Development of introgression lines and mapping of qGW2, a novel QTL that confers grain width, in rice (Oryza sativa L.).

Rice grain size is a key determinant of both grain yield and quality. Identification of favorable alleles for use in rice breeding may help to meet the demand for increased yield. In this study, we developed a set of 210 introgression lines (ILs) by using indica variety Huanghuazhan as the donor parent and erect-panicle japonica rice variety Wuyujing3R as the recurrent parent. A total of 133 ILs were selected for high-throughput sequencing. Using specific-locus amplified fragment (SLAF) sequencing technology, 10,103 high-quality SLAF labels evenly distributed on 12 chromosomes were obtained and selected for subsequent analysis. Using a high-density map, quantitative trait locus (QTL) mapping of grain size-related traits was performed, and a total of 38 QTLs were obtained in two environments. Furthermore, qGW2, a novel QTL that controls grain width on chromosome 2, was validated and delimited to a region of 309 kb via substitution mapping. These findings provide new genetic material and a basis for future fine mapping and cloning of favorable QTLs. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01453-0.

Mapping of Rf20(t), a minor fertility restorer gene for rice wild abortive cytoplasmic male sterility in the maintainer line 'Zhenshan97B'.

Three-line hybrid rice has primarily been developed on wild abortive (WA)-type cytoplasmic male sterility (CMS) and has helped increase the yield of rice globally. The development of WA-type CMS lines and hybrids was expedited through the identification and mapping of the fertility restorer gene (Rf) in maintainers. This study observed fertile plants in WA-TianfengA/Zhenshan97B//TianfengB population, indicating that the maintainer line 'Zhenshan97B' should carry Rfs for WA-type CMS. Several advanced backcross populations were generated with the genetic background of the 'WA-TianfengA,' and the pollen fertility levels of the backcrossed individuals in BC3F1, BC4F1 and BC4F2 populations are governed by a new gene, Rf20(t), from 'Zhenshan97B.' Employing bulk segregant analysis of fertile and sterile pools from the BC4F1 population, Rf20(t) was genetically mapped to a candidate region on chromosome 10. Subsequently, Rf20(t) was located between RM24883 and RM24919 through recombination analysis of molecular markers using the BC4F2 population. Implementing a substitution mapping strategy, Rf20(t) was ultimately mapped to a 245-kb region between the molecular markers STS10-122 and STS10-126 and obtained the most likely candidate gene LOC_Os10g02650, which is predicted to encode pentatricopeptide repeat-containing (PPR) protein. These results enhance our understanding of the fertility restoration of WA-type CMS lines, facilitating the development of high-quality pairs of WA-type CMS and maintainer lines.

A de novo evolved gene contributes to rice grain shape difference between indica and japonica.

The role of de novo evolved genes from non-coding sequences in regulating morphological differentiation between species/subspecies remains largely unknown. Here, we show that a rice de novo gene GSE9 contributes to grain shape difference between indica/xian and japonica/geng varieties. GSE9 evolves from a previous non-coding region of wild rice Oryza rufipogon through the acquisition of start codon. This gene is inherited by most japonica varieties, while the original sequence (absence of start codon, gse9) is present in majority of indica varieties. Knockout of GSE9 in japonica varieties leads to slender grains, whereas introgression to indica background results in round grains. Population evolutionary analyses reveal that gse9 and GSE9 are derived from wild rice Or-I and Or-III groups, respectively. Our findings uncover that the de novo GSE9 gene contributes to the genetic and morphological divergence between indica and japonica subspecies, and provide a target for precise manipulation of rice grain shape.

The Effects of Rf4 and the Genetic Mechanism Behind Fertility Restoration of Wild Abortive Cytoplasmic Male Sterility (WA-CMS) in Japonica Rice (Oryza sativa ssp. Japonica).

Wild abortive-type cytoplasmic male sterility (WA-type CMS) has been exclusively used in hybrid seed production in indica rice cultivars, and fertility restoration in WA-type CMS is controlled by two major restorer genes, Rf3 and Rf4, through a sporophytic mechanism. However, the genetic mechanism underlying fertility restoration in WA-type CMS in japonica cultivars is poorly understood. In the present study, C418, a leading Chinsurah Boro II- (BT)-type japonica restorer line, showed partial restoration ability in WA-type japonica CMS lines. The 1:1 segregation ratio of partially fertile to sterile plants in a three-cross F1 population indicated that fertility restoration is controlled by one dominant gene. Gene mapping and sequencing results revealed that the target gene should be Rf4. The Rf4 gene restores fertility through a sporophytic mechanism, but the Rf4 pollen grains show a preferential fertilization in the testcross F1 plants. Furthermore, Rf4 was confirmed to have only a minor effect on fertility restoration in WA-type japonica CMS lines, and Rf gene dosage effects influenced the fertility restoration of WA-type CMS in japonica rice. The results of our study not only provide valuable insights into the complex genetic mechanisms underlying fertility restoration of WA-type CMS but will also facilitate the efficient utilization of WA-type CMS in japonica rice lines.

Precise genetic mapping of Rf18(t), a new fertility restorer gene from 'Nipponbare' for wild abortive cytoplasmic male sterility in rice (Oryza sativa L.).

KEY MESSAGE: We mapped Rf18(t), a Restorer-of-fertility gene for wild abortive cytoplasmic male sterility from the japonica maintainer 'Nipponbare', to chromosome 1. The best candidate gene, LOC_Os01g71320, is predicted to encode hexokinase. Three-line hybrid rice obtained through cytoplasmic male sterility (CMS) has helped increase the yield of rice globally, and the wild abortive (WA)-type cytoplasm from wild rice (Oryza rufipogon Griff.) is used widely in three-line indica hybrids. The identification and mapping of the Restorer-of-fertility (Rf) genes in maintainer lines aided in uncovering the genetic basis of fertility restoration of WA-type CMS and the development of WA-type hybrids. In this study, we identified a new Rf gene, Rf18(t), for WA-type CMS from the japonica maintainer line 'Nipponbare' using a chromosome segment substitution line population derived from a cross between the indica line 9311 and 'Nipponbare.' Using a substitution mapping strategy, Rf18(t) was delimited to a 48-kb chromosomal region flanked by molecular marker loci ID01M28791 and ID01M28845 on chromosome 1. By comparative sequence analyses, we propose that LOC_Os01g71320 is the most likely candidate gene for Rf18(t), and it is predicted to encode hexokinase. Furthermore, Rf18(t) was found to function in fertility restoration probably by a posttranscriptional mechanism and its function is dependent on the genetic background of 9311. These results broaden our knowledge on the mechanism of fertility restoration of WA-type CMS lines and will facilitate the development of WA-type rice hybrids.

Characterization of qPL5: a novel quantitative trait locus (QTL) that controls panicle length in rice (Oryza sativa L.).

Panicle length (PL) is an important trait that determines panicle architecture and strongly affects grain yield and quality in rice. However, this trait has not been well characterized genetically, and its contribution to yield improvement is not well understood. Characterization of novel genes related to PL is of great significance for breeding high-yielding rice varieties. In our previous research, we identified qPL5, a quantitative trait locus for PL. In this study, we aimed to determine the exact position of qPL5 in the rice genome and identify the candidate gene. Through substitution mapping, we mapped qPL5 to a region of 21.86 kb flanked by the molecular marker loci STS5-99 and STS5-106 in which two candidate genes were predicted. By sequence analysis and relative expression analysis, LOC-Os05g41230, which putatively encodes a BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 precursor, was considered to be the most likely candidate gene for qPL5. In addition, we successfully developed a pair of near-isogenic lines (NILs) for qPL5 in different genetic backgrounds to evaluate the genetic effects of qPL5. Agronomic trait analysis of the NILs indicated that qPL5 positively contributes to plant height, grain number per panicle, panicle length, grain yield per plant, and flag leaf length, but it had no influence on heading date and grain-size-related traits. Therefore, qPL5 and the markers tightly linked to it should be available for molecular breeding of high-yielding varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01339-z.

The effects of Rf5 and Rf6 on fertility restoration in Honglian-type cytoplasmic male sterile (CMS) lines of japonica rice (Oryza sativa L. ssp. japonica).

Honglian (HL)-type cytoplasmic male sterility (CMS) has only been used in the development of three-line indica rice hybrids, and the fertility of HL-type indica CMS lines can be restored by two non-allelic fertility-restorer (Rf) genes, Rf5 and Rf6. For the development of HL-type japonica hybrid combinations, it is therefore necessary to determine whether Rf5 and Rf6 can restore the fertility of HL-type japonica CMS lines. Here, we genetically characterized HL-type japonica CMS lines and the ability of Rf5 and Rf6 to restore fertility for breeding HL-type japonica hybrids. I2-KI pollen staining revealed that HL-type japonica CMS lines and their derived testcross F1 hybrids had stained abortive pollen grains, unlike HL-type indica CMS lines. Crossing experiments showed that Rf5 and Rf6 partially restored the fertility of HL-type japonica CMS lines, and Rf6 showed higher restorability than Rf5. Furthermore, we found that there were additive and dosage effects of Rf5 and Rf6 with respect to fertility restoration in HL-type japonica CMS lines. These results give critical insight into the breeding of HL-type japonica CMS lines and restorers, which will be helpful for the development of commercial HL-type japonica hybrids. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01256-7.

Transcriptional changes of rice in response to rice black-streaked dwarf virus.

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus in the family Reoviridae, causes significant economic losses in rice production in China and many other Asian countries. Although a great deal of effort has been made to elucidate the interactions among the virus, insect vectors, host and environmental conditions, few RBSDV proteins involved in pathogenesis have been identified, and the biological basis of disease development in rice remains largely unknown. Transcriptomic information associated with the disease development in rice would be helpful to unravel the biological mechanism. To determine how the rice transcriptome changes in response to RBSDV infection, we carried out RNA-Seq to perform a genome-wide gene expression analysis of a susceptible rice cultivar KTWYJ3. The transcriptomes of RBSDV-infected samples were compared to those of RBSDV-free (healthy) at two time points (time points are represented by group I and II). The results derived from the differential expression analysis in RBSDV-infected libraries vs. healthy ones in group I revealed that 102 out of a total of 281 significant differentially expressed genes (DEGs) were up-regulated and 179 DEGs were down-regulated. Of the 2592 identified DEGs in group II, 1588 DEGs were up-regulated and 1004 DEGs were down-regulated. A total of 66 DEGs were commonly identified in both groups. Of these 66 DEGs, expression patterns for 36 DEGs were similar in both groups. Our analysis demonstrated that some genes related to disease defense and stress resistance were up-regulated while genes associated with chloroplast were down-regulated in response to RBSDV infection. In addition, some genes associated with plant-height were differentially expressed. This result indicates those genes might be involved in dwarf symptoms caused by RBSDV. Taken together, our results provide a genome-wide transcriptome analysis for rice plants in response to RBSDV infection which may contribute to the understanding of the regulatory mechanisms involved in rice-RBSDV interaction and the biological basis of rice black-streaked dwarf disease development in rice.

Ability of Rf5 and Rf6 to Restore Fertility of Chinsurah Boro II-type Cytoplasmic Male Sterile Oryza Sativa (ssp. Japonica) Lines.

BACKGROUND: Three-line Oryza sativa (ssp. japonica) hybrids have been developed mainly using Chinsurah Boro II (BT)-type cytoplasmic male sterility (CMS). The Rf1 gene restores the fertility of BT-type CMS lines, and is the only fertility restorer gene (Rf) that has been used to produce three-line japonica hybrids. Using more Rf genes to breed BT-type restorer lines may broaden the genetic diversity of the restorer lines, and represents a viable approach to improve the heterosis level of BT-type japonica hybrids. RESULTS: We identified two major Rf genes from '93-11' that are involved in restoring the fertility of BT-type CMS plants. These genes were identified from resequenced chromosome segment substitution lines derived from a cross between the japonica variety 'Nipponbare' and the indica variety '93-11'. Molecular mapping results revealed that these genes were Rf5 and Rf6, which are the Rf genes that restore fertility to Honglian-type CMS lines. The BT-type F1 hybrids with either Rf5 or Rf6 exhibited normal seed setting rates, but F1 plants carrying Rf6 showed more stable seed setting rates than those of plants carrying Rf5 under heat-stress conditions. Furthermore, the seed setting rates of F1 hybrids carrying both Rf5 and Rf6 were more stable than that of F1 plants carrying only one Rf gene. CONCLUSION: Rf6 is an important genetic resource for the breeding of BT-type japonica restorer lines. Our findings may be useful for breeders interested in developing BT-type japonica hybrids.

Simultaneous determination of kolliphor HS15 and miglyol 812 in microemulsion formulation by ultra-high performance liquid chromatography coupled with nano quantity analyte detector.

A novel method for simultaneous determination of kolliphor HS15 and miglyol 812 in microemulsion formulation was developed using ultra-high performance liquid chromatography coupled with a nano quantitation analytical detector (UHPLC-NQAD). All components in kolliphor HS15 and miglyol 812 were well separated on an Acquity BEH C18 column. Mobile phase A was 0.1% trifluoroacetic acid (TFA) in water and mobile phase B was acetonitrile. A gradient elution sequence was programed initially with 60% organic solvent, slowly increased to 100% within 8 min. The flow rate was 0.7 mL/min. Good linearity (r>0.95) was obtained in the range of 27.6-1381.1 µg/mL for polyoxyl 15 hydroxystearate in kolliphor HS15, 0.8-202.0 µg/mL for caprylic acid triglyceride and 2.7-221.9 µg/mL for capric acid triglyceride in miglyol 812. The relative standard deviations (RSD) ranged from 0.6% to 1.7% for intra-day precision and from 0.4% to 2.7% for inter-day precision. The overall recoveries (accuracy) were 99.7%-101.4% for polyoxyl 15 hydroxystearate in kolliphor HS15, 96.7%-99.6% for caprylic acid triglyceride, and 94.1%-103.3% for capric acid triglyceride in miglyol 812. Quantification limits (QL) were determined as 27.6 µg/mL for polyoxyl 15 hydroxystearate in kolliphor HS15, 0.8 µg/mL for caprylic acid triglyceride, and 2.7 µg/mL for capric acid triglyceride in miglyol 812. No interferences were observed in the retention time ranges of kolliphor HS15 and miglyol 812. The method was validated in terms of specificity, linearity, precision, accuracy, QL, and robustness. The proposed method has been applied to microemulsion formulation analyses with good recoveries (82.2%-103.4%).

Development and validation of a GC-FID method for quantitative analysis of oleic acid and related fatty acids.

Oleic acid is a common pharmaceutical excipient that has been widely used in various dosage forms. Gas chromatography (GC) has often been used as the quantitation method for fatty acids normally requiring a derivatization step. The aim of this study was to develop a simple, robust, and derivatization-free GC method that is suitable for routine analysis of all the major components in oleic acid USP-NF (United States Pharmacopeia-National Formulary) material. A gas chromatography-flame ionization detection (GC-FID) method was developed for direct quantitative analysis of oleic acid and related fatty acids in oleic acid USP-NF material. Fifteen fatty acids were separated using a DB-FFAP (nitroterephthalic acid modified polyethylene glycol) capillary GC column (30 m×0.32 mm i.d.) with a total run time of 20 min. The method was validated in terms of specificity, linearity, precision, accuracy, sensitivity, and robustness. The method can be routinely used for the purpose of oleic acid USP-NF material analysis.

Central region component1, a novel synaptonemal complex component, is essential for meiotic recombination initiation in rice.

In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated central region component1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID receptor-interacting protein13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with homologous pairing aberration in rice meiosis1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13.

Development of an orthogonal method for mometasone furoate impurity analysis using supercritical fluid chromatography.

While supercritical fluid chromatography (SFC) has received great popularity in chiral separation and purification, it has rarely been used for trace level pharmaceutical impurity analysis, partially due to the limitation of instrument sensitivity. In this study, a packed column SFC method has been developed for the quantitative analysis of mometasone furoate and its trace level impurities. The UV detection was optimized to improve the sensitivity by 2-4 fold. In combination with an increased sample concentration, this SFC method is capable of trace level (0.05% of the active) analysis of the impurities. The SFC method used a silica column and a mobile phase consisting of CO(2) and methanol. The new method provides an orthogonal selectivity complementary to the reversed phase HPLC (RP-HPLC) method. All of the impurities and the active were baseline separated within 12 min on SFC, which is less than one third of the RP-HPLC method run time. The method was also partially validated for linearity, accuracy, precision (repeatability), and limit of quantitation. This study demonstrated that the SFC method, with improved sensitivity, can be a valuable tool to provide orthogonal selectivity for trace level impurity separation. With further validation, the method may be suitable for release testing and stability testing for mometasone furoate drug substance.

Deletion in a quantitative trait gene qPE9-1 associated with panicle erectness improves plant architecture during rice domestication.

Rice plant architecture is an important agronomic trait and a major determinant in high productivity. Panicle erectness is the preferred plant architecture in japonica rice, but the molecular mechanism underlying domestication of the erect panicle remains elusive. Here we report the map-based cloning of a major quantitative trait locus, qPE9-1, which plays an integral role in regulation of rice plant architecture including panicle erectness. The R6547 qPE9-1 gene encodes a 426-amino-acid protein, homologous to the keratin-associated protein 5-4 family. The gene is composed of three Von Willebrand factor type C domains, one transmembrane domain, and one 4-disulfide-core domain. Phenotypic comparisons of a set of near-isogenic lines and transgenic lines reveal that the functional allele (qPE9-1) results in drooping panicles, and the loss-of-function mutation (qpe9-1) leads to more erect panicles. In addition, the qPE9-1 locus regulates panicle and grain length, grain weight, and consequently grain yield. We propose that the panicle erectness trait resulted from a natural random loss-of-function mutation for the qPE9-1 gene and has subsequently been the target of artificial selection during japonica rice breeding.

Serotonin transporter and tryptophan hydroxylase gene polymorphisms in Chinese patients with generalized anxiety disorder.

BACKGROUND: The serotonin transporter (5-HTT) and tryptophan hydroxylase (TPH) gene are important candidate genes for the psychiatric disorders. Many studies of patients with anxiety disorders have found abnormalities of serotonin metabolism and dysfunction of regulation in the transporter itself. In this study, we hypothesize that genetic variation in the 5-HTT and TPH gene may have an effect on the etiology of generalized anxiety disorder (GAD). METHODS: Using a polymerase chain reaction-based technique, the allele and genotype frequencies of three polymorphisms in the serotonin transporter gene (a deletion/insertion polymorphism in the transcriptional control region and a variable number of tandem repeats in intron 2) and TPH gene (A218C in intron 7) were analyzed in 138 patients with GAD and 90 healthy controls. These two groups were matched for ethnic and geographic origin. RESULTS: The frequencies of 5-HTT gene-linked functional polymorphic region (5-HTTLPR) SS (short/short) genotype were significantly higher in GAD patients than in control subjects (68% versus 49%, chi = 12.274, df = 2, P = 0.002), and the frequencies of S (short) allele observed in the GAD patients were higher than those in healthy subjects (79 versus 71%, chi = 4.063, df = 1, P = 0.044). The odds ratio for the SS genotype versus the other two genotypes was 2.33 (95% confidence interval, 1.29-3.86). Similarly, the odds ratio for the S allele versus L allele was 1.56 (95% confidence interval, 1.01-2.41). The genotypic and allelic distribution of 5-HTT VNTR and TPH A218C polymorphisms did not show statistically significant differences between patients and controls. CONCLUSION: Our findings support that the presence of 5-HTTLPR-SS genotype may increase the risk of GAD.