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The flowering period of rice significantly impacts variety adaptability and yield formation. Properly shortening the reproductive period of rice varieties can expand their ecological range without significant yield reduction. Targeted genome editing, like CRISPR/Cas9, is an ideal tool to fine-tune rice growth stages and boost yield synergistically. In this study, we developed a CRISPR/Cas9-mediated multiplex genome-editing vector containing five genes related to three traits, Hd2, Ghd7, and DTH8 (flowering-stage genes), along with the recessive rice blast resistance gene Pi21 and the aromatic gene BADH2. This vector was introduced into the high-quality japonica rice variety in Zhejiang province, Jiahe212 (JH212), resulting in 34 T0 plants with various effective mutations. Among the 17 mutant T1 lines, several displayed diverse flowering dates, but most exhibited undesirable agronomic traits. Notably, three homozygous mutant lines (JH-C15, JH-C18, and JH-C31) showed slightly earlier flowering dates without significant differences in yield-related traits compared to JH212. Through special Hyg and Cas marker selection of T2 plants, we identified seven, six, and two fragrant glutinous plants devoid of transgenic components. These single plants will serve as sib lines of JH212 and potential resources for breeding applications, including maintenance lines for indica–japonica interspecific three-line hybrid rice. In summary, our research lays the foundation for the creation of short-growth-period CMS (cytoplasmic male sterility, CMS) lines, and also provides materials and a theoretical basis for indica–japonica interspecific hybrid rice breeding with wider adaptability. [ABSTRACT FROM AUTHOR]

Grain size is one of key agronomic traits associated with grain yield and grain quality. Both major quantitative trait loci GS3 and GL3.1 play a predominant role in negative regulation of grain size. In this study, a CRISPR/Cas9-mediated multiplex genome editing system was used to simultaneously edit GS3 and GL3.1 in a typical japonica rice Nipponbare. In T1 generation, we found that gs3 formed slender grain with lower chalkiness percentage, while gs3gl3.1 produced larger grain with higher chalkiness percentage. In terms of other agronomic traits, flag leaf size, grain number and grain yield of both gs3 and gs3gl3.1 mutants were affected. It is noteworthy that gs3 and gs3gl3.1 mutants both led to dramatical reduction of grain number, thereby decreased grain yield. In conclusion, these results indicated that knockout of GS3 and GL3.1 could rapidly improve grain size, but probably have some negative influences on grain quality and grain yield.

Grain size is one of the critical agronomic traits governing grain yield and quality in rice. However, the underlying genetic mechanisms that control grain size in rice are poorly understood. We used an introgression line derived from Zhonghui 8015 and Oryza rufipogon Griff. This introgression line was evaluated under two different environmental conditions to dissect the quantitative trait loci controlling grain size. Genome-wide association study (GWAS) was performed using 28 193 SNPs through a general linear model, and 56 significant SNPs on different loci associated with the 4 grain size traits were detected. Cloned genes including GS3 and qGL3 showed substantial effects on grain length and size. Seven new stable loci were identified with pleiotropic effects on grain size. Haplotype, gene expression analyses, combined gene-based associations, and functional annotations permitted the shortlisting of important dominant genes including GS3 and qGL3. Keywords: grain size, association mapping, Oryza sativa, Oryza rufipogon, quantitative trait locus, genome-wide association analysis

Grain shape improvement, which determines grain yield, quality traits and commercial value, is an extremely important aspect of rice breeding. Grain size is controlled by multiple genes, and Maker Assistant Selection (MAS) breeding is effective for breeders in developing stable and efficient markers to aggregate these genes in order to speed up the selection of new lines with desirable traits during the breeding process. In this study, functional markers were developed based on the sequence differences of five grain-shaped genes (GL7, GW6a, GS6, GW5 and TGW6) between the long-grain japonica rice variety Zhendao and the indica-japonica restorer R2027. We then constructed a population of recombinant inbred lines (RILs) based on their cross. The newly designed functional markers were used to genotype grain-size genes, and a genetic effect analysis was conducted to screen high-quality long-grain restorers. Our results reveal diverse effects of different genes on grain size, and the five genotypes were distributed in the 36 selected BC1F8 lines. Specifically, gw5 positively regulates grain width and 1000-grain weight, gl7 and gs6 positively regulate grain length but negatively regulate grain width and 1000-grain weight, tgw6 positively regulates grain length and gw6a positively regulates 1000-grain weight. The most outstanding outcome is that 5 of the 36 lines achieved in this study showing an excellent performance of long grain and yield characters are ideal materials not only for studying the interaction and genetic effects between polygenes but also as restorers or donors for dominant genes in indica-japonica hybrid rice breeding. [ABSTRACT FROM AUTHOR]