Transgenic early japonica rice: Integration and expression characterization of stem borer resistance Bt gene.

BACKGROUND: Transgenic insect-resistant rice offers an environmentally friendly approach to mitigate yield losses caused by lepidopteran pests, such as stem borers. Bt (Bacillus thuringiensis) genes encode insecticidal proteins and are widely used to confer insect resistance to genetically modified crops. This study investigated the integration, inheritance, and expression characteristics of codon-optimised synthetic Bt genes, cry1C* and cry2A*, in transgenic early japonica rice lines. METHODS: The early japonica rice cultivar, Songgeng 9 (Oryza sativa), was transformed with cry1C* or cry2A*, which are driven by the ubi promoter via Agrobacterium tumefaciens-mediated transformation. Molecular analyses, including quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and Southern blot analysis were performed to confirm transgene integration, inheritance, transcriptional levels, and protein expression patterns across different tissues and developmental stages. RESULTS: Stable transgenic early japonica lines exhibiting single-copy transgene integration were established. Transcriptional analysis revealed variations in Bt gene expression among lines, tissues, and growth stages, with higher expression levels observed in leaves than in other organs. Notably, cry2A* exhibited consistently higher mRNA and protein levels than cry1C* across all examined tissues and developmental time points. Bt protein accumulation followed the trend of leaves > stem sheaths > young panicles > brown rice, with peak expression during the filling stage in the vegetative tissues. CONCLUSIONS: Synthetic cry2A* displayed markedly elevated transcription and translation compared to cry1C* in the transgenic early japonica rice lines examined. Distinct spatiotemporal patterns of Bt gene expression were elucidated, providing insights into the potential insect resistance conferred by these genes in rice. These findings will contribute to the development of insect-resistant japonica rice varieties and facilitate the rational deployment of Bt crops.

Alteration of soil pH induced by submerging/drainage and application of peanut straw biochar and its impact on Cd(II) availability in an acidic soil to indica-japonica rice varieties.

The effects of soil pH variations induced by submergence/drainage and biochar application on soil cadmium (Cd) availability to different rice (Oryza sativa L.) varieties are not well understood. This study aims to investigate the possible reasons for available Cd(II) reduction in paddy soil as influenced by biochar and to determine Cd(II) absorption and translocation rates in different parts of various rice varieties. A pot experiment in a greenhouse using four japonica and four indica rice varieties was conducted in Cd(II) contaminated paddy soil with peanut straw biochar. The results indicated that the submerging led to an increase in soil pH due to the consumption of protons (H+) by the reduction reactions of iron/manganese (Fe/Mn) oxides and sulfate (SO42-) and thus the decrease in soil available Cd(II) contents. However, the drainage decreased soil pH due to the release of protons during the oxidation of Fe2+, Mn2+, and S2- and thus the increase in soil available Cd(II) contents. Application of the biochar increased soil pH during soil submerging and inhibited the decline in soil pH during soil drainage, and thus decreased soil available Cd(II) contents under both submerging and drainage conditions. The indica rice varieties absorbed more Cd(II) in their roots and accumulated higher amounts of Cd(II) in their shoots and grains than the japonica rice varieties. The Cd(II) sensitive varieties exhibited a greater absorption and translocation rate of Cd(II) compared to the tolerant varieties of both indica and japonica rice. Biochar inhibited the absorption and accumulation of Cd(II) in the rice varieties, which ultimately lowered the Cd(II) contents in rice grains below the national food safety limit (0.2 mg kg-1). Overall, planting japonica rice varieties in Cd(II) polluted paddy soils combined with the use of biochar can effectively reduce Cd(II) content in rice grains which protects human health against Cd(II) toxicity.

Effect of Cd(II) adsorption onto rice roots on its uptake by different indica and japonica rice varieties and toxicity effect of Cd(II) under acidic conditions.

The rapid increase in soil acidity coupled with the deleterious effects of cadmium (Cd) toxicity had led to a decline in worldwide agricultural production. Rice absorbs and accumulates Cd(II) from polluted paddy soils, increasing human health risks throughout the food chain. A 35-day hydroponic experiment with four japonica and four indica (two each of them tolerant and sensitive cultivars) was conducted in this study to investigate the adsorption and absorption of Cd(II) by rice roots as related with surface chemical properties of the roots. The results showed that the three chemical forms of exchangeable, complexed, and precipitated Cd(II) increased with the increase in Cd(II) concentration for all rice cultivars. The roots of indica rice cultivars carried more negative charges and had greater functional groups and thus adsorbed more exchangeable and complexed Cd(II) than those of japonica rice cultivars. This led to more absorption of Cd(II) by the roots and greater toxicity of Cd(II) to the roots of indica rice cultivars and more inhibition of Cd(II) stress on the growth of the roots and whole plants of indica rice cultivars compared with japonica rice cultivars, which was one of the main reasons for more declines in the biomass and length of indica rice roots and shoots than japonica rice cultivars. Cd(II) stress showed more toxicity to the sensitive rice cultivars and thus greater inhibition on the growth of the cultivars due to more exchangeable and complexed Cd(II) adsorbed by their roots induced by more negative charges and functional groups on the roots compared with tolerant rice cultivar for both indica and japonica, which resulted in greater decreases in the biomass and length of roots and shoots as well as chlorophyll contents of the sensitive cultivars than the tolerant cultivars. The roots of sensitive rice cultivars also absorbed more Cd(II) than tolerant rice cultivars due to the same reasons as above. These findings will provide useful references for the safe utilization and health risk prevention of Cd-contaminated paddy fields.

Analysis of Erect-Panicle Japonica Rice in Northern China: Yield, Quality Status, and Quality Improvement Directions.

China is the only country that extensively cultivates the indica and japonica rice varieties, with the largest japonica rice production area being in northeast China. A study of the relationship between the yield and quality of japonica rice and the effect of nitrogen fertilizer application on this relationship is important. In this paper, we aimed to assess the current yield and quality of japonica rice in northeast China. We selected erect-panicle varieties as the test materials. Field experiments were conducted using different nitrogen fertilizer levels for two consecutive years to analyze the rice varieties' yield, quality, interrelationship, and nitrogen fertilizer response. The average yield following high- and low-nitrogen treatments exceeded 10,000.00 kg/hm2, with a maximum of 12,285.63 kg/hm2. The high-yield-high-nitrogen treatment group had more panicles, a higher seed-setting rate, and a higher 1000-grain weight than the other groups. The high-yield-low-nitrogen group had a higher number of panicles and seed-setting rate than the other groups. The low-yield-high-nitrogen group had a lower number of whole grains, grain length-to-width ratio, and taste value than the other groups. The low-yield-low-nitrogen group had fewer primary branches than the other groups; excluding the primary branch-setting rate and 1000-grain weight, the values of the other panicle traits of the group were significantly higher than those of the other groups. The high-nitrogen-high-flavor group had lower panicle and spikelet numbers and higher spikelet fertility rates than the other groups. The low-nitrogen-high-flavor group had higher spikelet fertility rates and 1000-grain weight than the other groups. Compared to the other groups, the low-nitrogen-high-flavor group had a higher head rice yield, and the high-nitrogen-high-flavor group had a lower chalkiness rate. The main goal of the breeding and cultivation of high-yield and high-quality erect-panicle japonica rice in northern China is to achieve "dual high, dual low, and one high and one low" conditions, signifying a high yield with high or low nitrogen levels, low protein and amylose contents, high head rice rates, and low chalkiness. This study provides a new technique for enhancing the taste of northern erect-panicle japonica rice to promote the sustainable, high-yield, and high-quality development of japonica rice in northern China.

A Novel Allele in the Promoter of Wx Decreases Gene Expression and Confers Lower Apparent Amylose Contents in Japonica Rice (Oryza sativa L.).

Wx is the key gene that controls amylose content (AC), and various alleles have been found in rice populations. Wxb is the major allele in japonica and produces moderate AC (15~18%). It was recently found that editing the promoter of Wx could produce a series of alleles that have different Wx activities. Although some studies have edited the promoter, few studies have focused on the natural variations in Wx. Here, we used the Rice3K database to investigate variations in the Wx promoter and found that the allele Wx1764178 (A/G) has a higher LD (linkage disequilibrium) with the two key SNPs (1765751, T/G; 1768006, A/C), which could produce different Wx alleles and influence AC, as reported previously. Further study showed that the Wx1764178 allele (A/G) is functional and influences the expression of Wx positively. Editing the A allele using CRISPR‒Cas9 produced 36 and 3 bp deletions and caused a decrease in the expression of Wx. The apparent amylose content (AAC) in the edited lines was decreased by 7.09% and 11.50% compared with that of the wild type, which was the japonica variety Nipponbare with Wxb and the A allele at 1764178, while a complementary line with the G allele showed a lower AAC than the A allele with no effect on other agronomic traits. The AAC of the edited lines showed a higher increase than that of the wild type (Nipponbare, Wxb) in low-nitrogen conditions relative to high-nitrogen conditions. We also developed a dCAPS marker to identify the allele and found that the G allele has widely been used (82.95%) in japonica-bred varieties from Jiangsu Province, China. Overall, we found a functional allele (Wx1764178, A/G) in the Wx promoter that could affect AAC in japonica cultivars and be developed as markers for quality improvement in rice breeding programs.

Pathogenicity Analyses of Rice Blast Fungus (Pyricularia oryzae) from Japonica Rice Area of Northeast China.

In order to understand the pathogenicity differentiation of rice blast fungus (Pyricularia oryzae Cavara), a total of 206 isolates of P. oryzae were collected from three Japonica rice regions in Jilin Province, northeast China. Pathogenicity test showed that the reaction pattern of 25 monogenic differential varieties (MDVs) of rice (Oryza sativa L.) demonstrated a wide pathogenic diversity among the isolates. Those MDVs harbor 23 resistance (R) genes with the susceptible variety Lijiangxintuanheigu (LTH) as control. Virulent isolates of MDVs harboring R genes Pish, Pit, Pia, Pii, Pik-s, Pik, Pita (two lines), and Pita-2 (two lines) had high frequencies ranging from 80 to 100%, to MDVs harboring R genes Pib, Pi5(t), Pik-m, Pi1, Pik-h, Pik-p, Pi7(t), Piz, Piz-5, and Piz-t showed intermediate frequencies ranging from 40 to 80%, and to MDVs with R genes Pi3, Pi9(t), Pi12(t), Pi19(t) and Pi20(t) presented low frequencies ranging only from 0 to 40%. The U-i-k-z-ta pattern of race-named criteria categorized the 206 isolates into 175 races. Sub-unit U73 for Pib, i7 for Pi3 and Pi5(t), k177 for Pik-m/Pik-h/Pik-p, z17 for Pi9(t), and ta332 for Pi20(t) were crucial on pathogenic differences in regions. Twenty-seven standard differential blast isolates (SDBIs) were selected to characterize resistance in rice accessions. This study could help to build a durable identification system against blast in the Japonica rice area of northeast China and enhance our understanding of the differentiation and diversity of blast races in the world.

CRISPR/Cas9-mediated knockout of Bsr-d1 enhances the blast resistance of rice in Northeast China.

KEY MESSAGE: The blast resistance allele of OsBsr-d1 does not exist in most japonica rice varieties of Jilin Province in China. The development of Bsr-d1 knockout mutants via CRISPR/Cas9 enhances broad-spectrum resistance to rice blast in Northeast China. Rice blast is a global disease that has a significant negative impact on rice yield and quality. Due to the complexity and variability of the physiological races of rice blast, controlling rice blast is challenging in agricultural production. Bsr-d1, a negative transcription factor that confers broad-spectrum resistance to rice blast, was identified in the indica rice cultivar Digu; however, its biological function in japonica rice varieties is still unclear. In this study, we analyzed the blast resistance allele of Bsr-d1 in a total of 256 japonica rice varieties from Jilin Province in Northeast China and found that this allele was not present in these varieties. Therefore, we generated Bsr-d1 knockout mutants via the CRISPR/Cas9 system using the japonica rice variety Jigeng88 (JG88) as a recipient variety. Compared with those of the wild-type JG88, the homozygous Bsr-d1 mutant lines KO#1 and KO#2 showed enhanced leaf blast resistance at the seedling stage to several Magnaporthe oryzae (M. oryzae) races collected from Jilin Province in Northeast China. Physiological and biochemical indices revealed that the homozygous mutant lines produced more hydrogen peroxide than did JG88 plants when infected with M. oryzae. Comparative RNA-seq revealed that the DEGs were mainly involved in the synthesis of amide compounds, zinc finger proteins, transmembrane transporters, etc. In summary, our results indicate that the development of Bsr-d1 knockout mutants through CRISPR/Cas9 can enhance the broad-spectrum resistance of rice in Northeast China to rice blast. This study not only provides a theoretical basis for disease resistance breeding involving the Bsr-d1 gene in Northeast China, but also provides new germplasm resources for disease-resistance rice breeding.

Effect of starch and protein on eating quality of japonica rice in Yangtze River Delta.

This study examined four types of japonica rice from Yangtze River Delta, categorized based on amylose content (AC) and protein content (PC): high AC with high PC, high AC with low PC, low AC with high PC, and low AC with low PC. It systematically explored the effect of starch, protein and their interactions on eating quality of japonica rice. Rheological analysis revealed that increased amylose, long chains amylopectin or protein levels during cooking strengthen starch-protein interactions (hydrogen bonding), forming a firm gel network. Scanning electron microscopy showed that increased amylose, long chains amylopectin or protein levels made protein and starch more stable in combination during cooking, limiting starch structure cleavage. Therefore, the eating quality of high AC in similar PC japonica rice and high PC in similar AC japonica rice were poor. Further, correlation and random-forest analysis (RFA) identified amylose as the most influential factor in starch-protein interactions affecting rice eating quality, followed by amylopectin and protein. RFA also revealed that in high AC japonica rice, the interactions of Fb3 and albumin with amylose were more conducive to forming good eating quality. In low AC japonica rice, the interactions of Fb2 and prolamin with amylose were more beneficial.

A comparative study of grain quality and physicochemical properties of premium japonica rice from three typical production regions.

Grain quality indicates rice commodity value. This research compared grain quality and physicochemical properties of premium japonica rice from three production regions, Yangtze River downstream of China (YRDCN), Northeast region of China (NECN) and Japan. Results showed that there were distinct quality and physicochemical characteristics variance among the three groups of japonica rice, while CVs of most quality parameters from low to high was Japan, YRDCN and NECN. YRDCN rice presented obvious lower apparent amylose content (AAC) and ratio of each chain-length sections of amylopectin, and showed higher protein contents especially glutelin and ratio in short and intermediate amylopectin molecules. Among three rice groups, YRDCN rice presented weaker appearance, whereas did not show inferior cooking and eating properties, which was primarily linked to lower AAC. Rice AAC and starch fine structure significantly correlated with pasting parameters, swelling power and solubility, while protein content had close relation with taste analyzer parameters. Results of this study indicated improvement direction for japonica rice of YRDCN, and also provided reference for consumers' rice purchasing selection in accordance with individual taste preference.

COG3 confers the chilling tolerance to mediate OsFtsH2-D1 module in rice.

The chilling stress induced by the global climate change harms rice production, especially at seedling and booting stage, which feed half the population of the world. Although there are key quantitative trait locus genes identified in the individual stage, few genes have been reported and functioned at both stages. Utilizing chromosome segment substitution lines (CSSLs) and a combination of map-based cloning and phenotypes of the mutants and overexpression lines, we identified the major gene Chilling-tolerance in Geng/japonica rice 3 (COG3) of q chilling-tolerance at the booting and seedling stage 11 (qCTBS11) conferred chilling tolerance at both seedling and booting stages. COG3 was significantly upregulated in Nipponbare under chilling treatment compared with its expression in 93-11. The loss-of-function mutants cog3 showed a reduced chilling tolerance. On the contrary, overexpression enhanced chilling tolerance. Genome evolution and genetic analysis suggested that COG3 may have undergone strong selection in temperate japonica during domestication. COG3, a putative calmodulin-binding protein, physically interacted with OsFtsH2 at chloroplast. In cog3-1, OsFtsH2-mediated D1 degradation was impaired under chilling treatment compared with wild-type. Our results suggest that COG3 is necessary for maintaining OsFtsH2 protease activity to regulate chilling tolerance at the booting and seedling stage.

A simplified and improved protocol of rice transformation to cater wide range of rice cultivars.

The latest CRISPR-Cas9-mediated genome editing technology is expected to bring about revolution in rice yield and quality improvement, and thus validation of rice transformation protocols using CRISPR-Cas9-gRNA constructs is the need of the hour. Moreover, regeneration of more number of transgenic rice plants is prerequisite for developing genome-edited rice lines, as recalcitrant rice varieties were shown to have lower editing efficiencies which necessities screening of large number of transgenic plants to find the suitable edits. In the present study, we have simplified the Agrobacterium-mediated rice transformation protocol for both Indica and Japonica rice cultivars using CRISPR/Cas9 empty vector construct, and the protocols have been suitably optimized for getting large numbers of the regenerated plantlets within the shortest possible time. The Japonica transgenic lines were obtained within 65 days and for the Indica cultivars, it took about 76-78 days. We also obtained about 90% regeneration efficiency for both Japonica and Indica cultivars. The transformation efficiency was about 97% in the case of Japonica and 69-83% in the case of Indica rice cultivars. Furthermore, we screened the OsWRKY24 gene editing efficiency by transforming rice cultivars with CRISPR/Cas9 construct harbouring sgRNA against OsWRKY24 gene and found about 90% editing efficiency in Japonica rice cultivars, while 30% of the transformed Indica cultivars were found to be edited. This implicated the presence of a robust repair mechanism in the Indica rice cultivars.

Differences in the metabolites of brown and milled rice grains of semiwaxy and conventional japonica varieties.

In this study, we analyzed the differences in metabolites between semiwaxy japonica rice (Yangnongxiang 28 [YNX28]) and conventional japonica rice (Hongyang 5 [HY5]) before and after brown rice milling. The metabolites of brown and milled rice grains from the two rice varieties were analyzed by LC-MS-based nontargeted metabolomics. A total of 266 differentially abundant metabolites (DMs) were tentatively identified in brown rice grains of YNX28 (YNX28B) compared with milled rice grains of YNX28 (YNX28H), and these included 248 upregulated and 12 downregulated DMs. A total of 273 (234 upregulated and 39 downregulated) DMs were tentatively identified in brown rice grains of HY5 (HY5B) compared with milled rice grains of this variety (HY5H). Kyoto Encyclopedia of Genes and Genomes pathway involved and enrichment analyses revealed that 53 and 7 metabolite pathways were enriched and significantly enriched (p < 0.05), respectively, in the DMs identified in YNX28B compared with YNX28H, and the main enriched pathways were related to starch and sucrose metabolism, glycerol phospholipid metabolism, arginine and proline metabolism, and glycine, serine and threonine metabolism. Forty-six metabolite pathways were enriched in DMs identified in HY5B compared with HY5H, and these included 16 pathways that were significantly enriched (p < 0.05); in addition, the main enriched pathways were related to starch and sucrose metabolism, glycerol phospholipid metabolism, arginine and proline metabolism, and glycine, serine and threonine metabolism. This study provides a theoretical reference for further on the changes in metabolites during rice processing and provides a basis for improving the nutritional quality in rice. PRACTICAL APPLICATION: Original data were obtained regarding the changes of different metabolites in semiwaxy japonica rice and conventional japonica rice before and after processing. The purpose of this study was to investigate the difference of metabolite loss in two rice varieties before and after processing. This paper reports on the differences of metabolites between the two types of japonica rice before and after processing, as well as the changes of key metabolites before and after processing, it also provides important theoretical basis for developing new rice varieties with good nutritional quality.

Effects of Lactic Acid Bacteria Fermentation on the Physicochemical Properties of Rice Flour and Rice Starch and on the Anti-Staling of Rice Bread.

In this study, Lactococcus lactis lactis subspecies 1.2472, Streptococcus thermophilus 1.2718, and thermostable Lactobacillus rhamnosus HCUL 1.1901-1912 were used to ferment rice flour for preparing rice bread. The characteristics of fermented rice bread were studied to elucidate the mechanism by which fermentation improves the anti-staling ability of rice bread. The amylose content of rice flour increased after fermentation. The peak viscosity, attenuation value, final viscosity, recovery value, and gelatinization temperature decreased. Amylopectin was partially hydrolyzed, and the amylose content decreased. The crystallinity of starch decreased, and the minimum crystallinity of Lactococcus lactis subsp. lactis fermented rice starch (LRS) was 11.64%. The thermal characteristics of fermented rice starch, including To, Tp, Tc, and ΔH, were lower than RS (rice starch), and the △H of LRS was the lowest. Meanwhile, LRS exhibited the best anti-staling ability, and with a staling degree of 43.22%. The T22 of the LRF rice flour dough was lower, and its moisture fluidity was the weakest, indicating that moisture was more closely combined with other components. The texture characteristics of fermented rice bread were improved; among these, LRF was the best: the hardness change value was 1.421 times, the elasticity decrease was 2.35%, and the chewability change was 47.07%. There, it provides a theoretical basis for improving the shelf life of bread.

[Adjuvant rice optimization of rice-steamed Rehmanniae Radix and its anti-osteoporosis effect].

The present study aimed to investigate the effect of various adjuvant rice on the quality of rice-steamed Rehmanniae Radix(RSRR) with Japonica rice, millet, yellow rice, black rice, and glutinous rice as raw materials, and analyze the anti-osteoporosis effect of RSRR by the optimal adjuvant rice. On the basis of the established UPLC-MS/MS method for the determination of the content of catalpol and rehmannioside D, comprehensive weighted scoring method was employed to evaluate the effect of various auxiliary rice on the quality of RSRR with the content of catalpol and rehmannioside D, character score, and taste score as indicators to optimize adjuvant rice. The osteoporosis model was induced by ovariectomy in rats. SD rats were randomly divided into a sham operation group, a model group, a positive control group, and low-dose and high-dose groups of Rehmanniae Radix, RSRR, steamed Rehmanniae Radix, and Epimedii Folium-RSRR. After treatment for 12 weeks, body weight, bone calcium content, and bone mineral density were mea-sured. The results showed that Japonica rice was selected as the optimal adjuvant due to the highest comprehensive score of RSRR steamed by Japonica rice. Rehmanniae Radix, RSRR, steamed Rehmanniae Radix, as well as Epimedii Folium-RSRR, could improve osteoporosis by increasing bone calcium content and bone mineral density. RSRR was superior to Rehmanniae Radix in improving osteo-porosis. However, there was no significant difference between RSRR and steamed Rehmanniae Radix. This study confirmed that Japo-nica rice was the optimal adjuvant rice of RSRR and verified the anti-osteoporosis effect of RSRR, which laid a foundation for further research on the pharmacological action and mechanism of RSRR.

Identification of candidate genes for salinity tolerance in Japonica rice at the seedling stage based on genome-wide association study and linkage mapping.

Background: Salinity tolerance plays a vital role in rice cultivation because the strength of salinity tolerance at the seedling stage directly affects seedling survival and final crop yield in saline soils. Here, we combined a genome-wide association study (GWAS) and linkage mapping to analyze the candidate intervals for salinity tolerance in Japonica rice at the seedling stage. Results: We used the Na+ concentration in shoots (SNC), K+ concentration in shoots (SKC), Na+/K+ ratio in shoots (SNK), and seedling survival rate (SSR) as indices to assess the salinity tolerance at the seedling stage in rice. The GWAS identified the lead SNP (Chr12_20864157), associated with an SNK, which the linkage mapping detected as being in qSK12. A 195-kb region on chromosome 12 was selected based on the overlapping regions in the GWAS and the linkage mapping. Based on haplotype analysis, qRT-PCR, and sequence analysis, we obtained LOC_Os12g34450 as a candidate gene. Conclusion: Based on these results, LOC_Os12g34450 was identified as a candidate gene contributing to salinity tolerance in Japonica rice. This study provides valuable guidance for plant breeders to improve the response of Japonica rice to salt stress.

Crop-Weed Introgression Plays Critical Roles in Genetic Differentiation and Diversity of Weedy Rice: A Case Study of Human-Influenced Weed Evolution.

As an important driving force, introgression plays an essential role in shaping the evolution of plant species. However, knowledge concerning how introgression affects plant evolution in agroecosystems with strong human influences is still limited. To generate such knowledge, we used InDel (insertion/deletion) molecular fingerprints to determine the level of introgression from japonica rice cultivars into the indica type of weedy rice. We also analyzed the impact of crop-to-weed introgression on the genetic differentiation and diversity of weedy rice, using InDel (insertion/deletion) and SSR (simple sequence repeat) molecular fingerprints. Results based on the STRUCTURE analysis indicated an evident admixture of some weedy rice samples with indica and japonica components, suggesting different levels of introgression from japonica rice cultivars to the indica type of weedy rice. The principal coordinate analyses indicated indica-japonica genetic differentiation among weedy rice samples, which was positively correlated with the introgression of japonica-specific alleles from the rice cultivars. In addition, increased crop-to-weed introgression formed a parabola pattern of dynamic genetic diversity in weedy rice. Our findings based on this case study provide evidence that human activities, such as the frequent change in crop varieties, can strongly influence weed evolution by altering genetic differentiation and genetic diversity through crop-weed introgression in agroecosystems.

Complete Genome Resource of a Hypervirulent Xanthomonas oryzae pv. oryzae Strain YNCX Isolated from Yunnan Plateau Japonica Rice.

Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight (BLB), is one of the most destructive bacterial pathogens in rice production worldwide. Although several complete genome sequences of Xoo strains have been released in public databases, they are mainly isolated from low-altitude indica rice cultivating areas. Here, a hypervirulent strain, YNCX, isolated from the high-altitude japonica rice-growing region in Yunnan Plateau, was used to extract genomic DNA for PacBio sequencing and Illumina sequencing. After assembly, a high-quality complete genome consisting of a circular chromosome and six plasmids was generated. The genome sequence of YNCX provides a valuable resource for high-altitude races and enables the identification of new virulence TALE effectors, contributing to a better understanding of rice-Xoo interactions.

Global Phosphoproteomic Analysis Reveals the Defense and Response Mechanisms of Japonica Rice under Low Nitrogen Stress.

Nitrogen-based nutrients are the main factors affecting rice growth and development. As the nitrogen (N) application rate increased, the nitrogen use efficiency (NUE) of rice decreased. Therefore, it is important to understand the molecular mechanism of rice plant morphological, physiological, and yield formation under low N conditions to improve NUE. In this study, changes in the rice morphological, physiological, and yield-related traits under low N (13.33 ppm) and control N (40.00 ppm) conditions were performed. These results show that, compared with control N conditions, photosynthesis and growth were inhibited and the carbon (C)/N and photosynthetic nitrogen use efficiency (PNUE) were enhanced under low N conditions. To understand the post-translational modification mechanism underlying the rice response to low N conditions, comparative phosphoproteomic analysis was performed, and differentially modified proteins (DMPs) were further characterized. Compared with control N conditions, a total of 258 DMPs were identified under low N conditions. The modification of proteins involved in chloroplast development, chlorophyll synthesis, photosynthesis, carbon metabolism, phytohormones, and morphology-related proteins were differentially altered, which was an important reason for changes in rice morphological, physiological, and yield-related traits. Additionally, inconsistent changes in level of transcription and protein modification, indicates that the study of phosphoproteomics under low N conditions is also important for us to better understand the adaptation mechanism of rice to low N stress. These results provide insights into global changes in the response of rice to low N stress and may facilitate the development of rice cultivars with high NUE by regulating the phosphorylation level of carbon metabolism and rice morphology-related proteins.

Combined QTL-sequencing, linkage mapping, and RNA-sequencing identify candidate genes and KASP markers for low-temperature germination in Oryza sativa L. ssp. Japonica.

MAIN CONCLUSION: Through QTL-seq, QTL mapping and RNA-seq, six candidate genes of qLTG9 can be used as targets for cold tolerance functional characterization, and six KASP markers can be used for marker-assisted breeding to improve the germination ability of japonica rice at low temperature. The development of direct-seeded rice at high latitudes and altitudes depends on the seed germination ability of rice under a low-temperature environment. However, the lack of regulatory genes for low-temperature germination has severely limited the application of genetics in improving the breeds. Here, we used cultivars DN430 and DF104 with significantly different low-temperature germination (LTG) and 460 F2:3 progeny derived from them to identify LTG regulators by combining QTL-sequencing, linkage mapping, and RNA-sequencing. The QTL-sequencing mapped qLTG9 within a physical interval of 3.4 Mb. In addition, we used 10 Kompetitive allele-specific PCR (KASP) markers provided by the two parents, and qLTG9 was optimized from 3.4 Mb to a physical interval of 397.9 kb and accounted for 20.4% of the phenotypic variation. RNA-sequencing identified qLTG9 as eight candidate genes with significantly different expression within the 397.9 kb interval, six of which possessed SNPs on the promoter and coding regions. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) completely validated the results of these six genes in RNA-sequencing. Subsequently, six non-synonymous SNPs were designed using variants in the coding region of these six candidates. Genotypic analysis of these SNPs in 60 individuals with extreme phenotypes indicated these SNPs determined the differences in cold tolerance between parents. The six candidate genes of qLTG9 and the six KASP markers could be used together for marker-assisted breeding to improve LTG.

Natural variations of chlorophyll fluorescence and ion transporter genes influenced the differential response of japonica rice germplasm with different salt tolerances.

Soil salinity seriously restricts rice growth, development, and production globally. Chlorophyll fluorescence and ion content reflect the level of injury and resistance of rice under salt stress. To understand the differences in the response mechanisms of japonica rice with varying degrees of salt tolerance, we analyzed the chlorophyll fluorescence characteristics and ion homeostasis of 12 japonica rice germplasm accessions by comprehensive evaluation of phenotype, haplotype, and expression of salt tolerance-related genes. The results revealed that salt-sensitive accessions were rapidly affected by the damage due to salinity. Salt tolerance score (STS) and relative chlorophyll relative content (RSPAD) were extremely significantly reduced (p<0.01), and chlorophyll fluorescence and ion homeostasis were influenced by various degrees under salt stress. The STS, RSPAD, and five chlorophyll fluorescence parameters of salt-tolerant accessions (STA) were significantly higher than that of salt-sensitive accessions (SSA). Principal component analysis (PCA) with 13 indices suggested three principal components (PCs), with a cumulative contribution rate of 90.254%, which were used to screen Huangluo (typical salt-tolerant germplasm) and Shanfuliya (typical salt-sensitive germplasm) based on the comprehensive evaluation D-value (DCI ). The expression characteristics of chlorophyll fluorescence genes (OsABCI7 and OsHCF222) and ion transporter protein genes (OsHKT1;5, OsHKT2;1, OsHAK21, OsAKT2, OsNHX1, and OsSOS1) were analyzed. The expressions of these genes were higher in Huangluo than in Shanfuliya under salt stress. Haplotype analysis revealed four key variations associated with salt tolerance, including an SNP (+1605 bp) within OsABCI7 exon, an SSR (-1231 bp) within OsHAK21 promoter, an indel site at OsNHX1 promoter (-822 bp), and an SNP (-1866 bp) within OsAKT2 promoter. Variation in OsABCI7 protein structure and differential expression of these three ion-transporter genes may contribute to the differential response of japonica rice to salt stress.