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. 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. 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.

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.

Background: The cold tolerance of rice is closely related to its production and geographic distribution. The identification of cold tolerance-related genes is of important significance for developing cold-tolerant rice. Dongxiang wild rice (Oryza rufipogon Griff.) (DXWR) is well-adapted to the cold climate of northernmost-latitude habitats ever found in the world, and is one of the most valuable rice germplasms for cold tolerance improvement. Results: Transcriptome analysis revealed genes differentially expressed between Xieqingzao B (XB; a cold sensitive variety) and 19H19 (derived from an interspecific cross between DXWR and XB) in the room temperature (RT), low temperature (LT), and recovery treatments. The results demonstrated that chloroplast genes might be involved in the regulation of cold tolerance in rice. A high-resolution SNP genetic map was constructed using 120 BC₅F₂ lines derived from a cross between 19H19 and XB based on the genotyping-by-sequencing (GBS) technique. Two quantitative trait loci (QTLs) for cold tolerance at the early seedling stage (CTS), qCTS12 and qCTS8, were detected. Moreover, a total of 112 candidate genes associated with cold tolerance were identified based on bulked segregant analysis sequencing (BSA-seq). These candidate genes were divided into eight functional categories, and the expression trend of candidate genes related to ‘oxidation-reduction process’ and ‘response to stress’ differed between XB and 19H19 in the RT, LT and recovery treatments. Among these candidate genes, the expression level of LOC_Os12g18729 in 19H19 (related to ‘response to stress’) decreased in the LT treatment but restored and enhanced during the recovery treatment whereas the expression level of LOC_Os12g18729 in XB declined during recovery treatment. Additionally, XB contained a 42-bp deletion in the third exon of LOC_Os12g18729, and the genotype of BC₅F₂ individuals with a survival percentage (SP) lower than 15% was consistent with that of XB. Weighted gene coexpression network analysis (WGCNA) and modular regulatory network learning with per gene information (MERLIN) algorithm revealed a gene interaction/coexpression network regulating cold tolerance in rice. In the network, differentially expressed genes (DEGs) related to ‘oxidation-reduction process’, ‘response to stress’ and ‘protein phosphorylation’ interacted with LOC_Os12g18729. Moreover, the knockout mutant of LOC_Os12g18729 decreased cold tolerance in early rice seedling stage signifcantly compared with that of wild type. Conclusions: In general, study of the genetic basis of cold tolerance of rice is important for the development of cold-tolerant rice varieties. In the present study, QTL mapping, BSA-seq and RNA-seq were integrated to identify two CTS QTLs qCTS8 and qCTS12. Furthermore, qRT-PCR, genotype sequencing and knockout analysis indicated that LOC_Os12g18729 could be the candidate gene of qCTS12. These results are expected to further exploration of the genetic mechanism of CTS in rice and improve cold tolerance of cultivated rice by introducing the cold tolerant genes from DXWR through marker-assisted selection.

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.

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.

Mineral malnutrition as a prevalent public health issue can be alleviated by increasing the intake of dietary minerals from major staple crops, such as rice. Identification of the gene responsible for mineral contents in rice would help breed cultivars enriched with minerals through marker‐assisted selection. Two segregating populations of backcross inbred lines (BIL) were employed to map quantitative trait loci (QTLs) for macronutrient contents in brown and milled rice, BC₁F₅, and BC₂F_4:5 derived from an interspecific cross of Xieqingzao B (Oryza sativa) and Dongxiang wild rice (O. rufipogon). Phenotyping the populations was conducted in multiple locations and years, and up to 169 DNA markers were used for the genotyping. A total of 17 QTLs for P, K, Na, Ca, and Mg contents in brown and milled rice distributed on eight regions were identified in the BC₁F₅ population, which is explained to range from 5.98% to 56.80% of phenotypic variances. Two regions controlling qCa1.1 and qCa4.1 were validated, and seven new QTLs for Ca and Mg contents were identified in the BC₂F_4:5 population. 18 of 24 QTLs were clustered across seven chromosomal regions, indicating that different mineral accumulation might be involved in common regulatory pathways. Of 24 QTLs identified in two populations, 16 having favorable alleles were derived from O. rufipogon and 10 were novel. These results will not only help understand the molecular mechanism of macronutrient accumulation in rice but also provide candidate QTLs for further gene cloning and grain nutrient improvement through QTL pyramiding.