QTL Analysis for Rice Quality-Related Traits and Fine Mapping of qWCR3.

The quality of rice, evaluated using multiple quality-related traits, is the main determinant of its market competitiveness. In this study, two japonica rice varieties with significant differences in quality-related traits were used as parents to construct two populations, BC3F2 and BC3F2:3, with Kongyu131 (KY131) as the recurrent parent. A genetic linkage map was constructed using the BC3F2 population based on 151 pairs of SSR/InDel polymorphic markers selected between the parents. Grain-shape-related traits (grain length GL, grain width GW, and length-to-width ratio LWR), chalkiness-related traits (white-core rate WCR, white-belly rate WBR, white-back rate BR, and chalkiness rate CR), and amylose content (AC) were investigated in the two populations in 2017 and 2018. Except for BR and CR, the traits showed similar characteristics with a normal distribution in both populations. Genetic linkage analysis was conducted for these quality-related traits, and a total of 37 QTLs were detected in the two populations. Further validation was performed on the newly identified QTLs with larger effects, and three grain shape QTLs and four chalkiness QTLs were successfully validated in different environments. One repeatedly validated QTL, qWCR3, was selected for fine mapping and was successfully narrowed down to a 100 kb region in which only two genes, LOC_0s03g45210 and LOC_0s03g45320, exhibited sequence variations between the parents. Furthermore, the variation of LOC_Os03g45210 leads to a frameshift mutation and premature protein termination. The results of this study provide a theoretical basis for positional cloning of the qWCR3 gene, thus offering new genetic resources for rice quality improvement.

QTL detection for rice grain storage protein content and genetic effect verifications.

Rice grain quality is a multifarious attribute mainly governed by multiple nutritional factors. Grain protein is the central component of rice grain nutrition dominantly affecting eating-cooking qualities. Grain protein content is quantitatively influenced by its protein fractions. Genetic quantification of five protein fractions-albumins, globulins, prolamins, glutelin, and grain protein content-were evaluated by exploiting two BC3F2 mapping populations, derived from Kongyu131/TKM9 (population-I) and Kongyu131/Bg94-1 (population-II), which were grown in a single environment. Correlation studies among protein fractions and grain protein content were thoroughly investigated. A genetic linkage map was developed by using 146 single sequence repeat (SSR) markers in population-I and 167 markers in population-II. In total, 40 QTLs were delineated for five traits in both populations. Approximately 22 QTLs were dissected in population-I, derived from Kongyu131/TKM9, seven QTLs for albumin content, four QTLs for globulin content, three QTLs for prolamin content, four QTLs for glutelin content, and four QTLs for grain protein content. In total, 18 QTLs were detected in population-II, derived from Kongyu131/Bg94-1, five QTLs for albumin content, three QTLs for globulin content, four QTLs for prolamin content, two QTLs for glutelin content, and four QTLs for grain protein content. Three QTLs, qAlb7.1, Alb7.2, and qGPC7.2, derived from population-II (Kongyu131/Bg94-1) for albumin and grain protein content were successfully validated in the near isogenic line (NIL) populations. The localized chromosomal locus of the validated QTLs could be helpful for fine mapping via map-based cloning to discover underlying candidate genes. The functional insights of the underlying candidate gene would furnish novel perceptivity for the foundation of rice grain protein content and trigger the development of nutritionally important rice cultivars by combining marker-assisted selection (MAS) breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01436-7.

Improvement of Rice Blast Resistance in TGMS Line HD9802S through Optimized Anther Culture and Molecular Marker-Assisted Selection.

Rice blast caused by Magnaporthe oryzae is one of the most serious rice diseases worldwide. The early indica rice thermosensitive genic male sterile (TGMS) line HD9802S has the characteristics of stable fertility, reproducibility, a high outcrossing rate, excellent rice quality, and strong combining ability. However, this line exhibits poor blast resistance and is highly susceptible to leaf and neck blasts. In this study, backcross introduction, molecular marker-assisted selection, gene chipping, anther culture, and resistance identification in the field were used to introduce the broad-spectrum blast-resistance gene R6 into HD9802S to improve its rice blast resistance. Six induction media were prepared by varying the content of each component in the culture medium. Murashige and Skoog's medium with 3 mg/L 2,4-dichlorophenoxyacetic acid, 2 mg/L 1-naphthaleneacetic acid, and 1 mg/L kinetin and N6 medium with 800 mg/L casein hydrolysate, 600 mg/L proline, and 500 mg/L glutamine could improve the callus induction rate and have a higher green seedling rate and a lower white seedling rate. Compared to HD9802S, two doubled haploid lines containing R6 with stable fertility showed significantly enhanced resistance to rice blast and no significant difference in spikelet number per panicle, 1000-grain weight, or grain shape. Our findings highlight a rapid and effective method for improving rice blast resistance in TGMS lines.

The genetic basis of grain protein content in rice by genome-wide association analysis.

The grain protein content (GPC) of rice is an important factor that determines its nutritional, cooking, and eating qualities. To date, although a number of genes affecting GPC have been identified in rice, most of them have been cloned using mutants, and only a few genes have been cloned in the natural population. In this study, 135 significant loci were detected in a genome-wide association study (GWAS), many of which could be repeatedly detected across different years and populations. Four minor quantitative trait loci affecting rice GPC at four significant association loci, qPC2.1, qPC7.1, qPC7.2, and qPC1.1, were further identified and validated in near-isogenic line F2 populations (NIL-F2), explaining 9.82, 43.4, 29.2, and 13.6% of the phenotypic variation, respectively. The role of the associated flo5 was evaluated with knockdown mutants, which exhibited both increased grain chalkiness rate and GPC. Three candidate genes in a significant association locus region were analyzed using haplotype and expression profiles. The findings of this study will help elucidate the genetic regulatory network of protein synthesis and accumulation in rice through cloning of GPC genes and provide new insights on dominant alleles for marker-assisted selection in the genetic improvement of rice grain quality. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01347-z.

Creation of Two-Line Fragrant Glutinous Hybrid Rice by Editing the Wx and OsBADH2 Genes via the CRISPR/Cas9 System.

Global food security has benefited from the development and promotion of the two-line hybrid rice system. Excellent eating quality determines the market competitiveness of hybrid rice varieties based on achieving the fundamental requirements of high yield and good adaptability. Developing sterile and restorer lines with improved quality for two-line hybrid breeding by editing quality genes with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is an efficient and practical alternative to the lengthy and laborious process of conventional breeding to improve rice quality. We edited Wx and OsBADH2 using CRISPR/Cas9 technology to produce both homozygous male sterile mutant lines and homozygous restorer mutant lines with Cas9-free. These mutants have a much lower amylose content while having a significantly higher 2-acetyl-1-pyrroline aroma content. Based on this, a fragrant glutinous hybrid rice was developed without too much effect on most agronomic traits. This study demonstrates the use of CRISPR/Cas9 in creating two-line fragrant glutinous hybrid rice by editing the components of the male sterile and the restorative lines.

GLW7.1, a Strong Functional Allele of Ghd7, Enhances Grain Size in Rice.

Grain size is a key determinant of both grain weight and grain quality. Here, we report the map-based cloning of a novel quantitative trait locus (QTL), GLW7.1 (Grain Length, Width and Weight 7.1), which encodes the CCT motif family protein, GHD7. The QTL is located in a 53 kb deletion fragment in the cultivar Jin23B, compared with the cultivar CR071. Scanning electron microscopy analysis and expression analysis revealed that GLW7.1 promotes the transcription of several cell division and expansion genes, further resulting in a larger cell size and increased cell number, and finally enhancing the grain size as well as grain weight. GLW7.1 could also increase endogenous GA content by up-regulating the expression of GA biosynthesis genes. Yeast two-hybrid assays and split firefly luciferase complementation assays revealed the interactions of GHD7 with seven grain-size-related proteins and the rice DELLA protein SLR1. Haplotype analysis and transcription activation assay revealed the effect of six amino acid substitutions on GHD7 activation activity. Additionally, the NIL with GLW7.1 showed reduced chalkiness and improved cooking and eating quality. These findings provide a new insight into the role of Ghd7 and confirm the great potential of the GLW7.1 allele in simultaneously improving grain yield and quality.

Natural variation in WHITE-CORE RATE 1 regulates redox homeostasis in rice endosperm to affect grain quality.

Grain chalkiness reduces the quality of rice (Oryza sativa) and is a highly undesirable trait for breeding and marketing. However, the underlying molecular cause of chalkiness remains largely unknown. Here, we cloned the F-box gene WHITE-CORE RATE 1 (WCR1), which negatively regulates grain chalkiness and improves grain quality in rice. A functional A/G variation in the promoter region of WCR1 generates the alleles WCR1A and WCR1G, which originated from tropical japonica and wild rice Oryza rufipogon, respectively. OsDOF17 is a transcriptional activator that binds to the AAAAG cis-element in the WCR1A promoter. WCR1 positively affects the transcription of the metallothionein gene MT2b and interacts with MT2b to inhibit its 26S proteasome-mediated degradation, leading to decreased reactive oxygen species production and delayed programmed cell death in rice endosperm. This, in turn, leads to reduced chalkiness. Our findings uncover a molecular mechanism underlying rice chalkiness and identify the promising natural variant WCR1A, with application potential for rice breeding.

Fine mapping of qTGW2b and qGL9, two minor QTL conferring grain size and weight in rice.

Rice grain size is a key determinant of both grain yield and quality. In this study, we conducted QTL mapping on grain size using a recombinant inbred line (RIL) population derived from a cross between japonica variety Beilu130 (BL130) and indica variety Jin23B (J23B). A total of twenty-two QTL related to grain length (GL), grain width (GW), grain length-to-width ratio (LWR), grain thickness (GT), and thousand grain weight (TGW) were detected under two environments, and 14 of them were repeatedly detected. Two minor QTL, qTGW2b and qGL9, were validated and further delimited to regions of 631 kb and 272 kb, respectively. Parental sequence comparison of genes expressed in inflorescence in corresponding candidate regions identified frameshifts in the exons of LOC_Os02g38690 and LOC_Os02g38780, both of which encode protein phosphatase 2C-containing protein, and LOC_Os09g29930, which encodes a BIM2 protein. Scanning electron microscopy (SEM) analysis revealed that the increase of cell size rather than cell number caused the differences in grain size between NILs of qTGW2b and qGL9. Quantitative RT-PCR analysis showed that the expression levels of EXPA4, EXPA5, EXPA6, EXPB3, EXPB4, and EXPB7 were significantly different in both qTGW2b NILs and qGL9 NILs. Our results lay the foundation for the cloning of qTGW2b and qGL9, and provide genetic materials for the improvement of rice yield and quality. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01328-2.

Biological functions of miR-183 on chemosensitivity of laryngeal cancer cells.

PURPOSE: The purpose of this study was to investigate the effect of miR-183 on the sensitivity of laryngeal cancer cells to 5-fluorouracil (5-Fu) and its mechanism, so as to provide certain references for the clinical prevention of drug resistance in laryngeal cancer cells. METHODS: Cell proliferation was determined via 5-ethynyl-2'-deoxyuridine (EdU) staining. Colony formation assay was applied to test the colony-formation ability in each group of cells. In addition, the expression levels of apoptosis-related proteins were measured through Western blotting. Wound-healing assay and Transwell assay were performed to examine the migratory and invasive abilities. Furthermore, the tumor-forming ability in vitro was detected by subcutaneous tumor formation assay. RESULTS: MiR-183 declined remarkably in 5-Fu-RES group compared with that in Control group. After overexpressing miR-183, the DNA replication and colony forming abilities of the resistant human primary laryngeal cancer cells were weakened notably. MiR-183 overexpression could obviously up-regulate Bax and inhibit Bcl-2 in the resistant human primary laryngeal cancer cells. Moreover, the overexpression of miR-183 was able to repress the migratory and invasive abilities of the resistant human primary laryngeal cancer cells. Further, overexpression of miR-183 restrained the in vitro tumor-forming ability of the resistant human primary laryngeal cancer cells markedly. Finally, it was revealed that TBX3 in the resistant human primary laryngeal cancer cells was suppressed distinctly, while that of PTEN was up-regulated evidently after overexpressing miR-183. CONCLUSIONS: The overexpression of miR-183 can inhibit the drug resistance of the human primary laryngeal cancer cells to 5-Fu, promote cancer cell apoptosis and inhibit their invasive and migratory abilities at the same time, whose mechanism may be associated with the targeted regulation of the TBX3/PTEN signaling pathway by miR-183.

FLOURY ENDOSPERM19 encoding a class I glutamine amidotransferase affects grain quality in rice.

As a staple food for more than half of the world's population, the importance of rice is self-evident. Compared with ordinary rice, rice cultivars with superior eating quality and appearance quality are more popular with consumers due to their unique taste and ornamental value, even if their price is much higher. Appearance quality and CEQ (cooking and eating quality) are two very important aspects in the evaluation of rice quality. Here, we performed a genome-wide association study on floury endosperm in a diverse panel of 533 cultivated rice accessions. We identified a batch of potential floury genes and prioritize one (LOC_Os03g48060) for functional analyses. Two floury outer endosperm mutants (flo19-1 and flo19-2) were generated through editing LOC_Os03g48060 (named as FLO19 in this study), which encodes a class I glutamine amidotransferase. The different performances of the two mutants in various storage substances directly led to completely different changes in CEQ. The mutation of FLO19 gene caused the damage of carbon and nitrogen metabolism in rice, which affected the normal growth and development of rice, including decreased plant height and yield loss by decreased grain filling rate. Through haplotype analysis, we identified a haplotype of FLO19 that can improve both CEQ and appearance quality of rice, Hap2, which provides a selection target for rice quality improvement, especially for high-yield indica rice varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01226-z.

An epidemiological survey of laryngopharyngeal reflux disease at the otorhinolaryngology-head and neck surgery clinics in China.

PURPOSE: Using the Reflux Symptom Index (RSI), this nationwide study aimed to investigate the incidence, diagnostic status, risk factors, and common symptoms of adult laryngopharyngeal reflux disease (LPRD) at otorhinolaryngology-head and neck surgery (OHNS) clinics in China. METHODS: This multicenter cross-sectional survey began at the different institutions ranged from July to October 2017, and the duration was 12 months. A total of 90,440 eligible patients were finally enrolled from 72 medical institutions in China. All these patients completed the questionnaire based on RSI. In this study, LPRD was defined as RSI > 13. RESULTS: There were 9182 with LPRD among the 90,440 eligible participants (10.15%). However, only 1294 had a history of LPRD diagnosis among those with LPRD (14.09%). There were regional differences in the frequency of LPRD (P < 0.001). The proportions of patients with LPRD in males (vs. females), middle- and old-aged patients (vs. young), with current smoking history (vs. no smoking), and current drinking history (vs. no drinking) were significantly higher (all P < 0.001). Middle and old age, current smoking, and drinking history were independent predictors of LPRD (all P < 0.001, OR 1.240, 1.261, and 1.481, respectively). "Sensations of something stuck in throat or a lump in throat", "clearing throat", and "excess throat mucus or postnasal drip" were the most frequent clinical symptoms in patients with LPRD. CONCLUSIONS: LPRD has a high incidence at the OHNS clinics in China. However, the diagnostic status of this disease is not optimistic. Older age, smoking, and drinking history were risk factors for LPRD.

Genetic Basis of Variation in Rice Seed Storage Protein (Albumin, Globulin, Prolamin, and Glutelin) Content Revealed by Genome-Wide Association Analysis.

Rice seed storage protein (SSP) is an important source of nutrition and energy. Understanding the genetic basis of SSP content and mining favorable alleles that control it will be helpful for breeding new improved cultivars. An association analysis for SSP content was performed to identify underlying genes using 527 diverse Oryza sativa accessions grown in two environments. We identified more than 107 associations for five different traits, including the contents of albumin (Alb), globulin (Glo), prolamin (Pro), glutelin (Glu), and total SSP (Total). A total of 28 associations were located at previously reported QTLs or intervals. A lead SNP sf0709447538, associated for Glu content in the indica subpopulation in 2015, was further validated in near isogenic lines NIL(Zhenshan97) and NIL(Delong208), and the Glu phenotype had significantly difference between two NILs. The association region could be target for map-based cloning of the candidate genes. There were 13 associations in regions close to grain-quality-related genes; five lead single nucleotide polymorphisms (SNPs) were located less than 20 kb upstream from grain-quality-related genes (PG5a, Wx, AGPS2a, RP6, and, RM1). Several starch-metabolism-related genes (AGPS2a, OsACS6, PUL, GBSSII, and ISA2) were also associated with SSP content. We identified favorable alleles of functional candidate genes, such as RP6, RM1, Wx, and other four candidate genes by haplotype analysis and expression pattern. Genotypes of RP6 and RM1 with higher Pro were not identified in japonica and exhibited much higher expression levels in indica group. The lead SNP sf0601764762, repeatedly detected for Alb content in 2 years in the whole association population, was located in the Wx locus that controls the synthesis of amylose. And Alb content was significantly and negatively correlated with amylose content and the level of 2.3 kb Wx pre-mRNA examined in this study. The associations or candidate genes identified would provide new insights into the genetic basis of SSP content that will help in developing rice cultivars with improved grain nutritional quality through marker-assisted breeding.