This study aimed to develop an aromatic thermosensitive genic male sterile (TGMS) line in indica rice using CRISPR/Cas9 technology. The TMS5 and FGR in the high-quality conventional rice variety Huahang 48 were targeted for editing using CRISPR/Cas9 technology. CRISPR/Cas9 vectors designed for TMS5 and FGR were constructed and introduced into rice calli through Agrobacterium-mediated transformation. Transgenic seedlings were subsequently regenerated, and the target sites of the edited plants were analyzed via sequencing. A total of fifteen T0 double mutants were successfully obtained. Three mutants without T-DNA insertion were screened in the T1 generation by the PCR detection of hygromycin gene fragments, and homozygous mutants without T-DNA insertion were screened in the T2 generation by the sequencing analysis of the mutation sites, named Huahang 48s. Huahang 48s exhibited complete sterility at 24 °C and pollen transfer at 23 °C. The 2-acetyl-1-pyrroline (2-AP) content was detected in the young panicles, leaves, and stems of Huahang 48s. The leaves of Huahang 48s had the highest 2-AP content, contrasting with the absence of 2-AP in HuaHang 48. F1 hybrids that crossed Huahang 48s with two high-quality restorer lines were superior to the two parents in terms of yield per plant and 1000-grain weight. Huahang 48s has a certain combining ability and application potential in two-line cross breeding. The successful application of CRISPR/Cas9 technology in Huahang 48 established a foundation for developing aromatic TGMS lines, providing both theoretical insights and practical materials for breeding efforts.

Grain shape in rice (Oryza sativa L.) is a complex trait governed by multiple quantitative trait loci (QTLs). To dissect the genetic basis of rice shape, QTL analysis was conducted for milled rice grain width (MGW), milled rice grain length (MGL), and milled rice length-to-width ratio (MLWR) using a recombinant inbred line (RIL) population of F10 and F11 generations derived from a cross between Yuexiangzhan and Shengbasimiao. A high-density genetic map consisting of 2412 bins was constructed by sequencing 184 RILs, spanning a total length of 2376.46 cM. A total of 19 QTLs related to MGL, MGW, and MLWR were detected under two environments. The range of phenotypic variation attributed to individual QTL ranged from 1.67% to 32.08%. Among those, a novel locus for MGL, MGW and MLWR, designated as qMLWR3.2, was pinpointed within a specific ~0.96-Mb region. Two novel loci for MGW and MLWR, qMLWR11.1 and qMLWR11.2, were verified within ~1.22-Mb and ~0.52-Mb regions using three RIL-developed populations, respectively. These findings lay the foundation for further map-based cloning and molecular design breeding in rice.

Abstract Heading date is one of the most important agronomic traits and a fundamental factor determining crop yield. Although many genes related to heading date have been reported in rice, the molecular mechanism of heading date is still poorly understood. The Small Auxin-Up RNA (SAUR) family genes regulate many aspects of plant growth and development. However, their functions involved in heading date of rice (Oryza sativa L.) are not characterized. Here, OsSAUR56 gene was edited by the CRISPR/Cas9 technology in the japonica cultivar Zhonghua11 (ZH11). We found that loss-of-function of OsSAUR56 led to early heading phenotype regardless of day length. OsSAUR56 was mainly expressed in anther, and its protein was localized in the cytoplasm and the nucleus. Gene expression analysis with quantitative RT-PCR showed that OsSAUR56 regulated flowering time and heading date by affecting the expression of a clock gene OsGI and two repressors, HEADING DATE 7 (Ghd7) and DTH8 (Ghd8/LHD1). Moreover, evolutionary analysis showed that OsSAUR56 presents divergence between indica and japonica, showing natural selection during the domestication of cultivated rice. These results indicate that OsSAUR56 plays an important role in the regulation of heading date, and may be an important target for rice breeding programs.

The plant architecture of rice is an important factor affecting yield. Strigolactones (SLs) are newly discovered carotenoid-derived plant hormones that play an important role in rice plant architecture. In this study, a high-tillering dwarf mutant, CHA-1, was identified by spatial mutagenesis. CHA-1 was located in the region of 31.52–31.55 MB on chromosome 1 by map-based cloning. Compared with the wild-type THZ, the CHA-1 mutant showed that ACCAC replaced TGGT in the coding region of the candidate gene LOC_Os01g54810, leading to premature termination of expression. Genetic complementation experiments proved that LOC_Os01g54810 was CHA-1, which encodes a putative member of Class III lipase. Expression analysis showed that CHA-1 was constitutively expressed in various organs of rice. Compared with those in THZ, the expression levels of the D17 and D10 genes were significantly downregulated in the CHA-1 mutant. In addition, the concentrations of ent-2′-epi-5-deoxystrigol (epi-5DS) in the root exudates of the CHA-1 mutant was significantly reduced compared with that of THZ, and exogenous application of GR24 inhibited the tillering of the CHA-1 mutant. These results suggest that CHA-1 influences rice architecture by affecting SL biosynthesis.

Grain size influences the yield and quality of rice (Oryza sativa L.), and grain length is one of the component traits of grain size. In this study, a near-isogenic line LB3 with long grain size was constructed using japonica rice cultivar 02428, with short grain size, as the recipient parent and indica rice cultivar ZYX, with long grain size, as the donor parent, by multi-generation backcrossing and selfing. BSA-seq was used for preliminary QTL mapping and InDel markers were developed to fine map the locus. The major QTL, tentatively named qGL10, for grain length was located in a 128.45 kb region of chromosome 10. Combined with haplotype analysis of rice varieties, expression pattern analysis of candidate genes suggested LOC_Os10g39130 (OsMADS56) as a candidate gene. Sequence alignment of OsMADS56 in 02428 and LB3 revealed that there were 15 SNPs in the promoter region and four in the coding region. Further haplotype analysis suggested that SNP9 (G/A) located in the TGTCACA motif might account for the different expression levels of OsMADS56 in 02428 and LB3. These results lay a foundation for the application of qGL10 in molecular breeding of new rice varieties.

Heading date is a critical agronomic trait that determines crop yield. Although numerous genes associated with heading date have been identified in rice, the mechanisms involving Small Auxin Up RNA (SAUR) family have not been elucidated. In this study, the biological function of several SAUR genes was initially investigated using the CRISPR- Cas9 technology in the Japonica cultivar Zhonghua11 (ZH11) background. Further analysis revealed that the loss-of-function of OsSAUR56 affected heading date in both NLD (natural long-day) and ASD (artificial short-day). OsSAUR56 exhibited predominant expression in the anther, with its protein localized in both the cytoplasm and nucleus. OsSAUR56 regulated flowering time and heading date by modulating the expression of the clock gene OsGI , as well as two repressors Ghd7 and DTH8 . Furthermore, haplotype-phenotype association analysis revealed a strong correlation between OsSAUR56 and heading date, suggesting its role in selection during the domestication of rice. In summary, these findings highlights the importance of OsSAUR56 in the regulation of heading date for further potential facilitating genetic engineering for flowering time during rice breeding.