The zinc finger protein DHHC09 S-acylates the kinase STRK1 to regulate H2O2 homeostasis and promote salt tolerance in rice.

Soil salinity results in oxidative stress and heavy losses to crop production. The S-acylated protein SALT TOLERANCE RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (STRK1) phosphorylates and activates CATALASE C (CatC) to improve rice (Oryza sativa L.) salt tolerance, but the molecular mechanism underlying its S-acylation involved in salt signal transduction awaits elucidation. Here, we show that the DHHC-type zinc finger protein DHHC09 S-acylates STRK1 at Cys5, Cys10, and Cys14 and promotes salt and oxidative stress tolerance by enhancing rice H2O2-scavenging capacity. This modification determines STRK1 targeting to the plasma membrane or lipid nanodomains and is required for its function. DHHC09 promotes salt signaling from STRK1 to CatC via transphosphorylation, and its deficiency impairs salt signal transduction. Our findings demonstrate that DHHC09 S-acylates and anchors STRK1 to the plasma membrane to promote salt signaling from STRK1 to CatC, thereby regulating H2O2 homeostasis and improving salt stress tolerance in rice. Moreover, overexpression of DHHC09 in rice mitigates grain yield loss under salt stress. Together, these results shed light on the mechanism underlying the role of S-acylation in RLK/RLCK-mediated salt signal transduction and provide a strategy for breeding highly salt-tolerant rice.

Rapid improvement of grain appearance in three-line hybrid rice via CRISPR/Cas9 editing of grain size genes.

KEY MESSAGE: Genetic editing of grain size genes quickly improves three-line hybrid rice parents to increase the appearance quality and yield of hybrid rice. Grain size affects rice yield and quality. In this study, we used CRISPR/Cas9 to edit the grain size gene GW8 in the maintainer line WaitaiB (WTB) and restorer line Guanghui998 (GH998). The new slender sterile line WTEA (gw8) was obtained in the BC2F1 generation by transferring the grain mutation of the maintainer plant to the corresponding sterile line WantaiA (WTA, GW8) in the T1 generation. Two slender restorer lines, GH998E1 (gw8(II)) and GH998E2 (gw8(I)), were obtained in T1 generation. In the early stage, new sterile and restorer lines in grain mutations were created by targeted editing of GS3, TGW3, and GW8 genes. These parental lines were mated to detect the impact of grain-type mutations on hybrid rice yield and quality. Mutations in gs3, gw8, and tgw3 had a minimal impact on agronomic traits except the grain size and thousand-grain weight. The decrease in grain width in the combination mainly came from gw8/gw8, gs3/gs3 increased the grain length, gs3/gs3-gw8/gw8 had a more significant effect on the grain length, and gs3/gs3-gw8/gw8(I) contributed more to grain length than gs3/gs3-gw8/gw8(II). The heterozygous TGW3/tgw3 may not significantly increase grain length. Electron microscopy revealed that the low-chalky slender-grain variety had a cylindrical grain shape, a uniform distribution of endosperm cells, and tightly arranged starch grains. Quantitative fluorescence analysis of endospermdevelopment-related genes showed that the combination of slender grain hybrid rice caused by gs3 and gw8 mutations promoted endosperm development and improved appearance quality. An appropriate grain size mutation resulted in hybrid rice varieties with high yield and quality.

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.

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.

Bacterial leaf streak 1 encoding a mitogen-activated protein kinase confers resistance to bacterial leaf streak in rice.

Bacterial leaf streak (BLS) is a major bacterial disease of rice. Utilization of host genetic resistance has become one of the most important strategies for controlling BLS. However, only a few resistance genes have been characterized. Previously, a recessive BLS resistance gene bls1 was roughly mapped on chromosome 6. Here, we further delineated bls1 to a 21 kb region spanning four genes. Genetic analysis confirmed that the gene encoding a mitogen-activated protein kinase (OsMAPK6) is the target of the allelic genes BLS1 and bls1. Overexpression of BLS1 weakened resistance to the specific Xanthomonas oryzae pv. oryzicola (Xoc) strain JZ-8, while low expression of bls1 increased resistance. However, both overexpression of BLS1 and low expression of bls1 could increase no-race-specific broad-spectrum resistance. These results indicate that BLS1 and bls1 negatively regulate race-specific resistance to Xoc strain JZ-8 but positively and negatively control broad-spectrum resistance, respectively. Subcellular localization demonstrated that OsMAPK6 was localized in the nucleus. RGA4, which is known to mediate resistance to Xoc, is the potential target of OsMAPK6. Overexpression of BLS1 and low expression of bls1 showed increase in salicylic acid and induced expression of defense-related genes, simultaneously increasing broad-spectrum resistance. Moreover, low expression of bls1 showed increase an in jasmonic acid and abscisic acid, in company with an increase in resistance to Xoc strain JZ-8. Collectively, our study provides new insights into the understanding of BLS resistance and facilitates the development of rice host-resistant cultivars.

OsTTG1, a WD40 repeat gene, regulates anthocyanin biosynthesis in rice.

Anthocyanins play an important role in the growth of plants, and are beneficial to human health. In plants, the MYB-bHLH-WD40 (MBW) complex activates the genes for anthocyanin biosynthesis. However, in rice, the WD40 regulators remain to be conclusively identified. Here, a crucial anthocyanin biosynthesis gene was fine mapped to a 43.4-kb genomic region on chromosome 2, and a WD40 gene OsTTG1 (Oryza sativa TRANSPARENT TESTA GLABRA1) was identified as ideal candidate gene. Subsequently, a homozygous mutant (osttg1) generated by CRISPR/Cas9 showed significantly decreased anthocyanin accumulation in various rice organs. OsTTG1 was highly expressed in various rice tissues after germination, and it was affected by light and temperature. OsTTG1 protein was localized to the nucleus, and can physically interact with Kala4, OsC1, OsDFR and Rc. Furthermore, a total of 59 hub transcription factor genes might affect rice anthocyanin biosynthesis, and LOC_Os01g28680 and LOC_Os02g32430 could have functional redundancy with OsTTG1. Phylogenetic analysis indicated that directional selection has driven the evolutionary divergence of the indica and japonica OsTTG1 alleles. Our results suggest that OsTTG1 is a vital regulator of anthocyanin biosynthesis, and an important gene resource for the genetic engineering of anthocyanin biosynthesis in rice and other plants.

The genetic editing of GS3 via CRISPR/Cas9 accelerates the breeding of three-line hybrid rice with superior yield and grain quality.

Grain size is one of the major traits that determine rice grain yield and quality. The GS3 gene is the first major quantitative trait locus (QTL) that was identified in regulating rice grain length and weight. It was reported that the gs3 allele with a mutation in the organ size regulation (OSR) domain of the GS3 protein produced longer grains. In this study, we used the CRISPR/Cas9 gene editing technology to introduce an edited gs3 allele into our indica maintainer line, Mei1B, to enhance its grain yield and quality. Through molecular analysis and sequencing, a homologous edited-gs3 mutant line without any transgene was obtained in the T1 generation and was named Mei2B. A superior male sterile line Mei2A was generated by backcrossing the cytoplasmic male sterile (CMS) line Mei1A with Mei2B. Mei2B had a higher grain quality and yield compared to its wild-type Mei1B. Its grain length increased by 7.9%, its length/width ratio increased from 3.89 to 4.19, TGW increased by 6.7%, and grain yield per plant increased by 14.9%. In addition, genetic improvement of other quality traits including brown rice length (6.83 mm), brown rice grain length/width ratio (3.61), matched the appearance standards set for traditional Simiao (silk seedling) type cultivars. Two restorer lines were outcrossed to both Mei1A and Mei2A to produce hybrid rice. Compared to two hybrids of Mei1A, the hybrids of Mei2A had longer grains, higher length/width ratio, TGW, and yield per plant. In addition, the hybrids of Mei2A showed a better grain appearance including better translucency, a lower chalky rice rate, and degree of chalkiness than the hybrids of Mei1A. These results demonstrated that the introduction of an elite gs3 allele into Mei1A via CRISPR/Cas9 gene editing technology led to significant genetic improvement of the rice grain. The resultant CMS line Mei2A(gs3) displayed much higher grain quality and yield than the original Mei1A. Therefore, our study demonstrated that the targeted genetic improvement via gene editing technology can enhance rice breeding, especially the breeding of three-line hybrid rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01290-z.

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.

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.

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.

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.

Rapid identification of a new gene influencing low amylose content in rice landraces (Oryza sativa L.) using genome-wide association study with specific-locus amplified fragment sequencing.

Starch is the major component of milled rice, and amylose content (AC) affects eating quality. In this study, a genome-wide association study (GWAS) with specific-locus amplified fragment sequencing (SLAF-seq) data was performed for AC on a core collection of 419 rice landraces. Using the compressed mixed linear method based on the Q+K model, we identified a new gene, LAC6 (Chr6: 5.65-6.04 Mb), associated with AC in the low amylose content panel. The LAC6 candidate gene was detected by qRT-PCR in rice panicles. Results indicate that LOC_Os06g11130 was up-regulated, and LOC_Os06g11340 was significantly down-regulated, making it most likely a key candidate gene of LAC6. In conclusion, the findings provide a certain theoretiacal basis of molecular biology for genetic improvement of AC in rice and rice quality variety breeding.

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.

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.

Identification of a new allele of soluble starch synthase IIIa involved in the elongation of amylopectin long chains in a chalky rice mutant.

A chalky endosperm mutant (GM03) induced from an indica rice GLA4 was used to investigate the functional gene in starch biosynthesis. Bulked segregant analysis and sanger sequencing determined that a novel mutation in soluble starch synthase IIIa (SSIIIa) is responsible for the chalky phenotype in GM03. Complementary test by transforming the active SSIIIa gene driven by its native promoter to GM03 recovered the phenotype to its wildtype. The expression of SSIIIa was significantly decreased, while SSIIIa protein was not detected in GM03. The mutation of SSIIIa led to increased expression of most of starch synthesis related genes and elevated the levels of most of proteins in GM03. The CRISPR/Cas9 technology was used for targeted disruption of SSIIIa, and the mutant lines exhibited chalky endosperm which phenocopied the GM03. Additionally, the starch fine structure in the knockout mutant lines ss3a-1 and ss3a-2 was similar with the GM03, which showed increased amylose content, higher proportions of B1 and B2 chains, much lower proportions of B3 chains and decreased degree of crystallinity, leading to altered thermal properties with lower gelatinization temperature and enthalpy. Collectively, these results suggested that SSIIIa plays an important role in starch synthesis by elongating amylopectin long chains in rice.

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.

QTL mapping and identification of candidate genes for cold tolerance at the germination stage in wild rice.

BACKGROUND: Cold damage stress significantly affects rice growth (germination and seedling) and causes serious losses in yield in temperate and high-altitude areas around the globe. OBJECTIVE: This study aimed to explore the cold tolerance (CT) locus of rice and create new cold-tolerant germplasm. We constructed a chromosome segment substitution line (CSSL) with strong CT and fine mapped quantitative trait loci (QTLs) associated with CT by performing the whole-genome resequencing of CSSL with phenotypes under cold treatment. METHODS: A chromosome CSSL, including 271 lines from a cross between the cold-tolerant wild rice Y11 (Oryza rufipogon Griff.) and the cold-sensitive rice variety GH998, was developed to map QTLs conferring CT at the germination stage. The whole-genome resequencing was performed on CSSL for mapping QTLs of associated with CT at the germination stage. RESULTS: A high-density linkage map of the CSSLs was developed using the whole-genome resequencing of 1484 bins. The QTL analysis using 615,466 single-nucleotide polymorphisms (SNPs) led to the identification of 2 QTLs related to germination rate at low-temperature on chromosome 8 (qCTG-8) and chromosome 11 (qCTG-11). The qCTG-8 and qCTG-11 explained 14.55% and 14.31% of the total phenotypic variation, respectively. We narrowed down qCTG-8 and qCTG-11 to 195.5 and 78.83-kb regions, respectively. The expression patterns of important candidate genes in different tissues, and of RNA-sequencing (RNA-seq) in CSSLs, were identified based on gene sequences in qCTG-8 and qCTG-11 cold-induced expression analysis. LOC_Os08g01120 and LOC_Os08g01390 were identified as candidate genes in qCTG-8, and LOC_Os11g32880 was identified as a candidate gene in qCTG-11. CONCLUSIONS: This study demonstrated a general method that could be used to identify useful loci and genes in wild rice and aid in the future cloning of candidate genes of qCTG-8 and qCTG-11. The CSSLs with strong CT were supported for breeding cold-tolerant rice varieties.