Mapping QTLs underpin nutrition components in aromatic rice germplasm.

As rice is an important staple food globally, research for development and enhancement of its nutritional value it is an imperative task. Identification of nutrient enriched rice germplasm and exploiting them for breeding programme is the easiest way to develop better quality rice. In this study, we analyzed 113 aromatic rice germplasm in order to identify quantitative trait loci (QTL) underpinning nutrition components and determined by measuring the normal frequency distribution for Fe, Zn, amylose, and protein content in those rice germplasm. Comparatively, the germplasm Radhuni pagal, Kalobakri, Thakurbhog (26.6 ppm) and Hatisail exhibited the highest mean values for Fe (16.9 ppm), Zn (34.1 ppm), amylose (26.6 ppm) and protein content (11.0 ppm), respectively. Moreover, a significant linear relationship (R2 = 0.693) was observed between Fe and Zn contents. Cluster analysis based on Mahalanobis D2 distances revealed four major clusters of 113 rice germplasm, with cluster III containing a maximum 37 germplasm and a maximum inter-cluster distance between clusters III and IV. The 45 polymorphic SSRs and four trait associations exhibited eight significant quantitative trait loci (QTL) located on eight different chromosomes using composite interval mapping (CIM). The highly significant QTL (variance 7.89%, LOD 2.02) for protein content (QTL.pro.1) was observed on chromosome 1 at 94.9cM position. Also, four QTLs for amylose content were observed with the highly significant QTL.amy.8 located on chromosome 8 exhibiting 7.2% variance with LOD 1.83. Only one QTL (QTL.Fe.9) for Fe content was located on chromosome 9 (LOD 1.24), and two (QTL.Zn.4 and QTL.Zn.5) for Zn on chromosome 4 (LOD 1.71) and 5 (LOD 1.18), respectively. Overall, germplasm from clusters III and IV might offer higher heterotic response with the identified QTLs playing a significant role in any rice biofortification breeding program and released with development of new varieties.

Combining Irrigation Scheme and Phosphorous Application Levels for Grain Yield and Their Impacts on Rhizosphere Microbial Communities of Two Rice Varieties in a Field Trial.

Root and rhizosphere is important for phosphorus (P) uptake in rice plants. However, little is known about the detailed regulation of irrigation regimes, especially frequently alternate wetting and drying (FAWD), on P usage of rice plants. Here, we found that compared with normal water and P dose, FAWD with a reduced P dose maintained the grain yield in two rice varieties. Compared to rice variety Gaoshan1, rice variety WufengyouT025 displayed a higher grain yield, shoot P content, rhizosphere acid phosphatase activity, abundance of bacteria, and bacterial acid phosphatase gene of rhizosphere. Moreover, the FAWD regime may increase the abundance of bacteria with acid phosphatase activity to release available phosphorus in the rhizosphere, which is associated with rice varieties. Our results suggest that an optimized management of irrigation and phosphorous application can enhance both water and phosphorus use efficiency without sacrificing the yield, which may contribute significantly to sustainable agriculture production.

Exploiting the bioactive properties of γ-oryzanol from bran of different exotic rice varieties.

The rice industry is one of the most significant food industries since rice is a widely consumed cereal in the world. As a result of this substantial production, the rice industry has a significant amount of side streams, including bran, representing millions of tons of raw materials mainly designated to animal feed. Rice bran is a rich source of γ-oryzanol, a bioactive compound with substantial health benefits. In this perspective, different bran rice samples from distinct germplasm origins (Philippines, Italy and Portugal) were studied for their γ-oryzanol content by HPLC-PDA, cytotoxicity in four human tumour cell lines, hepatotoxicity in a normal cell line and for their antimicrobial effects on different bacterial and fungal strains. The Ballatinao sample presented the strongest activity against all the tumour cell lines, and was also the sample showing the highest amount of γ-oryzanol, suggesting its contribution to the exhibited cytotoxic properties. Regarding the antimicrobial activity, the tested samples were able to inhibit the majority of bacterial and fungal strains, with the Portuguese Ceres sample being the one presenting the highest bacterial inhibition and the Maluit and Dinorado samples, the highest fungal inhibition. Overall, the results show that rice bran extracts may be considered as potential candidates for antimicrobial agents when incorporated into food matrices.

Effect of rice variety and reaction parameters on synthesis and antibacterial activity of silver nanoparticles.

In the present study, three different rice varieties; Jasmine (JM), Niaw Koko-6 (NKK), and Saohai (SH) were determined for reducing power using ferric reducing antioxidant power (FRAP) assay. SH showed the highest reducing property followed by JM and NKK, respectively. All modified rice samples were used to fabricate silver nanoparticles (AgNPs) by reducing silver nitrate (AgNO3) to metallic Ag. The obtained AgNPs from JM, NKK, and SH namely JM-AgNPs, NKK-AgNPs, and SH-AgNPs, respectively, showed maximum absorption at 410, 408, and 409 nm, respectively, which confirmed the spectra of AgNPs. Reaction parameters such as AgNO3 and modified rice concentration as well as the reaction period were investigated. It was found that increasing of these parameters gave better AgNPs until the concentration of modified rice and AgNO3 reached to 0.3% and 10 mM, respectively and the reaction period reached to 60 min, the most suitable AgNPs were obtained. Among the three rice varieties, SH showed the most potential for synthesis of AgNPs. SH-AgNPs showed the smallest size of 80.4 ± 2.8 nm and the highest zeta potential of - 45.9 ± 1.4 mV. The AgNPs obtained from all three rice varieties showed effective against Escherichia coli than Staphylococcus aureus and SH-AgNPs showed significantly higher antibacterial activity than JM- AgNPs and NKK-AgNPs.

Preparation and characterization of rice gels containing tooth bleaching agent.

In the present study, the modified white rice of Jasmine (JM) and Saohai (SH) were used to prepare the rice gels. Carbamide peroxide (CP) containing rice gels (CP rice gels) of JM (CP-JM) and SH (CP-SH) were prepared. The rice gels and CP rice gels show homogenous texture. Rice variety influences the characteristics and properties of the rice gels. Amylose content of JM was lower than SH. Rheological behavior of JM and CP-JM was pseudoplastic without thixotropy whereas that of SH and CP-SH was pseudoplastic with thixotropy. CP-SH showed higher adhesive property and viscosity than CP-JM whereas CP-JM showed faster in vitro drug release than CP-SH. For ex vivo efficacy evaluation, 55 normal human teeth were subjected to the CP rice gels. Samples were applied on tooth surface according to the dental bleaching techniques. For at-home bleaching technique, the CP rice gels with 10% and 20% CP were used with bleaching time of 8h and 4h, respectively. For in-office bleaching technique, the CP rice gels with 35% CP was used with bleaching time of 1 h. The developed CP rice gels showed significantly higher efficacy than the positive and negative controls. For at-home bleaching technique, CP-SH was the most effective gels whereas for in-office bleaching technique, CP-JM was the most effective gels.

Effect of rice variety and modification on antioxidant and anti-inflammatory activities.

The effects of variety and modification of rice on its antioxidant and anti-inflammatory activities were investigated. White rice varieties; Jasmine (JM) and Saohai (SH), and pigmented rice varieties; Doisket (DS) and Homnil (HN) were used. The modified rice samples were obtained from chemical modification using etherification reaction. The activities of the modified rice samples were compared with the ethanol extracts of the raw rice at the same rice concentration. Antioxidant activity was measured by the free radical scavenging activity tests and ferric reducing power assay. Results indicated that the ethanol extracts of raw rice had higher antioxidant activity than the modified rice. Among the raw rice tested, the pigmented rice showed higher antioxidant activity than white rice. Trolox equivalent antioxidant capacity values from free radical scavenging activity test were revealed that 50% ethanol extracts of HN and DS possessed the highest antioxidant activity. Ferric reducing power assay showed that 50% ethanol extracts of DS had the highest antioxidant activity. The anti-inflammatory activity was evaluated in vitro using a lipopolysaccharide-stimulated RAW264.7 macrophage cell model with enzyme-linked immunosorbent assay. Absolute ethanol extracts of HN reduced interleukin-6 secretion whereas that of DS suppressed interleukin-6 and tumor necrosis factor -α secretion. These results indicate that variety of rice, chemical modification, and extracting solvent were the factors that play an important role on antioxidant and anti-inflammatory activity. This study supports the potential use of the pigmented rice, especially DS, as a promising choice of a natural source because of its antioxidant and anti-inflammatory activities.

Compositional analyses of white, brown and germinated forms of popular Malaysian rice to offer insight into the growing diet-related diseases.

Diet-related metabolic diseases, and especially obesity, are metabolic disorders with multifactorial aetiologies. Diet has been a cornerstone in both the aetiology and management of this metabolic disorders. Rice, a staple food for over half of the world's population, could be exploited as part of the solution to check this menace which has been skyrocketing in the last decade. The present study investigated nine forms of rice from three widely grown Malaysian rice cultivars for in vitro and in vivo (glycaemic index and load) properties that could translate clinically into a lower predisposition to diet-related diseases. The germinated brown forms of MRQ 74 and MR 84 rice cultivars had high amylose content percentages (25.7% and 25.0%), high relative percentage antioxidant scavenging abilities of 85.0% and 91.7%, relatively low glycaemic indices (67.6 and 64.3) and glycaemic load (32.3 and 30.1) values, and modest glucose uptake capabilities of 33.69% and 31.25%, respectively. The results show that all things being equal, rice cultivars that are germinated and high in amylose content when compared to their white and low amylose counterparts could translate into a lower predisposition to diet-related diseases from the dietary point of view in individuals who consume this cereal as a staple food.

Optimized Mass Spectrometry-Based Metabolite Extraction and Analysis for the Geographical Discrimination of White Rice (Oryza sativa L.): A Method Comparison Study.

In this study, we examined the effects of different extraction methods for the GC-MS- and LC-MS-based metabolite profiling of white rice (Oryza sativa L.). In addition, the metabolite divergence of white rice cultivated in either Korea or China was also evaluated. The discrimination analysis of each extraction method for white rice from Korea and China and the corresponding discriminatory markers were estimated by unpaired t-test, principal component analysis, k-means cluster analysis, partial least-squares discriminant analysis (PLS-DA), and random forest (RF). According to the prediction parameters obtained from PLS-DA and RF classifiers as well as features that could be identified, the extraction method using 75% isopropanol heated at 100°C coupled with LC-MS analysis was confirmed to be superior to the other extraction methods. Noticeably, lysophospholipid concentrations were significantly different in white rice between Korea and China, and they are novel markers for geographical discrimination. In conclusion, our study suggests an optimized extraction and analysis method as well as novel markers for the geographical discrimination of white rice.

Microstructure and tensile properties of various varieties of rice husk.

BACKGROUND: Rice husk is a complex hierarchical assembly of hollow fibers consisting of cellulose, hemicellulose and lignin. In addition, it can also contain pectin and significant amounts of silica. Rice husk can be used in diverse applications and generally in the form of rice husk powder. This study aimed to investigate the structural features and mechanical properties of various varieties of whole rice husks. RESULTS: Rice husk consists of three sections: epidermis, sub-hypodermis and hypodermis. The thickness of these layers, the diameters of the hollow fibers and the wall thickness vary with the variety of rice husk. The elastic modulus is typically between 0.3 and 2.6 GPa, and the ultimate tensile strength varies from 19 to 135 MPa depending on the variety of rice husk. CONCLUSION: Rice husk has a unique hierarchical structure in which the fibers exhibit a staggered perpendicular arrangement and the entire fiber sections are covered by an external shell. The tensile properties vary with the variety of rice husk. The wide range in these tensile properties may be attributed to the size and orientation of the fibers. © 2017 Society of Chemical Industry.

Changes in mineral elements and starch quality of grains during the improvement of japonica rice cultivars.

BACKGROUND: The improvement of rice cultivars plays an important role in yield increase. However, little is known about the changes in starch quality and mineral elements during the improvement of rice cultivars. This study was conducted to investigate the changes in starch quality and mineral elements in japonica rice cultivars. RESULTS: Twelve typical rice cultivars, applied in the production in Jiangsu province during the last 60 years, were grown in the paddy fields. These cultivars were classified into six types according to their application times, plant types and genotypes. The nitrogen (N), phosphorus (P) and, and potassium (K) were mainly distributed in endosperm, bran and bran, respectively. Secondary and micromineral nutrients were distributed throughout grains. With the improvement of cultivars, total N contents gradually decreased, while total P, K and magnesium contents increased in grains. Total copper and zinc contents in type 80'S in grains were highest. The improvement of cultivars enhanced palatability (better gelatinisation enthalpy and amylose content), taste (better protein content) and protein quality (better protein components and essential amino acids). Correlation analysis indicated the close relationship between mineral elements and starch quality. CONCLUSION: The mineral elements and starch quality of grains during the improvement of japonica rice cultivars are improved. © 2017 Society of Chemical Industry.

Comparative proteomics and protein profile related to phenolic compounds and antioxidant activity in germinated Oryza sativa 'KDML105' and Thai brown rice 'Mali Daeng' for better nutritional value.

BACKGROUND: Brown rice (BR) and germinated brown rice (GBR) are considered as prime sources of carbohydrate and bioactive compounds for more than half of the populations worldwide. Several studies have reported on the proteomics of BR and GBR; however, the proteomic profiles related to the synthesis of bioactive compounds are less well documented. In the present study, BR and GBR were used in a comparative analysis of the proteomic and bioactive compound profiles for two famous Thai rice varieties: Khao Dawk Mali 105 (KDML) and Mali Daeng (MD). RESULTS: The proteomes of KDML and MD revealed differences in the expression patterns of proteins after germination. Total phenolic compound content, anthocyanin contents and antioxidant activity of red rice MD was approximately 2.6-, 2.2- and 9.2-fold higher, respectively, compared to that of the white rice KDML. Moreover, GBR of MD showed higher total anthocyanin content and greater antioxidant activity, which is consistent with the increase expression of several proteins involved in the biosynthesis of phenolic compounds and protection against oxidative stress. CONCLUSION: Red rice MD exhibits higher nutrient values compared to white rice KDML and the appropriate germination of brown rice could represent a method for improving health-related benefits. © 2017 Society of Chemical Industry.

Two rice receptor-like kinases maintain male fertility under changing temperatures.

Plants employ dynamic molecular networks to control development in response to environmental changes, yet the underlying mechanisms are largely unknown. Here we report the identification of two rice leucine-rich repeat receptor-like kinases, Thermo-Sensitive Genic Male Sterile 10 (TMS10) and its close homolog TMS10-Like (TMS10L), which redundantly function in the maintenance of the tapetal cell layer and microspore/pollen viability under normal temperature conditions with TMS10 playing an essential role in higher temperatures (namely, 28 °C). tms10 displays male sterility under high temperatures but male fertility under low temperatures, and the tms10 tms10l double mutant shows complete male sterility under both high and low temperatures. Biochemical and genetic assays indicate that the kinase activity conferred by the intracellular domain of TMS10 is essential for tapetal degeneration and male fertility under high temperatures. Furthermore, indica or japonica rice varieties that contain mutations in TMS10, created by genetic crosses or genome editing, also exhibit thermo-sensitive genic male sterility. These findings demonstrate that TMS10 and TMS10L act as a key switch in postmeiotic tapetal development and pollen development by buffering environmental temperature changes, providing insights into the molecular mechanisms by which plants develop phenotypic plasticity via genotype-environment temperature interaction. TMS10 may be used as a genetic resource for the development of hybrid seed production systems in crops.

Genomic structural variation-mediated allelic suppression causes hybrid male sterility in rice.

Hybrids between divergent populations commonly show hybrid sterility; this reproductive barrier hinders hybrid breeding of the japonica and indica rice (Oryza sativa L.) subspecies. Here we show that structural changes and copy number variation at the Sc locus confer japonica-indica hybrid male sterility. The japonica allele, Sc-j, contains a pollen-essential gene encoding a DUF1618-domain protein; the indica allele, Sc-i, contains two or three tandem-duplicated ~ 28-kb segments, each carrying an Sc-j-homolog with a distinct promoter. In Sc-j/Sc-i hybrids, the high-expression of Sc-i in sporophytic cells causes suppression of Sc-j expression in pollen and selective abortion of Sc-j-pollen, leading to transmission ratio distortion. Knocking out one or two of the three Sc-i copies by CRISPR/Cas9 rescues Sc-j expression and male fertility. Our results reveal the gene dosage-dependent allelic suppression as a mechanism of hybrid incompatibility, and provide an effective approach to overcome the reproductive barrier for hybrid breeding.

Comparative Study of the Phytoprostane and Phytofuran Content of indica and japonica Rice (Oryza sativa L.) Flours.

Phytoprostanes and phytofurans (PhytoPs and PhytoFs, respectively) are nonenzymatic lipid peroxidation products derived from α-linolenic acid (C18:3 n-3), considered biomarkers of oxidative degradation in plant foods. The present work profiled these compounds in white and brown grain flours and rice bran from 14 rice cultivars of the subspecies indica and japonica by ultrahigh performance liquid chromatography coupled to electrospray ionization and triple quadrupole mass spectrometry. For PhytoPs, the average concentrations were higher in rice bran (0.01-9.35 ng g-1) than in white and brown grain flours (0.01-1.17 ng g-1). In addition, the evaluation of rice flours for the occurrence PhytoFs evidenced average values 1.77, 4.22, and 10.30 ng g-1 dw in rice bran, brown grain flour, and white grain flour, respectively. A significant correlation was observed between total and individual compounds. The concentrations retrieved suggest rice bran as a valuable source of PhytoPs and PhytoFs that should be considered in further studies on bioavailability and bioactivity of such compounds.

Genetic analysis of a hybrid sterility gene that causes both pollen and embryo sac sterility in hybrids between Oryza sativa L. and Oryza longistaminata.

Oryza longistaminata originates from African wild rice and contains valuable traits conferring tolerance to biotic and abiotic stress. However, interspecific crosses between O. longistaminata and Oryza sativa cultivars are hindered by reproductive barriers. To dissect the mechanism of interspecific hybrid sterility, we developed a near-isogenic line (NIL) using indica variety RD23 as the recipient parent and O. longistaminata as the donor parent. Both pollen and embryo sac semi-sterility were observed in F1 hybrids between RD23 and NIL. Cytological analysis demonstrated that pollen abortion in F1 hybrids occurred at the early bi-nucleate stage due to a failure of the first mitosis in microspores. Partial embryo sacs in the F1 hybrids were defective during the functional megaspore formation stage. Most notably, nearly half of the male or female gametes were aborted in heterozygotes S40iS40l, regardless of their genotypes. Thus, S40 was indicated as a one-locus sporophytic sterility gene controlling both male and female fertility in hybrids between RD23 and O. longistaminata. A population of 16 802 plants derived from the hybrid RD23/NIL-S40 was developed to fine-map S40. Finally, the S40 locus was delimited to an 80-kb region on the short arm of chromosome 1 in terms with reference sequences of cv. 93-11. Eight open reading frames (ORFs) were localized in this region. On the basis of gene expression and genomic sequence analysis, ORF5 and ORF8 were identified as candidate genes for the S40 locus. These results are helpful in cloning the S40 gene and marker-assisted transferring of the corresponding neutral allele in rice breeding programs.

Physicochemical and Antioxidant Properties of Rice Bran Oils Produced from Colored Rice Using Different Extraction Methods.

This study investigated the physicochemical and antioxidant properties of rice bran oil (RBO) produced from the bran of three rice varities; Khao Dawk Mali 105 (white rice), Red Jasmine rice (red rice) and Hom-nin rice (black rice) using three extraction methods including cold-press extraction (CPE), solvent extraction (SE) and supercritical CO2 extraction (SC-CO2). Yields, color, acid value (AV), free fatty acid (FFA), peroxide value (PV), iodine value (IV), total phenolic compound (TPC), γ-oryzanol, α-tocopherol and fatty acid profile were analyzed. It was found that the yields obtained from SE, SC-CO2 and CPE extractions were 17.35-20.19%, 14.76-18.16% and 3.22-6.22%, respectively. The RBO from the bran of red and black rice samples exhibited high antioxidant activities. They also contained higher amount of γ-oryzanol and α-tocopherol than those of white rice sample. In terms of extraction methods, SC-CO2 provided better qualities of RBO as evidenced by their physicochemical and antioxidant properties. This study found that RBO produced from the bran of black rice samples using SC-CO2 extraction method showed the best physicochemical and antioxidant properties.

Phytochemical profiles and antioxidant activity of brown rice varieties.

The phytochemical content and antioxidant activity of eight varieties of brown rice (BR) are reported. The total phenolic contents of BR ranged from 72.45 to 120.13mg of gallic acid equiv./100g. The phenolics from bound fraction contributed 40.6-50.2% of the total phenolic content. The total flavonoid contents of BR ranged from 75.90 to 112.03mg catechin equiv./100g. The flavonoids from the bound fraction contributed 26.9-48.2% of total flavonoids. Trans-ferulic acid was the predominant phenolic acid in BR. Total trans-ferulic acid content ranged from 161.42 to 374.81µg/100g. The percentage of trans-ferulic acid in bound fraction ranged from 96.4% to 99.2%. Only α- and γ-tocopherols and -tocotrienols were detected in BR with α-tocopherol and γ-tocotrienol being the predominant. The total peroxyl radical scavenging capacity (PSC) of BR ranged from 18.29 to 40.33mg vitamin C equiv./100g. The bound fraction contributed 67.2-77.2% of total PSC.

Long-term balancing selection at the Phosphorus Starvation Tolerance 1 (PSTOL1) locus in wild, domesticated and weedy rice (Oryza).

BACKGROUND: The ability to grow in phosphorus-depleted soils is an important trait for rice cultivation in many world regions, especially in the tropics. The Phosphorus Starvation Tolerance 1 (PSTOL1) gene has been identified as underlying the ability of some cultivated rice varieties to grow under low-phosphorus conditions; however, the gene is absent from other varieties. We assessed PSTOL1 presence/absence in a geographically diverse sample of wild, domesticated and weedy rice and sequenced the gene in samples where it is present. RESULTS: We find that the presence/absence polymorphism spans cultivated, weedy and wild Asian rice groups. For the subset of samples that carry PSTOL1, haplotype sequences suggest long-term selective maintenance of functional alleles, but with repeated evolution of loss-of-function alleles through premature stops and frameshift mutations. The loss-of-function alleles have evolved convergently in multiple rice species and cultivated rice varieties. Greenhouse assessments of plant growth under low- and high-phosphorus conditions did not reveal significant associations with PSTOL1 genotype variation; however, the striking signature of balancing selection at this locus suggests that further phenotypic characterizations of PSTOL1 allelic variants is warranted and may be useful for crop improvement. CONCLUSIONS: These findings suggest balancing selection for both functional and non-functional PSTOL1 alleles that predates and transcends Asian rice domestication, a pattern that may reflect fitness tradeoffs associated with geographical variation in soil phosphorus content.

Instant rice made from white and pigmented giant embryonic rice reduces lipid levels and body weight in high fat diet-fed mice.

PROBLEM: With the growing health problem on obesity and its associated metabolic disorders, functional foods with hypolipidemic and body fat-lowering effects are greatly needed. The effects of instant rice made from a combination of pigmented giant embryonic rice (Keunnunjami) and white rice, in comparison with those of instant non-pigmented giant embryonic brown rice and instant ordinary brown rice, on the lipid metabolism and body weight in mice under high fat diet condition were investigated. METHODS: Male C57BL/6N mice (n=48) were given experimental diets for seven weeks: normal control (NC), high fat (HF), and HF supplemented with instant normal brown rice (HF-NB), non-pigmented giant embryonic brown rice (HF-GB), and white rice with 8% Keununnjami (HF-KJ8) or 18% Keunnunjami (HF-KJ18). Their body weight gain, plasma lipid profile, adipokine level, lipid-regulating enzymes activities, and mRNA expressions of peroxisome proliferator-activated receptors (PPARs) were determined. RESULTS: At the end of the experimental period, the HF mice exhibited significantly higher body weight and fat, plasma triglyceride and total cholesterol levels, and atherogenic index and lower high-density lipoprotein cholesterol concentration than the NC group. However, diet supplementation of instant rice counteracted this high fat-induced body weight gain and hyperlipidemia through inhibition of lipogenesis and adipokine production and alteration of PPARs expressions. CONCLUSION: The instant KJ8 and KJ18 rice showed generally similar hypolipidemic and body weight-lowering effects to instant NB and GB rice. These findings demonstrate that instant white rice with added 8% Keunnunjami may be beneficial as functional food with strong hypolipidemic and anti-obesity properties.

Cytoplasmic-genetic male sterility gene provides direct evidence for some hybrid rice recently evolving into weedy rice.

Weedy rice infests paddy fields worldwide at an alarmingly increasing rate. There is substantial evidence indicating that many weedy rice forms originated from or are closely related to cultivated rice. There is suspicion that the outbreak of weedy rice in China may be related to widely grown hybrid rice due to its heterosis and the diversity of its progeny, but this notion remains unsupported by direct evidence. We screened weedy rice accessions by both genetic and molecular marker tests for the cytoplasmic male sterility (CMS) genes (Wild abortive, WA, and Boro type, BT) most widely used in the production of indica and japonica three-line hybrid rice as a diagnostic trait of direct parenthood. Sixteen weedy rice accessions of the 358 tested (4.5%) contained the CMS-WA gene; none contained the CMS-BT gene. These 16 accessions represent weedy rices recently evolved from maternal hybrid rice derivatives, given the primarily maternal inheritance of this trait. Our results provide key direct evidence that hybrid rice can be involved in the evolution of some weedy rice accessions, but is not a primary factor in the recent outbreak of weedy rice in China.