the lack of validated genes underlying quantitative trait loci having small effects. In this study, a minor QTL for grain weight, qTGW1.2b, was fine-mapped into a 44.0 kb region using seven sets of near isogenic lines (NILs) developed from the indica rice cross (Zhenshan 97)³/Milyang 46, followed by validation of the causal gene using CRISPR/Cas9-targeted mutagenesis. In the NIL populations, 1000-grain weight of the Zhenshan 97 homozygous lines decreased by 0.9–2.0% compared with the Milyang 46 homozygous lines. A gene encoding VQ-motif protein, OsVQ4, was identified as the candidate gene based on parental sequence differences. The effect of OsVQ4 was confirmed by creating CRISPR/Cas9 knockout lines, whose 1000-grain weight decreased by 2.8–9.8% compared with the wild-type transgenic line and the recipient. These results indicate that applying genome editing system could create novel alleles with large phenotypic variation at minor QTLs, which is an effective way to validate causal genes of minor QTLs. Our study establishes a strategy for cloning minor QTLs, which could also be used to identify a large number of potential target genes for the application of CRISPR/Cas system.

Appropriate flowering time is crucial for successful grain production, which relies on not only the action of individual heading date genes, but also the gene-by-gene interactions. In this study, influences of interaction between Hd1 and Ghd7 on flowering time and yield traits were analyzed using near isogenic lines derived from a cross between indica rice cultivars ZS97 and MY46. In the non-functional ghd7^ZS97 background, the functional Hd1^ZS97 allele promoted flowering under both the natural short-day (NSD) conditions and natural long-day (NLD) conditions. In the functional Ghd7^MY46 background, Hd1^ZS97 remained to promote flowering under NSD conditions, but repressed flowering under NLD conditions. For Ghd7, the functional Ghd7^MY46 allele repressed flowering under both conditions, which was enhanced in the functional Hd1^ZS97 background under NLD conditions. With delayed flowering, spikelet number and grain weight increased under both conditions, but spikelet fertility and panicle number fluctuated. Rice lines carrying non-functional hd1^MY46 and functional Ghd7^MY46 alleles had the highest grain yield under both conditions. These results indicate that longer growth duration for a larger use of available temperature and light does not always result in higher grain production. An optimum heading date gene combination needs to be carefully selected for maximizing grain yield in rice.

Grains number is one of the most important agronomic traits in the determination of rice productivity. To explore the underlying genetic basis of grain number in rice, quantitative trait locus (QTL) analysis was performed using three recombinant inbred line populations derived from indica rice crosses of Teqing/IRBB lines, Zhenshan 97/Milyang 46, and Xieqingzao/Milyang 46, respectively. A total of 58 QTLs distributed on all 12 rice chromosomes were identified, including 22 for number of grains per panicle (NGP), 17 for number of spikelets per panicle, and 19 for spikelet fertility. The individual QTL counted for 1.5 to 22.1% of phenotypic variation. Among them, 15 QTLs shared by two or three populations and eight QTLs showed large effects with R² larger than 10%. Furthermore, three QTLs with minor effects for NGP, qNGP5.5, qNGP9.1, and qNGP12.1, were detected and validated by eliminating the segregation of major-effect QTL using four residual heterozygote-derived populations. These results not only enrich our understanding of the mechanism of grain number, but also provide a foundation for cloning and selecting candidate for marker-assisted selection breeding in rice.