Multiomics profiling of the therapeutic effect of Dan-deng-tong-nao capsule on cerebral ischemia-reperfusion injury.

BACKGROUND: Stroke is a complex physiological process associated with intestinal flora dysbiosis and metabolic disorders. Dan-deng-tong-nao capsule (DDTN) is a traditional Chinese medicine used clinically to treat cerebral ischemia-reperfusion injury (CIRI) for many years. However, little is known about the effects of DDTN in the treatment of CIRI from the perspective of gut microbiota and metabolites. PURPOSE: This study aimed to investigate the regulatory roles of DDTN in endogenous metabolism and gut microbiota in CIRI rats, thus providing a basis for clinical rational drug use and discovering natural products with potential physiological activities in DDTN for the treatment of CIRI. METHODS: The chemical composition of DDTN in vitro and in vivo was investigated using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLCHRMS), followed by target prediction using reverse molecular docking. Secondly, a biological evaluation of DDTN ameliorating neural damage in CIRI was performed at the whole animal level. Then, an integrated omics approach based on UHPLCHRMS and 16S rRNA sequencing was proposed to reveal the anti-CIRI effect and possible mechanism of DDTN. Finally, exploring the intrinsic link between changes in metabolite profiles, changes in the intestinal flora, and targets of components to reveal DDTN for the treatment of CIRI. RESULTS: A total of 112 chemical components of DDTN were identified in vitro and 10 absorbed constituents in vivo. The efficacy of DDTN in the treatment of CIRI was confirmed by alleviating cerebral infarction and neurological deficits. After the DDTN intervention, 21 and 26 metabolites were significantly altered in plasma and fecal, respectively. Based on the fecal microbiome, a total of 36 genera were enriched among the different groups. Finally, the results of the network integration analysis showed that the 10 potential active ingredients of DDTN could mediate the differential expression of 24 metabolites and 6 gut microbes by targeting 25 target proteins. CONCLUSION: This study was the first to outline the landscapes of metabolites as well as gut microbiota regulated by DDTN in CIRI rats using multi-omics data, and comprehensively revealed the systematic relationships among ingredients, targets, metabolites, and gut microbiota, thus providing new perspectives on the mechanism of DDTN in the treatment of CIRI.

Two ABCI family transporters, OsABCI15 and OsABCI16, are involved in grain-filling in rice.

Seed development is critical for plant reproduction and crop yield, with panicle seed-setting rate, grain-filling, and grain weight being key seed characteristics for yield improvement. However, few genes are known to regulate grain filling. Here, we identify two adenosine triphosphate (ATP)-binding cassette (ABC)I-type transporter genes, OsABCI15 and OsABCI16, involved in rice grain-filling. Both genes are highly expressed in developing seeds, and their proteins are localized to the plasma membrane and cytosol. Interestingly, knockout of OsABCI15 and OsABCI16 results in a significant reduction in seed-setting rate, caused predominantly by the severe empty pericarp phenotype, which differs from the previously reported low seed-setting phenotype resulting from failed pollination. Further analysis indicates that OsABCI15 and OsABCI16 participate in ion homeostasis and likely export ions between filial tissues and maternal tissues during grain filling. Importantly, overexpression of OsABCI15 and OsABCI16 enhances seed-setting rate and grain yield in transgenic plants and decreases ion accumulation in brown rice. Moreover, the OsABCI15/16 orthologues in maize exhibit a similar role in kernel development, as demonstrated by their disruption in transgenic maize. Therefore, our findings reveal the important roles of two ABC transporters in cereal grain filling, highlighting their value in crop yield improvement.

The OsSGS3-tasiRNA-OsARF3 module orchestrates abiotic-biotic stress response trade-off in rice.

Recurrent heat stress and pathogen invasion seriously threaten crop production, and abiotic stress often antagonizes biotic stress response against pathogens. However, the molecular mechanisms of trade-offs between thermotolerance and defense remain obscure. Here, we identify a rice thermo-sensitive mutant that displays a defect in floret development under high temperature with a mutation in SUPPRESSOR OF GENE SILENCING 3a (OsSGS3a). OsSGS3a interacts with its homolog OsSGS3b and modulates the biogenesis of trans-acting small interfering RNA (tasiRNA) targeting AUXIN RESPONSE FACTORS (ARFs). We find that OsSGS3a/b positively, while OsARF3a/b and OsARF3la/lb negatively modulate thermotolerance. Moreover, OsSGS3a negatively, while OsARF3a/b and OsARF3la/lb positively regulate disease resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) and the fungal pathogen Magnaporthe oryzae (M. oryzae). Taken together, our study uncovers a previously unknown trade-off mechanism that regulates distinct immunity and thermotolerance through the OsSGS3-tasiRNA-OsARF3 module, highlighting the regulation of abiotic-biotic stress response trade-off in plants.

UHPLC-HRMS-based metabolomic and lipidomic characterization of glioma cells in response to anlotinib.

Anlotinib, as a promising oral small-molecule antitumor drug, its role in glioma has been only reported in a small number of case reports. Therefore, anlotinib has been considered as a promising candidate in glioma. The aim of this study was to investigate the metabolic network of C6 cells after exposure to anlotinib and to identify anti-glioma mechanism from the perspective of metabolic reprogramming. Firstly, CCK8 method was used to evaluate the effects of anlotinib on cell proliferation and apoptosis. Secondly, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)-based metabolomic and lipidomic were developed to characterize the metabolite and lipid changes in cell and cell culture medium (CCM) caused by anlotinib in the treatment of glioma. As a result, anlotinib had concentration-dependent inhibitory effect with the concentration range. In total, twenty-four and twenty-three disturbed metabolites in cell and CCM responsible for the intervention effect of anlotinib were screened and annotated using UHPLC-HRMS. Altogether, seventeen differential lipids in cell were identified between anlotinib exposure and untreated groups. Metabolic pathways, including amino acid metabolism, energy metabolism, ceramide metabolism, and glycerophospholipid metabolism, were modulated by anlotinib in glioma cell. Overall, anlotinib has an effective treatment against the development and progression of glioma, and these remarkable pathways can generate the key molecular events in cells treated with anlotinib. Future research into the mechanisms underlying the metabolic changes is expected to provide new strategies for treating glioma.

Exogenous calcium regulates the growth and development of Pinus massoniana detecting by physiological, proteomic, and calcium-related genes expression analysis.

Pinus massoniana is an important industrial crop tree species commonly used for timber and wood pulp for papermaking, rosin, and turpentine. This study investigated the effects of exogenous calcium (Ca) on P. massoniana seedling growth, development, and various biological processes and revealed the underlying molecular mechanisms. The results showed that Ca deficiency led to severe inhibition of seedling growth and development, whereas adequate exogenous Ca markedly improved growth and development. Many physiological processes were regulated by exogenous Ca. The underlying mechanisms involved diverse Ca-influenced biological processes and metabolic pathways. Calcium deficiency inhibited or impaired these pathways and processes, whereas sufficient exogenous Ca improved and benefited these cellular events by regulating several related enzymes and proteins. High levels of exogenous Ca facilitated photosynthesis and material metabolism. Adequate exogenous Ca supply relieved oxidative stress that occurred at low Ca levels. Enhanced cell wall formation, consolidation, and cell division also played a role in exogenous Ca-improved P. massoniana seedling growth and development. Calcium ion homeostasis and Ca signal transduction-related gene expression were also activated at high exogenous Ca levels. Our study facilitates the elucidation of the potential regulatory role of Ca in P. massoniana physiology and biology and is of guiding significance in Pinaceae plant forestry.

Discovery and characterization of hydroxylysine O-glycosylation in an engineered IL-2 fusion protein.

In the present study, an engineered interleukin-2 (IL-2) fusion protein consisting of an anti-human serum albumin nanobody linked by ASTKG and a (G4S)2 linker to IL-2 was constructed. Liquid chromatography-mass spectrometry (LC-MS) characterization was performed on the intact molecule and at the peptide level. The LC-MS molecular mass analysis for the engineered fusion protein showed the appearance of unreported +340 Da peaks, apart from the expected O-glycosylation-related peaks in the IL-2 domain. Through a combination analysis of a K120R mutated molecule (The lysine at the position of 120 was mutated to arginine while the rest amino acid sequence remain unchanged), the possibility of a non-cleaved valine-histidine-serine signal peptide was ruled out and the presence of hydroxylysine (HyK) O-glycosylation in the ASTKG linker was confirmed. HyK O-glycosylation have been reported in other proteins such as collagen, which occurs in the conserved Gly-Xaa-HyK motif and is catalyzed by lysyl hydroxylase-3 complex. The present study showed high similar conserved motif of HyK-O-glycosylation in collagen, implying the HyK O-glycosylation in the engineered IL-2 possibly was catalyzed by the Chinese hamster ovary homolog of enzymes promoting HyK O-glycosylation in collagen. Bioactivity testing results revealed that HyK-O-glycosylation had no obvious effect on the in vitro activity of engineered IL-2. Our study is the first to report HyK-O-glycosylation modifications in therapeutic proteins through LC-MS characterization and in vitro activity analysis, which expands the scope of post-translational modification knowledge of therapeutic proteins.

Rapid and comprehensive identification of chemical constituents in Mai-Luo-Shu-Tong pill by UHPLC-Q-Orbitrap HRMS combined with a data mining strategy.

Mai-Luo-Shu-Tong pill is an effective traditional Chinese medicine formula for the treatment of superficial thrombophlebitis, but it was insufficiently chemically scrutinized. In this study, the mass spectral data of Mai-Luo-Shu-Tong pill were acquired by ultra-high performance liquid chromatography coupled with Q Exactive hybrid Quadrupole-Orbitrap high resolution mass spectrometry. Then, a data mining strategy combining multiple data processing methods was used to identify chemical constituents in Mai-Luo-Shu-Tong pill by constructing a database of precursor ions and summarizing the mass spectral fragmentation behaviors. As a result, a total of 211 compounds including 70 flavonoids, 56 terpenoids, 37 phenolic acids and 48 others were identified in positive and negative ion modes. Among them, 66 compounds have passed comparison verification with reference standards, 145 compounds were identified based on the data mining strategy combining the characteristic cleavage behaviour of homologous compounds and fragment ions and 4 compounds were potentially new compounds. This study provides a database for quality evaluation and further study of Mai-Luo-Shu-Tong pill in vivo. Moreover, it provides a reference for the characterization of the chemical constituents of other traditional Chinese medicine formulae.

On the effect of flavonoids and dietary fibre in lingonberries on atherosclerotic plaques, lipid profiles and gut microbiota composition in Apoe-/- mice.

It has not been clarified whether the anti-atherosclerotic effect of lingonberry can be ascribed to its content of flavonoids or dietary fibre or both. The aim of this study was to evaluate the metabolic effects of whole lingonberries compared with isolated flavonoid and fibre fractions on atherosclerotic plaques, plasma lipid profiles, gut microbiota and microbiota-dependent metabolites in an Apoe-/- mouse model. Mice fed whole lingonberries showed the lowest amount of atherosclerotic plaques, while mice fed the fibre fraction had the highest formation of caecal butyric acid. Flavonoids, rather than dietary fibre, were suggested to be the components that favour proliferation of Akkermansia, as judged by the lowest abundance of this bacterium in mice fed the fibre fraction. All groups fed lingonberry diets had both, lower Firmicutes/Bacteroidetes ratios and creatinine concentrations, compared with the control. To conclude, different components in lingonberries are associated with different physiological effects in Apoe-/- mice.

Interleukin-33: Metabolic checkpoints, metabolic processes, and epigenetic regulation in immune cells.

Interleukin-33 (IL-33) is a pleiotropic cytokine linked to various immune cells in the innate and adaptive immune systems. Recent studies of the effects of IL-33 on immune cells are beginning to reveal its regulatory mechanisms at the levels of cellular metabolism and epigenetic modifications. In response to IL-33 stimulation, these programs are intertwined with transcriptional programs, ultimately determining the fate of immune cells. Understanding these specific molecular events will help to explain the complex role of IL-33 in immune cells, thereby guiding the development of new strategies for immune intervention. Here, we highlight recent findings that reveal how IL-33, acting as an intracellular nuclear factor or an extracellular cytokine, alters metabolic checkpoints and cellular metabolism, which coordinately contribute to cell growth and function. We also discuss recent studies supporting the role of IL-33 in epigenetic alterations and speculate about the mechanisms underlying this relationship.

Exogenous hydrogen sulfide mediates Na+ and K+ fluxes of salt gland in salt-secreting mangrove plant Avicennia marina.

Hydrogen sulfide (H2S), is a crucial biological player in plants. Here, we primarily explored the interaction between sodium hydrosulfide (NaHS, a H2S donor) and the fluxes of Na+ and K+ from the salt glands of mangrove species Avicennia marina (Forsk.) Vierh. with non-invasive micro-test technology (NMT) and quantitative real-time PCR (qRT-PCR) approaches under salinity treatments. The results showed that under 400-mM NaCl treatment, the addition of 200-µM NaHS markedly increased the quantity of salt crystals in the adaxial epidermis of A. marina leaves, accompanied by an increase in the K+/Na+ ratio. Meanwhile, the endogenous content of H2S was dramatically elevated in this process. The NMT result revealed that the Na+ efflux was increased from salt glands, whereas K+ efflux was decreased with NaHS application. On the contrary, the effects of NaHS were reversed by H2S scavenger hypotaurine (HT), and DL-propargylglycine (PAG), an inhibitor of cystathionine-γ-lyase (CES, a H2S synthase). Moreover, enzymic assay revealed that NaHS increased the activities of plasma membrane and tonoplast H+-ATPase. qRT-PCR analysis revealed that NaHS significantly increased the genes transcript levels of tonoplast Na+/H+ antiporter (NHX1), plasma membrane Na+/H+ antiporter (SOS1), plasma membrane H+-ATPase (AHA1) and tonoplast H+-ATPase subunit c (VHA-c1), while suppressed above-mentioned gene expressions by the application of HT and PAG. Overall, H2S promotes Na+ secretion from the salt glands of A. marina by up-regulating the plasma membrane and tonoplast Na+/H+ antiporter and H+-ATPase.

An MKP-MAPK protein phosphorylation cascade controls vascular immunity in plants.

Global crop production is greatly reduced by vascular diseases. These diseases include bacterial blight of rice and crucifer black rot caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas campestris pv. campestris (Xcc). The molecular mechanisms that activate vascular defense against such pathogens remains underexplored. Here, we show that an Arabidopsis MAPK phosphatase 1 (MKP1) mutant has increased host susceptibility to the adapted pathogen Xcc and is compromised in nonhost resistance to the rice pathogen Xoo. MKP1 regulates MAPK-mediated phosphorylation of the transcription factor MYB4 that negatively regulates vascular lignification through inhibiting lignin biosynthesis. Induction of lignin biosynthesis is, therefore, an important part of vascular-specific immunity. The role of MKP-MAPK-MYB signaling in lignin biosynthesis and vascular resistance to Xoo is conserved in rice, indicating that these factors form a tissue-specific defense regulatory network. Our study likely reveals a major vascular immune mechanism that underlies tissue-specific disease resistance against bacterial pathogens in plants.

Effects of Whole Brown Bean and Its Isolated Fiber Fraction on Plasma Lipid Profile, Atherosclerosis, Gut Microbiota, and Microbiota-Dependent Metabolites in Apoe-/- Mice.

The health benefits of bean consumption are widely recognized and are largely attributed to the dietary fiber content. This study investigated and compared the effects of whole brown beans and an isolated bean dietary fiber fraction on the plasma lipid profile, atherosclerotic plaque amount, gut microbiota, and microbiota-dependent metabolites (cecal short-chain fatty acids (SCFAs) and plasma methylamines) in Apoe-/- mice fed high fat diets for 10.5 weeks. The results showed that both whole bean and the isolated fiber fraction had a tendency to lower atherosclerotic plaque amount, but not plasma lipid concentration. The whole bean diet led to a significantly higher diversity of gut microbiota compared with the high fat diet. Both bean diets resulted in a lower Firmicutes/Bacteroidetes ratio, higher relative abundance of unclassified S24-7, Prevotella, Bifidobacterium, and unclassified Clostridiales, and lower abundance of Lactobacillus. Both bean diets resulted in higher formation of all cecal SCFAs (higher proportion of propionic acid and lower proportion of acetic acid) and higher plasma trimethylamine N-oxide concentrations compared with the high fat diet. Whole beans and the isolated fiber fraction exerted similar positive effects on atherosclerotic plaque amount, gut microbiota, and cecal SCFAs in Apoe-/- mice compared with the control diets.

NLRs guard metabolism to coordinate pattern- and effector-triggered immunity.

Pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) in plants enable them to respond to pathogens by activating the production of defence metabolites that orchestrate immune responses1-4. How the production of defence metabolites is promoted by immune receptors and coordinated with broad-spectrum resistance remains elusive. Here we identify the deubiquitinase PICI1 as an immunity hub for PTI and ETI in rice (Oryza sativa). PICI1 deubiquitinates and stabilizes methionine synthetases to activate methionine-mediated immunity principally through biosynthesis of the phytohormone ethylene. PICI1 is targeted for degradation by blast fungal effectors, including AvrPi9, to dampen PTI. Nucleotide-binding domain, leucine-rich-repeat-containing receptors (NLRs) in the plant immune system, such as PigmR, protect PICI1 from effector-mediated degradation to reboot the methionine-ethylene cascade. Natural variation in the PICI1 gene contributes to divergence in basal blast resistance between the rice subspecies indica and japonica. Thus, NLRs govern an arms race with effectors, using a competitive mode that hinges on a critical defence metabolic pathway to synchronize PTI with ETI and ensure broad-spectrum resistance.

Ca2+ sensor-mediated ROS scavenging suppresses rice immunity and is exploited by a fungal effector.

Plant immunity is activated upon pathogen perception and often affects growth and yield when it is constitutively active. How plants fine-tune immune homeostasis in their natural habitats remains elusive. Here, we discover a conserved immune suppression network in cereals that orchestrates immune homeostasis, centering on a Ca2+-sensor, RESISTANCE OF RICE TO DISEASES1 (ROD1). ROD1 promotes reactive oxygen species (ROS) scavenging by stimulating catalase activity, and its protein stability is regulated by ubiquitination. ROD1 disruption confers resistance to multiple pathogens, whereas a natural ROD1 allele prevalent in indica rice with agroecology-specific distribution enhances resistance without yield penalty. The fungal effector AvrPiz-t structurally mimics ROD1 and activates the same ROS-scavenging cascade to suppress host immunity and promote virulence. We thus reveal a molecular framework adopted by both host and pathogen that integrates Ca2+ sensing and ROS homeostasis to suppress plant immunity, suggesting a principle for breeding disease-resistant, high-yield crops.

Egg white peptides ameliorate dextran sulfate sodium-induced acute colitis symptoms by inhibiting the production of pro-inflammatory cytokines and modulation of gut microbiota composition.

Egg white peptides (EWPs) can be effectively used to alleviate and treat inflammatory diseases due to their anti-oxidation, anti-inflammation, and microbiota regulation capabilities. A dextran sodium sulfate (DSS)-induced colitis model was used to clarify the regulatory effects of EWPs on colitis. Forty-three peptide sequences were identified from EWPs using LC-MS/MS. The results demonstrated that EWPs decreased the levels of pro-inflammatory cytokines and the extent of crypt damage in a dose-dependent manner. 16S rRNA gene sequencing results indicated that 200 mg/kg EWPs significantly increased the relative abundance of beneficial bacteria Lactobacillus and Candidatus_Saccharimonas, and reduced the relative abundance of pathogenic bacteria Ruminiclostridium and Akkermansia. In addition, the degree of correlation between pro-inflammatory cytokines and microbiota was as follows: interleukin (IL)-1ß > IL-8 > IL-6 > tumor necrosis factor-α To summarize, EWPs contributed to the alleviation of colitis symptoms and the intestinal injury through anti-inflammatory effects, repair of intestinal mucosa, and modulation of gut microbiota.

Proteomic analysis reveals the protective role of exogenous hydrogen sulfide against salt stress in rice seedlings.

Hydrogen sulfide (H2S) is an important gaseous signal molecule which participates in various abiotic stress responses. However, the underlying mechanism of H2S associated salt tolerance remains elusive. In this study, sodium hydrosulfide (NaHS, donor of H2S) was used to investigate the protective role of H2S against salt stress at the biochemical and proteomic levels. Antioxidant activity and differentially expressed proteins (DEPs) of rice seedlings treated by NaCl or/and exogenous H2S were investigated by the methods of biochemical approaches and comparative proteomic analysis. The protein-protein interaction (PPI) analysis was used for understanding the interaction networks of stress responsive proteins. In addition, relative mRNA levels of eight selected identified DEPs were analyzed by quantitative real-time PCR. The result showed that H2S alleviated oxidative damage caused by salt stress in rice seedling. The activities of some antioxidant enzymes and glutathione metabolism were mediated by H2S under salt stress. Proteomics analyses demonstrated that NaHS regulated antioxidant related proteins abundances and affected related enzyme activities under salt stress. Proteins related to light reaction system (PsbQ domain protein, plastocyanin oxidoreductase iron-sulfur protein), Calvin cycle (phosphoglycerate kinase, sedoheptulose-1,7-bisphosphatase precursor, ribulose-1,5-bisphosphate carboxylase/oxygenase) and chlorophyll biosynthesis (glutamate-1-semialdehyde 2,1-aminomutase, coproporphyrinogen III oxidase) are important for NaHS against salt stress. ATP synthesis related proteins, malate dehydrogenase and 2, 3-bisphosphoglycerate-independent phosphoglycerate mutase were up-regulated by NaHS under salt stress. Protein metabolism related proteins and cell structure related proteins were recovered or up-regulated by NaHS under salt stress. The PPI analysis further unraveled a complicated regulation network among above biological processes to enhance the tolerance of rice seedling to salt stress under H2S treatment. Overall, our results demonstrated that H2S takes protective roles in salt tolerance by mitigating oxidative stress, recovering photosynthetic capacity, improving primary and energy metabolism, strengthening protein metabolism and consolidating cell structure in rice seedlings.

A plasma membrane transporter coordinates phosphate reallocation and grain filling in cereals.

Phosphate (Pi) is essential to plant growth and crop yield. However, it remains unknown how Pi homeostasis is maintained during cereal grain filling. Here, we identified a rice grain-filling-controlling PHO1-type Pi transporter, OsPHO1;2, through map-based cloning. Pi efflux activity and its localization to the plasma membrane of seed tissues implicated a specific role for OsPHO1;2 in Pi reallocation during grain filling. Indeed, Pi over-accumulated in developing seeds of the Ospho1;2 mutant, which inhibited the activity of ADP-glucose pyrophosphorylase (AGPase), important for starch synthesis, and the grain-filling defect was alleviated by overexpression of AGPase in Ospho1;2-mutant plants. A conserved function was recognized for the maize transporter ZmPHO1;2. Importantly, ectopic overexpression of OsPHO1;2 enhanced grain yield, especially under low-Pi conditions. Collectively, we discovered a mechanism underlying Pi transport, grain filling and P-use efficiency, providing an efficient strategy for improving grain yield with minimal P-fertilizer input in cereals.

Grain Size Selection Using Novel Functional Markers Targeting 14 Genes in Rice.

BACKGROUND: Grain size is an extremely important aspect of rice breeding, affecting both grain yield and quality traits. It is controlled by multiple genes and tracking these genes in breeding schemes should expedite selection of lines with superior grain yield and quality, thus it is essential to develop robust, efficient markers. RESULT: In this study, 14 genes related to grain size (GW2, GS2, qLGY3, GS3, GL3.1, TGW3, GS5, GW5, GS6, TGW6, GW6a, GLW7, GL7 and GW8) were selected for functional marker development. Twenty-one PCR-gel-based markers were developed to genotype the candidate functional nucleotide polymorphisms (FNPs) of these genes, and all markers can effectively recognize the corresponding allele types. To test the allele effects of different FNPs, a global collection of rice cultivars including 257 accessions from the Rice Diversity Panel 1 was used for allele mining, and four grain-size-related traits were investigated at two planting locations. Three FNPs for GW2, GS2 and GL3.1 were genotyped as rare alleles only found in cultivars with notably large grains, and the allele contributions of the remaining FNPs were clarified in both the indica and japonica subspecies. Significant trait contributions were found for most of the FNPs, especially GS3, GW5 and GL7. Of note, GW5 could function as a key regulator to coordinate the performance of other grain size genes. The allele effects of several FNPs were also tested by QTL analysis using an F2 population, and GW5 was further identified as the major locus with the largest contribution to grain width and length to width ratio. CONCLUSIONS: The functional markers are robust for genotyping different cultivars and may facilitate the rational design of grain size to achieve a balance between grain yield and quality in future rice breeding efforts.