Background: In Asian rice production, an increasing number of countries now choose the direct seeding mode because of rising costs, labour shortages and water shortages. The ability of rice seeds to undergo anaerobic germination (AG) plays an important role in the success of direct seeding. Results: In this study, we used 2,123,725 single nucleotide polymorphism (SNP) markers based on resequencing to conduct a dynamic genome-wide association study (GWAS) of coleoptile length (CL) and coleoptile diameter (CD) in 209 natural rice populations. A total of 26 SNP loci were detected in these two phenotypes, of which 5 overlapped with previously reported loci (S1_ 39674301, S6_ 20797781, S7_ 18722403, S8_ 9946213, S11_ 19165397), and two sites were detected repeatedly at different time points (S3_ 24689629 and S5_ 27918754). We suggest that these 7 loci (−log₁₀ (P) value > 7.3271) are the key sites that affect AG tolerance. To screen the candidate genes more effectively, we sequenced the transcriptome of the flooding-tolerant variety R151 in six key stages, including anaerobic (AN) and the oxygen conversion point (AN-A), and obtained high-quality differential expression profiles. Four reliable candidate genes were identified: Os01g0911700 (OsVP1), Os05g0560900 (OsGA2ox8), Os05g0562200 (OsDi19–1) and Os06g0548200. Then qRT-PCR and LC-MS/ MS targeting metabolite detection technology were used to further verify that the up-regulated expression of these four candidate genes was closely related to AG. Conclusion: The four novel candidate genes were associated with gibberellin (GA) and abscisic acid (ABA) regulation and cell wall metabolism under oxygen-deficiency conditions and promoted coleoptile elongation while avoiding adverse effects, allowing the coleoptile to obtain oxygen, escape the low-oxygen environment and germinate rapidly. The results of this study improve our understanding of the genetic basis of AG in rice seeds, which is conducive to the selection of flooding-tolerant varieties suitable for direct seeding.

The development and utilization of genetic markers play a pivotal role in marker-assisted breeding of rice cultivars during pyramiding of valuable genes. Among molecular markers, SNPs have become the most promising due to their wide distribution within genomes and suitability for high -throughput automated genotyping. Although metadata of SNPs have been identified via next generation sequencing in rice, a large gap between the development of SNP markers and the application in breeding still exists. To promote the application of SNP markers based on the KASP (Kompetitive Allele-Specific PCR) method in rice breeding, a set of core SNP arrays was built via the screening of SNP databases and literature resources based on the KASP method. Five hundred and ninety six SNPs classified into eight subsets including quality control, indica-indica variation, highly polymorphic, functional genes, key genes targeting sites, gene cloned region, important trait associated and gap filling sites were chosen to design KASP primers and 565 out of them were successfully designed, and the assay design success rate was 94.8%. Finally, 467 out of the 565 successfully-designed SNPs can display diversity at the loci were used to develop a set of core SNP arrays. To evaluate the application value of the core SNP markers in rice breeding, 481 rice germplasms were genotyped with three functional KASP markers designed from the sequences of GBSSI, SSIIa, and Badh2 from the core SNP arrays for estimation of their grain quality performance. Eighteen rice lines, including Xiangwanxian 13, Basmati 370, Ruanhua A, and PR 33319–9–1-1-5-3-5-4-1, harbor all three favorable alleles. The core KASP arrays were also used for rice germplasm assessment, genetic diversity and population evaluation. Four hundred and eighty-one rice germplasms were divided into 3 groups: POP1, POP2 and POP3. POP1 and POP2 were indica rice subgroups consisting of 263 and 186 rice germplasms, respectively. POP3 was a japonica rice subgroup consisting of 32 rice germplasms. The average FST value for the three subgroups was 0.3501; the FST value of POP1 and POP3 was the largest (0.5482), while that of POP1 and POP2 was the smallest (0.0721). The results showed that the genetic distance between the japonica and indica rice subspecies was large, indicating that the core SNP markers were effective at discriminating the population structure of the germplasms. Finally, the core KASP arrays were used for association analysis with milled grain traits. A total of 31 KASP markers were significantly associated (P < 0.01) with ML and the LWR. Among the 31 markers, 13 were developed based on cloned genes or on identified loci related to yield traits. Notably, several KASP markers associated with grain quality were also found to be associated with brown planthopper resistance or green leafhopper resistance simultaneously. The core KASP arrays developed in our study were efficient and versatile for rice germplasm assessment, genetic diversity and population evaluation and are valuable for promoting SNP molecular breeding in rice. Our study demonstrated that useful assays combined with molecular breeding can be exploited for important economic trait improvement in rice breeding.

The metabolic profile of rice (Oryza sativa) during germination under low temperature (LT) has not been reported. In this study, the rice varieties 02428 (japonica) and YZX (indica) were subjected to experiments consisting of treatments including LT, normal temperature (NT) and a transition from LT to NT, and tissues were sampled at different time points during germination. A total of 730 metabolites were detected by a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based widely targeted metabolomics method. On the basis of the screening criteria of increased contents under LT and decreased contents under NT, we identified 35 different metabolites that responded to LT stress among the 730 metabolites. Furthermore, the content differences of the 35 metabolites were compared when the samples were transferred from LT to NT. According to a fold change 1 at the transition point, 7 out of the 35 metabolites responded significantly to LT stress and were defined as key metabolites. A partial least squares (PLS) regression model of seven key metabolites with seedling length (SL), seedling area (SSA), and seedling volume (SV) was constructed, and the fitting effect was good. These seven key metabolites participate in the biosynthesis of amino acids and phenylpropanoids and in the metabolism of glutathione and inositol phosphate. This study laid a foundation for an improved understanding of the LT-germination mechanism of rice seeds.

Class III peroxidases (CIII Prxs) play critical roles in plant immunity by scavenging reactive oxygen species (ROS). However, the functions of CIII Prxs in rice (Oryza sativa L.) immunity are largely unexplored. Here, we report a Prx precursor, OsPrx30, that is responsive to the bacterial blight Xanthomonas oryzae pv. oryzae (Xoo). OsPrx30 was primarily expressed in rice roots, leaves, and stems, and its protein product was mainly localized at the endoplasmic reticulum. Overexpression of OsPrx30 enhanced the plant's susceptibility to Xoo by maintaining a high level of peroxidase (POD) activity and reducing the content of H₂O₂, whereas depletion of OsPrx30 had the opposite effects. Furthermore, we identified an AT‐hook transcription factor, OsATH1, that is specifically bound to the OsPrx30 promoter. As observed in plants overexpressing OsPrx30, depletion of OsATH1 enhanced susceptibility to Xoo. Finally, we demonstrated that depletion of OsATH1 increased histone H3 acetylation at the AT‐rich region of the OsPrx30 promoter. Taken together, these results reveal a mechanism underlying the POD‐induced natural resistance to bacterial diseases and suggest a model for transcription regulation of Prx genes in rice.

Grain appearance quality is a main determinant of market value in rice and one of the highly important traits requiring improvement in breeding programs. The genetic basis of grain shape and endosperm chalkiness have been given significant attention because of their importance in affecting grain quality. Meanwhile, the introduction of NGS (Next Generation Sequencing) has a significant part to play in the area of genomics, and offers the possibility for high-resolution genetic map construction, population genetics analysis and systematic expression profile study. A RIL population derived from an inter-subspecific cross between indica rice PYZX and japonica rice P02428 was generated, based on the significant variations for the grain morphology and cytological structure between these two parents. Using the Genotyping-By-Sequencing (GBS) approach, 2711 recombination bin markers with an average physical length of 137.68 kb were obtained, and a high-density genetic map was constructed. Global genetic mapping of QTLs affecting grain shape and chalkiness traits was performed across four environments and the newly identified stable loci were obtained. Twelve important QTL clusters were detected, four of which were coincident with the genomic regions of cloned genes or fine mapped QTL reported. Eight novel QTL clusters (including six for grain shape, one for chalkiness, and one for both grain shape and chalkiness) were firstly obtained and highlighted the value and reliability of the QTL analysis. The important QTL cluster on chromosome 5 affects multiple traits including circularity (CS), grain width (GW), area size of grain (AS), percentage of grains with chalkiness (PGWC) and degree of endosperm chalkiness (DEC), indicating some potentially pleiotropic effects. The transcriptome analysis demonstrated an available gene expression profile responsible for the development of chalkiness, and several DEGs (differentially expressed genes) were co-located nearby the three chalkiness-related QTL regions on chromosomes 5, 7, and 8. Candidate genes were extrapolated, which were suitable for functional validation and breeding utilization. QTLs affecting grain shape (grain width, grain length, length-width ratio, circularity, area size of grain, and perimeter length of grain) and chalkiness traits (percentage of grains with chalkiness and degree of endosperm chalkiness) were mapped with the high-density GBS-SNP based markers. The important differentially expressed genes (DEGs) were co-located in the QTL cluster regions on chromosomes 5, 7 and 8 affecting PGWC and DEC parameters. Our research provides a crucial insight into the genetic architecture of rice grain shape and chalkiness, and acquired potential candidate loci for molecular cloning and grain quality improvement.