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The rapid expansion of cities has brought new challenges to sustainable development (SD). Therefore, how to balance between SD and the economic growth has become the key to regional development. With the aim of enhancing the research system of SD level, founded on the deep research of SD level from a new perspective of “input-output” and the dynamic evolution law of SD level, this paper has explored the path for cities to improve their level of SD to achieve regional coordinated development. By virtue of the Super-efficiency Slack-Based Measure model based on unexpected output (unexpected super efficiency SBM model), it has measured the SD level of 21 cities in Sichuan from 2010 to 2019. Using methods such as equilibrium coefficient, obstacle analysis and gravitational model, this paper has studied the spatiotemporal evolution law of SD level and constructed six scenarios to simulate the future trend of SD level. The results have indicated that the level of SD in Sichuan is showing a gradual decline with significant constraints. The focus of SD in Sichuan is gradually shifting from Chengdu to a city cluster centered around Chengdu, Ziyang, and Meishan. In an ideal development scenario, the level of SD has significantly improved. This study has revealed the current status and potential of SD in Sichuan. In the future, with more concentration on the role of core urban agglomerations, Sichuan should adjust policies to address input–output barriers to promote SD throughout the province.

The flowering period of rice significantly impacts variety adaptability and yield formation. Properly shortening the reproductive period of rice varieties can expand their ecological range without significant yield reduction. Targeted genome editing, like CRISPR/Cas9, is an ideal tool to fine-tune rice growth stages and boost yield synergistically. In this study, we developed a CRISPR/Cas9-mediated multiplex genome-editing vector containing five genes related to three traits, Hd2, Ghd7, and DTH8 (flowering-stage genes), along with the recessive rice blast resistance gene Pi21 and the aromatic gene BADH2. This vector was introduced into the high-quality japonica rice variety in Zhejiang province, Jiahe212 (JH212), resulting in 34 T0 plants with various effective mutations. Among the 17 mutant T1 lines, several displayed diverse flowering dates, but most exhibited undesirable agronomic traits. Notably, three homozygous mutant lines (JH-C15, JH-C18, and JH-C31) showed slightly earlier flowering dates without significant differences in yield-related traits compared to JH212. Through special Hyg and Cas marker selection of T2 plants, we identified seven, six, and two fragrant glutinous plants devoid of transgenic components. These single plants will serve as sib lines of JH212 and potential resources for breeding applications, including maintenance lines for indica–japonica interspecific three-line hybrid rice. In summary, our research lays the foundation for the creation of short-growth-period CMS (cytoplasmic male sterility, CMS) lines, and also provides materials and a theoretical basis for indica–japonica interspecific hybrid rice breeding with wider adaptability.

Abstract The multi-environment genomic selection enables plant breeders to select varieties resilient to diverse environments or particularly adapted to specific environments, which holds a great potential to be used in rice breeding. To realize the multi-environment genomic selection, a robust training set with multi-environment phenotypic data is of necessity. Considering the huge potential of genomic prediction enhanced sparse phenotyping on the cost saving of multi-environment trials (MET), the establishment of a multi-environment training set could also benefit from it. Optimizing the genomic prediction methods is also crucial to enhance the multi-environment genomic selection. Using haplotype-based genomic prediction models is able to capture local epistatic effects which could be conserved and accumulated across generations much like additive effects thereby benefitting breeding. However, previous studies often used fixed length haplotypes composed by a few adjacent molecular markers disregarding the linkage disequilibrium (LD) which is of essential role in determining the haplotype length. In our study, based on three rice populations with different sizes and compositions, we investigated the usefulness and effectiveness of multi-environment training sets with varying phenotyping intensities and different haplotype-based genomic prediction models based on LD-derived haplotype blocks for two agronomic traits, i.e., days to heading (DTH) and plant height (PH). Results showed that phenotyping merely 30% records in multi-environment training set is able to provide a comparable prediction accuracy to high phenotyping intensities; the local epistatic effects are much likely existent in DTH; dividing the LD-derived haplotype blocks into small segments with two or three single nucleotide polymorphisms (SNPs) helps to maintain the predictive ability of haplotype-based models in large populations; modelling the covariances between environments improves genomic prediction accuracy. Our study provides means to improve the efficiency of multi-environment genomic selection in rice.

Introduction: Circadian clocks coordinate internal physiology and external environmental factors to regulate cereals flowering, which is critical for reproductive growth and optimal yield determination. Objectives: In this study, we aimed to confirm the role of OsLUX in flowering time regulation in rice. Further research illustrates how the OsELF4s–OsELF3-1–OsLUX complex directly regulates flowering-related genes to mediate rice heading. Methods: We identified a circadian gene OsLUX by the MutMap method. The transcription levels of flowering-related genes were evaluated in WT and oslux mutants. OsLUX forms OsEC (OsELF4s–OsELF3-1–OsLUX) complex were supported by yeast two-hybrid, pull down, BiFC, and luciferase complementation assays (LCA). The EMSA, Chip-qPCR, luciferase luminescence images, and relative LUC activity assays were performed to examine the targeted regulation of flowering genes by the OsEC (OsELF4s–OsELF3-1–OsLUX) complex. Results: The circadian gene OsLUX encodes an MYB family transcription factor that functions as a vital circadian clock regulator and controls rice heading. Defect in OsLUX causes an extremely late heading phenotype under natural long-day and short-day conditions, and the function was further confirmed through genetic complementation, overexpression, and CRISPR/Cas9 knockout. OsLUX forms the OsEC (OsELF4s–OsELF3-1–OsLUX) complex by recruiting OsELF3-1 and OsELF4s, which were required to regulate rice heading. OsELF3-1 contributes to the translocation of OsLUX to the nucleus, and a compromised flowering phenotype results upon mutation of any component of the OsEC complex. The OsEC complex directly represses Hd1 and Ghd7 expression via binding to their promoter's LBS (LUX binding site) element. Conclusion: Our findings show that the circadian gene OsLUX regulates rice heading by directly regulating rhythm oscillation and core flowering-time-related genes. We uncovered a mechanism by which the OsEC target suppresses the expression of Hd1 and Ghd7 directly to modulate photoperiodic flowering in rice. The OsEC (OsELF4s–OsELF3-1–OsLUX)–Hd1/Ghd7 regulatory module provides the genetic targets for crop improvement.

Protein ubiquitination is common and crucial in cellular functions, however, little is known about how deubiquitinating enzymes (DUBs) reverse regulate the ubiquitination signaling process. PPPDE family proteins are a novel class of deubiquitinating peptidases with demonstrated deubiquitination/deSUMOylating activities. In this study, we identified 10 PPPDE genes from the rice (Oryza sativa L.) genome unevenly distributed on five chromosomes, where most of these members have not been reported to date. Based on the gene structure, the OsPPPDE family consists of three distinct subgroups within the phylogenetic tree. Cis-element analysis identified light/phytohormone-responsive, development, and abiotic stress-related elements in the promoters of OsPPPDE. Furthermore, we conducted and analyzed the transcript abundance of OsPPPDE under various tissues and stresses using the transcriptome data of 352 samples from the Rice Expression Database and GEO datasets. Moreover, OsPPPDE5 showed differential regulation of its transcript abundance during Cd and drought stress. Collinearity and syntenic analysis of 101 PPPDEs and PPPDE-like proteins in 10 plant genomes indicated that this family is evolutionarily conserved. Domestication analysis suggests that OsPPPDEs may contribute to indica–japonica divergence using the data from the 3K Rice Genome Project. Our study provides a foundation for further study on the function and molecular mechanism of the OsPPPDE gene family.

Leishmaniasis is a complex vector-borne disease caused by various Leishmania species, affecting humans and animals. Current diagnostic methods have limitations, leading to potential misdiagnosis. Therefore, there is an urgent need for specific and sensitive diagnostic tools. We evaluated the sensitivity of a quantitative real-time PCR (qPCR) assay targeting the 18S gene in diverse clinical sample matrices. The assay showed a wide dynamic range and a limit of detection (LoD) of 1 parasite equivalent per milliliter (eq-p/mL) for all tested species. It exhibited high specificity for Leishmania DNA, with no amplification against other microorganisms. When applied to samples from patients with visceral and cutaneous leishmaniasis, the qPCR assay provided results that matched the reference methods and allowed estimation of parasite burdens. This assay holds promise for diagnosing and monitoring leishmaniasis by offering high sensitivity, specificity, and the ability to estimate parasitemia. Further studies are needed to enhance Leishmania molecular diagnostics and expand their coverage for improved clinical impact.

Many excellent genes in wild rice have been lost during the domestication of wild rice to cultivated rice. In this study, introgression lines (ILs) were produced with a wild rice (Oryza rufipogon) accession, BJ194, as a donor parent and an indica restorer line, Zhonghui 8015 (ZH8015), as a recipient parent to map QTLs for plant height. We identified four QTLs (qPH3.1, qPH3.2, qPH2, and qPH8) related to plant height distributed on chromosomes 2, 3 and 8. Furthermore, we sequenced and analyzed qPH3.2 located in the interval of RM15753–RM3525, and found this QTL may be a new locus regulating rice plant height.

Heading date (flowering time) determines the adaptability of cultivars to different environments. We report the fine mapping and candidate gene analysis of qHD1b, a quantitative trait locus (QTL) responsible for early flowering that was derived from common wild rice (O. rufipogon) under both short-day and long-day conditions. The introgression line IL7391, which carried segments from common wild rice in a Zhonghui 8015 (ZH8015) background, exhibited early heading compared to the background and was crossed with ZH8015 to generate BC5F2:3 families for QTL analysis. This enabled the identification of two heading-date QTL, named qHD1b and qHD7, of which the first was selected for further research. High-resolution linkage analysis was performed in BC5F4:5 and BC5F6 populations, and the location of qHD1b was confined to a 112.7-kb interval containing 17 predicted genes. Five of these genes contained polymorphisms in the promoter or coding regions and were thus considered as candidates. Expression analysis revealed a positive association between LOC_Os01g11940 expression and early heading. This locus was annotated as OsFTL1, which encodes an ortholog of Arabidopsis Flowering Locus T and was the most likely candidate gene for qHD1b. Our study revealed that qHD1b acts as a floral activator that promotes flowering by up-regulating Ehd1, Hd3a, RFT1, OsMADS14, and OsMADS15 under both short-day and long-day conditions. Field experiments showed that qHD1b affected several yield-related agronomic traits including 1000-grain weight and grain length. qHD1b could be useful for marker-assisted selection and breeding of early-maturing cultivars.

Direct seeding has been widely adopted as an economical and labor-saving technique in rice production, though problems such as low seedling emergence rate, emergence irregularity and poor lodging resistance are existing. These problems are currently partially overcome by increasing seeding rate, however it is not acceptable for hybrid rice due to the high seed cost. Improving direct seeding by breeding is seen as the ultimate solution to these problems. For hybrid breeding, identifying superior hybrids among a massive number of hybrids from crossings between male and female parental populations by phenotypic evaluation is tedious and costly. Contrastingly, genomic selection/prediction (GS/GP) could efficiently detect the superior hybrids capitalizing on genomic data, which holds a great potential in plant hybrids breeding. In this study, we utilized 402 rice inbred varieties and 401 hybrids to investigate the effectiveness of GS on rice mesocotyl length, a representative indicative trait of direct seeding suitability. Several GP methods and training set designs were studied to seek the optimal scenario of hybrid prediction. It was shown that using half-sib hybrids as training set with the phenotypes of all parental lines being fitted as a covariate could optimally predict mesocotyl length. Partitioning the molecular markers into trait-associated and -unassociated groups based on genome-wide association study using all parental lines and hybrids could further improve the prediction accuracy. This study indicates that GS could be an effective and efficient method for hybrid breeding for rice direct seeding.

Chagas disease is a complex zoonosis caused by Trypanosoma cruzi. The diagnosis of this infection is complex and molecular tools are suggested to detect the parasite in blood samples. A long-standing question arises in Chagas disease molecular diagnostics and is related to the feasibility of using epimastigotes in standard curves to quantify parasitic loads. Herein, we conducted experiments running standard curves with all the known life stages of T. cruzi. Our results indicate that regardless of the life stage employed, there are no statistically significant differences when calculating parasitic loads in blood samples. Our results have practical implications from a laboratory perspective in terms of the usability of epimastigotes to build standard curves for T. cruzi pan-stage assessment. Future studies are needed to further improve T. cruzi molecular diagnostic methods and enhance their impact in clinical practice.

Drought poses a significant constraint on rice production, and, in this study, we have discovered a novel drought-sensitive mutant, designated as dsm3, arising from the progenies of indica rice variety Zhonghui8015 treated with ethyl methane sulphonate (EMS). Under drought stress conditions, dsm3 exhibited characteristic withered leaf tips, accompanied by increased levels of malondialdehyde (MDA) and H2O2, a reduced net photosynthetic rate (Pn), and decreased activity of peroxidase (POD) and superoxide dismutase (SOD). Genetic analysis revealed that the withered leaf tip phenotype was governed by a single recessive gene, designated as Osdsm3. To begin with, Osdsm3 was initially mapped to the short arm of chromosome 1 through a cross involving dsm3 and 02428. Subsequently, utilizing a population of 2591 F2 individuals, we narrowed down the location of Osdsm3 to a 78 Kb interval, encompassing 13 open reading frames (ORFs). Sequencing analysis unveiled a mutation (1275G → A) in the exon of the candidate gene (LOC_Os01g10680), leading to premature translation termination. Moreover, a quantitative RT-PCR assay demonstrated a high expression of OsDSM3 in the panicle and sheath, with a significant upregulation of drought-stress-related genes under drought conditions. Phylogenetic analyses indicated that Osdsm3 shares evolutionary homology with UNE1, an intracellular transport protein found in Arabidopsis thaliana. Subcellular studies further confirmed that OsDSM3 resides in the cytoplasm. In conclusion, the forthcoming cloning of Osdsm3 holds promise for delving deeper into the molecular mechanisms governing rice drought resistance.

Abstract Background Plant cell walls are the main physical barrier encountered by pathogens colonizing plant tissues. Alteration of cell wall integrity (CWI) can activate specific defenses by impairing proteins involved in cell wall biosynthesis, degradation and remodeling, or cell wall damage due to biotic or abiotic stress. Polygalacturonase (PG) depolymerize pectin by hydrolysis, thereby altering pectin composition and structures and activating cell wall defense. Although many studies of CWI have been reported, the mechanism of how PGs regulate cell wall immune response is not well understood. Results Necrosis appeared in leaf tips at the tillering stage, finally resulting in 3–5 cm of dark brown necrotic tissue. ltn-212 showed obvious cell death and accumulation of H2O2 in leaf tips. The defense responses were activated in ltn-212 to resist bacterial blight pathogen of rice. Map based cloning revealed that a single base substitution (G-A) in the first intron caused incorrect splicing of OsPG1, resulting in a necrotic phenotype. OsPG1 is constitutively expressed in all organs, and the wild-type phenotype was restored in complementation individuals and knockout of wild-type lines resulted in necrosis as in ltn-212. Transmission electron microscopy showed that thicknesses of cell walls were significantly reduced and cell size and shape were significantly diminished in ltn-212. Conclusion These results demonstrate that OsPG1 encodes a PG in response to the leaf tip necrosis phenotype of ltn-212. Loss-of-function mutation of ltn-212 destroyed CWI, resulting in spontaneous cell death and an auto-activated defense response including reactive oxygen species (ROS) burst and pathogenesis-related (PR) gene expression, as well as enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo). These findings promote our understanding of the CWI mediated defense response.

In this paper, an integrated design scheme of fault estimation (FE) and fault-tolerant control (FTC) is presented for a closed-loop system with uncertain short delay. Firstly, a closed-loop system with uncertain short delay is modeled as a system with uncertain parameters, and a bi-directional robustness interaction of uncertainty between the observer and the control system is analyzed. Then a fault estimation observer and a fault-tolerant controller are constructed, and the integrated design problem is converted into a robust control problem for the augmented system under H∞ performance index. Furthermore, the parameters of the fault estimation observer and fault-tolerant controller are solved by Lyapunov function and relaxation methods. Finally, the effectiveness and superiority of the proposed method are verified by an aircraft simulation.

Lesion mimic mutants (LMMs) exhibit spots on leaves without fungal infection pressure. The spots confer variable resistance to pathogens in different LMM, making them useful research materials. It is unclear how the rice immune system responds to infection with the fungal pathogen Magnaporthe oryzae (M. oryzae). Here, we identified a rice LMM, lm8015-3, which shows reduced resistance to M. oryzae. We used Quantitative Real-Time PCR (qRT-PCR) to observe the immune system response to M. oryzae–induced lm8015-3. Lm8015-3, obtained from an ethyl methane sulfonate (EMS)–induced Zhonghui8015 (ZH8015) library, showed orange-yellow spots starting in the seedling stage and accumulated more H2O2, resulting in severe degradation of the chloroplast. With map-based cloning, the target gene was located on chromosome 12. Once inoculated with M. oryzae, the expression level of pathogen-related genes of lm8015-3 was downregulated between 48 and 72 h. In addition, more germinating spores appeared in lm8015-3. Therefore, we conclude that M. oryzae weakening the immune system of lm8015-3 from 48 to 72 h makes lm8015-3 more susceptible to M. oryzae. These results suggested that understanding how LMMs defend against M. oryzae infection will contribute to improving rice breeding.

Tiller number (TN) is an important trait that contributes to yield in rice (Oryza sativa L.). A set of 76 chromosome segment substitution lines (CSSLs) derived from the super-hybrid cross between Zhonghui 9308 (ZH9308) and Xieqingzao B (XQZB) was used to map quantitative trait loci (QTL) controlling tiller number (TN). A total of four QTLs were detected in Fuyang, Zhejiang Province (30.15° N, 120° E). Two QTLs were detected in Lingshui, Hainan Province (18.5° N, 110° E) in our previous study. To further map the QTL on chromosome 4, namely qTN4, the line CSSL29 with a lower tiller number was selected to cross with ZH9308 to develop the secondary F2 population. In the F2:3 population, the qTN4 was validated and subsequently narrowed down to a 4.08 Mb region. What is more, combined phenotype with genotype, qTN4 was dissected into two QTLs, qTN4.1 and qTN4.2, in the F4:5 population. The qTN4.1 and qTN4.2 explained 34.31% and 32.05% of the phenotypic variance, with an additive effect of 1.47 and 1.38, respectively. Finally, the qTN4.1 and qTN4.2 were fine-mapped into a 193.55 Kb and 175.12 Kb intervals on chromosome 4, respectively. Based on genotype and phenotype, four near-isogenic lines (NILs) were selected in the mapping populations. Compared with NILCSSL29, tiller number (TN), grain setting rate, grain length (GL), the ratio of grain length to width (LWR) and grain yield per plant of NILZH9308, NIL-qTN4.1ZH9308 and NIL-qTN4.2ZH9308 were increased, and the heading date of these three lines were earlier than that of NILCSSL29. Interestingly, among the candidate genes of qTN4.1 and qTN4.2, except for LOC_Os04g23550, none of the other genes has been cloned, indicating the existence of a novel gene-controlling tiller number. These results lay a foundation for the analysis of QTL controlling tiller number in ZH9308 and provide a theoretical basis for the application of ZH9308 in super-hybrid breeding.

In angiosperms, anther development comprises of various complex and interrelated biological processes, critically needed for pollen viability. The transitory callose layer serves to separate the meiocytes. It helps in primexine formation, while the timely degradation of tapetal cells is essential for the timely callose wall dissolution and pollen wall formation by providing nutrients for pollen growth. In rice, many genes have been reported and functionally characterized that are involved in callose regulation and pollen wall patterning, including timely programmed cell death (PCD) of the tapetum, but the mechanism of pollen development largely remains ambiguous. We identified and functionally characterized a rice mutant dcet1, having a complete male-sterile phenotype caused by defects in anther callose wall, exine patterning, and tapetal PCD. DCET1 belongs to the RNA recognition motif (RRM)-containing family also called as the ribonucleoprotein (RNP) domain or RNA-binding domain (RBD) protein, having single-nucleotide polymorphism (SNP) substitution from G (threonine-192) to A (isoleucine-192) located at the fifth exon of LOC_Os08g02330, was responsible for the male sterile phenotype in mutant dcet1. Our cytological analysis suggested that DCET1 regulates callose biosynthesis and degradation, pollen exine formation by affecting exine wall patterning, including abnormal nexine, collapsed bacula, and irregular tectum, and timely PCD by delaying the tapetal cell degeneration. As a result, the microspore of dcet1 was swollen and abnormally bursted and even collapsed within the anther locule characterizing complete male sterility. GUS and qRT-PCR analysis indicated that DCET1 is specifically expressed in the anther till the developmental stage 9, consistent with the observed phenotype. The characterization of DCET1 in callose regulation, pollen wall patterning, and tapetal cell PCD strengthens our knowledge for knowing the regulatory pathways involved in rice male reproductive development and has future prospects in hybrid rice breeding.

Grain shape improvement, which determines grain yield, quality traits and commercial value, is an extremely important aspect of rice breeding. Grain size is controlled by multiple genes, and Maker Assistant Selection (MAS) breeding is effective for breeders in developing stable and efficient markers to aggregate these genes in order to speed up the selection of new lines with desirable traits during the breeding process. In this study, functional markers were developed based on the sequence differences of five grain-shaped genes (GL7, GW6a, GS6, GW5 and TGW6) between the long-grain japonica rice variety Zhendao and the indica-japonica restorer R2027. We then constructed a population of recombinant inbred lines (RILs) based on their cross. The newly designed functional markers were used to genotype grain-size genes, and a genetic effect analysis was conducted to screen high-quality long-grain restorers. Our results reveal diverse effects of different genes on grain size, and the five genotypes were distributed in the 36 selected BC1F8 lines. Specifically, gw5 positively regulates grain width and 1000-grain weight, gl7 and gs6 positively regulate grain length but negatively regulate grain width and 1000-grain weight, tgw6 positively regulates grain length and gw6a positively regulates 1000-grain weight. The most outstanding outcome is that 5 of the 36 lines achieved in this study showing an excellent performance of long grain and yield characters are ideal materials not only for studying the interaction and genetic effects between polygenes but also as restorers or donors for dominant genes in indica-japonica hybrid rice breeding.

Abstract Seed setting rate is one of the major components that determine rice (Oryza sativa L.) yield. Successful fertilization is necessary for normal seed setting. However, little is known about the molecular mechanisms governing this process. In this study, we report a novel rice gene, LOW SEED SETTING RATE1 (LSSR1), which regulates the seed setting rate by facilitating rice fertilization. LSSR1 encodes a putative GH5 cellulase, which is highly conserved in plants. LSSR1 is predominantly expressed in anthers during the microsporogenesis stage, and its encoded protein contains a signal peptide at the N-terminal, which may be a secretory protein that stores in pollen grains and functions during rice fertilization. To explore the physiological function of LSSR1 in rice, loss-of-function mutants of LSSR1 were created through the CRISPR-Cas9 system, which showed a significant decrease in rice seed setting rate. However, the morphology of the vegetative and reproductive organs appears normal in lssr1 mutant lines. In addition, lssr1 pollen grains could be normally stained by I2-KI solution. Cytological results demonstrate that the blockage of fertilization mostly accounted for the low seed setting rate in lssr1 mutant lines, which was most likely caused by abnormal pollen grain germination, failed pollen tube penetration, and retarded pollen tube elongation. Together, our results suggest that LSSR1 plays an important role in rice fertilization, which in turn is vital for maintaining rice seed setting rate.

Abstract Background Nitrogen (N) is a major input cost in rice production, in addition to causing severe pollution to agricultural and ecological environments. Root dry weight has been considered the most important component related to crop yields than the other root traits. Therefore, development of rice varieties/lines with low input of N fertilizer and higher root traits are essential for sustainable rice production. Results In this context, a main effect quantitative trait locus qRDWN6 XB on the long arm of chromosome 6 which positively confers tolerance to N deficiency in the Indica rice variety XieqingzaoB, was identified using a chromosomal segment substitution line (CSSL) population. qRDWN6 XB was determined to be located near marker InD90 on chromosome 6 based on association analysis of phenotype data from three N levels and 120 polymorphic molecular markers. The target chromosomal segment substitution line CSSL45, which has the higher root dry weight (RDW) than indica cultivar Zhonghui9308 and carry qRDWN6 XB , was selected for further study. A BC5F2:3 population derived from a cross between CSSL45 and Zhonghui9308 was constructed. To fine-map qRDWN6 XB , we used the homozygous recombinant plants and ultimately this locus was narrowed to a 52.3-kb between markers ND-4 and RM19771, which contains nine candidate genes in this region. One of these genes, LOC_Os06g15910 as a potassium transporter was considered a strong candidate gene for the RDWN6 XB locus. Conclusions The identification of qRDWN6 XB provides a new genetic resource for breeding rice varieties and a starting point to improve grain yield despite the decreased input of N fertilizers. The newly developed and tightly linked InDel marker ND-4 will be useful to improve the root system architecture under low N by marker-assisted selection (MAS) in rice breeding programs.

Heading date (HD) is one of the agronomic traits that influence maturity, regional adaptability, and grain yield. The present study was a follow-up of a previous quantitative trait loci (QTL) mapping study conducted on three populations, which uncovered a total of 62 QTLs associated with 10 agronomic traits. Two of the QTLs for HD on chromosome 7 (qHD7a and qHD7b) had a common flanking marker (RM3670) that may be due to tight linkage, and/or weakness of the statistical method. The objectives of the present study were to map QTLs associated with HD in a set of 76 chromosome segment substitution lines (CSSLs), fine map and validate one of the QTLs (qHD7b) using 2997 BC5F2:3 plants, and identify candidate genes using sequencing and expression analysis. Using the CSSLs genotyped with 120 markers and evaluated under two short-day and two long-day growing conditions, we uncovered a total of fourteen QTLs (qHD2a, qHD4a, qHD4b, qHD5a, qHD6a, qHD6b, qHD7b, qHD7c, qHD8a, qHD10a, qHD10b, qHD11a, qHD12a, and qHD12b). However, only qHD6a and qHD7b were consistently detected in all four environments. The phenotypic variance explained by qHD6a and qHD7b varied from 10.1% to 36.1% (mean 23.1%) and from 8.1% to 32.8% (mean 20.5%), respectively. One of the CSSL lines (CSSL52), which harbored a segment from the early heading XieqingzaoB (XQZB) parent at the qHD7b locus, was then used to develop a BC5F2:3 population for fine mapping and validation. Using a backcross population evaluated for four seasons under different day lengths and temperatures, the qHD7b interval was delimited to a 912.7-kb region, which is located between RM5436 and RM5499. Sequencing and expression analysis revealed a total of 29 candidate genes, of which Ghd7 (Os07g0261200) is a well-known gene that affects heading date, plant height, and grain yield in rice. The ghd7 mutants generated through CRISPR/Cas9 gene editing exhibited early heading. Taken together, the results from both the previous and present study revealed a consistent QTL for heading date on chromosome 7, which coincided not only with the physical position of a known gene, but also with two major effect QTLs that controlled the stigma exertion rate and the number of spikelets in rice. The results provide contributions to the broader adaptability of marker-assisted breeding to develop high-yield rice varieties.

Rice (Oryza sativa L.) occupies a very salient and indispensable status among cereal crops, as its vast production is used to feed nearly half of the world’s population. Male sterile plants are the fundamental breeding materials needed for specific propagation in order to meet the elevated current food demands. The development of the rice varieties with desired traits has become the ultimate need of the time. Genic male sterility is a predominant system that is vastly deployed and exploited for crop improvement. Hence, the identification of new genetic elements and the cognizance of the underlying regulatory networks affecting male sterility in rice are crucial to harness heterosis and ensure global food security. Over the years, a variety of genomics studies have uncovered numerous mechanisms regulating male sterility in rice, which provided a deeper and wider understanding on the complex molecular basis of anther and pollen development. The recent advances in genomics and the emergence of multiple biotechnological methods have revolutionized the field of rice breeding. In this review, we have briefly documented the recent evolution, exploration, and exploitation of genic male sterility to the improvement of rice crop production. Furthermore, this review describes future perspectives with focus on state-of-the-art developments in the engineering of male sterility to overcome issues associated with male sterility-mediated rice breeding to address the current challenges. Finally, we provide our perspectives on diversified studies regarding the identification and characterization of genic male sterility genes, the development of new biotechnology-based male sterility systems, and their integrated applications for hybrid rice breeding.

Rice (Oryza sativa L.) and Arabidopsis thaliana (L.) life cycles involve several major phase changes, throughout which MADS-box genes have a variety of functions. MADS-box genes are well recognized for their functions in floral induction and development, and some have multiple functions in apparently unrelated developmental stages. For example, in Arabidopsis, AGL15 and AGL6 play roles in both vegetative development and floral transition. Similarly, in rice, OsMADS1 is involved in flowering time and seed development, and OsMADS26 is expressed not only in the roots, but also in the leaves, shoots, panicles, and seeds. The roles of other MADS-box genes responsible for the regulation of specific traits in both rice and Arabidopsis are also discussed. Several are key components of gene regulatory networks involved in root development under diverse environmental factors such as drought, heat, and salt stress, and are also involved in the shift from vegetative to flowering growth in response to seasonal changes in environmental conditions. Thus, we argue that MADS-box genes are critical elements of gene regulation that underpin diverse gene expression profiles, each of which is linked to a unique developmental stage that occurs during root development and the shift from vegetative to reproductive growth.

Plant height (PH) is one of the most important agronomic traits determining plant architecture in rice. To investigate the genetic basis of plant height in the high-yielding hybrid rice variety Nei2You No.6, recombinant inbred sister lines (RISLs) were used to map quantitative trait loci (QTL) over four years. A total of 19 minor/medium-effect QTLs were mapped on eleven chromosomes except chromosome 10, totally explaining 44.61–51.15% phenotypic variance in four environments. Among these, qPH-1a, qPH-1b, qPH-2b, qPH-3b, qPH-6, and qPH-7b were repeatedly detected over four years. Among these, the qPH-6 was mapped to an interval of 22.11–29.41 Mb on chromosome 6L, which showed the highest phenotypic variation explained (PVE) of 10.22–14.05% and additive effect of 3.45–4.63. Subsequently, evaluation of near isogenic lines (NILs) showed that the qPH-6 allele from the restorer line (R8006) could positively regulate plant height, resulting in an 18.50% increase in grain yield. These results offered a basis for further mapping of qPH-6 and molecular breeding in improving plant architecture in rice.

The spikelet developmental processes that control structure and floral organ identity play critical roles in rice grain yield formation. In this study, we characterized a novel rice mutant, SLL1-ZH, which exhibits a variety of defective agronomic characters, including semi-dwarf, rolling leaf, deformed panicles, and reduced grains production. Morphological analysis also revealed that the SLL1-ZH mutant shows numerous defects of floral organs, such as cracked glumes, hooked and thin lemmas, shrunken but thickened paleas, an indeterminate number of stamens and stigmas, and heterotopic ovaries. Map-based cloning identified a single nucleotide substitution (C to G) in the first exon of LOC_Os09g23200 that is responsible for the SLL1-ZH phenotype. In addition, qPCR analysis showed a significant change in the relative expression of SLL1-ZH in the mutant during inflorescence differentiation and in the different floral organs. Transcription of rice floral organ development-related factors also changed significantly in the mutant. Therefore, our results suggested that SLL1-ZH plays a great role in plant growth, spikelet development, and grain yield in rice.

Abstract With the development and application of super rice breeding, elite rice hybrids with super high-yielding potential have been widely developed in last decades in China. Xieyou9308 is one of the most famous super hybrid rice varieties. To uncover the genetic mechanism of Xieyou9308’s high yield potential, a recombinant inbred line (RIL) population derived from cross of XieqingzaoB and Zhonghui9308 was re-sequenced and investigated on the grain yield (GYD) and its three component traits, number of panicles per plant (NP), number of filled grains per panicle (NFGP), and grain weight (GW). Unconditional and conditional genome-wide association analysis, based on a linear mixed model with epistasis and gene-environment interaction effects, were conducted, using ~0.7 million identified SNPs. There were six, four, seven, and seven QTSs identified for GYD, NP, NFGP, and GW, respectively, with accumulated explanatory heritability varying from 43.06% to 48.36%; additive by environment interactions were detected for GYD, some minor epistases were detected for NP and NFGP. Further, conditional genetic mapping analysis for GYD given its three components revealed several novel QTSs associated with yield than that were suppressed in our unconditional mapping analysis.

Mitogen-activated protein kinase (MAPK) cascades play central roles in response to biotic and abiotic stresses. However, the mechanisms by which various MAPK members regulate the plant immune response in rice remain elusive. In this article, to characterize the mechanisms, the knock-out and overexpression mutants of OsMPK15 were constructed and the disease resistance was investigated under the various fungal and bacterial inoculations. The knock-out mutant of OsMPK15 resulted in the constitutive expression of pathogenesis-related (PR) genes, increased accumulation of reactive oxygen species (ROS) triggered by the pathogen-associated molecular pattern (PAMP) elicitor chitin, and significantly enhanced the disease resistance to different races of Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo), which cause the rice blast and bacterial blight diseases, respectively. On contrary, the expression of PR genes and ROS were down-regulated in the OsMPK15-overexpressing (OsMPK15-OE) lines. Meanwhile, phytohormones such as salicylic acid (SA) and jasmonic acid (JA) were accumulated in the mpk15 mutant lines but decreased in the OsMPK15-OE lines. The expression of SA- and JA-pathway associated genes were significantly upregulated in the mpk15 mutant, whereas it was down regulated in the OsMPK15-OE lines. We conclude that OsMPK15 may negatively regulate the disease resistance through modulating SA- and JA-mediated signaling pathway.

Premature leaf senescence affects plant yield and quality, and numerous researches about it have been conducted until now. In this study, we identified an early senescent mutant es4 in rice (Oryza sativa L.); early senescence appeared approximately at 60 dps and became increasingly senescent with the growth of es4 mutant. We detected that content of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as activity of superoxide dismutase (SOD) were elevated, while chlorophyll content, soluble protein content, activity of catalase (CAT), activity of peroxidase (POD) and photosynthetic rate were reduced in the es4 mutant leaves. We mapped es4 in a 33.5 Kb physical distance on chromosome 4 by map-based cloning. Sequencing analysis in target interval indicated there was an eight bases deletion mutation in OsCPK12 which encoded a calcium-dependent protein kinase. Functional complementation of OsCPK12 in es4 completely restored the normal phenotype. We used CRISPR/Cas9 for targeted disruption of OsCPK12 in ZH8015 and all the mutants exhibited the premature senescence. All the results indicated that the phenotype of es4 was caused by the mutation of OsCPK12. Overexpression of OsCPK12 in ZH8015 enhanced the net photosynthetic rate (Pn) and chlorophyll content. OsCPK12 was mainly expressed in green organs. The results of qRT-PCR analysis showed that the expression levels of some key genes involved in senescence, chlorophyll biosynthesis, and photosynthesis were significantly altered in the es4 mutant. Our results demonstrate that the mutant of OsCPK12 triggers the premature leaf senescence; however, the overexpression of OsCPK12 may delay its growth period and provide the potentially positive effect on productivity in rice.

Grain shape is a key factor for yield and quality in rice. To investigate the genetic basis of grain shape in the high-yielding hybrid rice variety Nei2You No.6, a set of recombinant inbred sister lines (RISLs) were used to map quantitative trait loci (QTLs) determining grain length (GL), grain width (GW), and length-width ratio (LWR) in four environments. A total of 91 medium/minor-effect QTL were detected using a high-density genetic map consisting of 3203 Bin markers composed of single nucleotide polymorphisms, among which 64 QTL formed 15 clusters. Twelve of 15 clusters co-localized with QTL previously reported for grain shape/weight. Three new QTL were detected: qGL-7a, qGL-8, and qGL-11a. A QTL cluster, qLWR-12c/qGW-12, was detected across all four environments with phenotypic variation explained (PVE) ranging from 3.67% to 11.93%, which was subsequently validated in paired lines of F17 progeny and tightly linked marker assay in F10 generation. Subsequently, 17 candidate genes for qLWR-12c/qGW-12 were detected in the 431 Kb interval utilizing bulk segregant analysis (BSA). Among these, OsR498G1222170400, OsR498G1222171900, OsR498G1222185100, OsR498G1222173400, and OsR498G1222170500 were the best candidates, which lays the foundation for further cloning and will facilitate high-yield breeding in rice.

Rice is a pivotal cereal crop that provides the staple food for more than half of the world’s population. Along with improvements in the standard of living, people not only pay attention to the grain yield but also to the grain quality. Chalkiness is one of the most important index of grain quality. In this study, qPCG1, a QTL for percentage of chalky grain, was mapped in an interval with a physical distance about 139 kb on chromosome 1 by residual heterozygous line (RHL) method. qPCG1 was incomplete dominant and the additive effect plays a major role and explained 6.8–21.9% of phenotypic variance within the heterogeneous region on chromosome 1. The effect of allele from Zhonghui9308 was decreasing the percentage of chalky grains (PCG). Microscope observation results indicated that there are great differences in the shape, structure and arrangement of starch granule between the chalky part and transparent part. Analysis of starch physicochemical properties showed that the total starch content, amylose content and chain length distribution of amylopectin changed while the protein contents were not apparently affected with the changed chalkiness. qPCG1 had little influence on main agronomic traits and it might be useful in rice breeding for it did not bring negative effect on grain yield while reducing the chalkiness.

The rate of stigma exsertion (SE) is an important trait in rice breeding because the efficiency of hybrid rice seed production can be improved by increasing the percentage of stigmas that exsert. In this study, we developed a near isogenic line (NIL) from two parents, XieqingzaoB (XQZB) and Zhonghoi9308 (ZH9308), which have high and low SE rates in that order. In our previous study, we employed 75 chromosome segment substitution lines (CSSLs) and analyzed quantitative trait loci (QTLs) for their influence on SE rate. The single gene QTL (qSE11), which is located on chromosome 11, was responsible for this trait. In this study, we focused on one of the CSSLs (C65), namely, the NIL (qSE11XB). It contains an introgression segment of XQZB in the genetic background of ZH9308, and exhibits a significantly higher SE rate than that of the parents. We demonstrated that qSE11 regulated both the single and the dual SE rates. They both contribute to the total SE rate. Genetic analysis revealed that qSE11 acted as a single Mendelian factor and that the allele from XQZB increased the SE rate. The validity of our conclusions was established when C65 was used to develop secondary F2 (BC5F2) and F2:3 (BC5F2:3) populations by backcrossing to ZH9308, with subsequent selfing. We entered 3600 plants from the F2 population and 3200 from the F2:3 populations into a genetic dissection program and dissected the major QTL qSE11 to a 350.7-kb region located on chromosome 11. This study will contribute to the future isolation of candidate genes of SE and will play a vital role in future hybrid rice seed production programs.

Meiosis is crucial in reproduction of plants and ensuring genetic diversity. Although several genes involved in homologous recombination and DNA repair have been reported, their functions in rice (Oryza sativa) male meiosis remain poorly understood. Here, we isolated and characterized the rice OsFIGNL1 (OsFidgetin-like 1) gene, encoding a conserved AAA-ATPase, and explored its function and importance in male meiosis and pollen formation. The rice Osfignl1 mutant exhibited normal vegetative growth, but failed to produce seeds and displayed pollen abortion phenotype. Phenotypic comparisons between the wild-type and Osfignl1 mutant demonstrated that OsFIGNL1 is required for anther development, and that the recessive mutation of this gene causes male sterility in rice. Complementation and CRISPR/Cas9 experiments demonstrated that wild-type OsFIGNL1 is responsible for the male sterility phenotype. Subcellular localization showed that OsFIGNL1-green fluorescent protein was exclusively localized in the nucleus of rice protoplasts. Male meiosis in the Osfignl1 mutant exhibited abnormal chromosome behavior, including chromosome bridges and multivalent chromosomes at diakinesis, lagging chromosomes, and chromosome fragments during meiosis. Yeast two-hybrid assays demonstrated OsFIGNL1 could interact with RAD51A1, RAD51A2, DMC1A, DMC1B, and these physical interactions were further confirmed by BiFC assay. Taken together, our results suggest that OsFIGNL1 plays an important role in regulation of male meiosis and anther development.

Hybridization, a common process in nature, can give rise to a vast reservoir of allelic variants. Combination of these allelic variants may result in novel patterns of gene action and is thought to contribute to heterosis. In this study, we analyzed genome-wide allele-specific gene expression (ASGE) in the super-hybrid rice variety Xieyou9308 using RNA sequencing technology (RNA-Seq). We identified 9325 reliable single nucleotide polymorphisms (SNPs) distributed throughout the genome. Nearly 68% of the identified polymorphisms were CT and GA SNPs between R9308 and Xieqingzao B, suggesting the existence of DNA methylation, a heritable epigenetic mark, in the parents and their F1 hybrid. Of 2793 identified transcripts with consistent allelic biases, only 480 (17%) showed significant allelic biases during tillering and/or heading stages, implying that trans effects may mediate most transcriptional differences in hybrid offspring. Approximately 67% and 62% of the 480 transcripts showed R9308 allelic expression biases at tillering and heading stages, respectively. Transcripts with higher levels of gene expression in R9308 also exhibited R9308 allelic biases in the hybrid. In addition, 125 transcripts were identified with significant allelic expression biases at both stages, of which 74% showed R9308 allelic expression biases. R9308 alleles may tend to preserve their characteristic states of activity in the hybrid and may play important roles in hybrid vigor at both stages. The allelic expression of 355 transcripts was highly stage-specific, with divergent allelic expression patterns observed at different developmental stages. Many transcripts associated with stress resistance were differently regulated in the F1 hybrid. The results of this study may provide valuable insights into molecular mechanisms of heterosis.