Identifying QTLs for Grain Size in a Colossal Grain Rice (Oryza sativa L.) Line,and Analysis of Additive Effects of QTLs

Grain size is an important component of quality and harvest traits in the field of rice breeding. Although numerous quantitative trait loci (QTLs) of grain size in rice have been reported, the molecular mechanisms of these QTLs remain poorly understood, and further research on QTL observation and candidate gene identification is warranted. In our research, we developed a suite of F₂ intercross populations from a cross of 9311 and CG. These primary populations were used to map QTLs conferring grain size, evaluated across three environments, and then subjected to bulked-segregant analysis-seq (BSA-seq). In total, 4, 11, 12 and 14 QTLs for grain length (GL), grain width (GW), 1000-grain weight (TGW), and length/width ratio (LWR), respectively, were detected on the basis of a single-environment analysis. In particular, over 200 splicing-related sites were identified by whole-genome sequencing, including one splicing-site mutation with G>A at the beginning of intron 4 on Os03g0841800 (qGL3.3), producing a smaller open reading frame, without the third and fourth exons. A previous study revealed that the loss-of-function allele caused by this splicing site can negatively regulate rice grain length. Furthermore, qTGW2.1 and qGW2.3 were new QTLs for grain width. We used the near-isogenic lines (NILs) of these GW QTLs to study their genetic effects on individuals and pyramiding, and found that they have additive effects on GW. In summary, these discoveries provide a valuable genetic resource, which will facilitate further study of the genetic polymorphism of new rice varieties in rice breeding.     

The Function of DNA Demethylase Gene ROS1a Null Mutant on Seed Development in Rice (Oryza Sativa) Using the CRISPR/CAS9 System

The endosperm is the main nutrient source in cereals for humans, as it is a highly specialized storage organ for starch, lipids, and proteins, and plays an essential role in seed growth and development. Active DNA demethylation regulates plant developmental processes and is ensured by cytosine methylation (5-meC) DNA glycosylase enzymes. To find out the role of OsROS1a in seed development, the null mutant of OsROS1a was generated using the CRISPR/Cas9 system. The null mutant of OsROS1a was stable and heritable, which affects the major agronomic traits, particularly in rice seeds. The null mutant of OsROS1a showed longer and narrower grains, and seeds were deformed containing an underdeveloped and less-starch-producing endosperm with slightly irregularly shaped embryos. In contrast to the transparent grains of the wild type, the grains of the null mutant of OsROS1a were slightly opaque and rounded starch granules, with uneven shapes, sizes, and surfaces. A total of 723 differential expression genes (DEGs) were detected in the null mutant of OsROS1a by RNA-Seq, of which 290 were downregulated and 433 were upregulated. The gene ontology (GO) terms with the top 20 enrichment factors were visualized for cellular components, biological processes, and molecular functions. The key genes that are enriched for these GO terms include starch synthesis genes (OsSSIIa and OsSSIIIa) and cellulose synthesis genes (CESA2, CESA3, CESA6, and CESA8). Genes encoding polysaccharides and glutelin were found to be downregulated in the mutant endosperm. The glutelins were further verified by SDS-PAGE, suggesting that glutelin genes could be involved in the null mutant of OsROS1a seed phenotype and OsROS1a could have the key role in the regulation of glutelins. Furthermore, 378 differentially alternative splicing (AS) genes were identified in the null mutant of OsROS1a, suggesting that the OsROS1a gene has an impact on AS events. Our findings indicated that the function on rice endosperm development in the null mutant of OsROS1a could be influenced through regulating gene expression and AS, which could provide the base to properly understand the molecular mechanism related to the OsROS1a gene in the regulation of rice seed development.