Enterobacter ludwigii b3 in the rhizosphere of wild rice assists cultivated rice in mitigating drought stress by direct and indirect methods.

Drought is the primary factor limiting rice production in ecosystems. Wild rice rhizosphere bacteria possess the potential to assist in the stress resistance of cultivated rice. This study examines the impact of wild rice rhizosphere bacteria on cultivated rice under drought conditions. From the rhizosphere soil of wild rice, 20 potential drought-resistant strains were isolated. Subsequent to the screening, the most effective strain b3, was identified as Enterobacter ludwigii. Pot experiments were conducted on the cultivated Changbai 9 rice. It was found that inoculation with the E. ludwigii b3 strain improved the drought resistance of the rice, promotion of rice growth (shoot height increased by 13.47 %), increased chlorophyll content (chlorophyll a, chlorophyll b and carotenoid increased by 168.74 %, 130.68 % and 87.89 %), improved antioxidant system (content of glutathione was increased by 60.35 %), and accumulation of osmotic regulation substances (soluble sugar and soluble protein increased by 70.36 % and 142.03 %). Furthermore, E. ludwigii b3 had a transformative effect on the rhizosphere bacterial community of cultivated rice, increasing its abundance and diversity while simultaneously recruiting beneficial rhizosphere bacteria, resulting in a more complex community. Additionally, E. ludwigii b3 acted directly and indirectly on cultivated rice through its metabolites (organic acids, amino acids, flavonoids and other substances), which helped alleviate drought stress. In conclusion, the E. ludwigii b3 shows promise as a drought-resistant strain and has the potential to improve the growth and productivity of cultivated rice in arid agricultural ecosystems. This study represents the first investigation of E. ludwigii in the rhizosphere of wild rice under drought conditions on cultivated rice.

Identification of candidate genes controlling cold tolerance at the early seedling stage from Dongxiang wild rice by QTL mapping, BSA-Seq and RNA-Seq.

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.

Wild rice: unlocking the future of rice breeding.

Germplasm resources serve as the foundations of advancements in breeding and are crucial for maintaining food security. Wild rice species of the genus Oryza include rich sources of genetic diversity and high adaptability, making them a substantial resource for rice breeding. The discovery of wild-type cytoplasmic male sterility resources enabled the achievement of the 'three lines' goal in hybrid rice, significantly increasing rice yields. The application of resistance alleles from wild rice enables rice production to withstand losses caused by stress. Reduced genetic diversity due to rice breeding poses a significant limitation to further advances and can be alleviated through a systematic use of wild genetic resources that integrate geographic, climatic and environmental data of the original habitat, along with extensive germplasm collection and identification using advanced methods. Leveraging technological advancements in plant genomics, the understanding of genetic mechanisms and the application of artificial intelligence and gene editing can further enhance the efficiency and accuracy of this process. These advancements facilitate rapid isolation and functional studies of genes, and precise genome manipulation. This review systematically summarizes the utilization of superior genes and germplasm resources derived from wild rice sources, while also exploring the collection, conservation, identification and utilization of further wild rice germplasm resources. A focus on genome sequencing and biotechnology developments is leading to new breeding and biotechnology opportunities. These new opportunities will not only promote the development of rice varieties that exhibit high yields, superior stress resistance and high quality but also expand the genetic diversity among rice cultivars.

The superior allele LEA12OR in wild rice enhances salt tolerance and yield.

Soil salinity has negative impacts on food security and sustainable agriculture. Ion homeostasis, osmotic adjustment and reactive oxygen species scavenging are the main approaches utilized by rice to resist salt stress. Breeding rice cultivars with high salt tolerance (ST) and yield is a significant challenge due to the lack of elite alleles conferring ST. Here, we report that the elite allele LEA12OR, which encodes a late embryogenesis abundant (LEA) protein from the wild rice Oryza rufipogon Griff., improves osmotic adjustment and increases yield under salt stress. Mechanistically, LEA12OR, as the early regulator of the LEA12OR-OsSAPK10-OsbZIP86-OsNCED3 functional module, maintains the kinase stability of OsSAPK10 under salt stress, thereby conferring ST by promoting abscisic acid biosynthesis and accumulation in rice. The superior allele LEA12OR provides a new avenue for improving ST and yield via the application of LEA12OR in current rice through molecular breeding and genome editing.

Domestication of rice may have changed its arbuscular mycorrhizal properties by modifying phosphorus nutrition-related traits and decreasing symbiotic compatibility.

Modern cultivated rice (Oryza sativa) typically experiences limited growth benefits from arbuscular mycorrhizal (AM) symbiosis. This could be due to the long-term domestication of rice under favorable phosphorus conditions. However, there is limited understanding of whether and how the rice domestication has modified AM properties. This study compared AM properties between a collection of wild (Oryza rufipogon) and domesticated rice genotypes and investigated the mechanisms underlying their differences by analyzing physiological, genomic, transcriptomic, and metabolomic traits critical for AM symbiosis. The results revealed significantly lower mycorrhizal growth responses and colonization intensity in domesticated rice compared to wild rice, and this change of AM properties may be associated with the domestication modifications of plant phosphorus utilization efficiency at physiological and genomic levels. Domestication also resulted in a decrease in the activity of the mycorrhizal phosphorus acquisition pathway, which may be attributed to reduced mycorrhizal compatibility of rice roots by enhancing defense responses like root lignification and reducing carbon supply to AM fungi. In conclusion, rice domestication may have changed its AM properties by modifying P nutrition-related traits and reducing symbiotic compatibility. This study offers new insights for improving AM properties in future rice breeding programs to enhance sustainable agricultural production.

Mechanism of betterment towards growth and induction of defense in rice (Oryza sativa L.) by biopriming with bacterial endophytes isolated from wild rice.

Utilizing beneficial microorganisms associated with plants, particularly endophytes, is becoming more and more prevalent since it supports the physiological health and evolutionary adaption of the host. The range of enhanced endophytic bacteria found in wild rice makes it a promising resource for sustainable agriculture. Current study focused on bacterial endophytes the tissues of wild rice plants' roots, stems, and leaves for managing the health and development of rice (Oryza sativa L.) plants. Bacterial endophytes were characterized using 16S rRNA. Treatments with Priestia megaterium (NRRI EB 1) and Priestia aryabhattai (NRRI EB 2) outperformed the other isolates in rice growth enhancement activities significantly. The biocontrol efficacy of bacterial endophytes was tested against Xanthomonas oryzae pv. oryzae and Rhizoctonia solani and percentage of inhibition was the higher in NRRI EB 1 by 79.32-80.83% and in NRRI EB 2 by 79.69-80.45%. Bio-priming the seeds with specific endophytic bacterial strains led to a decrease in average germination time, an increase in seedling vigor, and total chlorophyll content. Additionally, they generated greater amounts of soluble phosphate (40.91-83.70 µg/mL) and indole acetic acid (28.10-60.18 µg/mL), which are in the midst of encouraging more plant development. Higher expressions of defense enzymes in comparison to the control, including catalase (>220% in root and shoot), peroxidase (>200% in shoot and root), and superoxide dismutase (> 150% in shoot and root) illustrates the rice crop's resilience to withstand stress. The activity of the mentioned enzymes was further validated through the activation of corresponding defense genes such as DEFENSIN (>2-fold), PAL (>3-fold), PR-3 (>2-fold), POX (>2-fold) and LOX (>1-fold) relative to the control's untreated plants. The possibility exists to extract advantageous endophytic bacteria from wild rice species, potentially rewilding the microbiome of farmed rice cultivators and fostering their development.

Identification of QTLs responsible for culturability, and fine-mapping of QTL qCBT9 related to callus browning derived from Dongxiang common wild rice (Oryza rufipogon Griff.).

Compared to japonica, the lower genetic transformation efficiency of indica is a technical bottleneck for rice molecular breeding. Specifically, callus browning frequently occurs during the culture of the elite indica variety 93-11, leading to poor culturability and lower genetic transformation efficiency. Here, 67 QTLs related to culturability were detected using 97 introgression lines (designated as 9DILs) derived from Dongxiang common wild rice (DXCWR, Oryza rufipogon Griff.) with 93-11 genetic background, explaining 4% ~12% of the phenotypic variations. The QTL qCBT9 on chromosome 9 was a primary QTL for reducing callus browning derived from DXCWR. Five 9DILs with light callus browning and high differentiation were screened. We evaluated the callus browning index (CBI) of 100 F2 population crossed of 93-11 and 9DIL71 and the recombinant plants screened from 3270 individuals. The qCBT9 was delimited to a ~148kb region between the markers X16 and X23. RNA-seq analysis of DEGs between 9DIL71 and 93-11 showed three upregulated DEGs (Os09g0526500, Os09g0527900, Os09g0528200,) and three downregulated DEGs (Os09g0526700, Os09g0526800, Os09g0527700) were located in the candidate region of qCBT9. Furthermore, callus browning may be involved in cell senescence and death caused by oxidative stress. The differentiation of indica and japonica in this region suggested that qCBT9 was possibly a vital QTL contributed to better culturability of japonica. Our results laid a foundation for further cloning of the gene for reduced callus browning in O. rufipogon, and also provided a new genetic resource and material basis for improving the culturability and genetic transformation efficiency of cultivated rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01470-z.

Oryza glumaepatula and calcium oxide nanoparticles enhanced Cr stress tolerance by maintaining antioxidant defense, chlorophyll and gene expression levels in rice.

Chromium (Cr), a potent heavy metal, threatens rice cultivation due to its escalating presence in soil from human activities. Wild rice contains useful genes for phytoremediation; however, it is difficult to use directly for metal mitigation. Here, a single segment substitution line (SSSL), SG001, was developed by crossing O. glumaepatula and Huajingxian74 (HJX) to evaluate the survival ability of plants against Cr. Further, we explored the potential effect of calcium oxide nanoparticles (CaO-NPs) (50 µM) to minimize the toxic effect of Cr (100 µM) in rice cultivars, SG001 and HJX. The findings of this study indicated that Cr toxicity led to increased oxidative stress. This was shown by higher levels of hydrogen peroxide (H2O2), which was increased by 104% in SG001 and 177% in HJX, and malondialdehyde (MDA) increased by 79% in SG001 and 135% in HJX. Furthermore, it also depicted that Cr toxicity considerably declined shoot and root length, shoot and root fresh weight by 30%, 27%, 25%, and 20% in SG001 and 44%, 51%, 42%, and 45% in HJX, respectively. This mitigation was evidenced by decreased Cr contents, increased calcium (Ca) levels in SG001, and the maintenance of chlorophyll, antioxidant defense, and gene expression levels. Moreover, there was a notable reduction in MDA and H2O2, while the defense mechanisms of key antioxidants, including ascorbate peroxidase, superoxide dismutase, glutathione, catalase, and peroxidase were upregulated, along with an increase in soluble protein contents in both rice cultivars after applying CaO-NPs. CaO-NPs effectively restored cellular and subcellular structural integrity and growth in both lines, which had been seriously disrupted by Cr toxicity. Overall, our findings suggest that SG001, in combination with CaO-NPs, could serve as an effective strategy to mitigate Cr toxicity in plants.

The GZnC1 variant from common wild rice influences grain Zn content.

Zinc (Zn) deficiency, caused by inadequate Zn intake in the human diet, has serious health implications. Rice (Oryza sativa) is the staple food in regions with a high incidence of Zn deficiency, so raising Zn levels in rice grain could help alleviate Zn deficiency. The wild relatives of cultivated rice vary widely in grain Zn content and thus are suitable resources for improving this trait. However, few loci underlying grain Zn content have been identified in wild rice relatives. Here, we identified a major quantitative trait locus for grain Zn content, Grain Zn Content 1 (qGZnC1), from Yuanjiang common wild rice (Oryza rufipogon Griff.) using map-based cloning. Down-regulating GZnC1 expression reduced the grain Zn content, whereas the presence of GZnC1 had the opposite effect, indicating that GZnC1 is involved in grain Zn content in rice. Notably, GZnC1 is identical to a previously reported gene, EMBRYO SAC ABORTION 1 (ESA1), involved in seed setting rate. The mutation in GZnC1/ESA1 at position 1819 (T1819C) causes delayed termination of protein translation. In addition, GZnC1 is specifically expressed in developing panicles. Several genes related to Zn-transporter genes were up-regulated in the presence of GZnC1. Our results suggest that GZnC1 activates Zn transporters to promote Zn distribution in panicles. Our work thus sheds light on the genetic mechanism of Zn accumulation in rice grain and provides a new genetic resource for improving Zn content in rice.

Actinoallomurus soli sp. nov. and Actinoallomurus rhizosphaericola sp. nov., two novel actinobacteria isolated from rhizosphere soil of Oryza rufipogon Griff.

The taxonomic position of two novel Actinoallomurus strains isolated from rhizosphere soil of wild rice (Oryza rufipogon Griff.) was established using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains WRP6H-15T and WRP9H-5T were closely related to Actinoallomurus spadix JCM 3146T and Actinoallomurus purpureus TTN02-30T. Chemotaxonomic and morphological characteristics of both strains were consistent with members of the genus Actinoallomurus, while phenotypic properties, genome-based comparisons and phylogenomic analyses distinguished strains WRP6H-15T and WRP9H-5T from their closest phylogenetic relatives. The two strains showed nearly identical 16S rRNA gene sequences (99.9 %). Strain WRP6H-15T showed 68.7 % digital DNA-DNA hybridization, 95.9 % average nucleotide identity (ANI) based on blast and 96.4 % ANI based on MUMmer to strain WRP9H-5T. A phylogenomic tree based on draft genome sequences of the strains and representative of the genus Actinoallomurus confirmed the phylogenetic relationships. The genomes sizes of strains WRP6H-15T and WRP9H-5T were 9.42 Mb and 9.68 Mb, with DNA G+C contents of 71.5 and 71.3 mol%, respectively. In silico analysis predicted that the strains contain biosynthetic gene clusters encoding for specialized metabolites. Characterization based on chemotaxonomic, phylogenetic, phenotypic and genomic evidence demonstrated that strains WRP6H-15T and WRP9H-5T represent two novel species of the genus Actinoallomurus, for which the names Actinoallomurus soli sp. nov. (type strain WRP6H-15T=TBRC 15726T=NBRC 115556T) and Actinoallomurus rhizosphaericola sp. nov. (type strain WRP9H-5T=TBRC 15727T=NBRC 115557T) are proposed.

Mapping quantitative trait loci associated with callus browning in Dongxiang common wild rice (Oryza rufipogon Griff.).

BACKGROUND: Genetic transformation of indica rice (Oryza sativa ssp. indica) is limited by callus browning, which results in poor in vitro tissue culturability. Elucidating the genes in common wild rice (Oryza rufipogon Griff.) that control callus browning is fundamental for improving the tissue culturability of indica rice varieties. METHODS AND RESULTS: We used a population of 129 O. rufipogon (Dongxiang common wild rice; DXCWR) introgression lines in the elite cultivar GC2 (Oryza sativa ssp. indica) background and 159 simple sequence repeat (SSR) markers to identify quantitative trait loci (QTLs) associated with callus browning. We evaluated callus browning based on the indices of callus browning rate (CBR), callus browning index (CBI), and standard callus browning index (SCBI). CONCLUSIONS: We detected 30 QTLs associated with callus browning across all lines, mapping to chromosomes 1, 2, 3, 4, 8, 9, and 12. These genomic regions were repeatedly associated with differences in CBR, CBI, and SCBI. The alleles from DXCWR showed additive effects in reducing callus browning. We identified new QTLs near the markers RM247 and RM7003 on chromosome 12, indicating that these QTLs are unique to DXCWR. Furthermore, we identified six introgression lines with significantly lower callus browning. These lines will be useful germplasms for genetic transformation and fine-mapping of the culturability trait.

Wild and cultivated allele effects on rice phenotypic traits in reciprocal backcross populations between Oryza rufipogon and two cultivars, O. sativa Nipponbare and IR36.

A total of four populations of reciprocal backcross recombinant inbred lines were produced from a cross between a wild accession of Oryza rufipogon W630 and two major cultivars, O. sativa Japonica Nipponbare and Indica IR36. Using these populations, quantitative trait locus (QTL) analysis for eight morphological traits (culm length, panicle length, days to heading, panicle shape, pericarp color, hull color, seed shattering and seed awning) was carried out, and the putative QTL regions were compared among the populations. The QTLs with strong allele effects were commonly detected for culm length, panicle shape, pericarp color and hull color in all four populations, and their peak locations were close to the major genes of sd1, Spr3, Rc and Bh4, respectively. For panicle length and days to heading, some QTL regions overlapped between two or three populations. In the case of seed shattering and seed awning, strong wild allele effects at major loci were observed only in the populations with cultivated backgrounds. Since the wild and cultivated alleles have never been evaluated in the reciprocal genetic backgrounds, the present results provide new information on gene effects in breeding and domestication studies.

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.

AP2/EREBP Pathway Plays an Important Role in Chaling Wild Rice Tolerance to Cold Stress.

Cold stress is the main factor limiting rice production and distribution. Chaling wild rice can survive in cold winters. AP2/EREBP is a known transcription factor family associated with abiotic stress. We identified the members of the AP2/EREBP transcription factor family in rice, maize, and Arabidopsis, and conducted collinearity analysis and gene family analysis. We used Affymetrix array technology to analyze the expression of AP2/EREBP family genes in Chaling wild rice and cultivated rice cultivar Pei'ai64S, which is sensitive to cold. According to the GeneChip results, the expression levels of AP2/EREBP genes in Chaling wild rice were different from those in Pei'ai64S; and the increase rate of 36 AP2/EREBP genes in Chaling wild rice was higher than that in Pei'ai64S. Meanwhile, the MYC elements in cultivated rice and Chaling wild rice for the Os01g49830, Os03g08470, and Os03g64260 genes had different promoter sequences, resulting in the high expression of these genes in Chaling wild rice under low-temperature conditions. Furthermore, we analyzed the upstream and downstream genes of the AP2/EREBP transcription factor family and studied the conservation of these genes. We found that the upstream transcription factors were more conserved, indicating that these upstream transcription factors may be more important in regulating cold stress. Meanwhile, we found the expression of AP2/EREBP pathway genes was significantly increased in recombinant inbred lines from Nipponbare crossing with Chaling wild rice, These results suggest that the AP2/EREBP signaling pathway plays an important role in Chaling wild rice tolerance to cold stress.

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.

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.

Genomic Survey of Flavin Monooxygenases in Wild and Cultivated Rice Provides Insight into Evolution and Functional Diversities.

The flavin monooxygenase (FMO) enzyme was discovered in mammalian liver cells that convert a carcinogenic compound, N-N'-dimethylaniline, into a non-carcinogenic compound, N-oxide. Since then, many FMOs have been reported in animal systems for their primary role in the detoxification of xenobiotic compounds. In plants, this family has diverged to perform varied functions like pathogen defense, auxin biosynthesis, and S-oxygenation of compounds. Only a few members of this family, primarily those involved in auxin biosynthesis, have been functionally characterized in plant species. Thus, the present study aims to identify all the members of the FMO family in 10 different wild and cultivated Oryza species. Genome-wide analysis of the FMO family in different Oryza species reveals that each species has multiple FMO members in its genome and that this family is conserved throughout evolution. Taking clues from its role in pathogen defense and its possible function in ROS scavenging, we have also assessed the involvement of this family in abiotic stresses. A detailed in silico expression analysis of the FMO family in Oryza sativa subsp. japonica revealed that only a subset of genes responds to different abiotic stresses. This is supported by the experimental validation of a few selected genes using qRT-PCR in stress-sensitive Oryza sativa subsp. indica and stress-sensitive wild rice Oryza nivara. The identification and comprehensive in silico analysis of FMO genes from different Oryza species carried out in this study will serve as the foundation for further structural and functional studies of FMO genes in rice as well as other crop types.

Genome-Wide Identification and Analysis of OsSPXs Revealed Its Genetic Influence on Cold Tolerance of Dongxiang Wild Rice (DXWR).

SPX-domain proteins (small proteins with only the SPX domain) have been proven to be involved in phosphate-related signal transduction and regulation pathways. Except for OsSPX1 research showing that it plays a role in the process of rice adaptation to cold stress, the potential functions of other SPX genes in cold stress are unknown. Therefore, in this study, we identified six OsSPXs from the whole genome of DXWR. The phylogeny of OsSPXs has a strong correlation with its motif. Transcriptome data analysis showed that OsSPXs were highly sensitive to cold stress, and real-time PCR verified that the levels of OsSPX1, OsSPX2, OsSPX4, and OsSPX6 in cold-tolerant materials (DXWR) during cold treatment were higher than that of cold-sensitive rice (GZX49). The promoter region of DXWR OsSPXs contains a large number of cis-acting elements related to abiotic stress tolerance and plant hormone response. At the same time, these genes have expression patterns that are highly similar to cold-tolerance genes. This study provides useful information about OsSPXs, which is helpful for the gene-function research of DXWR and genetic improvements during breeding.

Whole-genome assembly and annotation of northern wild rice, Zizania palustris L., supports a whole-genome duplication in the Zizania genus.

Zizania palustris L. (northern wild rice, NWR) is an aquatic grass native to North America that is notable for its nutritious grain. This is an important species with ecological, cultural and agricultural significance, specifically in the Great Lakes region of the USA. Using flow cytometry, we first estimated the NWR genome size to be 1.8 Gb. Using long- and short-range sequencing, Hi-C scaffolding and RNA-seq data from eight tissues, we generated an annotated whole-genome de novo assembly of NWR. The assembly was 1.29 Gb in length, highly repetitive (approx. 76.0%) and contained 46 421 putative protein-coding genes. The expansion of retrotransposons within the genome and a whole-genome duplication (WGD) after the Zizania-Oryza speciation event have both led to an increase in the genome size of NWR in comparison with Oryza sativa L. and Zizania latifolia. Both events depict a genome rapidly undergoing change over a short evolutionary time. Comparative analyses revealed the conservation of large syntenic blocks between NWR and O. sativa, which were used to identify putative seed-shattering genes. Estimates of divergence times revealed that the Zizania genus diverged from Oryza approximately 26-30 million years ago (26-30 MYA), whereas NWR and Z. latifolia diverged from one another approximately 6-8 MYA. Comparative genomics confirmed evidence of a WGD in the Zizania genus and provided support that the event occurred prior to the NWR-Z. latifolia speciation event. This genome assembly and annotation provides a valuable resource for comparative genomics in the Oryzeae tribe and provides an important resource for future conservation and breeding efforts of NWR.

Genome-Wide Identification of DUF26 Domain-Containing Genes in Dongxiang Wild Rice and Analysis of Their Expression Responses under Submergence.

The DUF26 domain-containing protein is an extracellular structural protein, which plays an important role in signal transduction. Dongxiang wild rice (Oryza rufipogon Griff.) is the northern-most common wild rice in China. Using domain analysis, 85 DUF26 domain-containing genes were identified in Dongxiang wild rice (DXWR) and further divided into four categories. The DUF26 domain-containing genes were unevenly distributed on chromosomes, and there were 18 pairs of tandem repeats. Gene sequence analysis showed that there were significant differences in the gene structure and motif distribution of the DUF26 domain in different categories. Motifs 3, 8, 9, 13, 14, 16, and 18 were highly conserved in all categories. It was also found that there were eight plasmodesmata localization proteins (PDLPs) with a unique motif 19. Collinearity analysis showed that DXWR had a large number of orthologous genes with wheat, maize, sorghum and zizania, of which 17 DUF26 domain-containing genes were conserved in five gramineous crops. Under the stress of anaerobic germination and seedling submergence treatment, 33 DUF26 domain-containing genes were differentially expressed in varying degrees. Further correlation analysis with the expression of known submergence tolerance genes showed that these DUF26 domain-containing genes may jointly regulate the submergence tolerance process with these known submergence tolerance genes in DXWR.