Effect of chemical fertilizer reduction on the quality of hybrid rice of different amylose contents.

To promote the reduction and efficiency of chemical fertilizers in rice production, two hybrid rice varieties with different amylose contents (16.43% and 27.58%) were selected to study the yield and quality performance of different quality rice varieties under reduced nitrogen/phosphorus conditions. Thus, the specific mechanism of the long-term nitrogen/phosphorus reduction effect on the quality of low- and high-amylose content (16.43% and 27.58%) rice was investigated by comparative analyses of the rapid visco analyzer and X-ray diffraction patterns, amylose contents, and starch structures of the samples. The results revealed that the effect of nitrogen fertilizer on the quality and yield of the hybrid rice was greater than that of phosphorus fertilizer. Indeed, reducing the former increased the Wx gene expression and amylose content of the rice varieties. Moreover, it reduced the starch crystallinity of Yixiangyou 1108 (2.16%), increasing the space between the starch grains and thus, chalkiness. Reducing nitrogen/phosphorus application did not significantly affect the yield and quality of the high-amylose rice but affected that of the high-quality rice with lower amylose contents. Thus, for high-amylose rice created for processing, appropriate chemical fertilizer reduction will not affect their yield and processing demand. PRACTICAL APPLICATIONS: Fertilization with different nitrogen and phosphorus fertilizers will seriously affect rice quality. The rice varieties with low amylose content (AC) could produce high taste quality rice by increasing nitrogen fertilizer and decreasing phosphorus fertilizer. The rice varieties with relatively high ACs should reduce the application of nitrogen/phosphorus fertilizer to appropriately increase AC, which can be used to produce healthy food with high resistant starch.

Disruption of EARLY LESION LEAF 1, encoding a cytochrome P450 monooxygenase, induces ROS accumulation and cell death in rice.

Lesion-mimic mutants (LMMs) provide a valuable tool to reveal the molecular mechanisms determining programmed cell death (PCD) in plants. Despite intensive research, the mechanisms behind PCD and the formation of lesions in various LMMs still remain to be elucidated. Here, we identified a rice (Oryza sativa) LMM, early lesion leaf 1 (ell1), cloned the causal gene by map-based cloning, and verified this by complementation. ELL1 encodes a cytochrome P450 monooxygenase, and the ELL1 protein was located in the endoplasmic reticulum. The ell1 mutant exhibited decreased chlorophyll contents, serious chloroplast degradation, upregulated expression of chloroplast degradation-related genes, and attenuated photosynthetic protein activity, indicating that ELL1 is involved in chloroplast development. RNA sequencing analysis showed that genes related to oxygen binding were differentially expressed in ell1 and wild-type plants; histochemistry and paraffin sectioning results indicated that hydrogen peroxide (H2 O2 ) and callose accumulated in the ell1 leaves, and the cell structure around the lesions was severely damaged, which indicated that reactive oxygen species (ROS) accumulated and cell death occurred in the mutant. TUNEL staining and comet experiments revealed that severe DNA degradation and abnormal PCD occurred in the ell1 mutants, which implied that excessive ROS accumulation may induce DNA damage and ROS-mediated cell death in the mutant. Additionally, lesion initiation in the ell1 mutant was light dependent and temperature sensitive. Our findings revealed that ELL1 affects chloroplast development or function, and that loss of ELL1 function induces ROS accumulation and lesion formation in rice.

Natural variation in the promoter of TGW2 determines grain width and weight in rice.

Understanding the genetic basis of natural variation in grain size among diverse rice varieties can help breeders develop high-yielding rice cultivars. Here, we report the discovery of qTGW2, a new semidominant quantitative trait locus for grain width and weight. The corresponding gene, TGW2, encodes CELL NUMBER REGULATOR 1 (OsCNR1) localized to the plasma membrane. A single nucleotide polymorphism (SNP) variation 1818 bp upstream of TGW2 is responsible for its different expression, leading to alteration in grain width and weight by influencing cell proliferation and expansion in glumes. TGW2 interacts with KRP1, a regulator of cell cycle in plants, to negatively regulate grain width and weight. Genetic diversity analysis of TGW2 in 141 rice accessions revealed it as a breeding target in a selective sweep region. Our findings provide new insights into the genetic mechanism underlying grain morphology and grain weight, and uncover a promising gene for improving rice yield.

AH2 encodes a MYB domain protein that determines hull fate and affects grain yield and quality in rice.

The palea and lemma (hull) are grass-specific organs, and determine grain size and quality. In the study, AH2 encodes a MYB domain protein, and functions in the development of hull and grain. Mutation of AH2 produces smaller grains and alters grain quality including decreased amylose content and gel consistency, and increased protein content. Meantime, part of the hull lost the outer silicified cells, and induces a transformation of the outer rough epidermis to inner smooth epidermis cells, and the body of the palea was reduced in the ah2 mutant. We confirmed the function of AH2 by complementation, CRISPR-Cas9, and cytological and molecular tests. Additionally, AH2, as a repressor, repress transcription of the downstream genes. Our results revealed that AH2 plays an important role in the determination of hull epidermis development, palea identity, and grain size.

Map-Based Cloning and Functional Analysis of YE1 in Rice, Which Is Involved in Light-Dependent Chlorophyll Biogenesis and Photoperiodic Flowering Pathway.

Light is one of the most important environmental factors that affect many aspects of plant growth, including chlorophyll (Chl) synthesis and flowering time. Here, we identified a rice mutant, yellow leaf and early flowering (ye1), and characterized the gene YE1 by using a map-based cloning method. YE1 encodes a heme oxygenase, which is localized to the chloroplasts. YE1 is expressed in various green tissues, especially in leaves, with a diurnal-rhythmic expression pattern, and its transcripts is also induced by light during leaf-greening. The mutant displays decreased Chl contents with less and disorderly thylakoid lamellar layers in chloroplasts, which reduced the photosynthesis rate. The early flowering phenotype of ye1 was not photoperiod-sensitive. Furthermore, the expression levels of Chl biosynthetic genes were downregulated in ye1 seedlings during de-etiolation responses to light. We also found that rhythmic expression patterns of genes involved in photoperiodic flowering were altered in the mutant. Based on these results, we infer that YE1 plays an important role in light-dependent Chl biogenesis as well as photoperiodic flowering pathway in rice.

Characterization of a major QTL for manganese accumulation in rice grain.

Some diets lack sufficient manganese (Mn), an essential mineral. Increasing Mn in grain by biofortification could prevent Mn deficiency, but may increase levels of the toxic element cadmium (Cd). Here, we investigated Mn in rice (Oryza sativa) grains in recombinant inbred lines (RILs) from the cross of 93-11 (low grain Mn) with PA64s (high grain Mn). Quantitative trait locus (QTL) analysis to identify loci controlling grain Mn identified a major QTL, qGMN7.1, on the short arm of chromosome 7; qGMN7.1 explained 15.6% and 22.8% of the phenotypic variation in the RIL populations grown in two distinct environments. We validated the QTL with a chromosome segment substitution line (CSSL), CSSL-qGMN7.1, in the 93-11 background harboring qGMN7.1 from PA64s. Compared to 93-11, CSSL-qGMN7.1 grain had increased Mn and decreased Cd concentrations; CSSL-qGMN7.1 roots also showed enhanced Mn uptake. Fine mapping delimited qGMN7.1 to a 49.3-kb region containing OsNRAMP5, a gene responsible for Mn and Cd uptake. Sequence variations in the OsNRAMP5 promoter caused changes in its transcript level, and in grain Mn levels. Our study thus cloned a major QTL for grain Mn concentration in rice, and identified materials for breeding rice for high Mn and low Cd concentrations in the grain.

Genetic analysis for rice seedling vigor and fine mapping of a major QTL qSSL1b for seedling shoot length.

Seedling vigor is an important agricultural trait as direct-seeded rice technology becomes widely applied. In order to investigate the genetic mechanisms underlying seedling vigor in rice, seeds of 132 recombinant inbred lines (RILs) derived from 93-11 and PA64s, harvested from Lingshui and Hangzhou were cultivated in the nutrient solution, and four indices for seedling vigor were measured including seedling shoot length (SSL), seedling root length (SRL), seedling wet weight (SWW) and seedling dry weight (SDW). Significant correlations were observed among the indices, and also between 1000-seed weight (TSW) and SWW or SDW. Combined with a high-resolution genetic map generated from sequencing of the RILs, 65 quantitative trait loci (QTLs) were detected on all chromosomes with interval of 1.93 Mb on average. Among 57 QTLs for seedling vigor, 28 were detected from seeds harvested in both sites and 33 were first identified. With BC3F2 derived from 93-11 and a CSSL harboring segments from PA64s in 93-11 background, a major QTL for SSL, qSSL1b was fine mapped within 80.5 kb between two InDel markers. Our study provides a platform for further cloning of the QTL and dissecting the molecular basis for seedling vigor at early seedling stage in rice.

[Genetic analysis of dense and erect panicle 2 allele DEP2-1388 and its application in hybrid rice breeding].

Using ethyl methanesulfonate (EMS) mutagenesis, we isolated an erect panicle mutant, R1338, from indica heavy-panicle restorer Shuhui498. Compared with wild type control, the mutant displayed dwarfism, erect and short panicle, short primary panicle branch, increased grain density, short grain length and increased grain thickness. In addition, the erect panicle architecture of R1388 resulted in significant decreased bending moment and increased resistance to panicle bending. Histocytological analysis indicated that the diameter of uppermost internode, cellulose content and lignin content play important roles in resistance to panicle bending. Genetic analysis revealed that the mutant phenotype was controlled by a semi-dominant nuclear gene. With resequencing and MutMap analysis strategy, we found that one SNP from A to G at the seventh exon of DEP2 resulted in the 928(th) amino acid substitution from arginine (AGG) to glycine (GGG) in R1338 mutant. Considering the phenotype of other dep2 mutants, the phenotype of R1338 was likely to be caused by the SNP in DEP2. The mutant R1338 and wild type were crossed with several sterile lines which respectively had different panicle types, the combinations generated from R1338 and curve panicle sterile lines showed semi-erect panicle, higher seed setting percentage and heterosis, and the combinations generated from R1388 and erect panicle sterile line with DEP1 showed erect panicle by gene additive effect. Moreover, the combinations with semi-erect panicle had superior light transmittance and stronger light intensity, which improved efficiency of light utilization to intermediate and subjacent leaves compared to the combinations with curved panicle. This study provides a good strategy to solve the problem of population density in three-line hybrid rice.

Genetic analysis of flag leaf size and candidate genes determination of a major QTL for flag leaf width in rice.

BACKGROUND: Flag leaf is the most essential organ for photosynthesis in rice and its size plays an important role in rice breeding for ideal plant-type. Flag leaf size affect photosynthesis to a certain extent, thereby influencing rice production. Several genes controlling leaf size and shape have been identified with mutants. Although a number of quantitative trait loci (QTLs) for leaf size and shape have been detected on 12 chromosomes with different populations of rice, few of them were cloned. RESULTS: The pair-wise correlation analysis was conducted on length, width and length-width ratio of the flag leaf, and yield per plant in the core recombinant inbred lines of Liang-You-Pei-Jiu (LYP9) developed in Hainan and Hangzhou. There were significant correlations among the three flag leaf size and shape traits. Interestingly, a positive correlation was found between flag leaf width and yield per plant. Based on the high-resolution linkage map we constructed before, 43 QTLs were detected for three flag leaf size and shape traits and yield per plant, among which 31 QTLs were unreported so far. Seven QTLs were identified common in two environments. And qFLW7.2, a new major QTL for flag leaf width, was fine mapped within 27.1 kb region on chromosome 7. Both qFLW7.2 and qPY7 were located in the interval of 45.30 ~ 53.34 cM on chromosome 7, which coincided with the relationship between yield per plant (PY) and flag leaf width (FLW). CONCLUSION: qFLW7.2, which explained 14% of the phenotypic variation, increased flag leaf width with 93-11 allele. Two candidate genes were selected based on sequence variation and expression difference between two parents, which facilitated further QTL cloning and molecular breeding in super rice.

A rice DEAD-box RNA helicase protein, OsRH17, suppresses 16S ribosomal RNA maturation in Escherichia coli.

DEAD-box proteins comprise a large protein family. These proteins function in all types of processes in RNA metabolism and are highly conserved among eukaryotes. However, the precise functions of DEAD-box proteins in rice physiology and development remain unclear. In this study, we identified a rice DEAD-box protein, OsRH17, that contains a DEAD domain and all of the common conserved motifs of DEAD-box RNA helicases. OsRH17 was specifically expressed in pollen and differentiated callus and upregulated by application of the plant hormones naphthyl acetic acid (NAA) and abscisic acid (ABA). The OsRH17:GFP fusion protein was localized to the nucleus. Tiny amounts of OsRH17 and partial fragments (N-427 and C-167) were detected when they were expressed in Escherichia coli, a prokaryote. Growth of the host cells was suppressed in E. coli by OsRH17, N-427 or C-167, and this suppression was independent of the concentration of the NaCl in the medium. Expression analysis of rRNAs in E. coli revealed that the 16S rRNA precursor accumulated in transgenic E. coli cells, and the relative growth rate was inversely proportional to the levels of pre-16S rRNA accumulation. Results suggested that OsRH17 may play a role in ribosomal biogenesis and suppress 16S rRNA maturation in E. coli. No visible phenotype was observed in transgenic yeast and rice (overexpressing OsRH17, N-427, and C-167, as well as OsRH17 knockdown), and even in some abiotic and biotic stresses, which could be due to the redundancy in rice under normal conditions.

Fine mapping and candidate gene analysis of a major QTL for panicle structure in rice.

KEY MESSAGE: A gene not only control tiller and plant height, but also regulate panicle structure by QTL dissection in rice. An ideal panicle structure is important for improvement of plant architecture and rice yield. In this study, using recombinant inbred lines (RILs) of PA64s and 93-11, we identified a quantitative trait locus (QTL), designated qPPB3 for primary panicle branch number. With a BC3F2 population derived from a backcross between a resequenced RIL carrying PA64s allele and 93-11, qPPB3 was fine mapped to a 34.6-kb genomic region. Gene prediction analysis identified four putative genes, among which Os03g0203200, a previously reported gene for plant height and tiller number, Dwarf 88 (D88)/Dwarf 14 (D14), had three nucleotide substitutions in 93-11 compared with PA64s. The T to G substitution resulted in one amino acid change from valine in 93-11 to glycine in PA64s. Real-time PCR analysis showed expression level of D88 was higher in 93-11 than PA64s. The expression of APO1 and IPA1 increased, while GN1a and DST decreased in 93-11 compared with PA64s. Therefore, D88/D14 is not only a key regulator for branching, but also affects panicle structure.

LSCHL4 from Japonica Cultivar, which is allelic to NAL1, increases yield of indica super rice 93-11.

The basic premise of high yield in rice is to improve leaf photosynthetic efficiency and coordinate the source-sink relationship in rice plants. Quantitative trait loci (QTLs) related to morphological traits and chlorophyll content of rice leaves were detected at the stages of heading to maturity, and a major QTL (qLSCHL4) related to flag leaf shape and chlorophyll content was detected at both stages in recombinant inbred lines constructed using the indica rice cultivar 93-11 and the japonica rice cultivar Nipponbare. Map-based cloning and expression analysis showed that LSCHL4 is allelic to NAL1, a gene previously reported in narrow leaf mutant of rice. Overexpression lines transformed with vector carrying LSCHL4 from Nipponbare and a near-isogenic line of 93-11 (NIL-9311) had significantly increased leaf chlorophyll content, enlarged flag leaf size, and improved panicle type. The average yield of NIL-9311 was 18.70% higher than that of 93-11. These results indicate that LSCHL4 had a pleiotropic function. Exploring and pyramiding more high-yield alleles resembling LSCHL4 for super rice breeding provides an effective way to achieve new breakthroughs in raising rice yield and generate new ideas for solving the problem of global food safety.

Dissecting yield-associated loci in super hybrid rice by resequencing recombinant inbred lines and improving parental genome sequences.

The growing world population and shrinkage of arable land demand yield improvement of rice, one of the most important staple crops. To elucidate the genetic basis of yield and uncover its associated loci in rice, we resequenced the core recombinant inbred lines of Liang-You-Pei-Jiu, the widely cultivated super hybrid rice, and constructed a high-resolution linkage map. We detected 43 yield-associated quantitative trait loci, of which 20 are unique. Based on the high-density physical map, the genome sequences of paternal variety 93-11 and maternal cultivar PA64s of Liang-You-Pei-Jiu were significantly improved. The large recombinant inbred line population combined with plentiful high-quality single nucleotide polymorphisms and insertions/deletions between parental genomes allowed us to fine-map two quantitative trait loci, qSN8 and qSPB1, and to identify days to heading8 and lax panicle1 as candidate genes, respectively. The quantitative trait locus qSN8 was further confirmed to be days to heading8 by a complementation test. Our study provided an ideal platform for molecular breeding by targeting and dissecting yield-associated loci in rice.