Grain quality is a key determinant of commercial value in rice. Efficiently improving grain quality, without compromising grain yield, is a challenge in rice breeding programs. Here we report on the identification and application of a grain quality gene, Chalk7, which causes a slender shape and decreases grain chalkiness in rice. Three allele-specific markers for Chalk7, and two other grain genes (GS3 and Chalk5) were developed, and used to stack the desirable alleles at these loci. The effects of individual or combined alleles at the loci were evaluated using a set of near-isogenic lines, each containing one to three favorable alleles in a common background of an elite variety. We found that the favorable allele combination of the three loci, which rarely occurs in natural rice germplasm, greatly reduces chalky grains without negatively impacting on grain yield. The data for newly developed allele-specific markers and pre-breeding lines will facilitate the improvement of grain appearance quality in rice.

Rice, a cold-sensitive crop, is a staple food for more than 50% of the world’s population. Low temperature severely compromises the growth of rice and challenges China’s food safety. Dongxiang wild rice (DXWR) is the most northerly common wild rice in China and has strong cold tolerance, but the genetic basis of its cold tolerance is still unclear. Here, we report quantitative trait loci (QTLs) analysis for seedling cold tolerance (SCT) using a high-density single nucleotide polymorphism linkage map in the backcross recombinant inbred lines that were derived from a cross of DXWR, and an indica cultivar, GZX49. A total of 10 putative QTLs were identified for SCT under 4 °C cold treatment, each explaining 2.0–6.8% of the phenotypic variation in this population. Furthermore, transcriptome sequencing of DXWR seedlings before and after cold treatment was performed, and 898 and 3413 differentially expressed genes (DEGs) relative to 0 h in cold-tolerant for 4 h and 12 h were identified, respectively. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) analysis were performed on these DEGs. Using transcriptome data and genetic linkage analysis, combined with qRT-PCR, sequence comparison, and bioinformatics, LOC_Os08g04840 was putatively identified as a candidate gene for the major effect locus qSCT8. These findings provided insights into the genetic basis of SCT for the improvement of cold stress potential in rice breeding programs.

SPX-domain proteins (small proteins with only the SPX domain) have been proven to be involved in phosphate-related signal transduction and regulation pathways. Except for OsSPX1 research showing that it plays a role in the process of rice adaptation to cold stress, the potential functions of other SPX genes in cold stress are unknown. Therefore, in this study, we identified six OsSPXs from the whole genome of DXWR. The phylogeny of OsSPXs has a strong correlation with its motif. Transcriptome data analysis showed that OsSPXs were highly sensitive to cold stress, and real-time PCR verified that the levels of OsSPX1, OsSPX2, OsSPX4, and OsSPX6 in cold-tolerant materials (DXWR) during cold treatment were higher than that of cold-sensitive rice (GZX49). The promoter region of DXWR OsSPXs contains a large number of cis-acting elements related to abiotic stress tolerance and plant hormone response. At the same time, these genes have expression patterns that are highly similar to cold-tolerance genes. This study provides useful information about OsSPXs, which is helpful for the gene-function research of DXWR and genetic improvements during breeding.

The DUF26 domain-containing protein is an extracellular structural protein, which plays an important role in signal transduction. Dongxiang wild rice (Oryza rufipogon Griff.) is the northern-most common wild rice in China. Using domain analysis, 85 DUF26 domain-containing genes were identified in Dongxiang wild rice (DXWR) and further divided into four categories. The DUF26 domain-containing genes were unevenly distributed on chromosomes, and there were 18 pairs of tandem repeats. Gene sequence analysis showed that there were significant differences in the gene structure and motif distribution of the DUF26 domain in different categories. Motifs 3, 8, 9, 13, 14, 16, and 18 were highly conserved in all categories. It was also found that there were eight plasmodesmata localization proteins (PDLPs) with a unique motif 19. Collinearity analysis showed that DXWR had a large number of orthologous genes with wheat, maize, sorghum and zizania, of which 17 DUF26 domain-containing genes were conserved in five gramineous crops. Under the stress of anaerobic germination and seedling submergence treatment, 33 DUF26 domain-containing genes were differentially expressed in varying degrees. Further correlation analysis with the expression of known submergence tolerance genes showed that these DUF26 domain-containing genes may jointly regulate the submergence tolerance process with these known submergence tolerance genes in DXWR.

The submergence tolerance of rice is a key factor in promoting rice direct seeding technology and resisting flood disasters. Dongxiang wild rice (DXWR) has strong submergence tolerance, but its genetic basis is still unclear. Here, we report quantitative trait loci (QTLs) analysis for hypoxic germination rate (HGR), hypoxic seedling rate (HSR), budlet submergence survival rate (BSSR) and seedling submergence survival rate (SSSR) using a linkage map in the backcross recombinant inbred lines (BRILs) that were derived from a cross of DXWR, and an indica cultivar, GZX49. A total of 20 QTLs related to submergence tolerance of rice were detected, explaining phenotypic variations ranging from 2% to 8.5%. Furthermore, transcriptome sequencing was performed on the seeds and seedlings of DXWR before and after submergence. During the seed hypoxic germination and seedling submergence stages, 6306 and 3226 differentially expressed genes (DEGs) were detected respectively. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses were conducted on these differentially expressed genes. Using genetic linkage analysis and transcriptome data, combined with qRT-PCR, sequence comparison, and bioinformatics, LOC_Os05g32820 was putatively identified as a candidate gene for qHGR5.2 co-located with HGR and SSSR. These results will provide insights into the mechanism of rice submergence tolerance and provide a basis for improving rice submergence tolerance.

Dongxiang wild rice (DXWR, Oryza rufipogon Griff.), the northernmost known wild rice species, exhibits exceptional tolerance to combined low-temperature and anaerobic stress during seed germination, providing a unique model for understanding plant adaptation to complex environmental constraints. Here, we employed an integrated multi-omics approach combining genomic, transcriptomic, and metabolomic analyses to unravel the synergistic regulatory mechanisms underlying this tolerance. Genomic comparative analysis categorized DXWR genes into three evolutionary groups: 18,480 core genes, 15,880 accessory genes, and 6822 unique genes. Transcriptomic profiling identified 10,593 differentially expressed genes (DEGs) relative to the control, with combined stress triggering the most profound changes, specifically inducing the upregulation of 5573 genes and downregulation of 5809 genes. Functional characterization revealed that core genes, including DREB transcription factors, coordinate energy metabolism and antioxidant pathways; accessory genes, such as glycoside hydrolase GH18 family members, optimize energy supply via adaptive evolution; and unique genes, including specific UDP-glycosyltransferases (UDPGTs), confer specialized stress resilience. Widely targeted metabolomics identified 889 differentially accumulated metabolites (DAMs), highlighting significant accumulations of oligosaccharides (e.g., raffinose) to support glycolytic energy production and a marked increase in flavonoids (153 compounds identified, e.g., procyanidins) enhancing antioxidant defense. Hormonal signals, including jasmonic acid and auxin, were reconfigured to balance growth and defense responses. We propose a multi-level regulatory network based on a “core-unique-adaptive” genetic framework, centered on ERF family transcriptional hubs and coordinated through a metabolic adaptation strategy of “energy optimization, redox homeostasis, and growth inhibition relief”. These findings offer innovative strategies for improving rice stress tolerance, particularly for enhancing germination of direct-seeded rice under early spring low-temperature and anaerobic conditions, by utilizing key genes such as GH18s and UDPGTs, thereby providing crucial theoretical and technological support for addressing food security challenges under climate change.