In Vitro Gamma Mutagenesis Techniques in Rice (Oryza sativa L. var. Lazarroz FL).

Gamma radiation (60Co)-induced mutagenesis offers an alternative to develop rice lines by accelerating the spontaneous mutation process and increasing the pool of allelic variants available for breeding. Ionizing radiation works by direct or indirect damage to DNA and subsequent mutations. The technique can take advantage of in vitro protocols to optimize resources and accelerate the development of traits. This is achieved by exposing mutants to a selection agent of interest in controlled conditions and evaluating large numbers of plants in reduced areas. This chapter describes the protocol for establishing gamma radiation dosimetry and in vitro protocols for optimization at the laboratory level using seeds as the starting material, followed by embryogenic cell cultures, somatic embryogenesis, and regeneration. The final product of the protocol is a genetically homogeneous population of Oryza sativa that can be evaluated for breeding against abiotic and biotic stresses.

CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).

Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.

GMOIT: a tool for effective screening of genetically modified crops.

BACKGROUND: Advancement in agricultural biotechnology has resulted in increasing numbers of commercial varieties of genetically modified (GM) crops worldwide. Though several databases on GM crops are available, these databases generally focus on collecting and providing information on transgenic crops rather than on screening strategies. To overcome this, we constructed a novel tool named, Genetically Modified Organisms Identification Tool (GMOIT), designed to integrate basic and genetic information on genetic modification events and detection methods. RESULTS: At present, data for each element from 118 independent genetic modification events in soybean, maize, canola, and rice were included in the database. Particularly, GMOIT allows users to customize assay ranges and thus obtain the corresponding optimized screening strategies using common elements or specific locations as the detection targets with high flexibility. Using the 118 genetic modification events currently included in GMOIT as the range and algorithm selection results, a "6 + 4" protocol (six exogenous elements and four endogenous reference genes as the detection targets) covering 108 events for the four crops was established. Plasmids pGMOIT-1 and pGMOIT-2 were constructed as positive controls or calibrators in qualitative and quantitative transgene detection. CONCLUSIONS: Our study provides a simple, practical tool for selecting, detecting, and screening strategies for a sustainable and efficient application of genetic modification.

Comparative analysis of Spodoptera frugiperda (J. E. Smith) (Lepidoptera, Noctuidae) corn and rice strains microbiota revealed minor changes across life cycle and strain endosymbiont association.

Background: Spodoptera frugiperda (FAW) is a pest that poses a significant threat to corn production worldwide, causing millions of dollars in losses. The species has evolved into two strains (corn and rice) that differ in their genetics, reproductive isolation, and resistance to insecticides and Bacillus thuringiensis endotoxins. The microbiota plays an important role in insects' physiology, nutrient acquisition, and response to chemical and biological controls. Several studies have been carried out on FAW microbiota from larvae guts using laboratory or field samples and a couple of studies have analyzed the corn strain microbiota across its life cycle. This investigation reveals the first comparison between corn strain (CS) and rice strain (RS) of FAW during different developmental insect stages and, more importantly, endosymbiont detection in both strains, highlighting the importance of studying both FAW populations and samples from different stages. Methods: The composition of microbiota during the life cycle of the FAW corn and rice strains was analyzed through high-throughput sequencing of the bacterial 16S rRNA gene using the MiSeq system. Additionally, culture-dependent techniques were used to isolate gut bacteria and the Transcribed Internal Spacer-ITS, 16S rRNA, and gyrB genes were examined to enhance bacterial identification. Results: Richness, diversity, and bacterial composition changed significantly across the life cycle of FAW. Most diversity was observed in eggs and males. Differences in gut microbiota diversity between CS and RS were minor. However, Leuconostoc, A2, Klebsiella, Lachnoclostridium, Spiroplasma, and Mucispirilum were mainly associated with RS and Colidextribacter, Pelomonas, Weissella, and Arsenophonus to CS, suggesting that FAW strains differ in several genera according to the host plant. Firmicutes and Proteobacteria were the dominant phyla during FAW metamorphosis. Illeobacterium, Ralstonia, and Burkholderia exhibited similar abundancies in both strains. Enterococcus was identified as a conserved taxon across the entire FAW life cycle. Microbiota core communities mainly consisted of Enterococcus and Illeobacterium. A positive correlation was found between Spiroplasma with RS (sampled from eggs, larvae, pupae, and adults) and Arsenophonus (sampled from eggs, larvae, and adults) with CS. Enterococcus mundtii was predominant in all developmental stages. Previous studies have suggested its importance in FAW response to B. thuringensis. Our results are relevant for the characterization of FAW corn and rice strains microbiota to develop new strategies for their control. Detection of Arsenophonus in CS and Spiroplasma in RS are promising for the improvement of this pest management, as these bacteria induce male killing and larvae fitness reduction in other Lepidoptera species.

Cross-kingdom RNA interference mediated by insect salivary microRNAs may suppress plant immunity.

Communication between insects and plants relies on the exchange of bioactive molecules that traverse the species interface. Although proteinic effectors have been extensively studied, our knowledge of other molecules involved in this process remains limited. In this study, we investigate the role of salivary microRNAs (miRNAs) from the rice planthopper Nilaparvata lugens in suppressing plant immunity. A total of three miRNAs were confirmed to be secreted into host plants during insect feeding. Notably, the sequence-conserved miR-7-5P is specifically expressed in the salivary glands of N. lugens and is secreted into saliva, distinguishing it significantly from homologues found in other insects. Silencing miR-7-5P negatively affects N. lugens feeding on rice plants, but not on artificial diets. The impaired feeding performance of miR-7-5P-silenced insects can be rescued by transgenic plants overexpressing miR-7-5P. Through target prediction and experimental testing, we demonstrate that miR-7-5P targets multiple plant genes, including the immune-associated bZIP transcription factor 43 (OsbZIP43). Infestation of rice plants by miR-7-5P-silenced insects leads to the increased expression of OsbZIP43, while the presence of miR-7-5P counteracts this upregulation effect. Furthermore, overexpressing OsbZIP43 confers plant resistance against insects which can be subverted by miR-7-5P. Our findings suggest a mechanism by which herbivorous insects have evolved salivary miRNAs to suppress plant immunity, expanding our understanding of cross-kingdom RNA interference between interacting organisms.

Identification of biochemical indices for brown spot (Bipolaris oryzae) disease resistance in rice mutants and hybrids.

Brown spot caused by Bipolaris oryzae is a major damaging fungal disease of rice which can decrease the yield and value of produce due to grain discoloration. The objectives of the current study were to investigate and understand the biochemical indices of brown spot disease resistance in rice. A total of 108 genotypes (mutant and hybrid) along with Super Basmati and parent RICF-160 were evaluated against brown spot disease. The genotypes exhibiting resistant and susceptible responses to brown spot disease according to the IRRI standard disease rating scale were screened and selected. To study the biochemical response mechanism, forty five selected genotypes along with Super Basmati and RICF-160 were analyzed using the biochemical markers. The physiological and biochemical analysis provided valuable insights and confirmed the resistance of rice hybrids and mutants against brown spot disease. Positive correlations were observed among stress bio-markers and disease response. Rice genotypes i.e. Mu-AS-8, Mu-AS-19, Mu-AS-20 and Mu-AS-35 exhibited moderate resistant response while Hy-AS-92, Hy-AS-98, Hy-AS-99, Hy-AS-101, Hy-AS-102 and Hy-AS-107 showed resistant response to brown spot disease. Brown spot resistant rice genotypes had lesser values of malondialdehyde and total oxidant status and higher antioxidant activities i.e. superoxide dismutase, peroxidase, total phenolic content and lycopene. The selected resistant rice genotypes had resistance capacity against Bipolaris oryzae stress. In conclusion, identified resistant mutants i.e. Mu-AS-8, Mu-AS-19, Mu-AS-20 and Mu-AS-35 and hybrids i.e. Hy-AS-92, Hy-AS-98, Hy-AS-99, Hy-AS-101, Hy-AS-102 and Hy-AS-107 could be used in rice breeding program to achieve sustainable rice production by coping the emerging challenge of brown spot disease under variable climate conditions.

Overexpression of OsNAR2.1 by OsNAR2.1 promoter increases drought resistance by increasing the expression of OsPLDα1 in rice.

BACKGROUND: pOsNAR2.1:OsNAR2.1 expression could significantly increase nitrogen uptake efficiency and grain yield of rice. RESULT: This study reported the effects of overexpression of OsNAR2.1 by OsNAR2.1 promoter on physiological and agronomic traits associated with drought tolerance. In comparison to the wild-type (WT), the pOsNAR2.1:OsNAR2.1 transgenic lines exhibited a significant improvement in survival rate when subjected to drought stress and then irrigation. Under limited water supply conditions, compared with WT, the photosynthesis and water use efficiency (WUE) of transgenic lines were increased by 39.2% and 28.8%, respectively. Finally, the transgenic lines had 25.5% and 66.4% higher grain yield than the WT under full watering and limited water supply conditions, respectively. Compared with the WT, the agronomic nitrogen use efficiency (NUE) of transgenic lines increased by 25.5% and 66.4% under full watering and limited water supply conditions, and the N recovery efficiency of transgenic lines increased by 29.3% and 50.2%, respectively. The interaction between OsNAR2.1 protein and OsPLDα1 protein was verified by yeast hybrids. After drought treatment, PLDα activity on the plasma membrane of the transgenic line increased 85.0% compared with WT. CONCLUSION: These results indicated that pOsNAR2.1:OsNAR2.1 expression could improve the drought resistance of rice by increasing nitrogen uptake and regulating the expression of OsPLDα1.

Country-wide, multi-location trials of Green Super Rice lines for yield performance and stability analysis using genetic and stability parameters.

Rice (Oryza sativa L.) is an important member of the family Poaceae and more than half of world population depend for their dietary nutrition on rice. Rice cultivars with higher yield, resilience to stress and wider adaptability are essential to ensure production stability and food security. The fundamental objective of this study was to identify higher-yielding rice genotypes with stable performance and wider adaptability in a rice growing areas of Pakistan. A triplicate RCBD design experiment with 20 Green Super Rice (GSR) advanced lines was conducted at 12 rice growing ecologies in four Provinces of Pakistan. Grain yield stability performance was assessed by using different univariate and multivariate statistics. Analysis of variance revealed significant differences among genotypes, locations, and G x E interaction for mean squares (p < 0.05) of major yield contributing traits. All the studied traits except for number of tillers per plant revealed higher genotypic variance than environmental variance. Broad sense heritability was estimated in the range of 44.36% to 98.60%. Based on ASV, ASI, bi, Wi2, σ2i and WAAS statistics, the genotypes G1, G4, G5, G8, G11 and G12 revealed lowest values for parametric statistics and considered more stable genotypes based on  paddy yield. The additive main effects and multiplicative interaction (AMMI) model revealed significant variation (p < 0.05) for genotypes, non-signification for environment and highly significant for G × E interaction. The variation proportion of PC1 and PC2 from interaction revealed 67.2% variability for paddy yield. Based on 'mean verses stability analysis of GGE biplot', 'Which-won-where' GGE Biplot, 'discriminativeness vs. representativeness' pattern of stability, 'IPCA and WAASB/GY' ratio-based stability Heat-map, and ranking of genotypes, the genotypes G1, G2, G3, G5, G8, G10, G11 and G13 were observed ideal genotypes with yield potential more than 8 tons ha-1. Discriminativeness vs. representativeness' pattern of stability identifies two environments, E5 (D.I Khan, KPK) and E6 (Usta Muhammad, Baluchistan) were best suited for evaluating genotypic yield performance. Based on these findings we have concluded that the genotypes G1, G2, G3, G5, G8, G10, G11 and G13 could be included in the commercial varietal development process and future breeding program.

PPanG: a precision pangenome browser enabling nucleotide-level analysis of genomic variations in individual genomes and their graph-based pangenome.

Graph-based pangenome is gaining more popularity than linear pangenome because it stores more comprehensive information of variations. However, traditional linear genome browser has its own advantages, especially the tremendous resources accumulated historically. With the fast-growing number of individual genomes and their annotations available, the demand for a genome browser to visualize genome annotation for many individuals together with a graph-based pangenome is getting higher and higher. Here we report a new pangenome browser PPanG, a precise pangenome browser enabling nucleotide-level comparison of individual genome annotations together with a graph-based pangenome. Nine rice genomes with annotations were provided by default as potential references, and any individual genome can be selected as the reference. Our pangenome browser provides unprecedented insights on genome variations at different levels from base to gene, and reveals how the structures of a gene could differ for individuals. PPanG can be applied to any species with multiple individual genomes available and it is available at https://cgm.sjtu.edu.cn/PPanG .

Characterization of SIPs-type aquaporins and their roles in response to environmental cues in rice (Oryza sativa L.).

BACKGROUND: Aquaporins (AQPs) facilitate water diffusion across biological membranes and are involved in all phases of growth and development. Small and basic intrinsic proteins (SIPs) belong to the fourth subfamily of the plant AQPs. Although SIPs are widely present in higher plants, reports on SIPs are limited. Rice is one of the major food crops in the world, and water use is an important factor affecting rice growth and development; therefore, this study aimed to provide information relevant to the function and environmental response of the rice SIP gene family. RESULTS: The rice (Oryza sativa L. japonica) genome encodes two SIP-like genes, OsSIP1 and OsSIP2, whose products are predominantly located in the endoplasmic reticulum (ER) membrane but transient localization to the plasma membrane is not excluded. Heterologous expression in a yeast aquaglyceroporin-mutant fps1Δ showed that both OsSIP1 and OsSIP2 made the cell more sensitive to KCl, sorbitol and H2O2, indicating facilitated permeation of water and hydrogen peroxide. In addition, the yeast cells expressing OsSIP2 were unable to efflux the toxic methylamine taken up by the endogenous MEP permeases, but OsSIP1 showed subtle permeability to methylamine, suggesting that OsSIP1 may have a wider conducting pore than OsSIP2. Expression profiling in different rice tissues or organs revealed that OsSIP1 was expressed in all tissues tested, whereas OsSIP2 was preferentially expressed in anthers and weakly expressed in other tissues. Consistent with this, histochemical staining of tissues expressing the promoter-ß-glucuronidase fusion genes revealed their tissue-specific expression profile. In rice seedlings, both OsSIPs were upregulated to varied levels under different stress conditions, including osmotic shock, high salinity, unfavorable temperature, redox challenge and pathogen attack, as well as by hormonal treatments such as GA, ABA, MeJA, SA. However, a reduced expression of both OsSIPs was observed under dehydration treatment. CONCLUSIONS: Our results suggest that SIP-like aquaporins are not restricted to the ER membrane and are likely to be involved in unique membrane functions in substrate transport, growth and development, and environmental response.

Rapid generation of fragrant thermo-sensitive genic male sterile rice with enhanced disease resistance via CRISPR/Cas9.

MAIN CONCLUSION: The three, by mutagenesis produced genes OsPi21, OsXa5, and OsBADH2, generated novel lines exhibiting desired fragrance and improved resistance. Elite sterile lines are the basis for hybrid rice breeding, and rice quality and disease resistance become the focus of new sterile lines breeding. Since there are few sterile lines with fragrance and high resistance to blast and bacterial blight at the same time in hybrid rice production, we here integrated the simultaneous mutagenesis of three genes, OsPi21, OsXa5, and OsBADH2, into Zhi 5012S, an elite thermo-sensitive genic male sterile (TGMS) variety, using the CRISPR/Cas9 system, thus eventually generated novel sterile lines would exhibit desired popcorn-like fragrance and improved resistance to blast and bacterial blight but without a loss in major agricultural traits such as yield. Collectively, this study develops valuable germplasm resources for the development of two-line hybrid rice with disease resistance, which provides a way to rapid generation of novel TGMS lines with elite traits.

Molecular characterization and evaluation of novel management options for Burkholderia glumae BG1, the causative agent of panicle blight of rice (Oryza sativa L.).

BACKGROUND: Bacterial panicle blight, incited by Burkholderia glumae, has impacted rice production globally. Despite its significance, knowledge about the disease and the virulence pattern of the causal agent is very limited. Bacterial panicle blight is a major challenge in the rice-growing belts of North-western India, resulting in yield reduction. However, the management of B. glumae has become a challenge due to the lack of proper management strategies. METHODOLOGY AND RESULTS: Twenty-one BG strains have been characterized using the 16S rRNA and the gyrB gene-based sequence approach in the present study. The gyrB gene-based phylogenetic analysis resulted in geographic region-specific clustering of the BG isolates. The virulence screening of twenty-one BG strains by inoculating the pathogenic bacterial suspension of 1 × 10-8 cfu/ml at the booting stage (55 DAT) revealed the variation in the disease severity and the grain yield of rice plants. The most virulent BG1 strain resulted in the highest disease incidence (82.11%) and lowest grain yield (11.12 g/plant), and the least virulent BG10 strain resulted in lowest disease incidence of 18.94% and highest grain yield (24.62 g/plant). In vitro evaluation of various biocontrol agents and nano copper at different concentrations by agar well diffusion method revealed that nano copper at 1000 mg/L inhibited the colony growth of B. glumae. Under net house conditions, nano copper at 1000 mg/L reduced the disease severity to 21.23% and increased the grain yield by 20.91% (31.76 g per plant) compared to the positive control (COC 0.25% + streptomycin 200 ppm). Remarkably, pre-inoculation with nano copper at 1000 mg/L followed by challenge inoculation with B. glumae enhanced the activity of enzymatic antioxidants viz., Phenyl ammonia-lyase (PAL), Polyphenol oxidase (PPO) and Peroxidase (POX) and non-enzymatic antioxidant phenol. Additionally, we observed a substantial transcript level upregulation of six defense-related genes to several folds viz., OsPR2, OsPR5, OsWRKY71, OsPAL1, OsAPX1, and OsPPO1 in comparison to the pathogen control and healthy control. CONCLUSIONS: Overall, our study provides valuable insights into the potential and practical application of nano copper for the mitigation of bacterial panicle blight, offering promising prospects for commercial utilization in disease management.

Oschib1 gene encoding a GH18 chitinase confers resistance against sheath blight disease of rice caused by Rhizoctonia solani AG1-IA.

Sheath blight disease of rice caused by Rhizoctonia solani AG1-IA, is a major fungal disease responsible for huge loss to grain yield and quality. The major limitation of achieving persistent and reliable resistance against R. solani is the governance of disease resistance trait by many genes. Therefore, functional characterization of new genes involved in sheath blight resistance is necessary to understand the mechanism of resistance as well as evolving effective strategies to manage the disease through host-plant resistance. In this study, we performed RNA sequencing of six diverse rice genotypes (TN1, BPT5204, Vandana, N22, Tetep, and Pankaj) from sheath and leaf tissue of control and fungal infected samples. The approach for identification of candidate resistant genes led to identification of 352 differentially expressed genes commonly present in all the six genotypes. 23 genes were analyzed for RT-qPCR expression which helped identification of Oschib1 showing differences in expression level in a time-course manner between susceptible and resistant genotypes. The Oschib1 encoding classIII chitinase was cloned from resistant variety Tetep and over-expressed in susceptible variety Taipei 309. The over-expression lines showed resistance against R. solani, as analyzed by detached leaf and whole plant assays. Interestingly, the resistance response was correlated with the level of transgene expression suggesting that the enzyme functions in a dose dependent manner. We report here the classIIIb chitinase from chromosome10 of rice showing anti-R. solani activity to combat the dreaded sheath blight disease.

Overexpression of rice OsNRT1.1A/OsNPF6.3 enhanced the nitrogen use efficiency of wheat under low nitrogen conditions.

MAIN CONCLUSION: Overexpression of OsNRT1.1A promotes early heading and increases the tolerance in wheat under nitrogen deficiency conditions. The application of inorganic nitrogen (N) fertilizers is a major driving force for crop yield improvement. However, the overuse of fertilizers significantly raises production costs and leads to environmental problems, making it critical to enhance crop nitrogen use efficiency (NUE) for the sake of sustainable agriculture. In this study, we created a series of transgenic wheat lines carrying the rice OsNRT1.1A gene, which encodes a nitrate transporter, to investigate its possible application in improving NUE in wheat. The transgenic wheat exhibited traits such as early maturation that were highly consistent with the overexpression of OsNRT1.1A in Arabidopsis and rice. However, we also observed that overexpression of the OsNRT1.1A gene in wheat can facilitate the growth of roots under low N conditions but has no effect on other aspects of growth and development under normal N conditions. Thus, it may lead to the improvement of wheat low N tolerance,which is different from the effects reported in other plants. A field trial analysis showed that transgenic wheat exhibited increased grain yield per plant under low N conditions. Moreover, transcriptome analysis indicated that OsNRT1.1A increased the expression levels of N uptake and utilization genes in wheat, thereby promoting plant growth under low N conditions. Taken together, our results indicated that OsNRT1.1A plays an important role in improving NUE in wheat with low N availability.

Purine permease (PUP) family gene PUP11 positively regulates the rice seed setting rate by influencing seed development.

KEY MESSAGE: Purine permease PUP11 is essential for rice seed development, regulates the seed setting rate, and influences the cytokinin content, sugar transport, and starch biosynthesis during grain development. The distribution of cytokinins in plant tissues determines plant growth and development and is regulated by several cytokinin transporters, including purine permease (PUP). Thirteen PUP genes have been identified within the rice genome; however, the functions of most of these genes remain poorly understood. We found that pup11 mutants showed extremely low seed setting rates and a unique filled seed distribution. Moreover, seed formation arrest in these mutants was associated with the disappearance of accumulated starch 10 days after flowering. PUP11 has two major transcripts with different expression patterns and subcellular locations, and further studies revealed that they have redundant positive roles in regulating the seed setting rate. We also found that type-A Response Regulator (RR) genes were upregulated in the developing grains of the pup11 mutant compared with those in the wild type. The results also showed that PUP11 altered the expression of several sucrose transporters and significantly upregulated certain starch biosynthesis genes. In summary, our results indicate that PUP11 influences the rice seed setting rate by regulating sucrose transport and starch accumulation during grain filling. This research provides new insights into the relationship between cytokinins and seed development, which may help improve cereal yield.

Identification of responsive genes to multiple abiotic stresses in rice (Oryza sativa): a meta-analysis of transcriptomics data.

Abiotic stresses limit the quantity and quality of rice grain production, which is considered a strategic crop in many countries. In this study, a meta-analysis of different microarray data at seedling stage was performed to investigate the effects of multiple abiotic stresses (drought, salinity, cold situation, high temperature, alkali condition, iron, aluminum, and heavy metal toxicity, nitrogen, phosphorus, and potassium deficiency) on rice. Comparative analysis between multiple abiotic stress groups and their control groups indicated 561 differentially expressed genes (DEGs), among which 422 and 139 genes were up-regulated and down-regulated, respectively. Gene Ontology analysis showed that the process of responding to stresses and stimuli was significantly enriched. In addition, pathways such as metabolic process and biosynthesis of secondary metabolites were identified by KEGG pathway analysis. Weighted correlation network analysis (WGCNA) uncovered 17 distinct co-expression modules. Six modules were significantly associated with genes involved in response to abiotic stresses. Finally, to validate the results of the meta-analysis, five genes, including TIFY9 (JAZ5), RAB16B, ADF3, Os01g0124650, and Os05g0142900 selected for qRT-PCR analysis. Expression patterns of selected genes confirmed the results of the meta-analysis. The outcome of this study could help introduce candidate genes that may be beneficial for use in genetic engineering programs to produce more tolerant crops or as markers for selection.

DNA methylation remodeling and the functional implication during male gametogenesis in rice.

BACKGROUND: Epigenetic marks are reprogrammed during sexual reproduction. In flowering plants, DNA methylation is only partially remodeled in the gametes and the zygote. However, the timing and functional significance of the remodeling during plant gametogenesis remain obscure. RESULTS: Here we show that DNA methylation remodeling starts after male meiosis in rice, with non-CG methylation, particularly at CHG sites, being first enhanced in the microspore and subsequently decreased in sperm. Functional analysis of rice CHG methyltransferase genes CMT3a and CMT3b indicates that CMT3a functions as the major CHG methyltransferase in rice meiocyte, while CMT3b is responsible for the increase of CHG methylation in microspore. The function of the two histone demethylases JMJ706 and JMJ707 that remove H3K9me2 may contribute to the decreased CHG methylation in sperm. During male gametogenesis CMT3a mainly silences TE and TE-related genes while CMT3b is required for repression of genes encoding factors involved in transcriptional and translational activities. In addition, CMT3b functions to repress zygotic gene expression in egg and participates in establishing the zygotic epigenome upon fertilization. CONCLUSION: Collectively, the results indicate that DNA methylation is dynamically remodeled during male gametogenesis, distinguish the function of CMT3a and CMT3b in sex cells, and underpin the functional significance of DNA methylation remodeling during rice reproduction.

Integrated meta-analysis and transcriptomics pinpoint genomic loci and novel candidate genes associated with submergence tolerance in rice.

BACKGROUND: Due to rising costs, water shortages, and labour shortages, farmers across the globe now prefer a direct seeding approach. However, submergence stress remains a major bottleneck limiting the success of this approach in rice cultivation. The merger of accumulated rice genetic resources provides an opportunity to detect key genomic loci and candidate genes that influence the flooding tolerance of rice. RESULTS: In the present study, a whole-genome meta-analysis was conducted on 120 quantitative trait loci (QTL) obtained from 16 independent QTL studies reported from 2004 to 2023. These QTL were confined to 18 meta-QTL (MQTL), and ten MQTL were successfully validated by independent genome-wide association studies from diverse natural populations. The mean confidence interval (CI) of the identified MQTL was 3.44 times narrower than the mean CI of the initial QTL. Moreover, four core MQTL loci with genetic distance less than 2 cM were obtained. By combining differentially expressed genes (DEG) from two transcriptome datasets with 858 candidate genes identified in the core MQTL regions, we found 38 common differentially expressed candidate genes (DECGs). In silico expression analysis of these DECGs led to the identification of 21 genes with high expression in embryo and coleoptile under submerged conditions. These DECGs encode proteins with known functions involved in submergence tolerance including WRKY, F-box, zinc fingers, glycosyltransferase, protein kinase, cytochrome P450, PP2C, hypoxia-responsive family, and DUF domain. By haplotype analysis, the 21 DECGs demonstrated distinct genetic differentiation and substantial genetic distance mainly between indica and japonica subspecies. Further, the MQTL7.1 was successfully validated using flanked marker S2329 on a set of genotypes with phenotypic variation. CONCLUSION: This study provides a new perspective on understanding the genetic basis of submergence tolerance in rice. The identified MQTL and novel candidate genes lay the foundation for marker-assisted breeding/engineering of flooding-tolerant cultivars conducive to direct seeding.

[Genetic dissection of rice resistance to bacterial blight].

Bacterial blight, a major disease in rice, poses a serious impact on rice production. In this study, a doubled haploid (DH) population derived from a cross between the introduced japonica cultivar 'Maybelle' and the indica landrace 'Baiyeqiu' was used to investigate the pathogenicity of four pathogen races causing bacterial blight. The results showed that the pathogenicity of all the pathogen races exhibited continuous, transgressive distribution in the DH population. Moreover, strong correlations existed between every two pathogen races, with the correlation coefficients ranging from 0.3 to 0.6. A total of 12 quantitative trait loci (QTLs) distributed on chromosomes 1, 2, 3, 5, 6, 7, 9, and 12 were detected for rice bacterial blight, explaining 4.95% to 16.05% of the phenotype. Among these QTLs, a major QTL located in the interval RM6024-RM163 on chromosome 5 was detected in three pathogen races. In addition, the pyramiding of the positive alleles can apparently improve the rice resistance to bacterial blight. This study is of great significance for broadening the genetic resources with resistance to bacterial blight in China.

[Identification and expression profile analysis of rice CRF gene family].

Cytokinin response factors (CRFs), as unique transcription factors in plants, play crucial roles in regulating development, phytohormone signaling pathway, and stress responses. In this study, we identified nine CRF genes from the rice genome by conducting a BLAST analysis using the protein sequences of twelve Arabidopsis AtCRFs. These genes are located on seven different rice chromosomes. We conducted a comprehensive analysis of the conserved domains, physicochemical properties, secondary structures, and phylogenetic relationships of rice CRF proteins using various online tools and local software. Additionally, we analyzed the exon-intron structures and cis-acting elements of OsCRFs, and found an abundance of elements relevant to phytohormone response and stress response on the promoters of rice CRF genes. Spatial-temporal expression pattern analysis revealed that four of the OsCRFs were barely expressed in all tested samples, while the other five were highly expressed in the leaf, panicle, or seed of rice. Microarray data showed that OsCRF genes are regulated to varying degrees by abscisic acid, auxin, cytokinin, and jasmonic acid. Furthermore, through analyzing the RNA-seq data, we found that OsCRFs are primarily involved in plant response to temperature stress (chilling and heat), with several OsCRFs also implicated in drought response, while hardly any respond to salt stress. This study provides an important basis for the functional characterization of rice CRF family genes.