MicroRNA2871b of Dongxiang Wild Rice (Oryza rufipogon Griff.) Negatively Regulates Cold and Salt Stress Tolerance in Transgenic Rice Plants.

Cold and salt stresses are major environmental factors that constrain rice production. Understanding their mechanisms is important to enhance cold and salt stress tolerance in rice. MicroRNAs (miRNAs) are a class of non-coding RNAs with only 21-24 nucleotides that are gene regulators in plants and animals. Previously, miR2871b expression was suppressed by cold stress in Dongxiang wild rice (DXWR, Oryza rufipogon Griff.). However, its biological functions in abiotic stress responses remain elusive. In the present study, miR2871b of DWXR was overexpressed to investigate its function under stress conditions. When miR2871b of DWXR was introduced into rice plants, the transgenic lines were more sensitive to cold and salt stresses, and their tolerance to cold and salt stress decreased. The increased expression of miR2871b in rice plants also increased the levels of reactive oxygen species (ROS) and malondialdehyde (MDA); however, it markedly decreased the activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) and the contents of proline (Pro) and soluble sugar (SS). These data suggested that miR2871b of DXWR has negative regulatory effects on cold and salt stress tolerance. Meanwhile, 412 differentially expressed genes (DEGs) were found in rice transgenic plants using transcriptome sequencing, among which 266 genes were up-regulated and 146 genes were down-regulated. Furthermore, the upstream cis-acting elements and downstream targets of miR2871b were predicted and analyzed, and several critical acting elements (ABRE and TC-rich repeats) and potential target genes (LOC_Os03g41200, LOC_Os07g47620, and LOC_Os04g30260) were obtained. Collectively, these results generated herein further elucidate the vital roles of miR2871b in regulating cold and salt responses of DXWR.

Genetic Analysis of Novel Fertility Restoration Genes (qRf3 and qRf6) in Dongxiang Wild Rice Using GradedPool-Seq Mapping and QTL-Seq Correlation Analysis.

The improvement of grain yield, quality, and resistance can be achieved through the utilization of heterosis. The combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) gene(s) greatly facilitates the commercial development of three-line hybrid rice based on heterosis. The basis for investigating the relationship between CMS and Rf genes lies in the rapid localization of wild rice fertility restoration genes. A set of the BC4F5 population derived from interspecific crosses between Xieqingzao B (XB) and the BC1F9 XB//Dongxiang wild rice (DWR)/XB line L5339 was used to detect quantitative trait loci (QTL) for fertility restoration. The population was then crossed with two male sterile lines, Zhong9A (Z9A) and DongB11A (DB11A), in order to generate a testcrossing population for investigating spikelet fertility. Based on the linkage mapping, seven QTLs were detected on chromosomes 1, 3, 5, 6, 8, and 10, explaining 2.76 to 12.46% of the phenotypic variation. Of them, two novel fertility restoration QTLs, qRf3 and qRf6, can restore fertility of the CMS-DWR line DB11A by 16.56% and 15.12%, respectively. By employing joint QTL-seq and GradedPool-Seq methods, two novel Rf QTLs for DB11A, qRf3 and qRf6, were identified at the physical locations of 10,900,001-11,700,000 bp and 28,016,785-31,247,556 bp, respectively. These findings are useful for exploring the natural variations of Rf genes in rice. Therefore, rice's new genetic resources for the selection and breeding of rice restorer lines provide promising candidates for QTL fine localization and clarification.

Genome-Wide Analysis of microRNAs and Their Target Genes in Dongxiang Wild Rice (Oryza rufipogon Griff.) Responding to Salt Stress.

Rice (Oryza sativa) is a staple food for more than half of the world's population, and its production is critical for global food security. Moreover, rice yield decreases when exposed to abiotic stresses, such as salinity, which is one of the most detrimental factors for rice production. According to recent trends, as global temperatures continue to rise due to climate change, more rice fields may become saltier. Dongxiang wild rice (Oryza rufipogon Griff., DXWR) is a progenitor of cultivated rice and has a high tolerance to salt stress, making it useful for studying the regulatory mechanisms of salt stress tolerance. However, the regulatory mechanism of miRNA-mediated salt stress response in DXWR remains unclear. In this study, miRNA sequencing was performed to identify miRNAs and their putative target genes in response to salt stress in order to better understand the roles of miRNAs in DXWR salt stress tolerance. A total of 874 known and 476 novel miRNAs were identified, and the expression levels of 164 miRNAs were found to be significantly altered under salt stress. The stem-loop quantitative real-time PCR (qRT-PCR) expression levels of randomly selected miRNAs were largely consistent with the miRNA sequencing results, suggesting that the sequencing results were reliable. The gene ontology (GO) analysis indicated that the predicted target genes of salt-responsive miRNAs were involved in diverse biological pathways of stress tolerance. This study contributes to our understanding of DXWR salt tolerance mechanisms regulated by miRNAs and may ultimately improve salt tolerance in cultivated rice breeding using genetic methods in the future.

Anti-rheumatoid arthritis potential of Rhododendron molle G. Don leaf extract in adjuvant induced arthritis rats.

AIM OF THE STUDY: The aim of this study was to test the anti-rheumatic arthritis effects of Rhododendron molle G. Don leaf extract in arthritis rats and inflammatory RAW 264.7 cells. Preliminary analysis and comparison of potential medicinal components of three polar extracts by HPLC and UHPLC-Q-TOF-MS. MATERIALS AND METHODS: SD rats were subcutaneously injected with complete Freund's adjuvant (CFA) to induce inflammation on the right hind paw. RAW 264.7 cells were induced by lipopolysaccharide (LPS) to established cell inflammatory model. The volume of rat hind paw was measured with a volume meter to detect swelling, and the weight of rats was measured with an electronic balance. The severity of arthritis in rats was evaluated by arthritis score. The pathological sections of rat hind paw joints were observed by hematoxylin-eosin staining, and the contents of IL-6 and IL-1ß in serum were detected. qRT-PCR was used to detect the expression of IL-1ß, IL-6, TNF-α and COX-2 genes in RAW 264.7 cells. The release of nitric oxide was measured by Griess reaction. The expression levels of IL-6 and IL-1ß were detected by Western-Blot. RESULTS: and discussion: The chloroform extract from R. molle leaves (CERL), Ethyl acetate extract from R. molle leaves (EERL), n-butanol extract from R. molle leaves (BERL) could significantly inhibit hind paws swelling and reduce arthritis index in arthritis rats. And it showed dose dependence. Compared with tripterygium glycosides (TG) tablets, an effective drug of RA treatment, CERL have better anti-RA effect after administration. In addition, the three kinds of the polar extracts of Rhododendron molle leaves (PERL) had lower toxicity, with the LD50 279.87, 239.65, 500.08 (mg/kg) respectively, while TG group's LD50 was 96.00 (mg/kg). In vitro experiments showed that the three PERLs can significantly inhibit the level of pro-inflammatory factors and inflammatory mediator, such as TNF-α, IL-1ß, IL-6, COX-2 and NO, which were consistent with their anti-RA ability. Among the three kinds of PERLs, CERL showed the best inhibitory activity. CONCLUSION: The R. molle leaf is a potential medicinal part for the treatment of RA. This study explored the anti-RA and anti-inflammatory activities of CERL, EERL, BERL, which laid a foundation for further promoting the clinical application of R. molle.

Development and verification of SSR markers from drought stress-responsive miRNAs in Dongxiang wild rice (Oryza rufipogon Griff.).

Rice production worldwide has continued to decline due to various environmental stresses, with drought stress being a prominent factor, as rice is a semi-aquatic plant. Thus, development of drought stress-resistant rice varieties is of great importance for rice production. In our previous study, we found that microRNAs (miRNAs) play a crucial role in the response to drought stress in Dongxiang wild rice (DXWR) (Oryza rufipogon Griff.). Developing drought stress-responsive miRNA-based single sequence repeat (SSR) markers for DXWR will be of great value for the efficient identification and utilization of miRNA genes to breed drought stress-resistant rice varieties. In this study, ninety-nine novel SSR markers were developed based on the drought stress-responsive miRNAs of DXWR. These markers were distributed in all 12 rice chromosomes, and most were in chromosomes 2 and 6, with di- and tri-nucleotides being the most abundant repeat motifs. Twelve out of fourteen synthesized markers displayed high levels of genetic diversity in the genomes of three populations of DXWR and 40 modern rice varieties worldwide. The number of alleles per locus ranged from 2 to 7, with an average of 4.67; the genetic diversity index ranged from 0.21 to 0.76, with an average of 0.58; and the polymorphism information content value ranged from 0.18 to 0.72, with an average of 0.53. These novel molecular markers developed from the drought stress-responsive miRNAs of DXWR could be additional tools for mapping elite miRNA genes and breeding drought stress-resistant rice varieties.

Flavonoid Metabolic Profiles and Gene Mapping of Rice (Oryza sativa L.) Purple Gradient Grain Hulls.

Rice (Oryza sativa L.) grain hull color is an easily observable trait and regarded as a crucial morphological marker in rice breeding. Here, a purple gradient grain hull mutant (pg) was found from natural mutations of a straw-white grain hull rice variety IARI 6184B (Orzya sativa L. subsp. indica). The color of the mutant grain hulls changed from straw-white to pink, then purple, and finally brownish-yellow. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) identified 217 flavonoids, including 18 anthocyanins, among which cyanidin O-syringic acid had the highest concentration in pink (66.2 × 106) and purple (68.0 × 106) grain hulls. The relative contents of hesperetin O-malonyl-hexoside, apigenin derivatives, genistein derivatives, and kaempferol 3-O derivatives were consistently downregulated during pg grain hull development. Conversely, 12 anthocyanins were upregulated in colored hulls, and cyanidin 3-O-malonylhexoside was abundant only in pink and purple grain hulls. Moreover, the candidate gene was mapped into a 1.38 Mb region on chromosome 4 through bulked segregant analysis based on deep sequencing (BSA-seq) and gene mapping approaches. These results increased our understanding of anthocyanin biosynthesis in rice grains, helping rice breeders to select new rice varieties with desirable grain traits.

Analysis of the genetic diversity and population structure of Monochasma savatieri Franch. ex Maxim using novel EST-SSR markers.

BACKGROUND: Monochasma savatieri Franch. ex Maxim is a medicinally valuable herb. However, the collection and protection of the wild germplasm resources of M. savatieri are still insufficient, and their genetic diversity and population structure have been poorly studied. RESULTS: We collected and examined 46 M. savatieri individuals from Fujian, Hunan, Jiangxi, and Zhejiang provinces for genetic diversity and population structure, using 33 newly developed expressed sequence tag-simple sequence repeat (EST-SSR) markers. Applying these markers, we detected a total of 208 alleles, with an average of 6.303 alleles per locus. The polymorphic information content varied from 0.138 to 0.884 (average: 0.668), indicating a high level of polymorphism. At the population level, there was a low degree of genetic diversity among populations (I = 0.535, He = 0.342), with Zhejiang individuals showing the highest genetic diversity among the four populations (Fst = 0.497), which indicated little gene flow within the M. savatieri populations (Nm = 0.253). Mantel test analysis revealed a significant positive correlation between geographical and genetic distance among populations (R2 = 0.3304, p < 0.05), and structure and principal coordinate analyses supported classification of populations into three clusters, which was consistent with the findings of cluster analysis. CONCLUSIONS: As a rare medicinal plants, the protection of M. savatieri does not look optimistic, and accordingly, protective efforts should be beefed up on the natural wild populations. This study provided novel tools and insights for designing effective collection and conservation strategies for M. savatieri.

Development and Characterization of Chromosome Segment Substitution Lines Derived from Oryza rufipogon in the Background of the Oryza sativa indica Restorer Line R974

Dongxiang wild rice (DXWR) (O. rufipogon Griff.), which has the northernmost worldwide distribution of a wild rice species, is a valuable genetic resource with respect to improving stress tolerance in cultivated rice (Oryza sativa L.). In the three-line hybrid rice breeding system, restorer lines play important roles in enhancing the tolerance of hybrid rice. However, restorer lines have yet to be used as a genomic background for development of substitution lines carrying DXWR chromosome segments. We developed a set of 84 chromosome segment substitution lines (CSSLs) from a donor parent DXWR × recurrent parent restorer line R974 (Oryza sativa indica) cross. On average, each CSSL carried 6.27 introgressed homozygous segments, with 93.37% total genome coverage. Using these CSSLs, we identified a single QTL, qDYST-1, associated with salt stress tolerance on chromosome 3. Furthermore, five CSSLs showing strong salt stress tolerance were subjected to whole-genome single-nucleotide polymorphism chip analyses, during which we detected a common substitution segment containing qDYST-1 in all five CSSLs, thereby implying the validity and efficacy of qDYST-1. These novel CSSLs could make a significant contribution to detecting valuable DXWR QTLs, and provide important germplasm resources for breeding novel restorer lines for use in hybrid rice breeding systems.

Identification of Phosphorus Stress Related Proteins in the Seedlings of Dongxiang Wild Rice (Oryza Rufipogon Griff.) Using Label-Free Quantitative Proteomic Analysis.

Phosphorus (P) deficiency tolerance in rice is a complex character controlled by polygenes. Through proteomics analysis, we could find more low P tolerance related proteins in unique P-deficiency tolerance germplasm Dongxiang wild rice (Oryza Rufipogon, DXWR), which will provide the basis for the research of its regulation mechanism. In this study, a proteomic approach as well as joint analysis with transcriptome data were conducted to identify potential unique low P response genes in DXWR during seedlings. The results showed that 3589 significant differential accumulation proteins were identified between the low P and the normal P treated root samples of DXWR. The degree of change was more than 1.5 times, including 60 up-regulated and 15 downregulated proteins, 24 of which also detected expression changes of more than 1.5-fold in the transcriptome data. Through quantitative trait locus (QTLs) matching analysis, seven genes corresponding to the significantly different expression proteins identified in this study were found to be uncharacterized and distributed in the QTLs interval related to low P tolerance, two of which (LOC_Os12g09620 and LOC_Os03g40670) were detected at both transcriptome and proteome levels. Based on the comprehensive analysis, it was found that DXWR could increase the expression of purple acid phosphatases (PAPs), membrane location of P transporters (PTs), rhizosphere area, and alternative splicing, and it could decrease reactive oxygen species (ROS) activity to deal with low P stress. This study would provide some useful insights in cloning the P-deficiency tolerance genes from wild rice, as well as elucidating the molecular mechanism of low P resistance in DXWR.

Identification, Analysis, and Confirmation of Seed Storability-Related Loci in Dongxiang Wild Rice (Oryza rufipogon Griff.).

Dongxiang wild rice (Oryza rufipogon Griff.) (DXWR) has strong seed storability and identifying its elite gene resources may facilitate genetic improvements in rice seed storability. In this study, we developed two backcross inbred lines (BILs) populations, with DXWR as a common donor parent and two rice varieties (F6 and R974) as recipient parents. Bulked segregant analysis via whole genome sequencing (BSA-seq) was used to identify seed storability-related loci in the DXWR and F6 population. Two main genomic regions containing 18,550,000-20,870,000 bp on chromosome 4 and 7,860,000-9,780,000 bp on chromosome 9 were identified as candidate loci of DXWR seed storability; these overlapped partially with seed storability-related quantitative trait loci (QTLs) discovered in previous studies, suggesting that these loci may provide important regions for isolating the responsible genes. In total, 448 annotated genes were predicted within the identified regions, of which 274 and 82 had nonsynonymous and frameshift mutations, respectively. We detected extensive metabolic activities and cellular processes during seed storability and confirmed the effects of the seed storability-related candidate loci using four BILs from DXWR and R974. These results may facilitate the cloning of DXWR seed storability-related genes, thereby elucidating rice seed storability and its improvement potential.

Metabolomic Profiling of Dongxiang Wild Rice Under Salinity Demonstrates the Significant Role of Amino Acids in Rice Salt Stress.

Dongxiang common wild rice is a precious rice germplasm resource for the study and improvement of salt tolerance in rice.The metabolism profile of Dongxiang wild rice (DXWR) under salinity was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS) to find differential metabolites and screen potential biomarkers for salt-tolerant rice varieties. A global untargeted metabolism analysis showed 4,878 metabolites accumulated in seedlings of Dongxiang wild rice. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) results provided a clear metabolism discrimination between DXWR under control and DXWR under salinity. A total of 90 metabolites were significantly changed (49 upregulated and 41 downregulated) under salinity, of which the largest increase was in DL-2-Aminoadipic acid (27.08-fold) and the largest decrease was in L-Carnitine (0.014-fold). Amino acids and nuclear glycosides were mainly upregulated, while carbohydrates and organic acids were mainly downregulated in the salt-treated group. Among the top 10 upregulated metabolites, five kinds of differential metabolites were amino acids. According to the survival rates of the seedlings under salinity, we selected three backcross inbred lines of DXWR with survival rates above 80% as salt-tolerant progenies (pro-DS) and three backcross inbred lines with survival rates below 10% as non-salt-tolerant progenies (pro-NDS) for an amino acid change analysis. This analysis found that the change in L-Asparagine (2.59-fold) was the biggest between pro-DS and pro-NDS under salinity, revealing that the contents of L-Asparagine may be one of the indices we can use to evaluate the salt tolerance of rice varieties.

In Vitro Propagation, Huperzine A Content and Antioxidant Activity of Three Genotypic Huperzia serrata.

Huperzia serrata is a traditional herb and endangered Chinese medicinal material, which has attracted much attention due to its production of Huperzine A (HupA). In vitro propagation of H. serrata is considered a new way to relieve the resource pressure of H. serrata. In this study, three different genotypic wild H. serrata were used for in vitro propagation. Then, the antioxidant activity and the content of HupA in the regenerated H. serrata were investigated. The results showed the survival rate of the explant was increased to 25.37% when using multiple sterilization processes. The best induction medium for H. serrata was the Schenk and Hildebrandt (SH) medium supplemented with 0.5 mg·L-1 Naphthalene acetic acid (NAA) and 0.1 mg·L-1 2,4-Dichlorophenoxyacetic acid (2,4-D), where the regeneration rate of the explant was to 57.04%. The best proliferation medium was the SH medium with NAA (1.0 mg·L-1), as the biomass of in vitro tissue increased 164.17 ± 0.41 times. High-performance liquid chromatography analysis showed that the in vitro culture of three genotypes could produce HupA and the content of HupA was 53.90-87.17 µg·g-1. The antioxidant experiment showed that the methanol extract of in vitro H. serrata had higher antioxidant activity than that of wild H. serrata. This study provides a reliable in vitro H. serrata culture protocol and laid an important foundation for the antioxidant capacity of the thallus and the content of HupA.

Cytological Observation and Transcriptome Comparative Analysis of Self-Pollination and Cross-Pollination in Dendrobium Officinale.

Dendrobium officinale is a rare and traditional medicinal plant with high pharmacological and nutritional value. The self-incompatibility mechanism of D. officinale reproductive isolation was formed in the long-term evolution process, but intraspecific hybridization of different germplasm resources leads to a large gap in the yield, quality, and medicinal value of D. officinale. To investigate the biological mechanism of self-incompatibility in D. officinale, cytological observation and the transcriptome analysis was carried out on the samples of self-pollination and cross-pollination in D. officinale. Results for self-pollination showed that the pollen tubes could grow in the style at 2 h, but most of pollen tubes stopped growing at 4 h, while a large number of cross-pollinated pollen tubes grew along the placental space to the base of ovary, indicating that the self-incompatibility of D. officinale may be gametophyte self-incompatibility. A total of 63.41 G basesum of D. officinale style samples from non-pollinated, self-pollination, and cross-pollination by RNA-seq were obtained, and a total of 1944, 1758, and 475 differentially expressed genes (DEGs) in the comparison of CK (non-pollinated) vs. HF (cross-pollination sample), CK vs. SF (self-pollination sample) and SF vs. HF were identified, respectively. Forty-one candidate genes related to self-incompatibility were found by function annotation of DEGs, including 6 Ca2+ signal genes, 4 armed repeat containing (ARC) related genes, 11 S-locus receptor kinase (SRK) related genes, 2 Exo70 family genes, 9 ubiquitin related genes, 1 fatty acid related gene, 6 amino acid-related genes, 1 pollen-specific leucine-rich repeat extensin-like protein (LRX) related gene and 1 lectin receptor-like kinases (RLKs) related gene, showed that self-incompatibility mechanism of D. officinale involves the interaction of multiple genes and pathways. The results can provide a basis for the study of the self-incompatibility mechanism of D. officinale, and provide ideas for the preservation and utilization of high-quality resources of D. officinale.

Identification and characterisation of cold stress-related proteins in Oryza rufipogon at the seedling stage using label-free quantitative proteomic analysis.

In this study, label-free quantitative proteomics were used to study cold stress-related proteins in Dongxiang wild rice (Oryza rufipogon Griff., DWR) and cold sensitive cultivated rice 'Xieqingzao B'(Oryza sativa L. ssp. indica cv., XB). The results demonstrated the presence of 101 and 216 differentially expressed proteins (DEPs) were detected in DWR and XB, respectively, after cold stress. Bioinformatics analysis showed that DWR and XB differed significantly in their ability to scavenge reactive oxygen species (ROS) and regulate energy metabolism. Of the 101 DEPs of DWR, 46 DEPs related to differential expressed genes were also detected by transcriptome analysis. And 13 out of 101 DEPs were located in previous cold related quantitative trait loci (QTL). Quantitative real-time PCR analysis indicated that protein expression and transcription patterns were not similar in XB and DWR. Protein-protein interaction (PPI) network was constituted using the DEPs of DWR and XB, and the following three centre proteins were identified: Q8H3I3, Q9LDN2, and Q2QXR8. Next, we selected a centre protein and two of the 37 DEPs with high levels of differential expression (fold change ≥ 2) were used for cloning and prokaryotic expression. We found that Q5Z9Q8 could significantly improve the cold tolerance of Escherichia coli.

Variability of Volatile Compounds in the Medicinal Plant Dendrobium officinale from Different Regions.

Dendrobium officinale Kimura et Migo, a rare and traditional medicinal plant, contains many nutrients such as polysaccharides, alkaloids, amino acids and so on. Different growth environment and intraspecific hybridization of different germplasm resources lead to large differences in the yield, quality and medicinal value of D. officinale. Here, the volatile compounds of D. officinale from four producing regions (Zhejiang, Fujian, Yunnan and Jiangxi) were analyzed to provide a certain reference value for the selection of a specific medicinal component in D. officinale breeding. Fresh stems of D. officinale germplasm resources were collected, and the chemical constituents were determined by gas chromatography-mass spectrometry. A total of 101 volatile compounds were identified, of which esters and alcohols accounted for 23 and 22. Hexacosane is the highest relative content of all volatile components. The highest content of hexacosane was observed in YA1 from Yunnan was 34.41%, and the lowest (23.41%) in JA1 from Jiangxi. Moreover, 5-10 unique substances were determined in different regions. A total of 17 medicinal components were detected, and three unique medicinal components were detected only in YA1, revealing that YA1 can provide raw materials for the application of specific medicinal substances extraction. A total of four toxic components were detected, but no toxic components were detected in JA1 from Jiangxi, suggested that the germplasm resources from Jiangxi could be exploited efficiently for breeding superior D. officinale specimens. The results provide a theoretical basis for the collection, protection and utilization of D. officinale germplasm resources in different regions.

The Anti-Inflammatory Properties of Rhododendron molle Leaf Extract in LPS-Induced RAW264.7.

Rhododendron molle G.Don is a well-known traditional medicine which has been used to treat rheumatic inflammation. In this study, an inflammatory model of lipopolysaccharide (LPS)-stimulated RAW264.7 cells was established to analyze the anti-inflammatory effect and potential mechanism of the methanol extract of R. molle leaves (RLE). The production of NO and the expression of tumor necrosis factor by LPS were detected by Griess reaction and enzyme linked immunosorbent assay (ELISA). The mRNA expression of TNF-α, IL-1ß, IL-6, COX-2 and iNOS was measured by qRT-PCR assay. Griess and qRT-PCR showed that the RLE could significantly concentration-dependently inhibit NO production and the expression of many pro-inflammatory cytokines and inflammatory-related enzymes. Scanning electron microscope (SEM) analysis indicated that RLE could inhibit LPS-stimulated RAW264.7 macrophages activation. The protein level of TNF-α and IL-1ß were decreased over 50 % at 100 µg/ml of RLE, as detected by ELISA. These results indicated that RLE had strong anti-inflammatory and immunomodulatory activity.

Ef-cd locus shortens rice maturity duration without yield penalty.

The contradiction between "high yielding" and "early maturing" hampers further improvement of annual rice yield. Here we report the positional cloning of a major maturity duration regulatory gene, Early flowering-completely dominant (Ef-cd), and demonstrate that natural variation in Ef-cd could be used to overcome the above contradictory. The Ef-cd locus gives rise to a long noncoding RNA (lncRNA) antisense transcript overlapping the OsSOC1 gene. Ef-cd lncRNA expression positively correlates with the expression of OsSOC1 and H3K36me3 deposition. Field test comparisons of early maturing Ef-cd near-isogenic lines with their wild types as well as of the derivative early maturing hybrids with their wild-type hybrids conducted under different latitudes determined that the early maturing Ef-cd allele shortens maturity duration (ranging from 7 to 20 d) without a concomitant yield penalty. Ef-cd facilitates nitrogen utilization and also improves the photosynthesis rate. Analysis of 1,439 elite hybrid rice varieties revealed that the 16 homozygotes and 299 heterozygotes possessing Ef-cd matured significantly earlier. Therefore, Ef-cd could be a vital contributor of elite early maturing hybrid varieties in balancing grain yield with maturity duration.

Development of a drought stress-resistant rice restorer line through Oryza sativa-rufipogon hybridization.

Restorer line F6 (Oryza sativa L. ssp. indica) has been widely used in hybrid rice breeding systems in southern China. However, line F6 is susceptible to drought stress, which restricts its utilization in many areas. Dongxiang wild rice (DXWR, Oryza rufipogon Griff.) has strong drought stress resistance, but the molecular factors responsible for drought resistance in DXWR remain unknown. In this study, we used the combination of phenotypic selection and molecular marker-assisted selection (MAS) to improve the drought stress resistance of line F6 by introgression of qSDT12-2, a large effect drought stress-related quantitative trait locus identified in DXWR. Molecular MAS was carried out using linked marker RM1226, which is associated with qSDT12-2. Genomic background assessmentwas performed using 112 polymorphic markers. Finally, a stable drought stress-resistant backcross inbred line (BIL) was developed from a BC5F5 population, termed BIL627. Genetic constitution analysis revealed that the genome of BIL627 is almost identical (99.1%) to that of the restorer line F6. Further, BIL627 showed no yield penalty and no decrease in restorationability under normal conditions. Taken together, our findings reveal the intrinsic value of using genetic resources present in wild species of Oryza to improve undesirable traits found in cultivated rice.

Transcriptome analysis of phosphorus stress responsiveness in the seedlings of Dongxiang wild rice (Oryza rufipogon Griff.).

BACKGROUND: Low phosphorus availability is a major factor restricting rice growth. Dongxiang wild rice (Oryza rufipogon Griff.) has many useful genes lacking in cultivated rice, including stress resistance to phosphorus deficiency, cold, salt and drought, which is considered to be a precious germplasm resource for rice breeding. However, the molecular mechanism of regulation of phosphorus deficiency tolerance is not clear. RESULTS: In this study, cDNA libraries were constructed from the leaf and root tissues of phosphorus stressed and untreated Dongxiang wild rice seedlings, and transcriptome sequencing was performed with the goal of elucidating the molecular mechanisms involved in phosphorus stress response. The results indicated that 1184 transcripts were differentially expressed in the leaves (323 up-regulated and 861 down-regulated) and 986 transcripts were differentially expressed in the roots (756 up-regulated and 230 down-regulated). 43 genes were up-regulated both in leaves and roots, 38 genes were up-regulated in roots but down-regulated in leaves, and only 2 genes were down-regulated in roots but up-regulated in leaves. Among these differentially expressed genes, the detection of many transcription factors and functional genes demonstrated that multiple regulatory pathways were involved in phosphorus deficiency tolerance. Meanwhile, the differentially expressed genes were also annotated with gene ontology terms and key pathways via functional classification and Kyoto Encyclopedia of Gene and Genomes pathway mapping, respectively. A set of the most important candidate genes was then identified by combining the differentially expressed genes found in the present study with previously identified phosphorus deficiency tolerance quantitative trait loci. CONCLUSION: The present work provides abundant genomic information for functional dissection of the phosphorus deficiency resistance of Dongxiang wild rice, which will be help to understand the biological regulatory mechanisms of phosphorus deficiency tolerance in Dongxiang wild rice.

Transcriptome analysis of phosphorus stress responsiveness in the seedlings of Dongxiang wild rice (Oryza rufipogon Griff.)

Abstract Background: Low phosphorus availability is a major factor restricting rice growth. Dongxiang wild rice (Oryza rufipogon Griff.) has many useful genes lacking in cultivated rice, including stress resistance to phosphorus deficiency, cold, salt and drought, which is considered to be a precious germplasm resource for rice breeding. However, the molecular mechanism of regulation of phosphorus deficiency tolerance is not clear. Results: In this study, cDNA libraries were constructed from the leaf and root tissues of phosphorus stressed and untreated Dongxiang wild rice seedlings, and transcriptome sequencing was performed with the goal of elucidating the molecular mechanisms involved in phosphorus stress response. The results indicated that 1184 transcripts were differentially expressed in the leaves (323 up-regulated and 861 down-regulated) and 986 transcripts were differentially expressed in the roots (756 up-regulated and 230 down-regulated). 43 genes were up-regulated both in leaves and roots, 38 genes were up-regulated in roots but down-regulated in leaves, and only 2 genes were down-regulated in roots but up-regulated in leaves. Among these differentially expressed genes, the detection of many transcription factors and functional genes demonstrated that multiple regulatory pathways were involved in phosphorus deficiency tolerance. Meanwhile, the differentially expressed genes were also annotated with gene ontology terms and key pathways via functional classification and Kyoto Encyclopedia of Gene and Genomes pathway mapping, respectively. A set of the most important candidate genes was then identified by combining the differentially expressed genes found in the present study with previously identified phosphorus deficiency tolerance quantitative trait loci. Conclusion: The present work provides abundant genomic information for functional dissection of the phosphorus deficiency resistance of Dongxiang wild rice, which will be help to understand the biological regulatory mechanisms of phosphorus deficiency tolerance in Dongxiang wild rice.