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.

基于辣椒基因组测序探究辣味在种群中的进化

辣椒(Capsicum annuum)是美洲最古老的驯化作物之一,在全世界被广泛种植。本研究对CM334(墨西哥地方品种)进行了全基因组测序和组装(覆盖度为186.6×),同时还对另外两个栽培种和一个野生种分别进行了重测序和从头(de novo)测序。结果表明辣椒基因组大小约为番茄的4倍,辣椒基因组上存在大量gypsy转座子和花椰菜病毒家族元件的积累。结合转录组综合分析认为,辣椒素合成酶基因表达模式的改变及新功能化是导致辣椒素生物合成的原因,研究还发现辣椒与番茄在乙烯合成及果实成熟调控方面存在明显不同的分子模式。辣椒参考基因组的发布为辣椒营养品质和药用价值的改善提供了重要平台。

Ortholog Alleles at Xa3/Xa26 Locus Confer Conserved Race-Specific Resistance against Xanthomonas oryzae in Rice

The rice disease resistance （R） gene Xa3/Xa26 （having also been named Xa3 and Xa26） against Xanthomonas oryzae pv. oryzae （Xoo）, which causes bacterial blight disease, belongs to a multiple gene family clustered in chromosome 11 and is from an AA genome rice cultivar （Oryza sativa L.）. This family encodes leucine-rich repeat （LRR） receptor kinase- type proteins, Here, we show that the orthologs （alleles） of Xa3/Xa26, Xa3/Xa26-2, and Xa3/Xa26-3, from wild Oryza spe- cies O. officinalis （CC genome） and O. minuta （BBCC genome）, respectively, were also R genes against Xoo. Xa3/Xa26-2 and Xa3/Xa26-3 conferred resistance to 16 of the 18 Xoo strains examined. Comparative sequence analysis of the Xa3/Xa26 families in the two wild Oryza species showed that Xa3/Xa26-3 appeared to have originated from the CC genome of O. minuta. The predicted proteins encoded by Xa3/Xa26, Xa3/Xa26-2, and Xa3/Xa26-3 share 91-99% sequence identity and 94-99% sequence similarity. Transgenic plants carrying a single copy of Xa3/Xa26, Xa3/Xa26-2, or Xa3PXa26-3, in the same genetic background, showed a similar resistance spectrum to a set of Xoo strains, although plants carrying Xa3/Xa26-2 or Xa3/Xa26-3 showed lower resistance levels than the plants carrying Xa3/Xa26. These results suggest that the Xa3/Xa26 locus predates the speciation of A and C genome, which is approximately 7.5 million years ago. Thus, the resistance spec- ificity of this locus has been conserved for a long time.

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.

Dynamic Intra-Japonica Subspecies Variation and Resource Application

We constructed a physical map of O. sativa ssp. japonica cv. ZH11 and compared it and its random sample sequences with the Nipponbare RefSeq derived from the same subspecies. This comparison showed that the two japonica genomes were highly syntenic but revealed substantial differences in terms of structural variations, rates of substitutions and indels, and transposable element content. For example, contractions/expansions as large as 450 kb and repeat sequences that were present in high copy numbers only in ZH11 were detected. In tri-alignment regions using the indica variety 93-11 sequence as an outgroup, we found that： （1） the substitution rates of the two japonica-indica inter- subspecies comparison combinations were close but almost a magnitude higher than the substitution rate between the japonica rice varieties ZH11 and Nipponbare; （2） of the substitutions found between ZH11 and Nipponbare, 47.2% occurred in ZH11 and 52.6% in Nipponbare; （3） of the indels found between ZH11 and Nipponbare, the indels that occurred in ZH11 were 15.8 times of those in Nipponbare. Of the indels that occurred in ZH11, 75.67% were insertions and 24.33% deletions. Of the indels that occurred in Nipponbare, 48.23% were insertions and 51.77% were deletions. The ZH11 com- parative map covered four Nipponbare physical gaps, detected assembly errors in the Nipponbare sequence, and was integrated with the FSTs of a large ZH11 T-DNA insertion mutant library. ZH11 BAC clones can be browsed, searched, and obtained at our website, http：//GResource.hzau.edu.cn.

The Subtelomere of Oryza sativa Chromosome 3 Short Arm as a Hot Bed of New Gene Origination in Rice

Despite general observations of non-random genomic distribution of new genes, it is unclear whether or not new genes preferentially occur in certain genomic regions driven by related molecular mechanisms. Using 1.5 Mb of genomic sequences from short arms of chromosome 3 of Oryza glaberrima and O. punctata, we conducted a comparative genomic analysis with the reference O. sativa ssp. japonica genome. We identified a 60-kb segment located in the middle of the subtelomeric region of chromosome 3, which is unique to the species O. sativa. The region contained gene duplicates that occurred in Asian cultivated rice species that diverged from the ancestor of Asian and African cultivated rice one million years ago （MYA）. For the 12 genes and one complete retrotransposon identified in this segment in O. sativa ssp. japonica, we searched for their parental genes. The high similarity between duplicated paralogs further supports the recent origination of these genes. We found that this segment was recently generated through multiple independent gene recombination and transposon insertion events. Among the 12 genes, we found that five had chimeric gene structures derived from multiple parental genes. Nine out of the 12 new genes seem to be functional, as suggested by Ka/Ks analysis and the presence of cDNA and/or MPSS data. Furthermore, for the eight transcribed genes, at least two genes could be classified as defense or stress response-related genes. Given these findings, and the fact that subtelomeres are associated with high rates of recombination and transcription, it is likely that subtelomeres may facilitate gene recombination and transposon insertions and serve as hot spots for new gene origination in rice genomes.

Sequencing and comparative analyses of Aegilops tauschii chromosome arm 3DS reveal rapid evolution of Triticeae genomes

Bread wheat （Triticum aestivum, AABBDD） is an allohexaploid species derived from two rounds of interspecific hybridizations. A high-quality genome sequence assembly of diploid Aegilops tauschii, the donor of the wheat D genome, will provide a useful platform to study polyploid wheat evolution. A combined approach of BAC pooling and next-generation sequencing technology was employed to sequence the minimum tiling path （MTP） of 3176 BAC clones from the short arm ofAe. tauschii chromosome 3 （At3DS）. The final assembly of 135 super-scaffolds with an N50 of 4.2 Mb was used to build a 247-Mb pseudomolecule with a total of 2222 predicted protein-coding genes. Compared with the orthologous regions of rice, Brachypodium, and sorghum, At3DS contains 38.67% more genes. In comparison to At3DS, the short arm sequence of wheat chromosome 3B （Ta3BS） is 95-Mb large in size, which is primarily due to the expansion of the non-centromeric region, suggesting that transposable element （TE） bursts in Ta3B likely occurred there. Also, the size increase is accompanied by a proportional increase in gene number in Ta3BS. We found that in the sequence of short arm of wheat chromosome 3D （Ta3DS）, there was only less than 0.27% gene loss compared to At3DS. Our study reveals divergent evolution of grass genomes and provides new insights into sequence changes in the polyploid wheat genome.

Fifteen Million Years of Evolution in the Oryza Genus Shows Extensive Gene Family Expansion

In analyzing gene families in the whole-genome sequences available for O. sativa （AA）, O. glaberrima （AA）, and O. brachyantha （FF）, we observed large size expansions in the AA genomes compared to FF genomes for the superfamilies F-box and NB-ARC, and five additional families： the Aspartic proteases, BTB/POZ proteins （BTB）, Glutaredoxins, Trypsin a-amylase inhibitor proteins, and Zf-Dof proteins. Their evolutionary dynamic was investigated to understand how and why such important size variations are observed between these closely related species. We show that expansions resulted from both amplification, largely by tandem duplications, and contraction by gene losses. For the F-box and NB-ARC gene families, the genes conserved in all species were under strong purifying selection while expanded orthologous genes were under more relaxed purifying selection. In F-box, NB-ARC, and BTB, the expanded groups were enriched in genes with little evidence of expression, in comparison with conserved groups. We also detected 87 loci under positive selection in the expanded groups. These results show that most of the duplicated copies in the expanded groups evolve neutrally after duplication because of functional redundancy but a fraction of these genes were preserved following neofunctionalization. Hence, the lineage-specific expansions observed between Oryza species were partly driven by directional selection.

Genome-Wide Prediction of Highly Specific Guide RNA Spacers for CRISPR-Cas9-Mediated Genome Editing in Model Plants and Major Crops

Dear Editor, RNA-guided genome editing （RGE） using the Streptococcus pyogenes CRISPR-Cas9 system （Jinek et al., 2012; Cong et al., 2013; Mall et al., 2013b） is emerging as a simple and highly efficient tool for genome editing in many organisms. The Cas9 nuclease can be programmed by dual or single guide RNA （gRNA） to cut target DNA at specific sites,

Genome and Comparative Transcriptomics of African Wild Rice Oryza Iongistaminata Provide Insights into Molecular Mechanism of Rhizomatousness and Self-Incompatibility

Dear Editor, Oryza Iongistaminata is an African wild rice species with AA genome type possessing special traits that are highly valued for improving cultivated rice, such as strong resistance to biotic and abiotic stresses （Song et al., 1995） for improving resistance of cultivars, rhizomatousness for perennial breeding （Glover et al., 2010）, and self-incompatibility （SI） for new ways to produce hybrid seeds （Ghesquiere, 1986）. Deciphering the genome of O. Iongistaminata will be the key to uncovering the mechanism of these hallmark traits and improving cultivated rice.