Mapping of Candidate Genes for Nitrogen Uptake and Utilization in Japonica Rice at Seedling Stage.

Nitrogen (N) is one of the essential nutrients for the growth and development of crops. The adequate application of N not only increases the yield of crops but also improves the quality of agricultural products, but the excessive application of N can cause many adverse effects on ecology and the environment. In this study, genome-wide association analysis (GWAS) was performed under low- and high-N conditions based on 788,396 SNPs and phenotypic traits relevant to N uptake and utilization (N content and N accumulation). A total of 75 QTLs were obtained using GWAS, which contained 811 genes. Of 811 genes, 281 genes showed different haplotypes, and 40 genes had significant phenotypic differences among different haplotypes. Of these 40 genes, 5 differentially expressed genes (Os01g0159250, Os02g0618200, Os02g0618400, Os02g0630300, and Os06g0619000) were finally identified as the more valuable candidate genes based on the transcriptome data sequenced from Longjing31 (low-N-tolerant variety) and Songjing 10 (low-N-sensitive variety) under low- and high-N treatments. These new findings enrich the genetic resources for N uptake and utilization in rice, as well as lay a theoretical foundation for improving the efficiency of N uptake and utilization in rice.

Joint QTL Mapping and Transcriptome Sequencing Analysis Reveal Candidate Genes for Salinity Tolerance in Oryza sativa L. ssp. Japonica Seedlings.

Salinity stress is one of the major abiotic stresses affecting crop growth and production. Rice is an important food crop in the world, but also a salt-sensitive crop, and the rice seedling stage is the most sensitive to salt stress, which directly affects the final yield formation. In this study, two RIL populations derived from the crosses of CD (salt-sensitive)/WD (salt-tolerant) and KY131 (salt-sensitive)/XBJZ (salt-tolerant) were used as experimental materials, and the score of salinity toxicity (SST), the relative shoot length (RSL), the relative shoot fresh weight (RSFW), and the relative shoot dry weight (RSDW) were used for evaluating the degree of tolerance under salt stress in different lines. The genetic linkage map containing 978 and 527 bin markers were constructed in two RIL populations. A total of 14 QTLs were detected on chromosomes 1, 2, 3, 4, 7, 9, 10, 11, and 12. Among them, qSST12-1, qSST12-2, and qRSL12 were co-localized in a 140-kb overlap interval on chromosome 12, which containing 16 candidate genes. Furthermore, transcriptome sequencing and qRT-PCR were analyzed in CD and WD under normal and 120 mM NaCl stress. LOC_Os12g29330, LOC_Os12g29350, LOC_Os12g29390, and LOC_Os12g29400 were significantly induced by salt stress in both CD and WD. Sequence analysis showed that LOC_Os12g29400 in the salt-sensitive parents CD and KY131 was consistent with the reference sequence (Nipponbare), whereas the salt-tolerant parents WD and XBJZ differed significantly from the reference sequence both in the promoter and exon regions. The salt-tolerant phenotype was identified by using two T3 homozygous mutant plants of LOC_Os12g29400; the results showed that the score of salinity toxicity (SST) of the mutant plants (CR-3 and CR-5) was significantly lower than that of the wild type, and the seedling survival rate (SSR) was significantly higher than that of the wild type, which indicated that LOC_Os12g29400 could negatively regulate the salinity tolerance of rice at the seedling stage. The results lay a foundation for the analysis of the molecular mechanism of rice salinity tolerance and the cultivation of new rice varieties.

BSA-Seq for the Identification of Major Genes for EPN in Rice.

Improving rice yield is one of the most important food issues internationally. It is an undeniable goal of rice breeding, and the effective panicle number (EPN) is a key factor determining rice yield. Increasing the EPN in rice is a major way to increase rice yield. Currently, the main quantitative trait locus (QTL) for EPN in rice is limited, and there is also limited research on the gene for EPN in rice. Therefore, the excavation and analysis of major genes related to EPN in rice is of great significance for molecular breeding and yield improvement. This study used japonica rice varieties Dongfu 114 and Longyang 11 to construct an F5 population consisting of 309 individual plants. Two extreme phenotypic pools were constructed by identifying the EPN of the population, and QTL-seq analysis was performed to obtain three main effective QTL intervals for EPN. This analysis also helped to screen out 34 candidate genes. Then, EPN time expression pattern analysis was performed on these 34 genes to screen out six candidate genes with higher expression levels. Using a 3K database to perform haplotype analysis on these six genes, we selected haplotypes with significant differences in EPN. Finally, five candidate genes related to EPN were obtained.

Fine Mapping and Candidate Gene Analysis of Rice Grain Length QTL qGL9.1.

Grain length (GL) is one of the crucial determinants of rice yield and quality. However, there is still a shortage of knowledge on the major genes controlling the inheritance of GL in japonica rice, which severely limits the improvement of japonica rice yields. Here, we systemically measured the GL of 667 F2 and 1570 BC3F3 individuals derived from two cultivated rice cultivars, Pin20 and Songjing15, in order to identify the major genomic regions associated with GL. A novel major QTL, qGL9.1, was mapped on chromosome 9, which is associated with the GL, using whole-genome re-sequencing with bulked segregant analysis. Local QTL linkage analysis with F2 and fine mapping with the recombinant plant revealed a 93-kb core region on qGL9.1 encoding 15 protein-coding genes. Only the expression level of LOC_Os09g26970 was significantly different between the two parents at different stages of grain development. Moreover, haplotype analysis revealed that the alleles of Pin20 contribute to the optimal GL (9.36 mm) and GL/W (3.31), suggesting that Pin20 is a cultivated species carrying the optimal GL variation of LOC_Os09g26970. Furthermore, a functional-type mutation (16398989-bp, G>A) located on an exon of LOC_Os09g26970 could be used as a molecular marker to distinguish between long and short grains. Our experiments identified LOC_Os09g26970 as a novel gene associated with GL in japonica rice. This result is expected to further the exploration of the genetic mechanism of rice GL and improve GL in rice japonica varieties by marker-assisted selection.

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.

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.

qCTB7 positively regulates cold tolerance at booting stage in rice.

KEY MESSAGE: LOC_Os07g07690 on qCTB7 is associated with cold tolerance at the booting stage in rice, and analysis of transgenic plants demonstrated that qCTB7 influenced cold tolerance by altering the morphology and cytoarchitecture of anthers and pollen. Cold tolerance at the booting stage (CTB) in rice can significantly affect yield in high-latitude regions. Although several CTB genes have been isolated, their ability to induce cold tolerance is insufficient to ensure adequate rice yields in cold regions at high latitudes. Here, we identified the PHD-finger domain-containing protein gene qCTB7 using QTL-seq and linkage analysis through systematic measurement of CTB differences and the spike fertility of the Longjing31 and Longdao3 cultivars, resulting in the derivation of 1570 F2 progeny under cold stress. We then characterized the function of qCTB7 in rice. It was found that overexpression of qCTB7 promoted CTB and the same yield as Longdao3 under normal growing conditions while the phenotype of qctb7 knockout showed anther and pollen failure under cold stress. When subjected to cold stress, the germination of qctb7 pollen on the stigma was reduced, resulting in lower spike fertility. These findings indicate that qCTB7 regulates the appearance, morphology, and cytoarchitecture of the anthers and pollen. Three SNPs in the promoter region and coding region of qCTB7 were identified as recognition signals for CTB in rice and could assist breeding efforts to improve cold tolerance for rice production in high latitudes.

Transcriptome and Co-Expression Network Analysis Reveals the Molecular Mechanism of Rice Root Systems in Response to Low-Nitrogen Conditions.

Nitrogen is an important nutrient for plant growth and essential metabolic processes. Roots integrally obtain nutrients from soil and are closely related to the growth and development of plants. In this study, the morphological analysis of rice root tissues collected at different time points under low-nitrogen and normal nitrogen conditions demonstrated that, compared with normal nitrogen treatment, the root growth and nitrogen use efficiency (NUE) of rice under low-nitrogen treatment were significantly improved. To better understand the molecular mechanisms of the rice root system's response to low-nitrogen conditions, a comprehensive transcriptome analysis of rice seedling roots under low-nitrogen and control conditions was conducted in this study. As a result, 3171 differentially expressed genes (DEGs) were identified. Rice seedling roots enhance NUE and promote root development by regulating the genes related to nitrogen absorption and utilization, carbon metabolism, root growth and development, and phytohormones, thereby adapting to low-nitrogen conditions. A total of 25,377 genes were divided into 14 modules using weighted gene co-expression network analysis (WGCNA). Two modules were significantly associated with nitrogen absorption and utilization. A total of 8 core genes and 43 co-expression candidates related to nitrogen absorption and utilization were obtained in these two modules. Further studies on these genes will contribute to the understanding of low-nitrogen adaptation and nitrogen utilization mechanisms in rice.

Mapping of Candidate Genes in Response to Low Nitrogen in Rice Seedlings.

Nitrogen is not only a macronutrient essential for crop growth and development, but also one of the most critical nutrients in farmland ecosystem. Insufficient nitrogen supply will lead to crop yield reduction, while excessive application of nitrogen fertilizer will cause agricultural and eco-environment damage. Therefore, mining low-nitrogen tolerant rice genes and improving nitrogen use efficiency are of great significance to the sustainable development of agriculture. This study was conducted by Genome-wide association study on a basis of two root morphological traits (root length and root diameter) and 788,396 SNPs of a natural population of 295 rice varieties. The transcriptome of low-nitrogen tolerant variety (Longjing 31) and low-nitrogen sensitive variety (Songjing 10) were sequenced between low and high nitrogen treatments. A total of 35 QTLs containing 493 genes were mapped. 3085 differential expressed genes were identified. Among these 493 genes, 174 genes showed different haplotype patterns. There were significant phenotype differences among different haplotypes of 58 genes with haplotype differences. These 58 genes were hypothesized as candidate genes for low nitrogen tolerance related to root morphology. Finally, six genes (Os07g0471300, Os11g0230400, Os11g0229300, Os11g0229400, Os11g0618300 and Os11g0229333) which expressed differentially in Longjing 31 were defined as more valuable candidate genes for low-nitrogen tolerance. The results revealed the response characteristics of rice to low-nitrogen, and provided insights into regulatory mechanisms of rice to nitrogen deficiency.

Response of Rice with Overlapping Growth Stages to Water Stress by Assimilates Accumulation and Transport and Starch Synthesis of Superior and Inferior Grains.

Drought stress at jointing-booting directly affects plant growth and productivity in rice. Limited by natural factors, the jointing and booting stages of short-growth-period rice varieties are highly overlapped in high-latitude areas, which are more sensitive to water deficit. However, little is known about the dry matter translocation in rice and the strategies of starch synthesis and filling of superior and inferior grains under different drought stress was unclear. In this study, the rice plants were subjected to three degrees of drought stress (-10 kPa, -25 kPa, -40 kPa) for 15 days during the jointing-booting stage; we investigated dry matter accumulation and translocation, grain filling and enzyme activities to starch synthesis of superior and inferior grains in rice with overlapping growth stages from 2016 to 2017. The results showed that drought stress significantly reduced dry matter accumulation in the stems and leaves. Mild and moderate drought increased dry matter translocation efficiency. However, severe drought stress largely limited the dry matter accumulation and translocation. A large amount of dry matter remains in vegetative organs under severe drought stress. The high content in NSC in stem and sheath plays a key role in resisting drought stress. The drought stress at jointing-booting directly caused a change in the grain filling strategy. Under moderate and severe drought, the grain-filling active period of the superior grains was shortened to complete the necessary reproductive growth. The grain-filling active period of the inferior grains was significantly prolonged to avoid a decrease in grain yield. The significant decrease in the grain-filling rate of the superior and inferior grains caused a reduction in the thousand-grain weight. In particular, the influence of the grain-filling rate of inferior grains on the thousand-grain weight was more significant. Drought stress changed the starch synthesis strategies of the superior and inferior grains. Soluble starch synthase and starch branching enzyme activities of inferior grains increased significantly under drought stress. GBSS activity was not sensitive to drought stress. Therefore, amylose content was decreased and amylopectin synthesis was enhanced under drought stress, especially in inferior grains.

Identification and Functional Analysis of the Caffeic Acid O-Methyltransferase (COMT) Gene Family in Rice (Oryza sativa L.).

Caffeic acid O-methyltransferase (COMT) is one of the core enzymes involved in lignin synthesis. However, there is no systematic study on the rice COMT gene family. We identified 33 COMT genes containing the methyltransferase-2 domain in the rice genome using bioinformatic methods and divided them into Group I (a and b) and Group II. Motifs, conserved domains, gene structure and SNPs density are related to the classification of OsCOMTs. The tandem phenomenon plays a key role in the expansion of OsCOMTs. The expression levels of fourteen and thirteen OsCOMTs increased or decreased under salt stress and drought stress, respectively. OsCOMTs showed higher expression levels in the stem. The lignin content of rice was measured in five stages; combined with the expression analysis of OsCOMTs and multiple sequence alignment, we found that OsCOMT8, OsCOMT9 and OsCOMT15 play a key role in the synthesis of lignin. Targeted miRNAs and gene ontology annotation revealed that OsCOMTs were involved in abiotic stress responses. Our study contributes to the analysis of the biological function of OsCOMTs, which may provide information for future rice breeding and editing of the rice genome.

OsWRKY115 on qCT7 links to cold tolerance in rice.

KEY MESSAGE: qCT7, a novel QTL for increasing seedling cold tolerance in rice, was fine-mapped to a 70.9-kb region on chromosome 7, and key OsWRKY115 was identified in transgenic plants. Cold stress caused by underground cold-water irrigation seriously limits rice productivity. We systemically measured the cold-responsive traits of 2,570 F2 individuals derived from two widely cultivated rice cultivars, Kong-Yu-131 and Dong-Nong-422, to identify the major genomic regions associated with cold tolerance. A novel major QTL, qCT7, was mapped on chromosome 7 associated with the cold tolerance and survival, using whole-genome re-sequencing with bulked segregant analysis. Local QTL linkage analysis with F2 and fine mapping with recombinant plant revealed a 70.9-kb core region on qCT7 encoding 13 protein-coding genes. Only the LOC_Os07g27670 expression level encoding the OsWRKY115 transcription factor on the locus was specifically induced by cold stress in the cold-tolerant cultivar. Moreover, haplotype analysis and the KASP8 marker indicated that OsWRKY115 was significantly associated with cold tolerance. Overexpression and knockout of OsWRKY115 significantly affected cold tolerance in seedlings. Our experiments identified OsWRKY115 as a novel regulatory gene associated with cold response in rice, and the Kong-Yu-131 allele with specific cold-induced expression may be an important molecular variant.

BSA-Seq and Fine Linkage Mapping for the Identification of a Novel Locus (qPH9) for Mature Plant Height in Rice (Oryza sativa).

BACKGROUND: Plant height is a key factor in the determination of rice yield since excessive height can easily cause lodging and reduce yield. Therefore, the identification and analysis of plant height-related genes to elucidate their physiological, biochemical, and molecular mechanisms have significant implications for rice breeding and production. RESULTS: High-throughput quantitative trait locus (QTL) sequencing analysis of a 638-individual F2:3 mapping population resulted in the identification of a novel height-related QTL (qPH9), which was mapped to a 2.02-Mb region of Chromosome 9. Local QTL mapping, which was conducted using 13 single nucleotide polymorphism (SNP)-based Kompetitive allele-specific PCR (KASP) markers for the qPH9 region, and traditional linkage analysis, facilitated the localization of qPH9 to a 126-kb region that contained 15 genes. Subsequent haplotype and sequence analyses indicated that OsPH9 was the most probable candidate gene for plant height at this locus, and functional analysis of osph9 CRISPR/Cas9-generated OsPH9 knockout mutants supported this conclusion. CONCLUSION: OsPH9 was identified as a novel regulatory gene associated with plant height in rice, along with a height-reducing allele in 'Dongfu-114' rice, thereby representing an important molecular target for rice improvement. The findings of the present study are expected to spur the investigation of genetic mechanisms underlying rice plant height and further the improvement of rice plant height through marker-assisted selection.

An integrated analysis of the rice transcriptome and lipidome reveals lipid metabolism plays a central role in rice cold tolerance.

BACKGROUND: Rice (Oryza sativa L.) is one of the most widely grown food crops, and its yield and quality are particularly important for a warm-saturated diet. Cold stress restricts rice growth, development, and yield; however, the specific mechanism of cold tolerance in rice remains unknown. RESULTS: The analysis of leaf physiological and photosynthetic characteristics showed that the two rice varieties were significantly affected by cold stress, but the cold-tolerant variety KY131 had more stable physiological characteristics, maintaining relatively good photosynthetic capacity. To better explore the transcriptional regulation mechanism and biological basis of rice response to cold stress, a comprehensive analysis of the rice transcriptome and lipidome under low temperature and control temperature conditions was carried out. The transcriptomic analysis revealed that lipid metabolism, including membrane lipid and fatty acid metabolism, may be an important factor in rice cold tolerance, and 397 lipid metabolism related genes have been identified. Lipidomics data confirmed the importance of membrane lipid remodeling and fatty acid unsaturation for rice adaptation to cold stress. This indicates that the changes in the fluidity and integrity of the photosynthetic membrane under cold stress lead to the reduction of photosynthetic capacity, which could be relieved by increased levels of monogalactosyldiacylglycerol that mainly caused by markedly increased expression of levels of 1,2-diacylglycerol 3-beta-galactosyltransferase (MGD). The upregulation of phosphatidate phosphatase (PAP2) inhibited the excessive accumulation of phosphatidate (PA) to produce more phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG), thereby preventing of membrane phase transition under cold stress. In addition, fatty acid ß-oxidation is worth further study in rice cold tolerance. Finally, we constructed a metabolic model for the regulatory mechanism of cold tolerance in rice, in which the advanced lipid metabolism system plays a central role. CONCLUSIONS: Lipidome analysis showed that membrane lipid composition and unsaturation were significantly affected, especially phospholipids and galactolipids. Our study provides new information to further understand the response of rice to cold stress.

QTL mapping and candidate gene mining of flag leaf size traits in Japonica rice based on linkage mapping and genome-wide association study.

BACKGROUND: As one of the most important factors of the japonica rice plant, leaf shape affects the photosynthesis and carbohydrate accumulation directly. Mining and using new leaf shape related genes/QTLs can further enrich the theory of molecular breeding and accelerate the breeding process of japonica rice. METHODS: In the present study, 2 RILs and a natural population with 295 japonica rice varieties were used to map QTLs for flag leaf length (FL), flag leaf width (FW) and flag leaf area (FLA) by linkage analysis and genome-wide association study (GWAS) throughout 2 years. RESULTS: A total of 64 QTLs were detected by 2 ways, and pleiotropic QTLs qFL2 (Chr2_33,332,579) and qFL10 (Chr10_10,107,835; Chr10_10,230,100) consisted of overlapping QTLs mapped by linkage analysis and GWAS throughout the 2 years were identified. CONCLUSIONS: The candidate genes LOC_Os02g54254, LOC_Os02g54550, LOC_Os10g20160, LOC_Os10g20240, LOC_Os10g20260 were obtained, filtered by linkage disequilibrium (LD), and haplotype analysis. LOC_Os10g20160 (SD-RLK-45) showed outstanding characteristics in quantitative real-time PCR (qRT-PCR) analysis in leaf development period, belongs to S-domain receptor-like protein kinases gene and probably to be a main gene regulating flag leaf width of japonica rice. The results of this study provide valuable resources for mining the main genes/QTLs of japonica rice leaf development and molecular breeding of japonica rice ideal leaf shape.

The Response of Grain Yield and Root Morphological and Physiological Traits to Nitrogen Levels in Paddy Rice.

Rice (Oryza sativa L.) is an important crop in China. Although it is known that its yield is restricted by nitrogen (N) supply, the response of the root system to N supply specifically has not been systematically explored. This study aimed to investigate the effect of N uptake on grain yield to clarify the relationships between root morphophysiological traits and N uptake, and to understand relation between phytohormones and root morphophysiological traits. Two N-efficient absorption cultivars (NEAs) and two N-inefficient absorption cultivars (NIAs) were grown in the field, and three N conditions, deficient N (60 kg ha-1), intermediate N (180 kg ha-1), and sufficient N (240 kg ha-1), were applied during the growing season. The results showed higher dry matter and grain yield in NEAs than in NIAs, which was mainly attributed to increased N uptake in the mid- and late growth stages under all N conditions. And NEAs have different root regulation methods to obtain higher N accumulation and yield under different N supply conditions. Under lower N conditions, compared with NIAs, NEAs shown greater total root length, root oxidation activity, and root active absorbing surface area and smaller root diameter owing to higher indole-3-acetic acid and cytokinin content and lower 1-aminocyclopropane-1-carboxylic acid content in the early growth stages to respond to low N stress faster, laying a morphophysiological basis for its high N-uptake capacity in the mid- and late growth stages. Under higher N conditions, NEAs had higher root oxidation activity and root active absorbing surface area for N uptake and yield formation owing to higher abscisic acid and cytokinin content in the mid- and late growth stages, which improved the seed setting rate, thereby increasing the rice grain yield. These results suggest that NEAs can optimize the morphophysiological characteristics of roots through phytohormone regulation to adapt to different nutrient conditions, thereby promoting N accumulation and yield formation in rice.

Combining QTL-seq and linkage mapping to fine map a candidate gene in qCTS6 for cold tolerance at the seedling stage in rice.

BACKGROUND: Cold stress caused by low temperatures is an important factor restricting rice production. Identification of cold-tolerance genes that can stably express in cold environments is crucial for molecular rice breeding. RESULTS: In this study, we employed high-throughput quantitative trait locus sequencing (QTL-seq) analyses in a 460-individual F2:3 mapping population to identify major QTL genomic regions governing cold tolerance at the seedling stage in rice. A novel major QTL (qCTS6) controlling the survival rate (SR) under low-temperature conditions of 9°C/10 days was mapped on the 2.60-Mb interval on chromosome 6. Twenty-seven single-nucleotide polymorphism (SNP) markers were designed for the qCST6 region based on re-sequencing data, and local QTL mapping was conducted using traditional linkage analysis. Eventually, we mapped qCTS6 to a 96.6-kb region containing 13 annotated genes, of which seven predicted genes contained 13 non-synonymous SNP loci. Quantitative reverse transcription PCR analysis revealed that only Os06g0719500, an OsbZIP54 transcription factor, was strongly induced by cold stress. Haplotype analysis confirmed that +376 bp (T>A) in the OsbZIP54 coding region played a key role in regulating cold tolerance in rice. CONCLUSION: We identified OsbZIP54 as a novel regulatory gene associated with rice cold-responsive traits, with its Dongfu-104 allele showing specific cold-induction expression serving as an important molecular variation for rice improvement. This result is expected to further exploration of the genetic mechanism of rice cold tolerance at the seedling stage and improve cold tolerance in rice varieties by marker-assisted selection.

Candidate Gene Analysis for Nitrogen Absorption and Utilization in Japonica Rice at the Seedling Stage Based on a Genome-Wide Association Study.

Over-application of nitrogen (N) fertilizer in fields has had a negative impact on both environment and human health. Domesticated rice varieties with high N use efficiency (NUE) reduce fertilizer requirements, enabling sustainable agriculture. Genome-wide association study (GWAS) analysis of N absorption and utilization traits under low and high N conditions was performed to obtain 12 quantitative trait loci (QTLs) based on genotypic data including 151,202 single-nucleotide polymorphisms (SNPs) developed by re-sequencing 267 japonica rice varieties. Eighteen candidate genes were obtained by integrating GWAS and transcriptome analyses; among them, the functions of OsNRT2.4, OsAMT1.2, and OsAlaAT genes in N transport and assimilation have been identified, and OsJAZ12 and OsJAZ13 also play important roles in rice adaptation to abiotic stresses. A NUE-related candidate gene, OsNAC68, was identified by quantitative real-time PCR (qRT-PCR) analyses. OsNAC68 encodes a NAC transcription factor and has been shown to be a positive regulator of the drought stress response in rice. Overexpression of OsNAC68 significantly increased rice NUE and grain yield under deficient N conditions, but the difference was not significant under sufficient N conditions. NUE and grain yield significantly decreased under both N supply conditions in the osbnac68 mutant. This study provides crucial insights into the genetic basis of N absorption and utilization in rice, and a NUE-related gene, OsNAC68, was cloned to provide important resources for rice breeding with high NUE and grain yield.

Identification of Candidate Genes Conferring Cold Tolerance to Rice (Oryza sativa L.) at the Bud-Bursting Stage Using Bulk Segregant Analysis Sequencing and Linkage Mapping.

Low-temperature tolerance during the bud-bursting stage is an important characteristic of direct-seeded rice. The identification of cold-tolerance quantitative trait loci (QTL) in species that can stably tolerate cold environments is crucial for the molecular breeding of rice with such traits. In our study, high-throughput QTL-sequencing analyses were performed in a 460-individual F2 : 3 mapping population to identify the major QTL genomic regions governing cold tolerance at the bud-bursting (CTBB) stage in rice. A novel major QTL, qCTBB9, which controls seed survival rate (SR) under low-temperature conditions of 5°C/9 days, was mapped on the 5.40-Mb interval on chromosome 9. Twenty-six non-synonymous single-nucleotide polymorphism (nSNP) markers were designed for the qCTBB9 region based on re-sequencing data and local QTL mapping conducted using traditional linkage analysis. We mapped qCTBB9 to a 483.87-kb region containing 58 annotated genes, among which six predicted genes contained nine nSNP loci. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed that only Os09g0444200 was strongly induced by cold stress. Haplotype analysis further confirmed that the SNP 1,654,225 bp in the Os09g0444200 coding region plays a key role in regulating the cold tolerance of rice. These results suggest that Os09g0444200 is a potential candidate for qCTBB9. Our results are of great significance to explore the genetic mechanism of rice CTBB and to improve the cold tolerance of rice varieties by marker-assisted selection.