Genetic Effects of Grain Quality Enhancement in Indica Hybrid Rice: Insights for Molecular Design Breeding.

Improving rice quality remains a crucial breeding objective, second only to enhancing yield, yet progress in quality improvement lags behind yield. The high temperature and ripening conditions in Southern China often result in poor rice quality, impacting hybrid rice production and utilization. Therefore, to address this challenge, analyzing the molecular basis of high-quality traits is essential for molecular design breeding of high-quality hybrid rice varieties. In this study, we investigated the molecular basis of grain shape, amylose content, gel consistency, gelatinization temperature, and aroma, which influence rice quality. We discovered that quality related alleles gs3, GW7TFA, gw8, chalk5, Wxb, ALKTT, and fgr can enhance rice quality when applied in breeding programs. Polymerization of gs3, GW7TFA, gw8, and chalk5 genes improves rice appearance quality. The gs3 and GW7TFA allele polymerization increasing the grain's length-width ratio, adding the aggregation of gw8 allele can further reducing grain width. The chalk5 gene regulates low chalkiness, but low correlation to chalkiness was exhibited with grain widths below 2.0 mm, with minimal differences between Chalk5 and chalk5 alleles. Enhancing rice cooking and eating quality is achieved through Wxb and ALKTT gene polymerization, while introducing the fgr(E7) gene significantly improved rice aroma. Using molecular marker-assisted technology, we aggregated these genes to develop a batch of indica hybrid rice parents with improved rice quality are obtained. Cross-combining these enhanced parents can generate new, high-quality hybrid rice varieties suitable for cultivation in Southern China. Therefore, our findings contribute to a molecular breeding model for grain quality improvement in high-quality indica hybrid rice. This study, along with others, highlights the potential of molecular design breeding for enhancing complex traits, particularly rice grain quality.

Cooked Rice Textural Properties and Starch Physicochemical Properties from New Hybrid Rice and Their Parents.

Although great progress has been made in the development of hybrid rice with increased yield, challenges for the improvement of grain quality still remain. In this study, the textural properties of cooked rice and physicochemical characteristics of starch were investigated for 29 new hybrid rice derived from 5 sterile and 11 restorer rice lines. Except for one sterile line Te A (P1) with high apparent amylose content (AAC) (26.9%), all other parents exhibited a low AAC. Gui 263 demonstrated the highest AAC (20.6%) among the restorer lines, so the Te A/Gui 263 hybrid displayed the highest AAC (23.1%) among all the hybrid rice. The mean AAC was similar between sterile, restorer lines and hybrid rice. However, the mean hardness of cooked rice and gels of sterile lines were significantly higher than that of restorer lines and hybrid rice (p < 0.05). Pasting temperature and gelatinization temperatures were significantly higher in the hybrids than in the restorer lines (p < 0.05). Cluster analysis based on the physicochemical properties divided the parents and hybrid rice into two major groups. One group included P1 (Te A), P12 and P14 and three hybrid rice derived from P1, while the other group, including 39 rice varieties, could be further divided into three subgroups. AAC showed significant correlation with many parameters, including peak viscosity, hot peak viscosity, cold peak viscosity, breakdown, setback, onset temperature, peak temperature, conclusion temperature, enthalpy of gelatinization, gel hardness and cooked rice hardness (p < 0.05). Principal component analysis revealed that the first component, comprised of the AAC, peak viscosity, breakdown, setback, onset temperature, peak temperature, conclusion temperature and gel hardness, explained 44.1% of variance, suggesting AAC is the most important factor affecting the grain quality of hybrid rice. Overall, this study enables targeted improvements to key rice grain quality attributes, particularly AAC and textural properties, that will help to develop superior rice varieties.

Molecular basis of genetic improvement for key rice quality traits in Southern China.

Grain qualities including milling quality, appearance quality, eating and cooking quality, and nutritional quality are important indicators in rice breeding. Significant achievements in genetic improvement of rice quality have been made. In this study, we analyzed the variation patterns of 16 traits in 1570 rice varieties and found significant improvements in appearance quality and eating and cooking quality, particularly in hybrid rice. Through genome-wide association study and allelic functional nucleotide polymorphisms analysis of quality trait genes, we found that ALK, FGR1, FLO7, GL7/GW7, GLW7, GS2, GS3, ONAC129, OsGRF8, POW1, WCR1, and Wx were associated with the genetic improvement of rice quality traits in Southern China. Allelic functional nucleotide polymorphisms analysis of 13 important rice quality genes, including fragrance gene fgr, were performed using the polymerase chain reaction amplification refractory mutation system technology. The results showed that Gui516, Gui569, Gui721, Ryousi, Rsimiao, Rbasi, and Yuehui9802 possessed multiple superior alleles. This study elucidates the phenotypic changes and molecular basis of key quality traits of varieties in Southern China. The findings will provide guidance for genetic improvement of rice quality and the development of new varieties.

Editing of rice (Oryza sativa L.) OsMKK3 gene using CRISPR/Cas9 decreases grain length by modulating the expression of photosystem components.

Grain size is one of the most important agronomic traits for grain yield determination in rice. To better understand the proteins that are regulated by the grain size regulatory gene OsMKK3, this gene was knocked out using the CRISPR/Cas9 system, and tandem mass tag (TMT) labeling combined with liquid chromatograph-tandem mass spectrometry analysis was performed to study the regulation of proteins in the panicle. Quantitative proteomic screening revealed a total of 106 differentially expressed proteins (DEPs) via comparison of the OsMKK3 mutant line to the wild-type YexiangB, including 15 and 91 up-regulated and down-regulated DEPs, respectively. Pathway analysis revealed that DEPs were enriched in metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and photosynthesis. Strong interactions were detected among seven down-regulated proteins related to photosystem components in the protein-protein interaction network, and photosynthetic rate was decreased in mutant plants. The results of the liquid chromatography-parallel reaction monitoring/mass spectromery analysis and western blot analysis were consistent with the results of the proteomic analysis, and the results of the quantitative reverse transcription polymerase chain reaction analysis revealed that the expression levels of most candidate genes were consistent with protein levels. Overall, OsMKK3 controls grain size by regulating the protein content in cells. Our findings provide new candidate genes that will aid the study of grain size regulatory mechanisms associated with the mitogen-activated protein kinase (MAPK) signaling pathway.

Test and Analysis of High-Permeability Material's Microstructure in Magnetic Shielding Device.

The magnetic shielding device is used to provide an extreme weak magnetic field, which plays a key role in variety of fields. Since the high-permeability material constituting the magnetic shielding device determines the magnetic shielding performance, it is important to evaluate the property of the high-permeability material. In this paper, the relationship between the microstructure and the magnetic properties of the high-permeability material is analyzed using minimum free energy principle based on magnetic domain theory, and the test method of the material's microstructure including the material composition, the texture and the grain structure to reflect the magnetic properties is put forward. The test result shows that the grain structure is closely related to the initial permeability and the coercivity, which is highly consistent with the theory. As a result, it provides a more efficient way to evaluate the property of the high-permeability material. The test method proposed in the paper has important significance in the high efficiency sampling inspection of the high-permeability material.

Utilization of natural alleles for heat adaptability QTLs at the flowering stage in rice.

BACKGROUND: Heat stress threatens rice yield and quality at flowering stage. In this study, average relative seed setting rate under heat stress (RHSR) and genotypes of 284 varieties were used for a genome-wide association study. RESULTS: We identified eight and six QTLs distributed on chromosomes 1, 3, 4, 5, 7 and 12 in the full population and indica, respectively. qHTT4.2 was detected in both the full population and indica as an overlapping QTL. RHSR was positively correlated with the accumulation of heat-tolerant superior alleles (SA), and indica accession contained at least two heat-tolerant SA with average RHSR greater than 43%, meeting the needs of stable production and heat-tolerant QTLs were offer yield basic for chalkiness degree, amylose content, gel consistency and gelatinization temperature. Chalkiness degree, amylose content, and gelatinization temperature under heat stress increased with accumulation of heat-tolerant SA. Gel consistency under heat stress decreased with polymerization of heat-tolerant SA. The study revealed qHTT4.2 as a stable heat-tolerant QTL that can be used for breeding that was detected in the full population and indica. And the grain quality of qHTT4.2-haplotype1 (Hap1) with chalk5, wx, and alk was better than that of qHTT4.2-Hap1 with CHALK5, WX, and ALK. Twelve putative candidate genes were identified for qHTT4.2 that enhance RHSR based on gene expression data and these genes were validated in two groups. Candidate genes LOC_Os04g52830 and LOC_Os04g52870 were induced by high temperature. CONCLUSIONS: Our findings identify strong heat-tolerant cultivars and heat-tolerant QTLs with great potential value to improve rice tolerance to heat stress, and suggest a strategy for the breeding of yield-balance-quality heat-tolerant crop varieties.

Testing and Analysis Method of Low Remanence Materials for Magnetic Shielding Device.

Magnetic shielding devices with a grid structure of multiple layers of highly magnetically permeable materials (such as permalloy) can achieve remanent magnetic fields at the nanotesla (nT) level or even lower. The remanence of the material inside the magnetic shield, such as the building materials used in the support structure, can cause serious damage to the internal remanence of the magnetic shield. Therefore, it is of great significance to detect the remanence of the materials used inside the magnetic shielding device. The existing test methods do not limit the test environment, the test process is vulnerable to additional magnetic field interference and did not consider the real results of the material in the weak magnetic environment. In this paper, a novel method of measuring the remanence of materials in a magnetic shielding cylinder is proposed, which prevents the interference of the earth's magnetic field and reduces the measurement error. This method is used to test concrete components, composite materials and metal materials commonly applicated in magnetic shielding devices and determine the materials that can be used for magnetic shielding devices with 1 nT, 10 nT and 100 nT as residual magnetic field targets.

TMT-based quantitative proteomic analysis of indica rice cultivars reveals that novel components of the signaling pathways might play a role in grain length regulation.

Grain length is one of the most important rice grain appearance components. To better understand the protein regulated by grain length in indica rice, the tandem mass tag (TMT) labeling combined with LC-MS/MS analysis was used for quantitative identification of differentially regulated proteins by comparing six long-grain cultivars (MeiB, LongfengB, YexiangB, FengtianB, WantaiB, and DingxiangB) to the short-grain cultivar BoB, respectively. A total of 6622 proteins were detected for quantitative analysis by comparing protein content of six long-grain cultivars to the short-grain cultivar, and 715 proteins were significantly regulated, consisting of 336 uniquely over-accumulated proteins and 355 uniquely down-accumulated proteins. KEGG pathway analysis revealed that most of accumulated proteins are involved in metabolic pathways, biosynthesis of secondary metabolites and phenylpropanoid biosynthesis. Four down-accumulated proteins maybe involved in the signaling pathways for grain length regulation. LC-PRM/MS quantitative analysis was used to analyze 10 differentially expressed proteins. The results were almost consistent with the TMT quantitative analysis. qRT-PCR analysis results showed that the transcription level was not always parallel to the protein content. This study identified many novel grain length accumulated proteins through the quantitative proteomics approach, providing candidate genes for further study of grain size regulatory mechanisms. SIGNIFICANCE: Rice grain length is one of the most important characteristics influencing appearance and yield. Six long-grain cultivars (MeiB, LongfengB, YexiangB, FengtianB, WantaiB, and DingxiangB obtained in Guangxi province of China from the 2000s to 2020s) and one short-grain cultivar (BoB obtained in Guangxi province of China in 1980s) were used for comparative analyses. Totally, 715 differentially expressed proteins (DEPs) were identified using TMT-base proteomic analysis. The numbers of DEPs increased as the grain length increased. 4 DEPs may be related to rice's signaling pathways for grain size regulation. A total of 85 DEPs regulated in at least four long-grain cultivars compared with the short-grain cultivar BoB, and 7 proteins were over-accumulated, and 3 proteins were down-accumulated in six long-grain cultivars. These findings provide valuable information to better understand the mechanisms of protein regulation by grain length in rice.

Evaluation of optical parameters for a microminiature Rb vapor cell in a dual-beam SERF magnetometer.

In the spin-exchange relaxation-free (SERF) magnetometer of a perpendicular pump-probe configuration, the pump and probe beam characteristics significantly affect the performance. In this paper, an efficient evaluation of optical parameters to improve the sensitivity of a miniature magnetometer has been presented. We have determined the pump light's optimal intensity and wavelength through theoretical analysis and the zero-field resonance experiments. Chirp signals are applied to measure the optical rotations at different probe intensities and frequencies. Through theoretical and experimental analysis of noise source characterization under different beam intensities and wavelengths, we demonstrate that dual-beam magnetometer performance is mainly limited by photon shot noise. Based on the optimum pump and probe beam parameters, we demonstrate magnetic field sensitivity of 6.3 fT/Hz in an 87Rb vapor cell filled with nitrogen gas, with an active measurement volume of 3 × 3 × 3 mm3.

ITRAQ-based quantitative proteomic analysis of japonica rice seedling during cold stress.

Low temperature is one of the important environmental factors that affect rice growth and yield. To better understand the japonica rice responses to cold stress, isobaric tags for a relative and absolute quantification (iTRAQ) labeling-based quantitative proteomics approach was used to detected changes in protein levels. Two-week-old seedlings of the cold tolerant rice variety Kongyu131 were treated at 8°C for 24, 48 and 72 h, then the total proteins were extracted from tissues and used for quantitative proteomics analysis. A total of 5082 proteins were detected for quantitative analysis, of which 289 proteins were significantly regulated, consisting of 169 uniquely up-regulated proteins and 125 uniquely down-regulated proteins in cold stress groups relative to the control group. Functional analysis revealed that most of the regulated proteins are involved in photosynthesis, metabolic pathway, biosynthesis of secondary metabolites and carbon metabolism. Western blot analysis showed that protein regulation was consistent with the iTRAQ data. The corresponding genes of 25 regulated proteins were used for quantitative real time PCR analysis, and the results showed that the mRNA level was not always parallel to the corresponding protein level. The importance of our study is that it provides new insights into cold stress responses in rice with respect to proteomics and provides candidate genes for cold-tolerance rice breeding.

Optimized gas pressure of an Rb vapor cell in a single-beam SERF magnetometer.

We present a theoretical and experimental study of a single-beam spin-exchange relaxation-free magnetometer in 87Rb vapor cells under different nitrogen gas pressures. The spin relaxation rate is a key component to limit the magnetic sensitivity, and the zero-field resonance method was used to measure the spin relaxation rates of different alkali metal cells. Simultaneously, in a single-beam spin-exchange-relaxation-free (SERF) magnetometer, we demonstrated that the fundamental magnetic field sensitivity was also limited by the pumping light intensity. Based on our theoretical analysis and experimental results, we determined the optimal pumping light intensity and optimal gas pressure. We experimentally demonstrated that the magnetic field sensitivity was 8.89 fT/Hz in the single-beam configuration, with an active measurement volume of 3 × 3 × 3~mm3.

Natural Variation in OsMKK3 Contributes to Grain Size and Chalkiness in Rice.

Rice (Oryza sativa L.) is an important staple food crop for more than half of the world's population. Enhancing the grain quality and yield of rice to meet growing demand remains a major challenge. Here, we show that OsMKK3 encode a MAP kinase kinase that controls grain size and chalkiness by affecting cell proliferation in spikelet hulls. We showed that OsSPL16, GS5, and GIF1 have a substantial effect on the OsMKK3-regulated grain size pathway. OsMKK3 has experienced strong directional selection in indica and japonica. Wild rice accessions contained four OsMKK3 haplotypes, suggesting that the OsMKK3 haplotypes present in cultivated rice likely originated from different wild rice accessions during rice domestication. OsMKK3-Hap1, gs3, and gw8 were polymerized to enhance the grain length. Polymerization of beneficial alleles, such as OsMKK3-Hap1, gs3, gw8, fgr, alk, chalk5, and wx, also improved the quality of hybrid rice. Overall, the results indicated that beneficial OsMKK3 alleles could be used for genomic-assisted breeding for rice cultivar improvement and be polymerized with other beneficial alleles.

Transcriptomic profiling of germinating seeds under cold stress and characterization of the cold-tolerant gene LTG5 in rice.

BACKGROUND: Low temperature is a limiting factor of rice productivity and geographical distribution. Wild rice (Oryza rufipogon Griff.) is an important germplasm resource for rice improvement. It has superior tolerance to many abiotic stresses, including cold stress, but little is known about the mechanism underlying its resistance to cold. RESULTS: This study elucidated the molecular genetic mechanisms of wild rice in tolerating low temperature. Comprehensive transcriptome profiles of two rice genotypes (cold-sensitive ce 253 and cold-tolerant Y12-4) at the germinating stage under cold stress were comparatively analyzed. A total of 42.44-68.71 million readings were obtained, resulting in the alignment of 29,128 and 30,131 genes in genotypes 253 and Y12-4, respectively. Many common and differentially expressed genes (DEGs) were analyzed in the cold-sensitive and cold-tolerant genotypes. Results showed more upregulated DEGs in the cold-tolerant genotype than in the cold-sensitive genotype at four stages under cold stress. Gene ontology enrichment analyses based on cellular process, metabolic process, response stimulus, membrane part, and catalytic activity indicated more upregulated genes than downregulated ones in the cold-tolerant genotype than in the cold-sensitive genotype. Quantitative real-time polymerase chain reaction was performed on seven randomly selected DEGs to confirm the RNA Sequencing (RNA-seq) data. These genes showed similar expression patterns corresponding with the RNA-Seq method. Weighted gene co-expression network analysis (WGCNA) revealed Y12-4 showed more positive genes than 253 under cold stress. We also explored the cold tolerance gene LTG5 (Low Temperature Growth 5) encoding a UDP-glucosyltransferase. The overexpression of the LTG5 gene conferred cold tolerance to indica rice. CONCLUSION: Gene resources related to cold stress from wild rice can be valuable for improving the cold tolerance of crops.

Genome-wide identification of the peptide transporter family in rice and analysis of the PTR expression modulation in two near-isogenic lines with different nitrogen use efficiency.

BACKGROUND: Nitrogen (N) is a major nutrient element for crop growth. In plants, the members of the peptide transporter (PTR) gene family may involve in nitrate uptake and transport. Here, we identified PTR gene family in rice and analyzed their expression profile in near-isogenic lines. RESULTS: We identified 96, 85 and 78 PTR genes in Nipponbare, R498 and Oryza glaberrima, and the phylogenetic trees were similar in Asian cultivated rice and African cultivated rice. The number of PTR genes was higher in peanut (125) and soybean (127). The 521 PTR genes in rice, maize, sorghum, peanut, soybean and Arabidopsis could be classified into 4 groups, and their distribution was different between monocots and dicots. In Nipponbare genome, the 25 PTR genes were distributed in 5 segmental duplication regions on chromosome 1, 2, 3, 4, 5, 7, 8, 9, and 10. The PTR genes in rice have 0-11 introns and 1-12 exons, and 16 of them have the NPF (NRT1/PTR family) domain. The results of RNA-seq showed that the number of differentially expressed genes (DEGs) between NIL15 and NIL19 at three stages were 928, 1467, and 1586, respectively. Under low N conditions, the number of differentially expressed PTR genes increased significantly. The RNA-seq data was analyzed using WGCNA to predict the potential interaction between genes. We classified the genes with similar expression pattern into one module, and obtained 25 target modules. Among these modules, three modules may be involved in rice N uptake and utilization, especially the brown module, in which hub genes were annotated as protein kinase that may regulate rice N metabolism. CONCLUSIONS: In this study, we comprehensively analyzed the PTR gene family in rice. 96 PTR genes were identified in Nippobare genome and 25 of them were located on five large segmental duplication regions. The Ka/Ks ratio indicated that many PTR genes had undergone positive selection. The RNA-seq results showed that many PTR genes were involved in rice nitrogen use efficiency (NUE), and protein kinases might play an important role in this process. These results provide a fundamental basis to improve the rice NUE via molecular breeding.

Mapping and Identifying a Candidate Gene Plr4, a Recessive Gene Regulating Purple Leaf in Rice, by Using Bulked Segregant and Transcriptome Analysis with Next-Generation Sequencing.

The anthocyanin biosynthesis of rice is a major concern due to the potential nutritional value. Purple appears in various organs and tissues of rice such as pericarp, flower organs, leaves, leaf sheaths, internodes, ligules, apex, and stigma. At present, there are many studies on the color of rice pericarp, but the gene and mechanism of other organs such as leaves are still unclear, and the gene regulatory network of specific organ coloring has not been systematically understood. In this study, genetic analysis demonstrated that the purple leaf traits of rice were regulated by a recessive gene. The green leaf cultivar Y58S and purple leaf cultivar XianHongB were used to construct the mapping population. A set of near isogenicline (NIL) (BC3F1) was bred via crossing and back-crossing. The generations of BC3F2 appeared to separate four phenotypes, pl1, pl2, pl3, and pl4, due to the occurrence of a purple color in different organs. We constructed three bulked segregant analysis (BSA) pools (pl1-pl2, pl1-pl3, and pl1-pl4) by using the separated generations of BC3F5 and mapped the purple leaf gene plr4 to the vicinity of 27.9-31.1 Mb on chromosome 4. Subsequently, transcriptome sequencing (RNA-Seq) for pl3 and pl2 was used to analyze the differentially expressed genes in the localization interval, where 12 unigenes exhibited differential expression in which two genes (Os04g0577800, Os04g0616400) were downregulated. The two downregulated genes (Os04g0577800 and Os04g0616400) are possible candidate genes because of the recessive genetic characteristics of the purple leaf genes. These results will facilitate the cloning of plr4 and illustrate the molecular mechanisms of the anthocyanin synthesis pathway.

Development of molecular marker and introgression of Bph3 into elite rice cultivars by marker-assisted selection.

The brown planthopper (BPH) is a serious insect pest of rice and a substantial threat to rice production. Identification of new BPH resistance genes and their transfer into modern rice cultivars are effective breeding approaches to reduce the damage caused by BPH. In this study, we mapped a BPH resistance gene to a 50-kb genomic interval between two InDel markers 4M03980 and 4M04041 on the short arm of chromosome 4 in indica rice cultivar BP60, where the BPH resistance gene was mapped in Rathu Heenati by Liu et al. (2015) and named "Bph3". This region contains two annotated genes Os04g0201900 and Os04g0202300, which encode lectin receptor kinases responsible for BPH resistance. We also developed a molecular marker "MM28T" for Bph3, and introgression Bph3 into susceptible rice restorer lines Guihui582 and Gui7571 by the marker-assisted selection (MAS) approach. The BPH resistance level is significantly enhanced in the Bph3-introgression lines, the resistance scores decrease from 8.2 to 3.6 for Guihui582 and decrease from 8.7 to around 3.8 for Gui7571. Therefore, developing molecular markers for the BPH resistance gene Bph3 and using them for molecular breeding will facilitate the creation of BPH-resistance rice cultivars to reduce damage caused by BPH.