High-quality genome assembly of Huazhan and Tianfeng, the parents of an elite rice hybrid Tian-you-hua-zhan.

High-quality rice reference genomes have accelerated the comprehensive identification of genome-wide variations and research on functional genomics and breeding. Tian-you-hua-zhan has been a leading hybrid in China over the past decade. Here, de novo genome assembly strategy optimization for the rice indica lines Huazhan (HZ) and Tianfeng (TF), including sequencing platforms, assembly pipelines and sequence depth, was carried out. The PacBio and Nanopore platforms for long-read sequencing were utilized, with the Canu, wtdbg2, SMARTdenovo, Flye, Canu-wtdbg2, Canu-SMARTdenovo and Canu-Flye assemblers. The combination of PacBio and Canu was optimal, considering the contig N50 length, contig number, assembled genome size and polishing process. The assembled contigs were scaffolded with Hi-C data, resulting in two "golden quality" rice reference genomes, and evaluated using the scaffold N50, BUSCO, and LTR assembly index. Furthermore, 42,625 and 41,815 non-transposable element genes were annotated for HZ and TF, respectively. Based on our assembly of HZ and TF, as well as Zhenshan97, Minghui63, Shuhui498 and 9311, comprehensive variations were identified using Nipponbare as a reference. The de novo assembly strategy for rice we optimized and the "golden quality" rice genomes we produced for HZ and TF will benefit rice genomics and breeding research, especially with respect to uncovering the genomic basis of the elite traits of HZ and TF.

Karrikin Signaling Acts Parallel to and Additively with Strigolactone Signaling to Regulate Rice Mesocotyl Elongation in Darkness.

Seedling emergence in monocots depends mainly on mesocotyl elongation, requiring coordination between developmental signals and environmental stimuli. Strigolactones (SLs) and karrikins are butenolide compounds that regulate various developmental processes; both are able to negatively regulate rice (Oryza sativa) mesocotyl elongation in the dark. Here, we report that a karrikin signaling complex, DWARF14-LIKE (D14L)-DWARF3 (D3)-O. sativa SUPPRESSOR OF MAX2 1 (OsSMAX1) mediates the regulation of rice mesocotyl elongation in the dark. We demonstrate that D14L recognizes the karrikin signal and recruits the SCFD3 ubiquitin ligase for the ubiquitination and degradation of OsSMAX1, mirroring the SL-induced and D14- and D3-dependent ubiquitination and degradation of D53. Overexpression of OsSMAX1 promoted mesocotyl elongation in the dark, whereas knockout of OsSMAX1 suppressed the elongated-mesocotyl phenotypes of d14l and d3 OsSMAX1 localizes to the nucleus and interacts with TOPLESS-RELATED PROTEINs, regulating downstream gene expression. Moreover, we showed that the GR24 enantiomers GR245DS and GR24 ent-5DS specifically inhibit mesocotyl elongation and regulate downstream gene expression in a D14- and D14L-dependent manner, respectively. Our work revealed that karrikin and SL signaling play parallel and additive roles in modulating downstream gene expression and negatively regulating mesocotyl elongation in the dark.

A genome-wide survey of copy number variations reveals an asymmetric evolution of duplicated genes in rice.

BACKGROUND: Copy number variations (CNVs) are an important type of structural variations in the genome that usually affect gene expression levels by gene dosage effect. Understanding CNVs as part of genome evolution may provide insights into the genetic basis of important agricultural traits and contribute to the crop breeding in the future. While available methods to detect CNVs utilizing next-generation sequencing technology have helped shed light on prevalence and effects of CNVs, the complexity of crop genomes poses a major challenge and requires development of additional tools. RESULTS: Here, we generated genomic and transcriptomic data of 93 rice (Oryza sativa L.) accessions and developed a comprehensive pipeline to call CNVs in this large-scale dataset. We analyzed the correlation between CNVs and gene expression levels and found that approximately 13% of the identified genes showed a significant correlation between their expression levels and copy numbers. Further analysis showed that about 36% of duplicate pairs were involved in pseudogenetic events while only 5% of them showed functional differentiation. Moreover, the offspring copy mainly contributed to the expression levels and seemed more likely to become a pseudogene, whereas the parent copy tended to maintain the function of ancestral gene. CONCLUSION: We provide a high-accuracy CNV dataset that will contribute to functional genomics studies and molecular breeding in rice. We also showed that gene dosage effect of CNVs in rice is not exponential or linear. Our work demonstrates that the evolution of duplicated genes is asymmetric in both expression levels and gene fates, shedding a new insight into the evolution of duplicated genes.

A Strigolactone Biosynthesis Gene Contributed to the Green Revolution in Rice.

Plant architecture is a complex agronomic trait and a major factor of crop yield, which is affected by several important hormones. Strigolactones (SLs) are identified as a new class hormoneinhibiting branching in many plant species and have been shown to be involved in various developmental processes. Genetical and chemical modulation of the SL pathway is recognized as a promising approach to modify plant architecture. However, whether and how the genes involved in the SL pathway could be utilized in breeding still remain elusive. Here, we demonstrate that a partial loss-of-function allele of the SL biosynthesis gene, HIGH TILLERING AND DWARF 1/DWARF17 (HTD1/D17), which encodes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), increases tiller number and improves grain yield in rice. We found that the HTD1 gene had been widely utilized and co-selected with Semidwarf 1 (SD1), both contributing to the improvement of plant architecture in modern rice varieties since the Green Revolution in the 1960s. Understanding how phytohormone pathway genes regulate plant architecture and how they have been utilized and selected in breeding will lay the foundation for developing the rational approaches toward improving crop yield.

The newly identified heat-stress sensitive albino 1 gene affects chloroplast development in rice.

High temperature, a major abiotic stress, significantly affects the yield and quality of crops in many parts of the world. Components of the photosynthetic apparatus are highly susceptible to thermal damage. Although the responses to acute heat stress have been studied intensively, the mechanisms that regulate chloroplast development under heat stress remain obscure, especially in crop plants. Here, we cloned and characterized the gene responsible for the heat-sensitive albino1 (hsa1) mutation in rice (Oryza sativa). The hsa1 mutant harbors a recessive mutation in a gene encoding fructokinase-like protein2 (FLN2); the mutation causes a premature stop codon and results in a severe albino phenotype, with defects in early chloroplast development. The color of hsa1 mutant plants gradually changed from albino to green at later stages of development at various temperatures and chloroplast biogenesis was strongly delayed at high temperature (32 °C). HSA1 expression was strongly reduced in hsa1 plants compared to wild type (WT). HSA1 localizes to the chloroplast and regulates chloroplast development. An HSA1 deletion mutant induced by CRISPR/Cas9 was heat sensitive but had a faster greening phenotype than the original hsa1 allele at all temperatures. RNA and protein levels of plastid-encoded RNA polymerase-dependent plastid genes were markedly reduced in hsa1 plants compared to WT. These results demonstrated that HSA1 plays important roles in chloroplast development at early stages, and functions in protecting chloroplasts under heat stress at later stages in rice.

Activation of ABA Receptors Gene GhPYL9-11A Is Positively Correlated with Cotton Drought Tolerance in Transgenic Arabidopsis.

The sensitivity to abscisic acid (ABA) by its receptors, pyrabactin resistance-like proteins (PYLs), is considered a most important factor in activating the ABA signal pathway in response to abiotic stress. However, it is still unknown which PYL is the crucial ABA receptor mediating response to drought stress in cotton (Gossypium hirsutum L.). Here, we reported the identification and characterization of highly induced ABA receptor GhPYL9-11A in response to drought in cotton. It is observed that GhPYL9-11A was highly induced by ABA treatment. GhPYL9-11A binds to protein phosphatase 2Cs (PP2Cs) in an ABA-independent manner. Moreover, the GhPYL-11A-PP2C interactions are partially disrupted by mutations, proline (P84) and histidine (H111), in the gate-latch region. Transgenic Arabidopsis overexpressing GhPYL9-11A plants were hypersensitive to ABA during seed germination and early seedling stage. Further, the increased in root growth and up regulation of drought stress-related genes in transgenic Arabidopsis as compared to wild type confirmed the potential role of GhPYL9-11A in abiotic stress tolerance. Consistently, the expression level of GhPYL9-11A is on average higher in drought-tolerant cotton cultivars than in drought-sensitive cottons under drought treatment. In conclusion, the manipulation of GhPYL9-11A expression could be a useful strategy for developing drought-tolerant cotton cultivars.

Functional Inactivation of Putative Photosynthetic Electron Acceptor Ferredoxin C2 (FdC2) Induces Delayed Heading Date and Decreased Photosynthetic Rate in Rice.

Ferredoxin (Fd) protein as unique electron acceptor, involved in a variety of fundamental metabolic and signaling processes, which is indispensable for plant growth. The molecular mechanisms of Fd such as regulation of electron partitioning, impact of photosynthetic rate and involvement in the carbon fixing remain elusive in rice. Here we reported a heading date delay and yellowish leaf 1 (hdy1) mutant derived from Japonica rice cultivar "Nipponbare" subjected to EMS treatment. In the paddy field, the hdy1 mutant appeared at a significantly late heading date and had yellow-green leaves during the whole growth stage. Further investigation indicated that the abnormal phenotype of hdy1 was connected with depressed pigment content and photosynthetic rate. Genetic analysis results showed that the hdy1 mutant phenotype was caused by a single recessive nuclear gene mutation. Map-based cloning revealed that OsHDY1 is located on chromosome 3 and encodes an ortholog of the AtFdC2 gene. Complementation and overexpression, transgenic plants exhibited the mutant phenotype including head date, leaf color and the transcription levels of the FdC2 were completely rescued by transformation with OsHDY1. Real-time PCR revealed that the expression product of OsHDY1 was detected in almost all of the organs except root, whereas highest expression levels were observed in seeding new leaves. The lower expression levels of HDY1 and content of iron were detected in hdy1 than WT's. The FdC2::GFP was detected in the chloroplasts of rice. Real-time PCR results showed that the expression of many photosynthetic electron transfer related genes in hdy1 were higher than WT. Our results suggest that OsFdC2 plays an important role in photosynthetic rate and development of heading date by regulating electron transfer and chlorophyll content in rice.

A Rare Allele of GS2 Enhances Grain Size and Grain Yield in Rice.

Grain size determines grain weight and affects grain quality. Several major quantitative trait loci (QTLs) regulating grain size have been cloned; however, our understanding of the underlying mechanism that regulates the size of rice grains remains fragmentary. Here, we report the cloning and characterization of a dominant QTL, grain size on chromosome 2 (GS2), which encodes Growth-Regulating Factor 4 (OsGRF4), a transcriptional regulator. GS2 localizes to the nucleus and may act as a transcription activator. A rare mutation of GS2 affecting the binding site of a microRNA, OsmiR396c, causes elevated expression of GS2/OsGRF4. The increase in GS2 expression leads to larger cells and increased numbers of cells, which thus enhances grain weight and yield. The introduction of this rare allele of GS2/OsGRF4 into rice cultivars could significantly enhance grain weight and increase grain yield, with possible applications in breeding high-yield rice varieties.