Rice Gene Index:A comprehensive pan-genome database for comparative and functional genomics of Asian rice

Dear Editor,Asian rice(Oryza sativa)is the staple food for half the world and is a model crop that has been extensively studied.It contributes20%of calories to the human diet(Stein et al.,2018).With the increase in global population and rapid changes in climate,rice breeders need to develop new and sustainable cultivars with higher yields,healthier grains,and reduced environmental footprints(Wing et al.,2018).Since the first gold-standard reference genome of rice variety Nipponbare was published(International Rice Genome Sequencing Project,2005),an increasing number of rice accessions have been sequenced,assembled,and annotated with global efforts.Nowadays,a single reference genome is obviously insufficient to perform the genetic difference analysis for rice accessions.Therefore,the pan-genome has been proposed as a solution,which allows the discovery of more presence-absence variants compared with single-reference genome-based studies(Zhao et al.,2018).Over the past years,several databases,such as RAP-db(https://rapdb.dna.affrc.go.jp),RGAP(http://rice.uga.edu),and Gramene(https://www.gramene.org),have long-term served rice genomic research by providing information based on one or a series of individual reference genomes.To integrate and utilize the genomic information of multiple accessions,we performed comparative analyses and established the user-friendly Rice Gene Index(RGI;https://riceome.hzau.edu.cn)platform.RGI is the first gene-based pan-genome database for rice.

A High-Density SNP Genotyping Array for Rice Biology and Molecular Breeding

A high-density single nucleotide polymorphism （SNP） array is critically important for geneticists and molecu- lar breeders. With the accumulation of huge amounts of genomic re-sequencing data and available technologies for accurate SNP detection, it is possible to design high-density and high-quality rice SNP arrays. Here we report the devel- opment of a high-density rice SNP array and its utility. SNP probes were designed by screening more than 10 000 000 SNP loci extracted from the re-sequencing data of 801 rice varieties and an array named RiceSNP50 was produced on the Illumina Infinium platform. The array contained 51 478 evenly distributed markers, 68% of which were within genic regions. Several hundred rice plants with parent/F1 relationships were used to generate a high-quality cluster file for accurate SNP calling. Application tests showed that this array had high genotyping accuracy, and could be used for dif- ferent objectives. For example, a core collection of elite rice varieties was clustered with fine resolution. Genome-wide association studies （GWAS） analysis correctly identified a characterized QTL. Further, this array was successfully used for variety verification and trait introgression. As an accurate high-throughput genotyping tool, RiceSNP50 will play an important role in both functional genomics studies and molecular breeding.

RNA-Directed DNA Methylation Is Involved in Regulating Photoperiod-Sensitive Male Sterility in Rice

Photoperiod-sensitive male sterility （PSMS） is a valuable germplasm for hybrid rice breeding. Recently, we cloned pros3, a locus controlling PSMS, which encodes a long non-coding RNA called LDMAR required for normal male fertility of the rice plant under long-day conditions. Increased methylation in the promoter of LDMAR in the PSMS rice （Nongken 58S） relative to the wild-type （Nongken 58） reduced expression of LDMAR leading to male sterility under long-day conditions. In this study, we identified a siRNA named Psi-LDMAR in the LDMAR promoter region that was more abundant in Nongken 58S than in Nongken 58. We showed that Psi-LDMAR was likely derived from AKl11270, a transcript obtained from the sense strand of the LDMAR promoter with its 3＇-end having a 110-base overlap with the 5＇-end of LDMAR. Overexpressing AKl11270 in Nongken 58S greatly enriched Psi-LDMAR, which induced RNA-directed DNA methylation in the LDMAR promoter and repressed the expression of LDMAR. Reduction of LDMAR in Nongken 58S changed the critical day length for fertility recovery and delayed the fertility recovery under short-day conditions. This result added to our understanding of the molecular mechanism for PSMS.

Genetic Architecture of Natural Variation in Rice Chlorophyll Content Revealed by a Genome-Wide Association Study

Chlorophyll content is one of the most important physiological traits as it is closely related to leaf photo- synthesis and crop yield potential. So far, few genes have been reported to be involved in natural variation of chlorophyll content in rice （Oryza sativa） and the extent of variations explored is very limited. We con- ducted a genome-wide association study （GWAS） using a diverse worldwide collection of 529 O. sativa accessions. A total of 46 significant association loci were identified. Three F2 mapping populations with parents selected from the association panel were tested for validation of GWAS signals. We clearly demon- strated that Grain number, plant height, andheading date7 （GhdT） was a major locus for natural variation of chlorophyll content at the heading stage by combining evidence from near-isogenic lines and transgenic plants. The enhanced expression of Ghd7 decreased the chlorophyll content, mainly through down- regulating the expression of genes involved in the biosynthesis of chlorophyll and chloroplast. In addition, Narrow leaf1 （NAL1） corresponded to one significant association region repeatedly detected over two years. We revealed a high degree of polymorphism in the 5＇ UTR and four non-synonymous SNPs in the cod- ing region of NAL1, and observed diverse effects of the major haplotypes. The loci or candidate genes iden- tified would help to fine-tune and optimize the antenna size of canopies in rice breeding.

Two gap-free reference genomes and a global view of the centromere architecture in rice

Rice(Oryza sativa),a major staple throughout the world and a model system for plant genomics and breeding,was the first crop genome sequenced almost two decades ago.However,reference genomes for all higher organisms to date contain gaps and missing sequences.Here,we report the assembly and analysis of gap-free reference genome sequences for two elite O.sativa xian/indica rice varieties,Zhenshan 97 and Minghui 63,which are being used as a model system for studying heterosis and yield.Gap-free reference genomes provide the opportunity for a global view of the structure and function of centromeres.We show that all rice centromeric regions share conserved centromere-specific satellite motifs with different copy numbers and structures.In addition,the similarity of CentO repeats in the same chromosome is higher than across chromosomes,supporting a model of local expansion and homogenization.Both genomes have over 395 non-TE genes located in centromere regions,of which∼41%are actively transcribed.Two large structural variants at the end of chromosome 11 affect the copy number of resistance genes between the two genomes.The availability of the two gap-free genomes lays a solid foundation for further understanding genome structure and function in plants and breeding climate-resilient varieties.

Genome-and transcriptome-wide association studies provide insights into the genetic basis of natural variation of seed oil content in Brassica napus

Seed oil content(SOC)is a highly important and complex trait in oil crops.Here,we decipher the genetic basis of natural variation in SOC of Brassica napus by genome-and transcriptome-wide association studies using 505 inbred lines.We mapped reliable quantitative trait loci(QTLs)that control SOC in eight environments,evaluated the effect of each QTL on SOC,and analyzed selection in QTL regions during breeding.Six-hundred and ninety-two genes and four gene modules significantly associated with SOC were identified by analyzing population transcriptomes from seeds.A gene prioritization framework,POCKET(prioritizing the candidate genes by incorporating information on knowledge-based gene sets,effects of variants,genome-wide association studies,and transcriptome-wide association studies),was implemented to determine the causal genes in the QTL regions based on multi-omic datasets.A pair of homologous genes,BnPMT6s,in two QTLs were identified and experimentally demonstrated to negatively regulate SOC.This study provides rich genetic resources for improving SOC and valuable insights toward understanding the complex machinery that directs oil accumulation in the seeds of B.napus and other oil crops.

Bract suppression regulated by the miR156/529-SPLs-NL1-PLA1 module is required for the transition from vegetative to reproductive branching in rice

Reproductive transition of grasses is characterized by switching the pattern of lateral branches,featuring the suppression of outgrowth of the subtending leaves(bracts)and rapid formation of higher-order branches in the inflorescence(panicle).However,the molecular mechanisms underlying such changes remain largely unknown.Here,we show that bract suppression is required for the reproductive branching in rice.We identified a pathway involving the intrinsic time ruler microRNAI56/529,their targets SQUAMOSA PROMOTER BINDING PROTEIN LIKE(SPL)genes,NECK LEAF1(NL1),and PLASTOCHRON1(PLA1),which regulates the bract outgrowth and thus affects the pattern switch between vegetative and reproductive branching.Suppression of the bract results in global reprogramming of transcriptome and chromatin accessibility following the reproductive transition,while these processes are largely dysregu-lated in the mutants of these genes.These discoveries contribute to our understanding of the dynamic plant architecture and provide novel insights for improving crop yields.

A Nonresonant Hybridized Electromagnetic-Triboelectric Nanogenerator for Irregular and Ultralow Frequency Blue Energy Harvesting

As a promising renewable energy source,it is a challenging task to obtain blue energy,which is irregular and has an ultralow frequency,due to the limitation of technology.Herein,a nonresonant hybridized electromagnetic-triboelectric nanogenerator was presented to efficiently obtain the ultralow frequency energy.The instrument adopted the flexible pendulum structure with a precise design and combined the working principle of electromagnetism and triboelectricity to realize the all-directional vibration energy acquisition successfully.The results confirmed that the triboelectric nanogenerator(TENG)had the potential to deliver the maximum power point of about 470μW while the electromagnetic nanogenerator(EMG)can provide 523 mW at most.The conversion efficiency of energy of the system reached 48.48%,which exhibited a remarkable improvement by about 2.96 times,due to the elastic buffering effect of the TENG with the double helix structure.Furthermore,its ability to collect low frequency wave energy was successfully proven by a buoy in Jialing River.This woke provides an effective candidate to harvest irregular and ultralow frequency blue energy on a large scale.

Fixation of hybrid sterility genes and favorable alleles of key yield-related genes with dominance contribute to the high yield of the Yongyou series of intersubspecific hybrid rice

In rice,the Yongyou series of Xian-Geng intersubspecific hybrids have excellent production performance,as shown by their extremely high yield.However,the mechanisms underlying the success of these rice hybrids are unclear.In this study,three F2 populations are generated from three Yongyou hybrids to determine the genetic basis of the extremely high yield of intersubspecific hybrids.Genome constitution analysis reveals that the female and male parental lines belong to the Geng and Xian subspecies,respectively,although introgression of 20%of the Xian ancestry and 14%of the Geng ancestry are observed.Twenty-five percent of the hybrid genomes carries homozygous Xian or Geng fragments,which harbors hybrid sterility genes such as Sd,Sc,f5,and q S12 and favorable alleles of key yield-related genes,including NAL1,Ghd7,and Ghd8.None of the parents carries the S5+killer of the S5 killer-protector system.Compatible allele combinations of hybrid sterility genes ensure the fertility of these intersubspecific hybrids and overcome the bottleneck in applying intersubspecific hybrids.Additive effects of favorable alleles of yield-related genes fixed in both parents enhances midparent values.Many QTLs for yield and its key component spikelets per panicle shows dominance and the net positive dominant effects lead to heterosis.These factors result in an extremely high yield of the hybrids.These findings will aid in the development of new intersubspecific rice hybrids with diverse genetic backgrounds.

An inferred functional impact map of genetic variants in rice

Interpreting the functional impacts of genetic variants(GVs)is an important challenge for functional genomic studies in crops and next-generation breeding.Previous studies in rice(Oryza sativa)have focused mainly on the identification of GVs,whereas systematic functional annotation of GVs has not yet been performed.Here,we present a functional impact map of GVs in rice.We curated haplotype information for 17397026 GVs from sequencing data of 4726 rice accessions.We quantitatively evaluated the effects of missense mutations in coding regions in each haplotype based on the conservation of amino acid residues and obtained the effects of 918848 non-redundant missense GVs.Furthermore,we generated high-quality chromatin accessibility(CA)data from six representative rice tissues and used these data to train deep convolutional neural network models to predict the impacts of 5067405 GVs for CA in regulatory regions.We characterized the functional properties and tissue specificity of the GV effects and found that large-effect GVs in coding and regulatory regions may be subject to selection in different directions.Finally,we demonstrated how the functional impact map could be used to prioritize causal variants in mapping populations.This impact map will be a useful resource for accelerating gene cloning and functional studies in rice,and can be freely queried in RiceVarMap V2.0(http://ricevarmap.ncpgr.cn).

Natural Variation in the Sequence of SNAC1 and Its Expression Level Polymorphism in Rice Germplasms under Drought Stress

Water is a major limiting factor for food production and many countries fail to produce sufficient food for their population due to severe water scarcity （Jury and Vaux, 2005）. Rice is the main staple food worldwide. More than 50% of rice in the world is rain-fed and drought causes severe reduction in rice grain yield in rain-fed environments （Venuprasad et al., 2007; Zhang, 2007; Sandhu et al., 2014）. Therefore, enhancing drought resistance （DR） of rice is important for food security. However, DR is a complex trait, which is controlled by a large number of loci with small effect and is also affected by different genetic background, genotype-by-environment interaction and other stresses such as heat （Hu and Xiong, 2014）.

Origination and Establishment of a Trigenic Reproductive Isolation System in Rice

Dear Editor,Reproductive isolation is both the indicator and a primary force of speciation, and plays a key role in maintaining species identity. Understanding the origin and mechanisms of reproductive isola- tion is of fundamental importance in evolutionary biology. In recent years, a number of genes that induce reproductive barriers have been identified in several model organisms such as Drosophila, rodents, yeast, Arabidopsis.

QTL Scanning for Rice Yield Using a Whole Genome SNP Array

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 ofl.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.