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Y900 is one of the top hybrid rice (Oryza sativa) varieties, with its yield exceeding 15 t·hm−2. To dissect the mechanism of heterosis, we sequenced the male parent line R900 and female parent line Y58S using long-read and Hi-C technology. High-quality reference genomes of 396.41 Mb and 398.24 Mb were obtained for R900 and Y58S, respectively. Genome-wide variations between the parents were systematically identified, including 1,367,758 single-nucleotide polymorphisms, 299,149 insertions/deletions, and 4,757 structural variations. The level of variation between Y58S and R900 was the lowest among the comparisons of Y58S with other rice genomes. More than 75% of genes exhibited variation between the two parents. Compared with other two-line hybrids sharing the same female parent, the portion of Geng/japonica (GJ)-type genetic components from different male parents increased with yield increasing in their corresponding hybrids. Transcriptome analysis revealed that the partial dominance effect was the main genetic effect that constituted the heterosis of Y900. In the hybrid, both alleles from the two parents were expressed, and their expression patterns were dynamically regulated in different tissues. The cis-regulation was dominant for young panicle tissues, while trans-regulation was more common in leaf tissues. Overdominance was surprisingly prevalent in stems and more likely regulated by the trans-regulation mechanism. Additionally, R900 contained many excellent GJ haplotypes, such as NARROW LEAF1, Oryza sativa SQUAMOSA PROMOTER BINDING PROTEIN-LIKE13, and Grain number, plant height, and heading date8, making it a good complement to Y58S. The fine-tuned mechanism of heterosis involves genome-wide variation, GJ introgression, key functional genes, and dynamic gene/allele expression and regulation pattern changes in different tissues and growth stages.

Y900 is one of the top hybrid rice varieties with a yield exceeding 15 t/hm2. To dissect the mechanism of heterosis, the male parent line R900 and female parent line Y58S were sequenced using long-read and Hi-C technology. High-quality reference genomes of sizes of 396.41 Mb and 398.24 Mb were obtained for R900 and Y58S, respectively. Genome-wide variations between the parents were systematically identified, including 1,367,758 SNPs and 299,149 Indels. No megabase level structural variations exist. >75% of genes exhibited variation between the two parents. Compared with other two-line hybrids sharing the same female parent, the Geng/japonica-type genetic components from different male parents showed an increasing trend from phase 2-4 super-hybrid rice; Transcriptome analysis revealed that additive and dominance effects are the main genetic effects that constitute the heterosis of Y900. Allele-specific expression patterns and expression regulation patterns are quite dynamic in different tissues. For young panicle tissues, cis-regulation is dominant, while trans-regulation is more popular in leaf issues. Overdominance is more likely regulated by the trans-regulation mechanism. The differential gene expression and regulation pattern are closely related to Geng/japonica introgression. Additionally, R900 contained several excellent japonica haplotypes, such as NAL1, OsSPL13, Ghd8, OsBRI1, and DTH2, which make a good complement to Y58S. The fine tune mechanism through dynamic expression or regulation pattern change, especially on some key functional genes, is the base for heterosis.

Summary The development of embryo sacs is crucial for seed production in plants, but the genetic basis regulating the meiotic crossover formation in the macrospore and microspore mother cells remains largely unclear. Here, we report the characterization of a spontaneous rice female sterile variation 1 mutant (fsv1) that showed severe embryo sacs abortion with low seed‐setting rate. Through map‐based cloning and functional analyses, we isolated the causal gene of fsv1, OsMLH3 encoding a MutL‐homolog 3 protein, an ortholog of HvMLH3 in barley and AtMLH3 in Arabidopsis. OsMLH3 and OsMLH1 (MutL‐homolog 1) interact to form a heterodimer (MutLγ) to promote crossover formation in the macrospore and microspore mother cells and development of functional megaspore during meiosis, defective OsMLH3 or OsMLH1 in fsv1 and CRISPR/Cas9‐based knockout lines results in reduced type I crossover and bivalent frequency. The fsv1 and OsMLH3‐knockout lines are valuable germplasms for development of female sterile restorer lines for mechanized seed production of hybrid rice.

The brown planthopper (BPH) (Nilaparvata lugens Stal) is a highly destructive pest that seriously damages rice (Oryza sativa L.) and causes severe yield losses. To better understand the physiological and metabolic mechanisms through which BPHs respond to resistant rice, we combined mass-spectrometry-based lipidomics with transcriptomic analysis and gene knockdown techniques to compare the lipidomes of BPHs feeding on either of the two resistant (NIL-Bph6 and NIL-Bph9) plants or a wild-type, BPH susceptible (9311) plant. Insects that were fed on resistant rice transformed triglyceride (TG) to phosphatidylcholine (PC) and digalactosyldiacylglycerol (DGDG), with these lipid classes showing significant alterations in fatty acid composition. Moreover, the insects that were fed on resistant rice were characterized by prominent expression changes in genes involved in lipid metabolism processes. Knockdown of the NlBmm gene, which encodes a lipase that regulates the mobilization of lipid reserves, significantly increased TG content and feeding performance of BPHs on resistant plants relative to dsGFP-injected BPHs. Our study provides the first detailed description of lipid changes in BPHs fed on resistant and susceptible rice genotypes. Results from BPHs fed on resistant rice plants reveal that these insects can accelerate TG mobilization to provide energy for cell proliferation, body maintenance, growth and oviposition.

The enhancement of rice production numbers can be achieved by using quality rice cultivars and fertilizers. The double rice cropping model has given rise to an important rice production system in southern China. Exploring the possibility of whether hybrid vigor could make a substantial contribution to early and late season rice production, and how the heterosis expression of hybrid rice functions under different levels of fertilizer application is of great significance. The objective of this study was to evaluate the grain yield and associated plant traits of popular hybrid and inbred rice varieties with large–scale promotion under conditions of customary (high) and combined (low) fertilization in the early and late seasons of 2017–18 in Changsha County, Hunan Province, China. We found that hybrid rice varieties displayed their respective advantages in the early and late rice seasons, but the advantages in their relative yield traits varied. The leading advantages of early season rice were effective panicle number per hill (EPN), 1000–grain weight (KGW), harvest index (HI), yield, and nitrogen use efficiency (NUE), whereas in late season rice, the foremost advantages were grain number per panicle (GNP), HI, yield, and NUE. The EPN was the prime advantage of early season hybrid rice with a short growth period, and the GNP was the main advantage of late season hybrid rice with a long growth period. Notably, the main yield advantage of hybrid rice was stronger under combined (low) fertilization than under customary (high) fertilization. Hence, high yield can be achieved by selecting the best hybrid rice varieties supported by combined fertilization (lower fertilizer use with higher efficiency).

Obtaining genetic variation information from indica rice hybrid parents and identification of loci associated with heterosis are important for hybrid rice breeding. Here, we resequence 1,143 indica accessions mostly selected from the parents of superior hybrid rice cultivars of China, identify genetic variations, and perform kinship analysis. We find different hybrid rice crossing patterns between 3- and 2-line superior hybrid lines. By calculating frequencies of parental variation differences (FPVDs), a more direct approach for studying rice heterosis, we identify loci that are linked to heterosis, which include 98 in superior 3-line hybrids and 36 in superior 2-line hybrids. As a proof of concept, we find two accessions harboring a deletion in OsNramp5, a previously reported gene functioning in cadmium absorption, which can be used to mitigate rice grain cadmium levels through hybrid breeding. Resource of indica rice genetic variation reported in this study will be valuable to geneticists and breeders., Hybrid rice cultivars are widely planted around the world. Here, the authors resequence 1,143 indica accessions, focusing on the parents of superior hybrid rice lines in China, and reveal genetic loci that are associated with heterosis via measuring frequency of parental variation difference (FPVD).

Significance Developing a male-sterility system that is effective in multiple species is essential for hybrid seed production in different plants, especially for plants without cloned male-sterility genes. Here, we identified the transcriptional regulation mechanism for maize male-sterility gene ZmMs7 and thereby developed a dominant male-sterility system that was proved to be effective in maize, rice, and Arabidopsis. Compared with current male-sterility systems, this system has potential advantages, e.g., utilization of a single transgene cassette, high stability of male sterility under different genetic backgrounds, and producing fluorescent transgenic and normal color nontransgenic F1 hybrid seeds which can be used flexibly in different countries where transgenic crop cultivation is prohibited or allowed. Therefore, it is a simple, cost-effective, and multiple-crop-applicable biotechnology., Understanding the molecular basis of male sterility and developing practical male-sterility systems are essential for heterosis utilization and commercial hybrid seed production in crops. Here, we report molecular regulation by genic male-sterility gene maize male sterility 7 (ZmMs7) and its application for developing a dominant male-sterility system in multiple species. ZmMs7 is specifically expressed in maize anthers, encodes a plant homeodomain (PHD) finger protein that functions as a transcriptional activator, and plays a key role in tapetal development and pollen exine formation. ZmMs7 can interact with maize nuclear factor Y (NF-Y) subunits to form ZmMs7-NF-YA6-YB2-YC9/12/15 protein complexes that activate target genes by directly binding to CCAAT box in their promoter regions. Premature expression of ZmMs7 in maize by an anther-specific promoter p5126 results in dominant and complete male sterility but normal vegetative growth and female fertility. Early expression of ZmMs7 downstream genes induced by prematurely expressed ZmMs7 leads to abnormal tapetal development and pollen exine formation in p5126-ZmMs7 maize lines. The p5126-ZmMs7 transgenic rice and Arabidopsis plants display similar dominant male sterility. Meanwhile, the mCherry gene coupled with p5126-ZmMs7 facilitates the sorting of dominant sterility seeds based on fluorescent selection. In addition, both the ms7-6007 recessive male-sterility line and p5126-ZmMs7M dominant male-sterility line are highly stable under different genetic germplasms and thus applicable for hybrid maize breeding. Together, our work provides insight into the mechanisms of anther and pollen development and a promising technology for hybrid seed production in crops.

Summary Heterosis utilization is the most effective way to improve rice yields. The cytoplasmic male‐sterility (CMS) and photoperiod/thermosensitive genic male‐sterility (PTGMS) systems have been widely used in rice production. However, the rate of resource utilization for the CMS system hybrid rice is low, and the hybrid seed production for the PTGMS system is affected by the environment. The technical limitations of these two breeding methods restrict the rapid development of hybrid rice. The advantages of the genic male‐sterility (GMS) rice, such as stable sterility and free combination, can fill the gaps of the first two generations of hybrid rice technology. At present, the third‐generation hybrid rice breeding technology is being used to realize the application of GMS materials in hybrid rice. This study aimed to use an artificial CMS gene as a pollen killer to create a smart sterile line for hybrid rice production. The clustered regularly interspaced short palindromic repeats/CRISPR‐associated 9 (CRISPR/Cas9) technology was used to successfully obtain a CYP703A3‐deficient male‐sterile mutant containing no genetically modified component in the genetic background of indica 9311. Through young ear callus transformation, this mutant was transformed with three sets of element‐linked expression vectors, including pollen fertility restoration gene CYP703A3, pollen‐lethality gene orfH79 and selection marker gene DsRed2. The maintainer 9311‐3B with stable inheritance was obtained, which could realize the batch breeding of GMS materials. Further, the sterile line 9311‐3A and restorer lines were used for hybridization, and a batch of superior combinations of hybrid rice was obtained.

It is a matter of fact that essentially derived varieties (EDV) exist in rice breeding in China, which impact breeding innovation negatively. This study explores the potential of using identity score (IS) method, which was originally developed by animal breeding and population genetic society, in determining essentially deviated rice male sterile (MS) lines released by “two-way hybrid rice system”. Six MS lines plus a control line, Nongken 58, which is an autonomous mutation donor of Nongken 58S, are analyzed with IS. Our results show that IS can determine similarity between rice lines to a very good level, with superiority on genome-wide coverage and accuracy when compared with NYT 1433-2007, which is the present rice authenticity standard. It is noteworthy that the 2 EDV lines identified by our study show enough polymorphisms when tested by simple sequence repeat (SSR), which suggests that it should call for challenges for SSR based authenticity test as required by the present national standards. Discussions on how to push forward rice breeding with implementation of IS are also covered.

Brown planthopper (BPH) is one of the most destructive insects affecting rice ( Oryza sativa L.) production. Phenylalanine ammonia-lyase (PAL) is a key enzyme involved in plant defense against pathogens, but the role of PAL in insect resistance is still poorly understood. Here we show that expression of the majority of PALs in rice is significantly induced by BPH feeding. Knockdown of Os PALs significantly reduces BPH resistance, whereas overexpression of OsPAL8 in a susceptible rice cultivar significantly enhances its BPH resistance. We found that OsPALs mediate resistance to BPH by regulating the biosynthesis and accumulation of salicylic acid and lignin. Furthermore, we show that expression of OsPAL6 and OsPAL8 in response to BPH attack is directly up-regulated by OsMYB30, an R2R3 MYB transcription factor. Taken together, our results demonstrate that the phenylpropanoid pathway plays an important role in BPH resistance response, and provide valuable targets for genetic improvement of BPH resistance in rice.

Significance Rice breeding programs aim to develop cultivars with improved traits, including high grain yield and superior quality. In rice, OsPDCD5 encodes a programmed cell death 5 protein. Targeted mutagenesis of OsPDCD5 enhanced grain yield and plant architecture. Statistical analysis indicated that plot grain yield of OsPDCD5 knockout lines was enhanced by 6.25 to 20.13% in 11 popular or newly bred rice cultivars compared with the corresponding wild types. The OsPDCD5 knockout lines showed increases in milled rice percentage and gel consistency, and a decrease in amylose content. Our results provide insight into the molecular mechanism by which OsPDCD5 influences grain yield and plant architecture, and highlight a promising candidate gene for use in breeding programs designed to develop super rice cultivars.

The brown planthopper (BPH) (Nilaparvata lugens Stål) is a highly destructive pest that seriously damages rice (Oryza sativa L.) and causes severe yield losses. To better understand the physiological and metabolic mechanisms through which BPHs respond to resistant rice, we combined mass-spectrometry-based lipidomics with transcriptomic analysis and gene knockdown techniques to compare the lipidomes of BPHs feeding on either of the two resistant (NIL-Bph6 and NIL-Bph9) plants or a wild-type, BPH susceptible (9311) plant. Insects that were fed on resistant rice transformed triglyceride (TG) to phosphatidylcholine (PC) and digalactosyldiacylglycerol (DGDG), with these lipid classes showing significant alterations in fatty acid composition. Moreover, the insects that were fed on resistant rice were characterized by prominent expression changes in genes involved in lipid metabolism processes. Knockdown of the NlBmm gene, which encodes a lipase that regulates the mobilization of lipid reserves, significantly increased TG content and feeding performance of BPHs on resistant plants relative to dsGFP-injected BPHs. Our study provides the first detailed description of lipid changes in BPHs fed on resistant and susceptible rice genotypes. Results from BPHs fed on resistant rice plants reveal that these insects can accelerate TG mobilization to provide energy for cell proliferation, body maintenance, growth and oviposition.

The brown planthopper (BPH), Nilaparvata lugens Stal, is one of the major pests of rice. It uses its stylet to penetrate rice phloem, feeding on rice sap and causing direct damage to rice or even plant death. During the feeding process, BPHs secrete saliva into plant tissues, which plays crucial roles in the plant-insect interactions. However, little is known about how the salivary proteins secreted by BPH affect feeding ability and how they induce plant immune responses. Here, we identified an N. lugens Salivary Protein 1 (NlSP1) by screening salivary proteome and characterized its functions in BPH and plants. NlSP1 induces cell death, H2O2 accumulation, the expression of defense-related genes, and callose deposition in planta. The active region of NlSP1 that induces plant cell death is located in its N-terminal region. Inhibition of NlSP1 expression in BPHs reduced their feeding ability and had a lethal effect on them. Most importantly, we demonstrated that NlSP1 was able to be secreted into rice plant during feeding process and form a complex with certain interacting partner of rice. These results provide a detailed characterization of a salivary protein from BPHs and offers new insights into our understanding of rice-BPH interaction.

Two‐line hybrid rice with high efficiency and yield was successfully and widely being planted in China. HHZ is an elite inbred rice which is widely cultivated in Southern China. The latest developed engineered hybrid is promising for the future of hybrid rice breeding and production. In order to explore its heterosis performance, the main yield traits of two different groups of hybrid rice varieties and a inbred variety were investigated, specifically: (1) a large‐scale extension of high‐yield varieties of two‐line hybrid rice and their parents (restorer lines), (2) a new generation of hybrid rice created by genetic engineered with HHZ as a background genome and its parental lines, and (3) a inbred indica rice variety, HHZ. We postulate that the performance of the main yield traits of genetically engineered hybrid rice may be better than that of commercial high‐yielding hybrid rice in the same paddy field. To test our assumption, field experiments were conducted to investigate the performance of the main yield traits in the different types of hybrid rice and to compare and analyze the differences of the yield heterosis traits. The hybrid rice varieties showed obvious high‐parent heterosis for effective panicle number (EPN) per hill, grain number per panicle (GNP), seed set rate (SSR), and harvest index (HI), but high‐parent heterosis for the 1000‐grain weight (KGW) was negative. Standard heterosis for GNP and KGW was found in the hybrid rice varieties, and the standard heterosis for EPN, SSR, and HI was negative. The grain number per panicle (GNP) is the largest contributor to heterotic performance. The genetically engineered hybrid rice showed similar heterotic performance to but not greater than that of two‐line hybrid rice varieties. This study provides important insights for the breeding, cultivation, and popularization of different types of hybrid rice varieties.

Background Increasing rice yield with fewer external inputs is critical to ensuring food security, reducing environmental costs, and improving returns. Use of hybrid rice has expanded greatly in China due to its higher yield potential. Meanwhile, large and increasing amounts of nitrogen (N) fertilizers have been used for expanding rice production in China. It is not clear to what extent the success of hybrid rice in China is associated with N fertilizer inputs. Findings We observed that the higher grain yield with N fertilizer in hybrid rice was driven more by a higher yield without N fertilizer than by increases in grain yield with N fertilizer under moderate to high soil fertility conditions. Conclusions Our results suggest that greater application of N fertilizers is not needed to benefit from hybrid rice production under moderate to high soil fertility conditions, and that improving and maintaining soil fertility should be a focus for sustaining hybrid rice production. Moreover, our study also indicates that zero-N testing may be a potentially useful tool to develop hybrid rice with high yield and without requirement of greater external N inputs under moderate to high soil fertility conditions. Electronic supplementary material The online version of this article (10.1186/s12284-017-0182-1) contains supplementary material, which is available to authorized users.

Background The breeding and large-scale adoption of hybrid rice is an important achievement in modern agriculture. Mechanized seed production is urgently needed for widespread adoption of hybrid rice because it can compensate for the shortage of manual labor to meet the growing food demands in China. Results Here, we report the development of a mechanized hybrid rice seed production method using a female sterile rice. In this method, three closely linked gene expression cassettes were introduced into female sterile rice. The three expression cassettes are: 1) a rice female fertility gene expression cassette; 2) a pollen-lethal gene expression cassette; and 3) a red fluorescence protein gene expression cassette. During the self-fertilization process of a heterozygous transgenic rice plant, pollen grains carrying the transgene die off and cannot participate in fertilization; pollen grains not carrying a transgene can normally fertilize the female gamete, leading to fructification. By means of fluorescence-assisted sorting, homogeneous female sterile rice seeds are sorted out from other seeds carrying the transgene and are used for mechanized hybrid rice seed production; heterozygous seeds carrying the transgene can then be used in the multiplication of female sterile rice. Conclusions This technology solves the difficulty of multiplying female-sterile rice, allows for mechanized production of hybrid rice seed, and will prove especially valuable in systems using a mixed-planting, mixed-harvesting approach. Moreover, it uses transgenic technology that has not yet been employed in a seed production process in which the output is non-transgenic seeds.

Rice ( Oryza sativa L.) is a chilling-sensitive staple crop that originated in subtropical regions of Asia. Introduction of the chilling tolerance trait enables the expansion of rice cultivation to temperate regions. Here we report the cloning and characterization of HAN1 , a quantitative trait locus (QTL) that confers chilling tolerance on temperate japonica rice. HAN1 encodes an oxidase that catalyzes the conversion of biologically active jasmonoyl-L-isoleucine (JA-Ile) to the inactive form 12-hydroxy-JA-Ile (12OH-JA-Ile) and fine-tunes the JA-mediated chilling response. Natural variants in HAN1 diverged between indica and japonica rice during domestication. A specific allele from temperate japonica rice, which gained a putative MYB cis-element in the promoter of HAN1 during the divergence of the two japonica ecotypes, enhances the chilling tolerance of temperate japonica rice and allows it to adapt to a temperate climate. The results of this study extend our understanding of the northward expansion of rice cultivation and provide a target gene for the improvement of chilling tolerance in rice.

The Pi-ta gene deployed in southern U.S. rice germplasm is effective in preventing the infection by strains of Magnaporthe oryzae isolates that carry the avirulence (AVR) gene AVR-Pita1. In the present study, 169 isolates from rice (Oryza sativa) cultivars, with and without Pi-ta, were analyzed for their genetic identity using an international differential system, repetitive element-based polymerase chain reaction (Rep-PCR), and sequence analysis of PCR products of AVR-Pita1. These isolates belong to the races IA1, IB1, IB17, IC1, and IC17 of M. oryzae. These isolates were further classified into 15 distinct groups by Rep-PCR. There was a predominant group within each race. Pathogenicity assays on ‘Katy’ (Pi-ta) and ‘M202’ (pi-ta) rice determined that IC1 was virulent to Katy and M202; IB17, IC17, and most of IA1 and IB1 were avirulent to Katy and virulent to M202, suggesting that the Pi-ta gene in Katy is responsible for preventing infection by these isolates. Consistently, AVR-Pita1 was not amplified from 28 virulent isolates. One AVR-Pita1 allele was amplified by AVR-Pita1-specific primers in 78 avirulent isolates. Interestingly, different AVR-Pita1 alleles were found in each of the 12 avirulent isolates, as determined by DNA sequencing. Sequence analysis of 90 PCR products revealed 10 AVR-Pita1 haplotypes, 4 of which were new. In total, 12 amino acid changes were identified in the new variants when compared with the first described AVR-Pita sequence (AF207841). The finding of isolates with altered AVR-Pita1 from rice cultivars with and without Pi-ta suggests that these virulent isolates were adapted to the field environments in the southern United States. Further research will be needed to verify this prediction.

Traits such as grain shape, panicle length and seed shattering, play important roles in grain yield and harvest. In this study, the cloning and functional analysis of PANICLE TRAITS 2 (PT2), a novel gene from the Indica rice Chuandali (CDL), is reported. PT2 is synonymous with Growth-Regulating Factor 4 (OsGRF4), which encodes a growth-regulating factor that positively regulates grain shape and panicle length and negatively regulates seed shattering. Higher expression of OsGRF4 is correlated with larger grain, longer panicle and lower seed shattering. A unique OsGRF4 mutation, which occurs at the OsmiRNA396 target site of OsGRF4, seems to be associated with high levels of OsGRF4 expression, and results in phenotypic difference. Further research showed that OsGRF4 regulated two cytokinin dehydrogenase precursor genes (CKX5 and CKX1) resulting in increased cytokinin levels, which might affect the panicle traits. High storage capacity and moderate seed shattering of OsGRF4 may be useful in high-yield breeding and mechanized harvesting of rice. Our findings provide additional insight into the molecular basis of panicle growth.

Quantitative trait loci (QTL) play important roles in controlling rice blast disease. In the present study, 10 field isolates of the races IA1, IB1, IB17, and IC1 of US rice blast fungus Magnaporthe oryzae collected in 1996 and 2009 were used to identify blast resistance QTL with a recombinant inbred line (RIL) population consisting of 227 F₇ individuals derived from the cross of rice (Oryza sativa L.) cultivars Lemont and Jasmine 85. Jasmine 85 is an indica cultivar that is moderately resistant, and Lemont is a tropical japonica cultivar susceptible to rice blast in greenhouse inoculation. Disease reactions of the parents and RILs were evaluated under greenhouse conditions. A total of six resistance QTL, qBLR8, qBLR10-1, qBLR10-2, qBLR10-3, qBLR12-1, and qBLR12-2, were identified on chromosomes 8, 10, and 12, respectively. Phenotypic variation, conditioned by these six resistance QTL, ranged from 5.37 to 39.18%. Among them, qBLR12-1 and qBLR12-2 provided the strongest resistance to the newest isolates of the most virulent race IA1 of M. oryzae. Three of these resistance QTL have been identified using different blast isolates in a previous study. qBLR10-1, qBLR10-2, and qBLR10-3 have not been previously found in this cross. These confirmed and new resistance QTL will be useful for the development of rice cultivars with improved effective resistance to rice blast via a marker-assisted selection (MAS) approach.

Table S2.Indigenous soil N uptake and labeled-N uptake in rice cultivars in a micro-plot experiment with application of 15Â N labeled urea in Changsha, Hunan Province in China in 2013. (DOC 31 kb)

Materials and Methods (DOC 52 kb)

Table S1.Grain yield in rice cultivars grown under two N fertilizer rates in Xingyi, Guizhou Province, China in 2012â 2014 (Experiment I), and in Ningxiang, Hunan Province, China in 2015 and 2016 (Experiment II). (DOC 53 kb)

In this study, we reported that a F-box protein, OsADF, as one of the direct targets of TDR , plays a critical role in rice tapetum cell development and pollen formation. The tapetum, the innermost sporophytic tissue of anther, plays an important supportive role in male reproduction in flowering plants. After meiosis, tapetal cells undergo programmed cell death (PCD) and provide nutrients for pollen development. Previously we showed that tapetum degeneration retardation (TDR), a basic helix-loop-helix transcription factor, can trigger tapetal PCD and control pollen wall development during anther development. However, the comprehensive regulatory network of TDR remains to be investigated. In this study, we cloned and characterized a panicle-specific expression F-box protein, anther development F-box (OsADF). By qRT-PCR and RNA in situ hybridization, we further confirmed that OsADF expressed specially in tapetal cells from stage 9 to stage 12 during anther development. In consistent with this specific expression pattern, the RNAi transgenic lines of OsADF exhibited abnormal tapetal degeneration and aborted microspores development, which eventually grew pollens with reduced fertility. Furthermore, we demonstrated that the TDR, a key regulator in controlling rice anther development, could regulate directly the expression of OsADF by binding to E-box motifs of its promoter. Therefore, this work highlighted the possible regulatory role of TDR, which regulates tapetal cell development and pollen formation via triggering the possible ADF-mediated proteolysis pathway.

Genetic diversity within parental lines of hybrid rice is the foundation of heterosis utilization and yield improvement. Previous studies have suggested that genetic diversity was narrow in cytoplasmic male sterile (CMS/A line) and restorer lines (R line) for Three-line hybrid rice. However, the genetic diversity within maintainer lines (B line), especially at a genome-wide scale, remains largely unknown. In the present study, we performed deep re-sequencing of the elite maintainer line V20B (Oryza sativa L. ssp. indica). We then compared the V20B sequence with the 93-11 (Oryza sativa L. ssp. indica) genome sequence. 112.1 × 106 paired-end reads (PE reads) were generated with approximately 30-fold sequencing depth. The V20B PE reads uniquely covered 87.6 % of the 93-11 genome sequence. Overall, a total of 660,778 single-nucleotide polymorphism (SNPs) and 266,301 insertions and deletions (InDels) were identified, yielding an average of 2.1 SNPs/kb and 0.8 InDels/kb. Genome-wide distribution of the SNPs and InDels was non-random, and variation-rich and variation-poor regions were identified in all chromosomes. A total of 20,562 non-synonymous SNPs spanning 8,854 genes were annotated. Our results identified DNA polymorphisms at the genome-wide scale and uncovered the high level of genetic diversity between V20B and 93-11. Our results proved that next-generation sequencing technologies can be powerful tools to study genome-wide DNA polymorphisms, to query genetic diversity, and to enable molecular improvement efforts with Three-line hybrid rice. Further, our results also indicated that 93-11 could be used as core germplasm for the improvement of wild-abortive CMS lines and the maintainer lines.

Molecular mapping of new blast resistance genes is important for developing resistant rice cultivars using marker-assisted selection. In this study, 259 recombinant inbred lines (RILs) were developed from a cross between Nipponbare and 93-11, and were used to construct a 1165.8-cM linkage map with 131 polymorphic simple sequence repeat (SSR) markers. Four major quantitative trait loci (QTLs) for resistance to six isolates of Magnaporthe oryzae were identified: qPi93-1, qPi93-2, qPi93-3, and qPiN-1. For the three genes identified in 93-11, qPi93-1 is linked with SSR marker RM116 on the short arm of chromosome 11 and explains 33% of the phenotypic variation in resistance to isolate CHE86. qPi93-2 is linked with SSR marker RM224 on the long arm of chromosome 11 and accounts for 31% and 25% of the phenotypic variation in resistance to isolates 162-8B and ARB50, respectively. qPi93-3 is linked with SSR marker RM7102 on chromosome 12 and explains 16%, 53%, and 28% of the phenotypic variation in resistance to isolates CHE86, ARB52, and ARB94, respectively. QTL qPiN-1 from Nipponbare is associated with SSR marker RM302 on chromosome 1 and accounts for 34% of the phenotypic variation in resistance to isolate PO6-6. These new genes can be used to develop new varieties with blast resistance via marker-aided selection and to explore the molecular mechanism of rice blast resistance.

Immature embryos from immature seeds of rice (Oryza sativa L.) were transformed by biolistic bombardment with the plasmid carrying the coding region of the hygromycin phosphotransferase gene under the control of the 5' region of the cauliflower mosaic virus 35S promoter and the synthetic green fluorescence protein gene (sgfp) under the control of the maize ubiquitine promoter. Southern blot analysis confirmed the stable integration of hpt and sgfp genes in transformants. Subsequently leaves from regenerated plants were resistant to hygromycin, and microscopic observation of the green fluorescence and immunoblotting analysis revealed that green fluorescence protein was not only detected in the leaf and pollen of primary transformants but also in mature seeds. The results bear out the importance of the suitability of GFP as an in vivo marker to follow the processes of selection of somatic hybrid embryos and plants.

Hybrid rice is the dominant form of rice planted in China, and its use has extended worldwide since the 1970s. It offers great yield advantages and has contributed greatly to the world’s food security. However, the molecular mechanisms underlying heterosis have remained a mystery. In this study we integrated genetics and omics analyses to determine the candidate genes for yield heterosis in a model two-line rice hybrid system, Liang-you-pei 9 (LYP9) and its parents. Phenomics study revealed that the better parent heterosis (BPH) of yield in hybrid is not ascribed to BPH of all the yield components but is specific to the BPH of spikelet number per panicle (SPP) and paternal parent heterosis (PPH) of effective panicle number (EPN). Genetic analyses then identified multiple quantitative trait loci (QTLs) for these two components. Moreover, a number of differentially expressed genes and alleles in the hybrid were mapped by transcriptome profiling to the QTL regions as possible candidate genes. In parallel, a major QTL for yield heterosis, rice heterosis 8 (RH8), was found to be the DTH8/Ghd8/LHD1 gene. Based on the shared allelic heterozygosity of RH8 in many hybrid rice cultivars, a common mechanism for yield heterosis in the present commercial hybrid rice is proposed.

Chloroplast is a new hotspot in the field of plant transformation system of plant genetic engineering. Plastid transformation has several advantages: high expression, multiple expressed genes in a single transformation event, absence of gene silencing, et al. A series of elements for construction of dicistronic site-specific integration expression vector of rice chloroplast have been cloned, including trnl-trnA (rice chloroplast homologous recombination fragments), Prrn (16S rRNA operon promotor), PpsbA (the 3' untranslated region of the chloroplastpsbA gene), hptll gene (encoding hygromycin phosphotransferase) and EGFP (encoding enhanced green fluorescence protein). All the elements were constructed into a rice chloroplast dicistronic expression vector pCTE04 (-trnl-Prrn-RBS-hptlI-RBS-EGFP-PpsbA- trnA-). Then pCTE04 was introduced into chloroplasts of Dunaliella salina through particle bombardment. Strong green fluorescence was observed in chloroplasts of some bombarded Dunaliella salina cells under a stereo fluorescence microscope, indicating that pCTE04 could be expressed in Dunaliella salina chloroplasts transiently. It provides a solid foundation for further genetic engineering in rice chloroplast transformation.

Hybrid rice plays an important role in China's aim to improve rice production as it accounts for some 50% of rice planting area but produces about 60% of the total rice grain. However, the existing three-line system used in hybrid rice production has its limitations. The two-line system, which makes use of photoperiod-sensitive genic male-sterile (PGMS) and thermo-sensitive genic male-sterile (TGMS) lines to generate the male-sterile parental line, was developed to overcome some of these limitations. The sterility of the male-sterile line of two-line hybrid rice, however, fluctuates when the temperature-sensitive phase of fertility encounters abnormal low temperatures during hybrid seed production, which induces selfing and decreases the purity of hybrid. We describe here the strategy of utilizing a herbicide resistance gene in two-line hybrid rice to eliminate this fluctuation in the sterility of the P/TGMS lines during hybrid seed production and reports the development of the herbicide resistance restorer line Bar68-1 and its herbicide-resistant early season hybrid rice Xiang125s/Bar68-1. When the restorer line and its derived hybrid are herbicide resistant, the selfed seeds can be removed easily from the hybrid by herbicide spraying. A herbicide resistance gene bar was transferred into a restorer line by particle bombardment. The resulting transgenic restorer line Bar68-1 and its hybrid Xiang125 s/Bar68-1 inherited stable herbicide resistance. The purity of Xiang125s/Bar68-1 was increased by spraying the seed bed with herbicide, which resulted in a significant increase in yield, grain quality, and disease resistance in comparison to the controls in a regional trial.

The future of rice breeding will likely be built on the basis of the further utilization of heterosis between elite cultivars and genetic resources from distant subspecies of rice. Previous studies have proved that exogenous genomic DNA transformation methods can be used to transfer genetic information from distant relatives (donor) into cultivated rice (recipient). However, the mechanism underlying this form of genetic transfer is poorly characterized, and the genes that cause the phenotypic changes in these variants are typically difficult to identify. This study examined YVB, a stable variant line with greatly improved grain quality traits that was derived from an indica variety (V20B) by transferring genomic DNA of O.minuta through the "spike-stalk injection method (SIM)". We used restriction-site associated DNA sequencing technology (RAD-seq) to evaluate a population of BC1F5 backcross lines (YVB × V20B); the RAD-seq data were used to construct a genetic linkage map with high-density SNPs for use in association analysis exploring genotype-phenotype relationships at the whole-genome level. A total of 17 quantitative trait loci (QTLs) for rice quality traits were mapped to chromosomes 3, 5, 6, 8, and 9. 8 major QTLs controlling different phenotypic variations were mapped to the same region of chromosome 5. This region contained the GS5 gene for grain weight and the qSW5/GW5 gene for grain width. This study provides new resources and insights into the molecular mechanisms of grain trait phenotypic variation and the transmission of genetic information via the introduction of genomic DNA to a distantly related crop relative species.

Significance Flowering time is one of the best studied ecologically important traits under natural or human selection for adaptation of plants to specific local environments. Photoperiodic sensitivity is a major agronomic trait that tailors vegetative and reproductive growth to local climates and is thus particularly important for crop yield and quality. This study not only identifies a major quantitative trait locus underlying photoperiod sensitivity in rice ( Days to heading 7 , DTH7 ) but also demonstrates that various haplotype combinations of DTH7 with Grain number, plant height, and heading date 7 ( Ghd7 ) and DTH8 correlate well with the flowering time and grain yield of rice varieties under diverse cultivating conditions. Our results build a foundation for breeding of high-yield rice varieties with desired photosensitivity and optimum adaptation to the target environments.

We have produced a draft sequence of the rice genome for the most widely cultivated subspecies in China, Oryza sativa L. ssp. indica , by whole-genome shotgun sequencing. The genome was 466 megabases in size, with an estimated 46,022 to 55,615 genes. Functional coverage in the assembled sequences was 92.0%. About 42.2% of the genome was in exact 20-nucleotide oligomer repeats, and most of the transposons were in the intergenic regions between genes. Although 80.6% of predicted Arabidopsis thaliana genes had a homolog in rice, only 49.4% of predicted rice genes had a homolog in A. thaliana . The large proportion of rice genes with no recognizable homologs is due to a gradient in the GC content of rice coding sequences.