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Summary Safflower (Carthamus tinctorius L.), a member of the Asteraceae, is a popular crop due to its high linoleic acid (LA) and flavonoid (such as hydroxysafflor yellow A) contents. Here, we report the first high‐quality genome assembly (contig N50 of 21.23 Mb) for the 12 pseudochromosomes of safflower using single‐molecule real‐time sequencing, Hi‐C mapping technologies and a genetic linkage map. Phyloge nomic analysis showed that safflower diverged from artichoke (Cynara cardunculus) and sunflower (Helianthus annuus) approximately 30.7 and 60.5 million years ago, respectively. Comparative genomic analyses revealed that uniquely expanded gene families in safflower were enriched for those predicted to be involved in lipid metabolism and transport and abscisic acid signalling. Notably, the fatty acid desaturase 2 (FAD2) and chalcone synthase (CHS) families, which function in the LA and flavonoid biosynthesis pathways, respectively, were expanded via tandem duplications in safflower. CarFAD2‐12 was specifically expressed in seeds and was vital for high‐LA content in seeds, while tandemly duplicated CarFAD2 genes were up‐regulated in ovaries compared to CarFAD2‐12, which indicates regulatory divergence of FAD2 in seeds and ovaries. CarCHS1, CarCHS4 and tandem‐duplicated CarCHS5˜CarCHS6, which were up‐regulated compared to other CarCHS members at early stages, contribute to the accumulation of major flavonoids in flowers. In addition, our data reveal multiple alternative splicing events in gene families related to fatty acid and flavonoid biosynthesis. Together, these results provide a high‐quality reference genome and evolutionary insights into the molecular basis of fatty acid and flavonoid biosynthesis in safflower.

Seed dormancy of cultivated rice was largely weakened during the progress of domestication. Correct timing and uniformity of seed germination are important for rapid seedling establishment and high-yield production. In the present study, we found that the heading-date gene Ghd7 acted as a negative regulator of germination. A mutant of ghd7 showed low sensitivity to exogenous ABA treatment during seed germination. Further investigation revealed reduced accumulation of ABA in mature ghd7 seeds as a consequence of dampened expression of OsNCED genes. Moreover, elevated GA3 level was detected in seeds of ghd7 mutant during imbibition course, which was attributed to the induction of genes responsible for the synthesis pathways of bioactive GAs. Thus, Ghd7 inhibits seed germination by increasing the ABA/GA3 ratio. Besides revealing pleiotropic effects of Ghd7, our results indicate its role in linking seed germination to growth-phase transition in rice, which would enrich the theoretical basis for future breeding practices.

Graphical abstract, Highlights • 4-way Multiparental population covered the limitations of the biparental structure. • The combination of SNP and bin-GWAS showed a powerful tool for QTL mapping. • qPGWC8.2 harbored a novel predicted gene for rice chalkiness quality., Introduction The multiparental population provides us the chance to identify superior alleles controlling a trait for genetic improvement. Genome wide association studies at bin level (bin-GWAS) are expected to be more power in QTL mapping than GWAS at SNP level (SNP-GWAS). Objectives This study is to estimate genetic effects of QTL conferring grain appearance quality in rice by SNP-GWAS and bin-GWAS, compare their power in QTL mapping and identify the superior alleles of all detected QTL from 4 parents for genetic improvement. Methods A 4-way MAGIC population and its four founders were cultivated in two environments to dissect the genetic basis of rice grain appearance quality. Both SNP-GWAS and bin-GWAS were conducted for QTL mapping. Multiple comparison among 4 parental bin/alleles was used to identify the superior alleles. Results A total of 16 and 20 QTL associated with grain appearance quality were identified by SNP- and bin-GWAS, respectively. A minor chalkiness QTL qPGWC8.2/qDEC8 was assigned to a 30-kb genomic region, in which OsMH_08T0121900 is the potential candidate gene because its encoded protein, glucan endo-1,3-beta-glucosidase precursor is involved in the starch and sucrose metabolism pathway. The superior parental alleles for GS3, GL3.1, GW5, GW7, and Chalk5 and two QTLs were almost carried by the high-quality parents Cypress and Yuejingsimiao (YJSM), while the poor-quality parent Guichao-2 (GC2) always carried the inferior alleles. The top five recombinant inbred lines with the highest quality of grain shape and chalkiness traits all carried gene combinations of superior alleles. Conclusions Both SNP- and bin-GWAS methods are encouraged for joint QTL mapping with MAGIC population. qPGWC8.2/qDEC8 is a novel candidate gene strongly associated with chalkiness. The superior alleles of GS3, GW5, GL3.1, GW7, Chalk5 and qPGWC8.2 were identified, and the pyramiding of these superior alleles is helpful to improve rice appearance quality.

The phase transition from vegetative to reproductive growth is triggered by internal and external signals that participate in circadian clock in plants. We identified a rice floral inhibitor OsPRR73 encoding a CONSTANS protein. Overexpression of OsPRR73 resulted in late heading under both long-day (LD) and short-day (SD) conditions. Knockout mutants led to early heading under LD conditions but no change under SD. OsPRR73 mRNA accumulated at noon and exhibited a robust oscillation under constant light (LL) and constant darkness (DD) conditions. OsPRR73 overexpression exerted negative feedback on endogenous OsPRR73 expression and altered diurnal expressions of key flowering genes and circadian clock genes. OsPRR73 bound to the promoters of the floral gene Ehd1 and the circadian gene OsLHY, and significantly suppressed their expression at dawn. In LL and DD, the oscillatory patterns of the circadian genes OsLHY, OsTOC1, OsGI and OsELF3 were varied in OsPRR73OX and osprr73 mutants. OsPRR73 expression was decreased in osphyb mutants, and overexpression of OsPRR73 complemented the early heading date phenotype of osphyb, indicating OsPRR73 works downstream of OsPhyB. Therefore, OsPRR73 is involved in a feedback loop of the rice clock and connects the photoperiod flowering pathway by binding to the Ehd1 promoter in rice.

Summary Lodging reduces grain yield in cereal crops. Understanding the genetic basis of lodging resistance (LR) benefits LR breeding. In the study, 524 accessions from a rice germplasm collection and 193 recombinant inbred lines were phenotyped for 17 LR‐related traits. Height and culm strength (the magnitude of applied force necessary to break the culm) were two major factors affecting LR. We conducted genome‐wide association study (GWAS) and identified 127 LR‐associated loci. Significant phenotypic correlations between culm‐strength traits and yield‐related traits were observed. To reveal the genetic relationship between them, we conducted GWAS of culm‐strength traits with adding yield‐related trait as a covariate and detected 63 loci linking culm strength and yield. As a proof, a near‐isogenic line for an association locus on chromosome 7 showed enhanced LR and yield. Strikingly, 58 additional loci were identified in the covariate‐added GWAS. Several LR‐associated loci had undergone divergent selection. Linkage analysis supported the GWAS results. We propose that introgression of alleles beneficial for both culm strength and panicle weight without negative effects on panicle number or pyramiding high‐yielding alleles and lodging‐resistant alleles without effects on yield can be employed for the post‐Green‐Revolution breeding.

The narrow base of genetic diversity of modern rice varieties is mainly attributed to the overuse of the common backbone parents that leads to the lack of varied favorable alleles in the process of breeding new varieties. Introgression lines (ILs) developed by a backcross strategy combined with marker-assisted selection (MAS) are powerful prebreeding tools for broadening the genetic base of existing cultivars. They have high power for mapping quantitative trait loci (QTLs) either with major or minor effects, and are used for precisely evaluating the genetic effects of QTLs and detecting the gene-by-gene or gene-by-environment interactions due to their low genetic background noise. ILs developed from multiple donors in a fixed background can be used as an IL platform to identify the best alleles or allele combinations for breeding by design. In the present paper, we reviewed the recent achievements from ILs in rice functional genomics research and breeding, including the genetic dissection of complex traits, identification of elite alleles and background-independent and epistatic QTLs, analysis of genetic interaction, and genetic improvement of single and multiple target traits. We also discussed how to develop ILs for further identification of new elite alleles, and how to utilize IL platforms for rice genetic improvement.

Tiller angle largely determines plant architecture, which in turn substantially influences crop production by affecting planting density. A recent study revealed that HEAT STRESS TRANSCRIPTION FACTOR2D (HSFA2D) acts upstream of LAZY1 (LA1) to regulate tiller angle establishment in rice (Oryza sativa). However, the mechanisms underlying transcriptional regulation of HSFA2D remain unknown. In this study, two class II homeodomain-Leu zipper genes, OsHOX1 and OsHOX28, were identified as positive regulators of tiller angle by affecting shoot gravitropism. OsHOX1 and OsHOX28 showed strong transcriptional suppressive activity in rice protoplasts and formed intricate self- and mutual-transcriptional negative feedback loops. Moreover, OsHOX1 and OsHOX28 bound to the pseudopalindromic sequence CAAT(C/G)ATTG within the promoter of HSFA2D, thus suppressing its expression. In contrast to HSFA2D and LA1, OsHOX1 and OsHOX28 attenuated lateral auxin transport, thus repressing the expression of WUSCHEL-RELATED HOMEOBOX 6 (WOX6) and WOX11 in the lower side of the shoot base of plants subjected to gravistimulation. Genetic analysis further confirmed that OsHOX1 and OsHOX28 act upstream of HSFA2D. Additionally, both OsHOX1 and OsHOX28 inhibit the expression of multiple OsYUCCA genes and decrease auxin biosynthesis. Taken together, these results demonstrated that OsHOX1 and OsHOX28 regulate the local distribution of auxin, and thus tiller angle establishment, through suppression of the HSFA2D-LA1 pathway and reduction of endogenous auxin content. Our finding increases the knowledge concerning fine tuning of tiller angles to optimize plant architecture in rice.

CONSTANS, CONSTANS-LIKE, and TIMING OF CAB EXPRESSION1 (CCT) domain-containing proteins are a large family unique to plants. They transcriptionally regulate photoperiodic flowering, circadian rhythms, vernalization, and other related processes. Through their CCT domains, CONSTANS and HEADING DATE1 (HD1) coordinate with the NUCLEAR FACTOR Y (NF-Y) B/C dimer to specifically target a conserved ‘CCACA’ motif within the promoters of their target genes. However, the mechanism underlying DNA recognition by the CCT domain remains unclear. Here we determined the crystal structures of the rice (Oryza sativa) NF-YB/YC dimer and the florigen gene Heading date 3a (Hd3a)-bound HD1CCT/NF-YB/YC trimer with resolutions of 2.0 Å and 2.55 Å, respectively. The CCT domain of HD1 displays an elongated structure containing two α-helices and two loops, tethering Hd3a to the NF-YB/YC dimer. Helix α2 and loop 2 are anchored into the minor groove of the ‘CCACA’ motif, which determines the specific base recognition. Our structures reveal the interaction mechanism among the CCT domain, NF-YB/YC dimer, and the target DNA. These results not only provide insight into the network between the CCT proteins and NF-Y subunits, but also offer potential approaches for improving productivity and global adaptability of crops by manipulating florigen expression.

SG3 , an R2R3 MYB protein coding gene that tightly linked to a major QTL GS3 , negatively regulates grain length while dependent on the status of GS3 in rice. It is often very difficult to isolate a minor QTL that is closely linked to a major QTL in rice. In this study, we focused on the isolation of a minor grain length QTL, small grain 3 (SG3), which is closely linked to the major QTL grain size 3 (GS3). The genetic effect of SG3 on grain length was dependent on GS3 status. Its genetic effect was larger in the presence of nonfunctional sg3 than functional SG3. A large number of near-isogenic F2 plants in which GS3 was fixed with nonfunctional alleles were developed to clone SG3. A gene encoding an R2R3 MYB domain transcriptional regulator was identified as the candidate gene for SG3. SG3 overexpression and knockdown plants showed shortened and elongated grains, respectively, which demonstrated that SG3 acts as a negative regulator of grain length. SG3 was preferentially expressed in panicles after flowering, and SG3 acted as a transcription activator. Comparative sequencing analysis identified a 12-bp insertion in the third exon of NYZ that led to a frameshift and resulted in a premature stop codon. The insertion/deletion was associated with grain length in the presence of functional GS3 in the indica subspecies. SG3 and GS3 were frequently in coupling phase in indica rice, making them good targets for the breeding of cultivars with short or long grains. The isolation of the SG3 MYB gene provides new gene resource and contributes to the regulatory network of grain length in rice.

The review summarizes the functions of the plant special transcription factors CCT family genes in multiple traits and discusses the molecular breeding strategies with CCT family genes in the future. Plants integrate circadian clock and external signals such as temperature and photoperiod to synchronize flowering with seasonal environmental changes. This process makes cereal crops including short-day crops, such as rice and maize, and long-day crops, such as wheat and barley, better adapt to varied growth zones from temperate to tropical regions. CCT family genes involve circadian clock and photoperiodic flowering pathways and help plants set a suitable flowering time to produce offspring. Beyond the flowering time, CCT family genes in cereal crops are associated with biomass and grain yield. Moreover, recent studies showed that they also associate with photosynthesis, nutrition use efficiency and stress tolerance. Here, we systematically review the progress in functional characterization of CCT family genes in flowering, geographical adaptation and grain yield formation, raise the core questions related to their molecular mechanisms and discuss how to practice them in genetic improvement in cereal crops by combining gene diagnosis and top-level design.

Grain size is a major determinant of grain weight, a key component of grain yield of rice. Here, we identified the grain size gene WIDE GRAIN 7 (WG7) from a T-DNA insertion mutant. The grain size of WG7 knockout mutants and WG7 overexpression lines indicated that WG7 is a positive regulator of grain size. WG7 encodes a cysteine-tryptophan (CW) domain-containing transcriptional activator. EMSAs and ChIP-qPCR assay confirmed that WG7 directly bound to the promoter of OsMADS1, a grain size gene, and thereby significantly activated its expression. Point mutations showed that the cis-element CATTTC motif in the promoter was the binding site of WG7. Compared with the wild-type, deletion mutants of the cis-element motif exhibited lower expression of OsMADS1 and produced narrower grains, implicating the requirement of this motif for WG7 function. ChIP-qPCR assays showed that WG7 enhanced histone H3K4me3 enrichment in the promoter of OsMADS1. WG7 underwent directional selection due to the poor fertility of the non-functional mutant. These findings demonstrated that WG7 upregulated OsMADS1 expression by directly binding to its promoter, enhanced histone H3K4me3 enrichment in the promoter and ultimately increased grain width. This study will enrich the knowledge concerning the regulatory network of grain size formation in rice.

Root-derived mobile signals play critical roles in coordinating a shoot’s response to underground conditions. However, the identification of root-to-shoot long-distance mobile signals has been scant. In this study, we aimed to characterize root-to-shoot endogenous mobile miRNAs by using an Arabidopsis/Nicotiana interfamilial heterograft in which these two taxonomically distant species with clear genetic backgrounds had sufficient diversity in differentiating miRNA sources. Small RNA deep sequencing analysis revealed that 82 miRNAs from the Arabidopsis scion could travel through the graft union to reach the rootstock, whereas only a very small subset of miRNA (6 miRNAs) preferred the root-to-shoot movement. We demonstrated in an ex vivo RNA imaging experiment that the root-to-shoot mobile Nb-miR164, Nb-miR395 and Nb-miR397 were targeted to plasmodesmata using the bacteriophage coat protein MS2 system. Furthermore, the Nb-miR164 was shown to move from the roots to the shoots to induce phenotypic changes when its overexpressing line was used as rootstock, strongly supporting that root-derived Nb-miR164 was able to modify the scion trait via its long-distance movement.

Inflorescence architecture is a major determinant of spikelet numbers per panicle, a key component of grain yield in rice. In this study, Short Panicle 3 (SP3) was identified from a short panicle 3 (sp3) mutant in which T-DNA was inserted in the promoter of SP3, resulting in a knockdown mutation. SP3 encodes a DNA binding with one finger (Dof) transcriptional activator. Quantitative real time (qRT)-PCR and RNA in situ hybridization assays confirmed that SP3 is preferentially expressed in the young rice inflorescence, specifically in the branch primordial regions. SP3 acts as a negative regulator of inflorescence meristem abortion by upregulating APO2/RFL. SP3 both up- and downregulates expression of genes involved in cytokinin biosynthesis and catabolism, respectively. Consequently, cytokinin concentrations are decreased in young sp3 panicles, thereby leading to small panicles having fewer branches and spikelets. Our findings support a model in which SP3 regulates panicle architecture by modulating cytokinin homeostasis. Potential applications to rice breeding, through gene-editing of the SP3 promoter are assessed.

Pentatricopeptide repeat (PPR) proteins regulate organellar gene expression in plants, through their involvement in organellar RNA metabolism. In rice (Oryza sativa), 477 genes are predicted to encode PPR proteins; however, the majority of their functions remain unknown. In this study, we identified and characterized a rice mutant, pale-green leaf12 (pgl12); at the seedling stage, pgl12 mutants had yellow-green leaves, which gradually turned pale green as the plants grew. The pgl12 mutant had significantly reduced Chl contents and increased sensitivity to changes in temperature. A genetic analysis revealed that the pgl12 mutation is recessive and located within a single nuclear gene. Map-based cloning of PGL12, including a transgenic complementation test, confirmed the presence of a base substitution (C to T), generating a stop codon, within LOC_Os12g10184 in the pgl12 mutant. LOC_Os12g10184 encodes a novel PLS-type PPR protein containing 17 PPR motifs and targeted to the chloroplasts. A quantitative real-time PCR analysis showed that PGL12 was expressed in various tissues, especially the leaves. We also showed that the transcript levels of several nuclear- and plastid-encoded genes associated with chloroplast development and photosynthesis were significantly altered in pgl12 mutants. The mutant exhibited defects in the 16S rRNA processing and splicing of the plastid transcript ndhA. Our results indicate that PGL12 is a new PLS-type PPR protein required for proper chloroplast development and 16S rRNA processing in rice.

The fungus Magnaporthe oryzae threatens the rice production of Kongyu 131 (KY131), a leading japonica variety in Northeast China. In this study, two rice lines, KP1 and KP2-Hd1, were obtained by introgressing the blast resistance genes Pi1 and Pi2 into KY131, respectively. However, both lines headed later than KY131. RICE60K SNP array analysis showed that Hd1 closely linked to Pi2 was introgressed into KP2-Hd1, and the linkage drag of Hd1 was broken by recombination. On the other hand, no known flowering genes were introgressed into KP1. Gene diagnosis by resequencing six flowering genes showed that KP1 carried functional Hd16 and Ghd8 alleles. Due to its suppression role in heading under long-day conditions, Ghd8 was chosen as the target for gene editing to disrupt its function. Four sgRNAs targeting different sites within Ghd8 were utilized to induce large-deletion mutations, which were easy to detect via agarose gel electrophoresis. All the ghd8-mutated KP1 lines were resistant to rice blast disease and headed earlier than the control KP1, even than KY131, under natural long-day conditions, which ensures its growth in Northeast China. This study confirmed that a combination of gene diagnosis and targeted gene editing is a highly efficient way to quickly eliminate undesired traits in a breeding line.

There are 41 members of the CCT (CO, CO-like, and TOC1) domain-containing gene family in rice, which are divided into three subfamilies: COL (CONSTANS-like), CMF (CCT motif family), and PRR (pseudoresponse regulator). The first flowering gene to be isolated by map-based cloning, Heading date 1 (Hd1), which is the orthologue of CO in rice, belongs to COL. The central regulator of plant development, Ghd7, belongs to CMF. The major role in controlling rice distribution to high latitudes, Ghd7.1/PRR37, belongs to PRR. Both of Hd1, Ghd7 and Ghd7.1 simultaneously control grain number, plant height, and the heading date. To date, 13 CCT family genes from these three subfamilies have been shown to regulate flowering. Some of them have pleiotropic effects on grain yield, plant height, and abiotic stresses, and others function as circadian oscillators. There are two independent photoperiod flowering pathways that are mediated by GI-Hd1-Hd3a/RFT and GI-Ehd1-Hd3a/RFT in rice. CCT family genes are involved in both pathways. The latest study reveals that protein interaction between Hd1 and Ghd7 integrates the two pathways. CCT family genes are rich in natural variation because rice cultivars have been subjected to natural and artificial selection for different day lengths in the process of domestication and improvement. Alleles of several crucial CCT family genes such as Hd1, Ghd7, and Ghd7.1 exhibit geographic distribution patterns and are highly associated with yield potentials. In addition, CCT family genes are probably involved in the responses to abiotic stress, which should be emphasized in future work. In general, CCT family genes play important roles in regulating flowering, plant growth, and grain yield. The functional identification and elucidation of the molecular mechanisms of CCT family genes would help construct a flowering regulatory network and maximize their contribution to rice production.

FLOWERING LOCUS T2 (FT2) is expressed in the distal part of the developing wheat spike and contributes to the regulation of the number of spikelets per spike., FLOWERING LOCUS T2 (FT2) is the closest paralog of the FT1 flowering gene in the temperate grasses. Here we show that overexpression of FT2 in Brachypodium distachyon and barley results in precocious flowering and reduced spikelet number, while down-regulation by RNA interference results in delayed flowering and a reduced percentage of filled florets. Similarly, truncation mutations of FT2 homeologs in tetraploid wheat delayed flowering (2–4 d) and reduced fertility. The wheat ft2 mutants also showed a significant increase in the number of spikelets per spike, with a longer spike development period potentially contributing to the delayed heading time. In the wheat leaves, FT2 was expressed later than FT1, suggesting a relatively smaller role for FT2 in the initiation of the reproductive phase. FT2 transcripts were detected in the shoot apical meristem and increased during early spike development. Transversal sections of the developing spike showed the highest FT2 transcript levels in the distal part, where new spikelets are formed. Our results suggest that, in wheat, FT2 plays an important role in spike development and fertility and a limited role in the timing of the transition between the vegetative and reproductive shoot apical meristem.

Root-derived mobile signals play critical roles in coordinating a shoot’s response to underground conditions. However, the identification of root-to-shoot long-distance mobile signals has been scant. In this study, we aimed to characterize root-to-shoot endogenous mobile miRNAs by using an Arabidopsis/Nicotiana interfamilial heterograft in which these two taxonomically distant species with clear genetic backgrounds had sufficient diversity in differentiating miRNA sources. Small RNA deep sequencing analysis revealed that 82 miRNAs from the Arabidopsis scion could travel through the graft union to reach the rootstock, whereas only a very small subset of miRNA (6 miRNAs) preferred the root-to-shoot movement. We demonstrated in an ex vivo RNA imaging experiment that the root-to-shoot mobile Nb-miR164, Nb-miR395 and Nb-miR397 were targeted to plasmodesmata using the bacteriophage coat protein MS2 system. Furthermore, the Nb-miR164 was shown to move from the roots to the shoots to induce phenotypic changes when its overexpressing line was used as rootstock, strongly supporting that root-derived Nb-miR164 was able to modify the scion trait via its long-distance movement.

Both heading date and plant height are important traits related to grain yield in rice. In this study, a recombinant inbred lines (RILs) population was used to map quantitative trait loci (QTLs) for both traits under 3 long-day (LD) environments and 1 short-day (SD) environment. A total of eight QTLs for heading date and three QTLs for plant height were detected by composite interval mapping under LD conditions. Additional one QTL for heading date and three QTLs for plant height were identified by Two-QTL model under LD conditions. Among them, major QTLs qHd7.1, qHd7.2 and qHd8 for heading date, and qPh1 and qPh7.1 for plant height were commonly detected. qHd7.1 and qHd7.2 were mapped to small regions of less than 1 cM. Genome position comparison of previously cloned genes with QTLs detected in this study revealed that qHd5 and qPh3.1 were two novel QTLs. The alleles of these QTLs increasing trait values were dispersed in both parents, which well explained the transgressive segregation observed in this population. In addition, the interaction between qHd7.1 and qHd8 was detected under all LD conditions. Multiple-QTL model analysis revealed that all QTLs and their interactions explained over 80% of heading date variation and 50% of plant height variation. Two heading date QTLs were detected under SD condition. Of them, qHd10 were commonly identified under LD condition. The difference in QTL detection between LD and SD conditions indicated most heading date QTLs are sensitive to photoperiod. These findings will benefit breeding design for heading date and plant height in rice.

The transition from the vegetative to the reproductive stage, which is called as flowering, is an important process of plant growth. Many studies had investigated the regulation of flowering in Arabidopsis, but few studies addressed rice and other crops, particularly neglecting the effects of nutrients on rice flowering. This study was conducted to clarify the effects of different nutrients on the characteristic parameters of rice flowering. Five gradients of nitrogen (N), phosphorus (P) and potassium (K) fertilizer were set respectively to study the effects of nutrients on rice flowering time and flowering duration. The results showed that N, P, and K fertilizers affected the growth and the flowering time of rice. The grain numbers per panicle increased by 31.4%, 23.9%, and 48.2%, and the panicle numbers increased by 55.1%, 29.2%, and 6.7% after application of N, P, K fertilizers, respectively. N application delayed the rice flowering time by 1–4 days, and further increasing the N application led to even later flowering. Compared to no nitrogen application (N0), N application treatments prolonged the flowering duration per panicle, per hill, and group by 0.5–1.2 days, 0.7–1.7 days, and 0.7–2.0 days, respectively. After application of P fertilizer, rice flowering was 1 day ahead of schedule, and no significant difference was found between P levels. K application clearly promoted early flowering by 1–3 days, and the higher the application rate of K fertilizer, the earlier the flowering. The application of P and K fertilizers had no significant effects on the duration of rice flowering. The above evidence indicated that fertilization is an important measure to regulate flowering in rice production. This can serve as an optimized protocol for future studies about the physiological and molecular mechanism of flower induction by nutrients. Keywords: Rice, Flowering time, Flowering duration, Regulation, Fertilization

Seed germination is a complex process involving various physical and biochemical cues, determined by exogenous and endogenous factors. Here, we identified a gene, OsMFT2, that negatively regulates seed germination in rice. OsMFT2 knock-out lines exhibited pre-harvest sprouting, whereas OsMFT2 overexpression lines showed delayed germination. RNA expression profiling showed that OsMFT2 was specifically expressed in seeds. Subcellular localization indicated that OsMFT2 was a nuclear protein. Exogenous abscisic acid (ABA) treatment of imbibed seeds and seedlings indicated that OsMFT2 altered ABA sensitivity during seed germination and post-germination growth. In vivo and in vitro assays showed that three bZIP transcription factors, OsbZIP23, OsbZIP66 and OsbZIP72, interacted with OsMFT2. OsbZIP23/66/72 bound to the promoter of Rab16A, a typical gene containing the ABA-responsive element, and OsMFT2 enhanced the binding to the Rab16A promoter. Moreover, several ABA-responsive genes were differentially expressed in the imbibed seeds of OsMFT2 transgenic lines and the wild type. The performance of the transgenic plants demonstrated that overexpressing OsbZIP23 rescued the pre-harvest sprouting phenotype and the decrease in ABA-signaling genes expression caused by OsMFT2 knock-out. All of these results demonstrate that OsMFT2 positively regulates ABA-responsive genes through interacting with OsbZIP23/66/72 and functions in seed germination.

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.

Background Heading date is crucial for rice reproduction and geographic expansion. Many heading date genes are sensitive to photoperiod and jointly regulate flowering time in rice. However, it is not clear how these genes coordinate rice heading. Results Here, we performed a genetic interaction analysis among four major rice heading date genes Ghd7, Ghd8, OsPRR37/Ghd7.1 (hereafter PRR37) and Hd1 in the near-isogenic background under both natural long-day (NLD) and natural short-day (NSD) conditions. The 4-gene segregating population exhibited a large heading date variation with more than 95 days under NLD and 42 days under NSD conditions. Tetragenic, trigenic and digenic interactions among these four genes were observed under both conditions but more significant under NLD conditions. In the functional Hd1 backgrounds, the strongest digenic interaction was Ghd7 by Ghd8 under NLD but was Ghd7 by PRR37 under NSD conditions. Interestingly, PRR37 acted as a flowering suppressor under NLD conditions, while it functioned alternatively as an activator or a suppressor under NSD conditions depending on the status of the other three genes. Based on the performances of 16 homozygous four-gene combinations, a positive correlation between heading date and spikelets per panicle (SPP) was found under NSD conditions, but changed to a negative correlation when heading date was over 90 days under NLD conditions. Conclusions These results demonstrate the importance of genetic interactions in the rice flowering regulatory network and will help breeders to select favorable combinations to maximize rice yield potential for different ecological areas. Electronic supplementary material The online version of this article (10.1186/s12284-019-0314-x) contains supplementary material, which is available to authorized users.

Several major heading date genes are sensitive to photoperiod and jointly regulate heading date in rice. However, it is not clear how these genes coordinate rice heading. In this study, a near-isogenic F2 population with Ghd7, Ghd7.1, Ghd8 and Hd1 segregating in the Zhenshan 97 (ZS97) background was used to evaluate the genetic interactions among these four genes under natural long-day (NLD) and natural short-day (NSD) conditions, and a series of Ghd7.1-segregating populations in different backgrounds were developed to estimate the genetic effects of Ghd7.1 on heading date under both conditions. Tetragenic, trigenic and digenic interactions among these four genes were observed under both conditions. In the functional Hd1 backgrounds, the strongest digenic interaction was Ghd7 by Ghd8 under NLD conditions but was Ghd7 by Ghd7.1 under NSD conditions. Interestingly, Ghd7.1 acted as a flowering suppressor under NLD conditions, while it functioned alternatively as an activator or a suppressor under NSD conditions depending on the status of the other three genes. Based on the performances of 16 homozygous four-gene combinations, a positive correlation between heading date and yield was found under NSD conditions, but changed to a negative correlation when heading date was over 90 days under NLD conditions. These results demonstrate the importance of genetic interactions in the rice flowering regulatory network and will help breeders to select favorable combinations to maximize rice yield potential for different ecological areas.

Awn is one of the most important domesticated traits in rice (Oryza sativa). Understanding the genetic basis of awn length is important for grain harvest and production, because long awn length is disadvantageous for both grain harvest and milling. We investigated the awn length of 529 rice cultivars and performed a Genomewide association studies (GWAS) in the indica and japonica subpopulations, and the whole population. In total, we found 17 loci associated with awn length. Of these loci, seven were linked to previously reported quantitative trait loci, and one was linked to the awn gene An-1. Nine novel loci were repeatedly identified in different environments. One of the nine associations was identified in both the whole and japonica populations. Special interest was the detection of the most significant association SNP, sf0136352825, which was less than 95 kb from the seed shattering gene qSH1. These results may provide potentially favourable haplotypes for molecular breeding in rice.

Highlight Gene duplication has led to the existence of a large HAP gene family. In this study, three HAP genes were identified that regulate flowering in rice in addition to the previously reported Ghd8/OsHAP3H., Heterotrimeric Heme Activator Protein (HAP) family genes are involved in the regulation of flowering in plants. It is not clear how many HAP genes regulate heading date in rice. In this study, we identified 35 HAP genes, including seven newly identified genes, and performed gene duplication and candidate gene-based association analyses. Analyses showed that segmental duplication and tandem duplication are the main mechanisms of HAP gene duplication. Expression profiling and functional identification indicated that duplication probably diversifies the functions of HAP genes. A nucleotide diversity analysis revealed that 13 HAP genes underwent selection. A candidate gene-based association analysis detected four HAP genes related to heading date. An investigation of transgenic plants or mutants of 23 HAP genes confirmed that overexpression of at least four genes delayed heading date under long-day conditions, including the previously cloned Ghd8/OsHAP3H. Our results indicate that the large number of HAP genes in rice was mainly produced by gene duplication, and a few HAP genes function to regulate heading date. Selection of HAP genes is probably caused by their diverse functions rather than regulation of heading.

Gelatinization temperature (GT), an important parameter for rice cooking quality, is mainly regulated by the ALK gene encoding starch synthase IIa. Here, we reported the nucleotide diversity of the ALK gene in 122 cultivated accessions and 199 wild rice accessions that were collected around the Pearl River Basin in China. A total of 93 single nucleotide ploymorphisms (SNPs) were identified, with an average of one SNP per 40 bp. Tajima D statistics revealed that the DNA sequences covering the last exon have probably evolved under balancing selection. Based on two functional SNPs (an A to G substitution at 4198 bp and a GC to TT dinucleotide substitution at 4330/4331 bp), three haplotypes, G/GC, G/TT, and A/GC, were identified in both wild and cultivated accessions, with the G/GC haplotype being predominant. Interestingly, the A/GC haplotype was exclusively found in the wild accessions from Guangdong province, while the G/TT haplotype was only present in the wild accessions from Jiangxi province and Hainan Island. This suggests that the G/TT and A/GC variants may have arisen independently and undergone balancing selection on separate haplotypes in multiple populations. Our result supports earlier hypothesis that cultivated rice was independently domesticated from multiple domestication events in China. Our study aids in the understanding of the domestication process that led to the improvement of rice grain quality.

Asian rice (Oryza sativa) and African rice (Oryza glaberrima) are separately domesticated from their wild ancestors Oryza rufipogon and Oryza barthii, which are very sensitive to daylength. In the process of domestication, some traits that are favorable for the natural survival of wild rice such as seed dormancy and shattering have become favorable ones for human consumption due to the loss-of-function mutations in the genes that are underlying these traits. As a consequence, many genes that are related to these kinds of traits have been fixed with favorable alleles in modern cultivars by artificial selection. After domestication, Oryza sativa cultivars gradually spread to temperate and cool regions from the tropics and subtropics due to the loss of their photoperiod sensitivity. In this paper, we review the characteristics of domestication-related seed traits and heading dates in rice, including the key genes controlling these traits, the differences in allelic diversity between wild rice and cultivars, the geographic distribution of alleles, and the regulatory pathways of these traits. A comprehensive comparison shows that these genes contributed to rice domestication and its global expansion. In addition, these traits have also experienced parallel evolution by artificial selection on the homologues of key genes in other cereals.

Auxin plays critical roles in many developmental processes of plants. The auxin signaling pathway is a series of plant responses to auxin stimuli. However, the functions of many genes in this pathway are still obscure. As auxin receptors, TIR/AFB family genes encode F-Box proteins that directly bind auxin and then transduce the stimulus through the signaling pathway. In this paper, we generated an overexpression line of Auxin-signaling F-Box 6 (OsAFB6) in rice, which largely delayed heading, greatly increased spikelets per panicle and primary branch number and ultimately enhanced grain yield by 50%. OsAFB6 is preferentially expressed in young tissues with stronger meristem activities and suppresses flowering by upregulating OsRR1 and downregulating Ehd1 expression levels. Overexpression of OsAFB6 delayed heading, increased cytokinin (CK) by suppressing the expression level of Gn1a and simultaneously decreased the IAA concentration in the young panicle, which promoted inflorescence meristem development and resulted in large panicles with more spikelets per panicle, primary branches and increased grain yield. It would be a beneficial strategy to generate lines with varied expression levels of OsAFB6 to breed high-yielding cultivars for specific regions that can fully utilize the local sunlight and temperature resources.

Rice (Oryza sativa) is an important dietary source of both essential micronutrients and toxic trace elements for humans. The genetic basis underlying the variations in the mineral composition, the ionome, in rice remains largely unknown. Here, we describe a comprehensive study of the genetic architecture of the variation in the rice ionome performed using genome-wide association studies (GWAS) of the concentrations of 17 mineral elements in rice grain from a diverse panel of 529 accessions, each genotyped at ∼6.4 million single nucleotide polymorphism loci. We identified 72 loci associated with natural ionomic variations, 32 that are common across locations and 40 that are common within a single location. We identified candidate genes for 42 loci and provide evidence for the causal nature of three genes, the sodium transporter gene Os-HKT1;5 for sodium, Os-MOLYBDATE TRANSPORTER1;1 for molybdenum, and Grain number, plant height, and heading date7 for nitrogen. Comparison of GWAS data from rice versus Arabidopsis (Arabidopsis thaliana) also identified well-known as well as new candidates with potential for further characterization. Our study provides crucial insights into the genetic basis of ionomic variations in rice and serves as an important foundation for further studies on the genetic and molecular mechanisms controlling the rice ionome.

As a major component of ideal plant architecture, leaf angle especially flag leaf angle (FLA) makes a large contribution to grain yield in rice. We utilized a worldwide germplasm collection to elucidate the genetic basis of FLA that would be helpful for molecular design breeding in rice. Genome-wide association studies (GWAS) identified a total of 40 and 32 QTLs for FLA in Wuhan and Hainan, respectively. Eight QTLs were commonly detected in both conditions. Of these, 2 and 3 QTLs were identified in the indica and japonica subpopulations, respectively. In addition, the candidates of 5 FLA QTLs were verified by haplotype-level association analysis. These results indicate diverse genetic bases for FLA between the indica and japonica subpopulations. Three candidates, OsbHLH153, OsbHLH173 and OsbHLH174, quickly responded to BR and IAA involved in plant architecture except for OsbHLH173, whose expression level was too low to be detected; their overexpression in plants increased rice leaf angle. Together with previous studies, it was concluded that all 6 members in bHLH subfamily 16 had the conserved function in regulating FLA in rice. A comparison with our previous GWAS for tiller angle (TA) showed only one QTL had pleiotropic effects on FLA and TA, which explained low similarity of the genetic basis between FLA and TA. An ideal plant architecture is expected to be efficiently developed by combining favorable alleles for FLA from indica with favorable alleles for TA from japonica by inter-subspecies hybridization., Author summary Rice leaf angle is a major component of ideal plant architecture that determines plant density. Many leaf angle-related genes have been characterized based on mutants, but natural variations of these genes and potential values in genetic improvement have not been evaluated. Here we explore the genetic basis of rice FLA in rice by GWAS. Dozens of quantitative trait loci (QTLs) have been identified, but few FLA QTLs have been commonly detected between indica and japonica subpopulations. Alleles for small leaf angle mostly come from indica rice. Three novel OsbHLH genes have been confirmed to regulate leaf angle by overexpression. Together with previous studies, all 6 members of bHLH subfamily 16 have a conserved function in regulating leaf angle. Only one QTL has been identified with pleiotropic effects on FLA and TA in the same collection, indicated the diverse genetic basis between FLA and TA. Alleles for small TA mostly come from japonica rice. We suggest to breed compact plant type cultivars by simultaneously utilizing favorable alleles for FLA and TA via inter-subspecies hybridization.

New strategy of breeding by modulating key heading date gene Ehd1 to enhance the variations of heading date regardless of genetic background for better adaptation to local environment in rice. Flowering time (or heading date) is an important quantitative trait in rice (Oryza sativa) that determines its adaptation to specific cultivation areas and growing seasons. However, breeding of flowering time is currently relying on laborious selections and combinations of different alleles of various genes. Here, we cloned a cis-variant allele of Ehd1 that regulated not only heading date but also yield potential. Genetic analysis revealed that Ehd1 acted downstream of Ghd7 as a negative regulator of yield potential, and expression divergence of Ehd1 negatively correlates with phenotype variations including heading date and grain yield. Moreover, regardless of genetic background, manipulations of the expression of a single gene, Ehd1, are sufficient for recreating beneficial heading date variations which could be subjected to the selection of best suitable lines for local environment conditions. Beyond a deeper understanding of transcriptional control of quantitative traits, this study provided an effective and flexible strategy for breeding rice cultivars to maximize grain production for any region of cultivation.

A new rice gene, OsMFT1, was identified that both regulates rice heading date and panicle architecture and is different from its homologs in other species., Heading date and panicle architecture are important agronomic traits in rice. Here, we identified a gene MOTHER OF FT AND TFL1 (OsMFT1) that regulates rice heading and panicle architecture. Overexpressing OsMFT1 delayed heading date by over 7 d and greatly increased spikelets per panicle and the number of branches. In contrast, OsMFT1 knockout mutants had an advanced heading date and reduced spikelets per panicle. Overexpression of OsMFT1 significantly suppressed Ehd1 expression, and Ghd7 up-regulated OsMFT1 expression. Double mutants showed that OsMFT1 acted downstream of Ghd7. In addition, transcription factor OsLFL1 was verified to directly bind to the promoter of OsMFT1 via an RY motif and activate the expression of OsMFT1 in vivo and in vitro. RNA-seq and RNA in situ hybridization analysis confirmed that OsMFT1 repressed expression of FZP and five SEPALLATA-like genes, indicating that the transition from branch meristem to spikelet meristem was delayed and thus more panicle branches were produced. Therefore, OsMFT1 is a suppressor of flowering acting downstream of Ghd7 and upstream of Ehd1, and a positive regulator of panicle architecture.

Rice cultivars have been adapted to favorable ecological regions and cropping seasons. Although several heading date genes have separately made contributions to this adaptation, the roles of gene combinations are still unclear. We employed a map-based cloning approach to isolate a heading date gene, which coordinated the interaction between Ghd7 and Ghd8 to greatly delay rice heading. We resequenced these three genes in a germplasm collection to analyze natural variation. Map-based cloning demonstrated that the gene largely affecting the interaction between Ghd7 and Ghd8 was Hd1. Natural variation analysis showed that a combination of loss-of-function alleles of Ghd7, Ghd8 and Hd1 contributes to the expansion of rice cultivars to higher latitudes; by contrast, a combination of pre-existing strong alleles of Ghd7, Ghd8 and functional Hd1 (referred as SSF) is exclusively found where ancestral Asian cultivars originated. Other combinations have comparatively larger favorable ecological scopes and acceptable grain yield. Our results indicate that the combinations of Ghd7, Ghd8 and Hd1 largely define the ecogeographical adaptation and yield potential in rice cultivars. Breeding varieties with the SSF combination are recommended for tropical regions to fully utilize available energy and light resources and thus produce greater yields.

Intragenic recombination is one of the most important sources of genetic variability. In our previous study, RI92 a tall line (160 cm of plant height) was observed in the cross progeny between two semi-dwarf indica cultivars Zhenshan 97 and Minghui 63. Genome-wide genotyping and sequencing indicated that the genome constitution of RI92 was completely from both parents. Bulk segregant analysis in a BC3F2 population revealed that "green revolution gene" semi-dwarf 1 (sd1) was most likely the gene controlling the tall plant height in RI92. Sequencing analysis of SD1 revealed that an intragenic recombination occurred between two parental non-functional sd1 alleles and generated a functional SD1 in RI92. Four-fold high recombination rate in SD1 located bins to the genome-wide average was observed in two RIL populations, indicating recombination hotspot in the SD1 region. Intragenic recombination creates new alleles in the progeny distinct from parental alleles and diversifies natural variation.

Rice (Oryza sativa L.) is a staple food crop for people worldwide, and a key goal has been to increase its grain yield. An increasing population that relies on a decreasing level of farmland has rendered the traditional method for the isolation and use of genetic loci in rice breeding unsatisfactory. Recently, the rapid development in next generation sequencing (NGS) has boosted the number of genome sequences for hundreds to thousands of rice varieties. A MutMap strategy and bulk segregation analysis (BSA) has been developed to directly identify candidate genes based on NGS. The genome-wide association analysis (GWAS) has become a commonly used approach toward identifying the genetic loci and candidate genes for several traits that are closely associated with grain yield. The Multi-parent Advanced Generation Inter-Cross population (MAGIC) is introduced here to discuss potential applications for mapping QTLs for rice varietal development. These strategies broaden the capacity of functional gene identification and its application as a complementary method to insert mutants that comprise T-DNA and transposons. High-throughput SNP analysis platforms, such as the SNP array, provide novel strategies for genomic-assisted selections (GAS) for rice genetic improvements. Moreover, accurate genome sequence information enables genome editing for the utilization of key recessive genes that control important agronomic traits. This review summarizes how NGS accelerates rice genetic improvements through the identification and utilization of key functional genes that regulate agronomic traits.

Transcriptional silencer and copy number variants (CNVs) are associated with gene expression. However, their roles in generating phenotypes have not been well studied. Here we identified a rice quantitative trait locus, SGDP7 (Small Grain and Dense Panicle 7). SGDP7 is identical to FZP (FRIZZY PANICLE), which represses the formation of axillary meristems. The causal mutation of SGDP7 is an 18-bp fragment, named CNV-18bp, which was inserted ~5.3 kb upstream of FZP and resulted in a tandem duplication in the cultivar Chuan 7. The CNV-18bp duplication repressed FZP expression, prolonged the panicle branching period and increased grain yield by more than 15% through substantially increasing the number of spikelets per panicle (SPP) and slightly decreasing the 1,000-grain weight (TGW). The transcription repressor OsBZR1 binds the CGTG motifs in CNV-18bp and thereby represses FZP expression, indicating that CNV-18bp is the upstream silencer of FZP. These findings showed that the silencer CNVs coordinate a trade-off between SPP and TGW by fine-tuning FZP expression, and balancing the trade-off could enhance yield potential.

Rice Variation Map (RiceVarMap, http:/ricevarmap.ncpgr.cn) is a database of rice genomic variations. The database provides comprehensive information of 6,551,358 single nucleotide polymorphisms (SNPs) and 1,214,627 insertions/deletions (INDELs) identified from sequencing data of 1479 rice accessions. The SNP genotypes of all accessions were imputed and evaluated, resulting in an overall missing data rate of 0.42% and an estimated accuracy greater than 99%. The SNP/INDEL genotypes of all accessions are available for online query and download. Users can search SNPs/INDELs by identifiers of the SNPs/INDELs, genomic regions, gene identifiers and keywords of gene annotation. Allele frequencies within various subpopulations and the effects of the variation that may alter the protein sequence of a gene are also listed for each SNP/INDEL. The database also provides geographical details and phenotype images for various rice accessions. In particular, the database provides tools to construct haplotype networks and design PCR-primers by taking into account surrounding known genomic variations. These data and tools are highly useful for exploring genetic variations and evolution studies of rice and other species.

Exploitation of heterosis in rice (Oryza sativa L.) has contributed greatly to global food security. In this study, we generated three sets of reciprocal F1 hybrids of indica and japonica subspecies to evaluate the relationship between yield heterosis and the circadian clock. There were no differences in trait performance or heterosis between the reciprocal hybrids, indicating no maternal effects on heterosis. The indica-indica and indica-japonica reciprocal F1 hybrids exhibited pronounced heterosis for chlorophyll and starch content in leaves and for grain yield/biomass. In contrast, the japonica-japonica F1 hybrids showed low heterosis. The three circadian clock genes investigated expressed in an above-high-parent pattern (AHP) at seedling stage in all the hybrids. The five genes downstream of the circadian clock, and involved in chlorophyll and starch metabolic pathways, were expressed in AHP in hybrids with strong better-parent heterosis (BPH). Similarly, three of these five genes in the japonica-japonica F1 hybrids showing low BPH were expressed in positive overdominance, but the other two genes were expressed in additive or negative overdominance. These results indicated that the expression patterns of circadian clock genes and their downstream genes are associated with heterosis, which suggests that the circadian rhythm pathway may be related to heterosis in rice.

Seed vigor is an index of seed quality that is used to describe the rapid and uniform germination and the establishment of strong seedlings in any environmental conditions. Strong seed vigor in low-temperature germination conditions is particularly important in direct-sowing rice production systems. However, seed vigor has not been selected as an important breeding trait in traditional breeding programs due to its quantitative inherence. In this study, we identified and mapped eight quantitative trait loci (QTLs) for seed vigor by using a recombinant inbred population from a cross between rice (Oryza sativa L. ssp. indica) cultivars ZS97 and MH63. Conditional QTL analysis identified qSV-1, qSV-5b, qSV-6a, qSV-6b, and qSV-11 influenced seedling establishment and that qSV-5a, qSV-5c, and qSV-8 influenced only germination. Of these, qSV-1, qSV-5b, qSV-6a, qSV-6b, and qSV-8 were low-temperature-specific QTLs. Two major-effective QTLs, qSV-1, and qSV-5c were narrowed down to 1.13-Mbp and 400-kbp genomic regions, respectively. The results provide tightly linked DNA markers for the marker-assistant pyramiding of multiple positive alleles for increased seed vigor in both normal and low-temperature germination environments.

Grain chalkiness is a highly undesirable quality trait in the marketing and consumption of rice grain. However, the molecular basis of this trait is poorly understood. Here we show that a major quantitative trait locus (QTL), Chalk5, influences grain chalkiness, which also affects head rice yield and many other quality traits. Chalk5 encodes a vacuolar H(+)-translocating pyrophosphatase (V-PPase) with inorganic pyrophosphate (PPi) hydrolysis and H(+)-translocation activity. Elevated expression of Chalk5 increases the chalkiness of the endosperm, putatively by disturbing the pH homeostasis of the endomembrane trafficking system in developing seeds, which affects the biogenesis of protein bodies and is coupled with a great increase in small vesicle-like structures, thus forming air spaces among endosperm storage substances and resulting in chalky grain. Our results indicate that two consensus nucleotide polymorphisms in the Chalk5 promoter in rice varieties might partly account for the differences in Chalk5 mRNA levels that contribute to natural variation in grain chalkiness.

The genetic basis of heterosis has been debated for over 100 years regarding whether dominance or overdominance plays a more important role and the answer is still unclear. The major limitation to assess the contribution of a single locus has been the genetic background noise due to genome-wide segregation of multiple loci. To dissect the genetic basis of heterosis at a single locus for plant height, we developed a set of 202 chromosome segment substitution lines (CSSLs) of an elite hybrid, Shanyou 63, the best hybrid rice in China in the 1990s. Fifteen CSSLs had varied plant heights within lines. A total of 15 partial dominance QTLs for plant height were detected in these 15 CSSL-F2 populations. All hybrids between the 15 CSSLs and the recurrent parent, Zhenshan 97, were shorter than the corresponding CSSLs, but taller than Zhenshan 97. These indicated that these 15 QTLs were also heterosis loci (HLs) contributed to heterosis acted in dominance. Each HL contributed from -7.4 to 14.4% of midparent heterosis. Additive by additive (AA) and additive by dominance (AD) interactions were detected in the Tetra-F2 population segregating at the four major QTLs with the largest effects on plant height. Substantial negative AA effects were detected between two major QTLs QPH7.2 and QPH7.3, which increased heterosis in the study. Thus we concluded that dominance and epistasis are the major genetic basis of plant height heterosis, which could explain the better parent heterosis in Shanyou 63.

Grain number, plant height, and heading date7 (Ghd7) has been regarded as an important regulator of heading date and yield potential in rice (Oryza sativa). In this study, we investigated functions of Ghd7 in rice growth, development, and environmental response. As a long-day dependent negative regulator of heading date, the degree of phenotypic effect of Ghd7 on heading date and yield traits is quantitatively related to the transcript level and is also influenced by both environmental conditions and genetic backgrounds. Ghd7 regulates yield traits through modulating panicle branching independent of heading date. Ghd7 also regulates plasticity of tiller branching by mediating the PHYTOCHROME B-TEOSINTE BRANCHED1 pathway. Drought, abscisic acid, jasmonic acid, and high-temperature stress strongly repressed Ghd7 expression, whereas low temperature enhanced Ghd7 expression. Overexpression of Ghd7 increased drought sensitivity, whereas knock-down of Ghd7 enhanced drought tolerance. Gene chip analysis of expression profiles revealed that Ghd7 was involved in the regulation of multiple processes, including flowering time, hormone metabolism, and biotic and abiotic stresses. This study suggests that Ghd7 functions to integrate the dynamic environmental inputs with phase transition, architecture regulation, and stress response to maximize the reproductive success of the rice plant.

Rice production in China has abused chemical fertilizers and pesticides for a long time, causing serious environmental pollution. In response to this major social issue, Chinese scientists proposed the second green revolution concept of "less investment, more output and better environment" at the end of the 20th century. In 2007, Chinese scientists proposed the concept of green super rice with the characters of "fewer pesticides, less chemical fertilizers, water saving and drought resistance, high yield and superior quality, and comfortable environment ". In 2010 and 2014, Huazhong Agricultural University as the coordinator hosted the key project of 863 Program of Ministry of Science and Technology "cultivation of new green super rice varieties". They suggested that Green super rice cultivation should divided into three phases. The first phase is to cultivate rice varieties resistant to many major diseases and pests, and thereby without spraying pesticides. The second phase is to cultivate varieties with a higher utilization efficiency and absorption efficiency of water and nitrogen and phosphorus fertilizers, thereby reducing the use of chemical fertilizers. The third phase is to cultivate a significantly enhanced drought resistance of new rice varieties, to achieve the goal of drought and water conservation through biotechnology, molecular marker-assisted selection and genome-wide selection technology. After the three-phase goal is reached, rice production will eventually achieve "less pesticides, less chemical fertilizers, water saving and drought resistance, high yield and super quality". Over the past 20 years, several reference genome sequences have been released one after another, which promotes significant progress of rice functional genomics. Special resources have been identified by establishing a rice core collection. For example, resources are resistance to pests and diseases, low nitrogen and phosphorus and drought stress, or show good performance for other target traits. Dozens of genes for grain yield, quality, plant architecture, biotic stress and abiotic stress have been isolated, which provide gene resources for the cultivation of green super rice through genome-wide selection to pyramid different good genes in the background of high-yielding varieties. More than 20 varieties (combinations) have been released at provincial or national levels. About 70 lines with green traits have been selected to adapt to different rice growing areas. Super green rice new varieties have been grown 1.4 million hectares, and reduced amounts of pesticides, fertilizers and other labor inputs, saved irrigation water, and brought significant social and economic benefits, significant ecological and environmental benefits.

High-throughput SNP genotyping is widely used for plant genetic studies. Recently, a RICE6K SNP array has been developed based on the Illumina Bead Array platform and Infinium SNP assay technology for genome-wide evaluation of allelic variations and breeding applications. In this study, the RICE6K SNP array was used to genotype a recombinant inbred line (RIL) population derived from the cross between the indica variety, Zhenshan 97, and the japonica variety, Xizang 2. A total of 3324 SNP markers of high quality were identified and were grouped into 1495 recombination bins in the RIL population. A high-density linkage map, consisting of the 1495 bins, was developed, covering 1591.2 cM and with average length of 1.1 cM per bin. Segregation distortions were observed in 24 regions of the 11 chromosomes in the RILs. One half of the distorted regions contained fertility genes that had been previously reported. A total of 23 QTLs were identified for yield. Seven QTLs were firstly detected in this study. The positive alleles from about half of the identified QTLs came from Zhenshan 97 and they had lower phenotypic values than Xizang 2. This indicated that favorable alleles for breeding were dispersed in both parents and pyramiding favorable alleles could develop elite lines. The size of the mapping population for QTL analysis using high throughput SNP genotyping platform is also discussed.

The size of cultivated rice (Oryza sativa) grains has been altered by both domestication and artificial selection over the course of evolutionary history. Several quantitative trait loci (QTLs) for grain size have been cloned in the past 10 yr. To explore the natural variation in these QTLs, resequencing of grain width and weight 2 (GW2), grain size 5 (GS5) and QTL for seed width 5 (qSW5) and genotyping of grain size 3 (GS3) were performed in the germplasms of 127 varieties of rice (O. sativa) and 10-15 samples of wild rice (Oryza rufipogon). Ten, 10 and 15 haplotypes were observed for GW2, GS5 and qSW5. qSW5 and GS3 had the strongest effects on grain size, which have been widely utilized in rice production, whereas GW2 and GS5 showed more modest effects. GS5 showed small sequence variations in O. sativa germplasm and that of its progenitor O. rufipogon. qSW5 exhibited the highest level of nucleotide diversity. GW2 showed signs of purifying selection. The four grain size genes experienced different selection intensities depending on their genetic effects. In the indica population, linkage disequilibrium (LD) was detected among GS3, qSW5 and GS5. The substantial genetic variation in these four genes provides the flexibility needed to design various rice grain shapes. These findings provide insight into the evolutionary features of grain size genes in rice.

In plants, flowering is a major biological phenomenon, which is regulated by an array of interactions occurring between biotic and abiotic factors. In our study, we have compared the expression profiles of flowering genes involved in the flowering pathway, which are influenced by conditions like photoperiod and temperature from seedling to heading developmental stages in two Oryza sativa indica varieties, viz., Zhenshan 97 and Minghui 63 using a expression network approach. Using the network expression approach, we found 17 co-expressed genes having the same expression profile pattern as three key photoperiod flowering genes Hd1, Ehd1 and Hd3a. We also demonstrated that these three co-expressed genes have a similar simulation pattern as temperature flowering genes. Based on our observations, we hypothesize that photoperiod and temperature regulate flowering pathways independently. The present study provides a basis for understanding the network of co-expressed genes involved in flowering pathway and presents a way to demonstrate the behavior of specific gene sets in specific cultivars.

A quantitative trait locus (QTL) that affects heading date (HD) and the number of spikelets per panicle (SPP) was previously identified in a small region on chromosome 7 in rice (Oryza sativa L.). In order to further characterize the QTL region, near isogenic lines (NILs) were quickly obtained by self-crossing recombinant inbred line 189, which is heterozygous in the vicinity of the target region. The pleiotropic effects of QTL Ghd7.1 on plant height (PH), SPP, and HD, were validated using an NIL-F2 population. Ghd7.1 explained 50.2%, 45.3%, and 76.9% of phenotypic variation in PH, SPP, and HD, respectively. Ghd7.1 was precisely mapped to a 357-kb region on the basis of analysis of the progeny of the NIL-F2 population. Day-length treatment confirmed that Ghd7.1 is sensitive to photoperiod, with long days delaying heading up to 12.5 d. Identification of panicle initiation and development for the pair of NILs showed that Ghd7.1 elongated the photoperiod-sensitive phase more than 10 d, but did not change the basic vegetative phase and the reproductive growth phase. These findings indicated that Ghd7.1 regulates SPP by controlling the rate of panicle differentiation rather than the duration of panicle development.

Tiller angle is one of the most important components of the ideal plant architecture that can greatly enhance rice grain yield. Understanding the genetic basis of tiller angle and mining favorable alleles will be helpful for breeding new plant-type varieties. Here, we performed genome-wide association studies (GWAS) to identify genes controlling tiller angle using 529 diverse accessions of Oryza sativa including 295 indica and 156 japonica accessions in two environments. We identified 7 common quantitative trait loci (QTLs), including the previously reported major gene Tiller Angle Control 1 (TAC1), in the two environments, 10 and 13 unique QTLs in Hainan and Wuhan, respectively. More QTLs were identified in indica than in japonica, and three major QTLs (qTA3, qTA1b/DWARF2 (D2) and qTA9c/TAC1) were fixed in japonica but segregating in indica, which explained the wider variation observed in indica compared with that in japonica. No common QTLs were identified between the indica and japonica subpopulations. Mutant analysis for the candidate gene of qTA3 on chromosome 3 indicated a novel gene, Tiller Angle Control 3 (TAC3), encoding a conserved hypothetical protein controlling tiller angle. TAC3 is preferentially expressed in the tiller base. The ebisu dwarf (d2) mutant exhibited a decreased tiller angle, in addition to its previously described abnormal phenotype. A nucleotide diversity analysis revealed that TAC3, D2 and TAC1 have been subjected to selection during japonica domestication. A haplotype analysis identified favorable alleles of TAC3, D2 and TAC1, which may be used for breeding plants with an ideal architecture. In conclusion, there is a diverse genetic basis for tiller angle between the two subpopulations, and it is the novel gene TAC3 together with TAC1, D2, and other newly identified genes in this study that controls tiller angle in rice cultivars., Author Summary Tiller angle is the key component of plant architecture that greatly affect grain yield. However, few tiller angle-related genes that can be used for improving rice plant architecture have been isolated based on natural variation. Here, we identified 7 common tiller angle-related QTLs by a genome-wide association study, including the previously reported major gene TAC1, in two environments in the 529 diverse rice accessions and dozens of QTLs specially identified in one environment. Two QTLs were validated by mutant analysis: A novel gene TAC3, encoding a conserved hypothetical protein and preferentially expressing in the tiller base, was the candidate gene of qTA3; d2 mutant exhibited a decreased tiller angle, in addition to its previously described abnormal phenotype. A haplotype analysis identified favorable alleles of TAC3, D2 and TAC1 in indica, which may be used for breeding plants with an ideal architecture, while they were all subjected to selection and fixed in japonica. In conclusion, there is a diverse genetic basis for tiller angle between the two subpopulations, and it is the novel gene TAC3, together with TAC1 and D2 that greatly controls tiller angle in rice cultivars.

Asian cultivated rice consists of two subspecies: Oryza sativa subsp. indica and O. sativa subsp. japonica Despite the fact that indica rice accounts for over 70% of total rice production worldwide and is genetically much more diverse, a high-quality reference genome for indica rice has yet to be published. We conducted map-based sequencing of two indica rice lines, Zhenshan 97 (ZS97) and Minghui 63 (MH63), which represent the two major varietal groups of the indica subspecies and are the parents of an elite Chinese hybrid. The genome sequences were assembled into 237 (ZS97) and 181 (MH63) contigs, with an accuracy99.99%, and covered 90.6% and 93.2% of their estimated genome sizes. Comparative analyses of these two indica genomes uncovered surprising structural differences, especially with respect to inversions, translocations, presence/absence variations, and segmental duplications. Approximately 42% of nontransposable element related genes were identical between the two genomes. Transcriptome analysis of three tissues showed that 1,059-2,217 more genes were expressed in the hybrid than in the parents and that the expressed genes in the hybrid were much more diverse due to their divergence between the parental genomes. The public availability of two high-quality reference genomes for the indica subspecies of rice will have large-ranging implications for plant biology and crop genetic improvement.