Understanding the genetic basis of rice heterosis: Advances and prospects

Heterosis,which describes the superior vigor and yield of F_1 hybrids with respect to their parents,is observed in many rice hybrid crosses.The exploitation of heterosis is a great leap in the history of rice breeding.With advances in genomics and genetics,high-resolution mapping and functional identification of heterosis-associated loci have been performed in rice.Here we summarize advances in understanding the genetic basis of grain yield heterosis in hybrid rice and provide a vision for the genetic study and breeding application of rice heterosis in the future.

Transcriptome Analysis of Reaction Wood in Gymnosperms by Next-Generation Sequencing

Special xylem tissue called “compression wood” is formed on the lower side of inclined stems when gymnosperms grow on a slope. We investigated the molecular mechanism of compression wood formation. Transcriptome analysis by next-generation sequencing (NGS) was applied to the xylem of Chamaecyparis obtusa to develop a catalog of general gene expression in differentiating xylem during compression and normal wood formation. The sequencing output generated 234,924,605 reads and 40,602 contigs (mean size = 529 bp). Based on a sequence similarity search with known proteins, 54.2% (22,005) of the contigs showed homology with sequences in the databases. Of these annotated contigs, 19,293 contigs were assigned to Gene Ontology categories. Differential gene expression between the compression and normal wood libraries was analyzed by mapping the reads from each library to the assembled contigs. In total, 2875 contigs were identified as differentially expressed, including 1207 that were up-regulated and 1668 that were down-regulated in compression wood. We selected 30 genes and compared the transcript abundance between compression and normal wood by quantitative polymerase chain reaction analysis to validate the NGS results. We found that 27 of the 30 genes showed the same expression patterns as the original NGS results.

Computational tools for plant genomics and breeding

Plant genomics and crop breeding are at the intersection of biotechnology and information technology.Driven by a combination of highthroughput sequencing,molecular biology and data science,great advances have been made in omics technologies at every step along the central dogma,especially in genome assembling,genome annotation,epigenomic profiling,and transcriptome profiling.These advances further revolutionized three directions of development.One is genetic dissection of complex traits in crops,along with genomic prediction and selection.The second is comparative genomics and evolution,which open up new opportunities to depict the evolutionary constraints of biological sequences for deleterious variant discovery.The third direction is the development of deep learning approaches for the rational design of biological sequences,especially proteins,for synthetic biology.All three directions of development serve as the foundation for a new era of crop breeding where agronomic traits are enhanced by genome design.

ALK,the Key Gene for Gelatinization Temperature,is a Modifier Gene for Gel Consistency in Rice

Gelatinization temperature（GT） is an important parameter in evaluating the cooking and eating quality of rice.Indeed,the phenotype,biochemistry and inheritance of GT have been widely studied in recent times.Previous map-based cloning revealed that GT was controlled by ALK gene,which encodes a putative soluble starch synthase II-3.Complementation vector and RNAi vector were constructed and transformed into Nipponbare mediated by Agrobacterium.Phenotypic and molecular analyses of transgenic lines provided direct evidence for ALK as a key gene for GT.Meanwhile,amylose content,gel consistency and pasting properties were also affected in transgenic lines.Two of four nonsynonymous single nucleotide polymorphisms in coding sequence of ALK were identified as essential for GT.Based on the single nucleotide polymorphisms（SNPs）,two new sets of SNP markers combined with one cleaved amplified polymorphic sequence marker were developed for application in rice quality breeding.

Cyclic Bis(3'-5')diadenylic Acid (c-di-AMP) Analogs Promote the Activities of Photosynthesis and Respiration of <i>Chlamydomonas reinhardtii</i>

Physiological changes in the photosynthesis, respiration and cell division of Chlamydomonas reinhardtii, a freshwater green alga, in response to adenine nucleotides were investigated. In advance of this investigation, two adenine nucleotides, di(2'-O-methyl)-cyclic bis(3'-5')diadenylic acid (1) and its N-benzoyl-protected analog 2 were synthesized from the commercially available adenosine phosphoramidite. The respective analogs significantly promoted the cell division (cell number) of C. reinhardtii strains 137c mt+ and BR mt+. Moreover, they significantly enhanced the O2 evolution (photosynthesis) and O2 uptake (respiration) of both strains. c-di-AMP analogs seem to play an effective role as a physiological activator in planta.

Rice Functional Genomics Research： Past Decade and Future

Rice （Oryza sativa） is a major staple food crop for more than 3.5 billion people worldwide. Under- standing the regulatory mechanisms of complex agronomic traits in rice is critical for global food security. Rice is also a model plant for genomics research of monocotyledonso Thanks to the rapid development of functional genomic technologies, over 2000 genes controlling important agronomic traits have been cloned, and their molecular biological mechanisms have also been partially char- acterized. Here, we briefly review the advances in rice functional genomics research during the past 10 years, including a summary of functional genomics platforms, genes and molecular regulatory networks that regulate important agronomic traits, and newly developed tools for gene identification. These achievements made in functional genomics research will greatly facilitate the development of green super rice. We also discuss future challenges and prospects of rice functional genomics research.

Expressional and Biochemical Characterization of Rice Disease Resistance Gene Xa3/Xa26 Family

The rice （Oryza sativa L.） Xa3/Xa26 gene, conferring race-specific resistance to bacterial blight disease and encoding a leucine-rich repeat （LRR） receptor kinase-like protein, belongs to a multigene family consisting of tandem clustered homologous genes, colocalizing with several uncharacterized genes for resistance to bacterial blight or fungal blast. To provide more information on the expressional and biochemical characteristics of the Xa3/Xa26 family, we analyzed the family members. Four Xa3/Xa26 family members in the indica rice variety Teqing, which carries a bacterial blight resistance gene with a chromosomal location tightly linked to Xa3/Xa26, and five Xa3/Xa26 family members in the japonica rice variety Nipponbare, which carries at least one uncharacterized blast resistance gene, were constitutively expressed in leaf tissue. The result suggests that some of the family members may be candidates of these uncharacterized resistance genes. At least five putative N-glycosylation sites in the LRR domain of XA3/XA26 protein are not glycosylated. The XA3/XA26 and its family members MRKa and MRKc all possess the consensus sequences of paired cysteines, which putatively function in dimerization of the receptor proteins for signal transduction, immediately before the first LRR and immediately after the last LRR. However, no homo-dimer between the XA3/XA26 molecules or hetero-dimer between XA3/XA26 and MRKa or MRKc were formed, indicating that XA3/XA26 protein might function either as a monomer or a hetero-dimer formed with other protein outside of the XA3/XA26 family. These results provide valuable information for further extensive investigation into this multiple protein family.

Identification of a novel tillering dwarf mutant and fine mapping of the TDDL(T) gene in rice (Oryza sativa L.)

Rice plant architecture is an important agronomic trait that affects the grain yield. To understand the molecular mechanism that controls plant architecture, a tillering dwarf mutant with darker-green leaves derived from an indica cultivar IR64 treated with EMS is characterized. The mutant, designated as tddl(t), is nonallelic to the known tillering dwarf mutants. It is controlled by one recessive nuclear gene, TDDL(T), and grouped into the dn-type dwarfism according to Takeda's definition. The dwarfism of the mutant is independent of gibberellic acid based on the analyses of two GA-mediated processes. The independence of brassinosteroid (BR) and naphthal-3-acetic acid (NAA) of the tddl(t) mutant, together with the decreased size of parenchyma cells in the vascular bundle, indicates that the TDDL(T) gene might participate in another hormone pathway. TDDL(T) is fine mapped within an 85.51 kb region on the long arm of rice chromosome 4, where 20 ORFs are predicted by RiceGAAS (http://ricegaas.dna.affrc. go.jp/rgadb/). Further cloning of TDDL(T) will benefit both marker assisted selection (MAS) of plant architecture and dissection of the molecular mechanism underlying tillering dwarf in rice.

Differentiation of a Miniature Inverted Transposable Element （MITE） System in Asian Rice Cultivars and Its Inference for a Diphyletic Origin of Two Subspecies of Asian Cultivated Rice

In the present study, we report a survey on a Miniature Inverted Transposable Element （MITE） system known as mPing in 102 varieties of Asian cultivated rice （Oryza sativa L.）. We found that mPing populations could be generalized Into two families, mPing-1 and mPing-2, according to their sequence structures. Further analysis showed that these two families of mPing had significant bias in their distribution pattern in two subspecies of rice, namely O. sativa ssp. japonica and indica. 0. sativa japonica has a higher proportion of mPing-1 as a general trait, whereas 0. sativa indica has a higher proportion of roPing-2. We also examined the mPing system In a doubled haploid （DH） cross-breeding population of jingxi 17 （japonica） and zhaiyeqing 8 （indica） varieties and observed that the mPing system was not tightly linked to major subspecies-determining genes. Furthermore, we checked the mPing system in 28 accessions of Asian common wild rice O. rufipogon and found the roPing system in 0. rufipogon. The distribution pattern of the roPing system in O. rufipogon indicated a diphyletlc origin of the Asian cultivated rice O. sativa species. We did not find the mPing system in another 20 Oryza species. These results substantiated a previous hypothesis that O. ruflpogon and O. nivara species were the closest relatives of O. sativa and that the two extant subspecies of O. sativa were evolved independently from corresponding ecotypes of O. ruflpogon.

Twenty years of rice genomics research: From sequencing and functional genomics to quantitative genomics

Since the completion of the rice genome sequencing project in 2005,we have entered the era of rice genomics,which is still in its ascendancy.Rice genomics studies can be classified into three stages:structural genomics,functional genomics,and quantitative genomics.Structural genomics refers primarily to genome sequencing for the construction of a complete map of rice genome sequence.This is fundamental for rice genetics and molecular biology research.Functional genomics aims to decode the functions of rice genes.Quantitative genomics is large-scale sequence-and statistics-based research to define the quantitative traits and genetic features of rice populations.Rice genomics has been a transformative influence on rice biological research and contributes significantly to rice breeding,making rice a good model plant for studying crop sciences.

Rice functional genomics:decades'efforts and roads ahead

Rice(Oryza sativa L.)is one of the most important crops in the world.Since the completion of rice reference genome sequences,tremendous progress has been achieved in understanding the molecular mechanisms on various rice traits and dissecting the underlying regulatory networks.In this review,we summarize the research progress of rice biology over past decades,including omics,genome-wide association study,phytohormone action,nutrient use,biotic and abiotic responses,photoperiodic flowering,and reproductive development(fertility and sterility).For the roads ahead,cutting-edge technologies such as new genomics methods,high-throughput phenotyping platforms,precise genome-editing tools,environmental microbiome optimization,and synthetic methods will further extend our understanding of unsolved molecular biology questions in rice,and facilitate integrations of the knowledge for agricultural applications.

Glycosylation engineering of therapeutic IgG antibodies： challenges for the safety, functionality and efficacy

Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic antibodies. While the biantennary complex.type oligosaccharide attached to Asn297 of the Fc is essen- tial for antibody effector functions, fucose and outer-arm sugars attached to the core heptasaccharide that gen- erate structural heterogeneity （glycoforms） exhibit unique biological activities. Hence, efficient and quan- titative glycan analysis techniques have been increas- ingly important for the development and quality control of therapeutic antibodies, and g｜ycan profiles of the Fc are recognized as critical quality attributes. In the past decade our understanding of the influence of glycosy- lation on the structure/function of IgG-Fc has grown rapidly through X-ray crystallographic and nuclear magnetic resonance studies, which provides possibili- ties for the design of novel antibody therapeutics. Fur- thermore, the chemoenzymatic glycoengineering approach using endoglycosidase-based glycosyn- thases may facilitate the development of homogeneous IgG glycoforms with desirable functionality as next- generation therapeutic antibodies. Thus, the Fc glycans are fertile ground for the improvement of the safety,functionality, and efficacy of therapeutic IgG antibodies in the era of precision medicine.

Rice Protein Tagging Project:A Call for International Collaborations on Genome-wide In-Locus Tagging of Rice Proteins

In September 1997,the International Rice Genome Sequencing Project was launched.This initiative pooled the resources of ten nations to obtain the first complete rice genome sequence,and promoted rice research and breeding into the post-genomics era(Li et al.,2018).In 2008,an internationally coordinated project named "RICE2020" was proposed to systematically and fully characterize all rice genes,transcripts,and proteins(Zhang et al.,2008).While genes and their transcripts can be readily characterized by sequencing-and PCR-based methods,the characterization of protein dynamics including protein levels,subcellular localizations,post-transla-tional modifications,and interactions with macromolecules(e.g.,proteins,DNA,RNA,carbohydrates,and lipids)and small molecules(e.g.,metabolites and ligands)is much more challenging and usually requires antibodies that specifically recognize the protein of interest.Because it is very difficult to systematically produce reliable antibodies for the specific recognition of individual plant proteins,a common practice is to transform a tag-fused open reading frame(ORF)of a gene to the corresponding loss-of-function mutant plants.However,such an ectopically expressed tagged protein may not fully reca-pitulate the properties of the endogenous protein due to the random insertion of the transgene,even when the transgene is expressed under the endogenous gene promoter.A preferred so-lution is to genetically label the coding sequence of the gene of interest,at its in vivo locus,with a sequence encoding a fluores-cent protein tag or an affinity tag such as FLAG or HA.Such "in-locus" protein tagging,as we are naming it here,has been carried out genome-wide in yeast,Caenorhabditis elegans,fly,and mammalian cultured cells,greatly facilitating the characterization of proteins in these organisms(Jiang et al.,2018).In 2017,a Genome Tagging Project was launched in mice,aiming to label every protein using a CRISPR/Cas9-based "artificial spermatids"method(Jiang et al.,2018).Significant funding and efforts have been put into this project,which is expected to provide valuable mouse resources to accelerate biomedical research.In higher plants,in-locus tagging of proteins has been extremely challenging technically.

Cytoplasmic and nuclear genome variations of rice hybrids and their parents inform the trajectory and strategy of hybrid rice breeding

The male sterility(MS)line is a prerequisite for efficient production of hybrid seeds in rice,a self-pollinating species.MS line breeding is pivotal for hybrid rice improvement.Understanding the historical breeding tra-jectory will help to improve hybrid rice breeding strategies.Maternally inherited cytoplasm is an appro-priate tool for phylogenetic reconstruction and pedigree tracing in rice hybrids.In this study,we analyzed the cytoplasmic genomes of 1495 elite hybrid rice varieties and identified five major types of cytoplasm,which correspond to different hybrid production systems.As the cytoplasm donors for hybrids,461 MS lines were also divided into five major types based on cytoplasmic and nuclear genomic architecture.Spe-cific core accessions cooperating with different fertility-associated genes drove the sequence divergence of MS lines.Dozens to hundreds of convergent and divergent selective sweeps spanning several agro-nomic trait-associated genes were identified among different types of MS lines.We further analyzed the cross patterns between different types of MS lines and their corresponding restorers.This study system-atically analyzed the cytoplasmic genomes of rice hybrids revealed their relationships with nuclear ge-nomes of MS lines,and illustrated the trajectory of hybrid rice breeding and the strategies for breeding different types of MS lines providing new insights for future improvement of hybrid rice.

Rice 2020： A Call For An International Coordinated Effort In Rice Functional Genomics

We describe a call for an international coordinated effort in rice functional genomics in the form of a project named RICE2020. The mission of the project will be： to determine the function of every gene in the rice genome by the year 2020, to identify functional diversity of alleles for agriculturally useful genes from the primary gene pool of rice, and to apply the findings of functional genomics research to rice genetic improvement.

Cutting the Gordian knot of plant genetics:retrieval of missing heritability in tomato

A pan-genome can be defined as the non-redundant collection of genomic diversity within a single species. Although the single reference genomes have achieved extremely high continuity and completeness owing to the recent advances in single-molecule sequencing and physical mapping technologies, it is hard for a linear reference genome to capture all the diversity within a species.

From Genetic Mapping to Molecular Breeding： Genomics Have Paved the Highway

Owing to the recent advances in sequencing technology, genetic mapping of mutations has been revolutionized by whole-genome deep sequencing and bulk segregant analysis （Schneeberger et al., 2009; Abe et al., 2012）. Once the mapping population for a mutant is available, genetic analysis can be simplified by using the pool of F2 plants with mutant phenotype and the pool of those showing wild-type phenotype. Since most mutant phe- notypes are of qualitative character and follow simple inheritance patterns, genome-wide comparisons between the two DNA pools could localize the causal gene within a small genomic region. In most cases, the causative mutation （e.g., an in-frame indel） can be detected as well because there are only a limited number of sequence differences between the mutant and its wild-type. Hence, rapid mutant mapping via genomics ap- proaches is now becoming practical and cost-effective.

Photoperiodic flowering in Arabidopsis:Multilayered regulatory mechanisms of CONSTANS and the florigen FLOWERING LOCUS T

The timing of flowering affects the success of sexual reproduction.This developmental event also determines crop yield,biomass,and longevity.Therefore,this mechanism has been targeted for improvement along with crop domestication.The underlying mechanisms of flowering are highly conserved in angiosperms.Central to these mechanisms is how environmental and endogenous conditions control transcriptional regulation of the FLOWERING LOCUS T(FT)gene,which initiates floral development under long-day conditions in Arabidopsis.Since the identification of FT as florigen,efforts have been made to understand the regulatory mechanisms of FT expression.Although many transcriptional regulators have been shown to directly influence FT,the question of how they coordinately control the spatiotemporal expression patterns of FT still requires further investigation.Among FT regulators,CONSTANS(CO)is the primary one whose protein stability is tightly controlled by phosphorylation and ubiquitination/proteasome-mediated mechanisms.In addition,various CO interaction partners,some of them previously identified as FT transcriptional regulators,positively or negatively modulate CO protein activity.The FT promoter possesses several transcriptional regulatory"blocks,"highly conserved regions among Brassicaceae plants.Different transcription factors bind to specific blocks and affect FT expression,often causing topological changes in FT chromatin structure,such as the formation of DNA loops.We discuss the current understanding of the regulation of FT expression mainly in Arabidopsis and propose future directions related to this topic.

An-2 Encodes a Cytokinin Synthesis Enzyme that Regulates Awn Length and Grain Production in Rice

A wide range of morphological and physiological traits have changed between cultivated rice Oryza sativa and wild rice Oryza rufipogon under domestication. Here, we report cloning of the An-2 gene, encoding the Lonely Guy Like protein 6 （OsLOGL6）, which catalyzes the final step of cytokinin synthesis in O. rufipogon. The near-isogenic line harboring a wild allele of An-2 in the genetic background of the awnless indica Guangluai 4 shows that An-2 promotes awn elongation by enhancing cell division, but decreases grain production by reducing grains per panicle and tillers per plant. We reveal that a genetic variation in the An-2 locus has a large impact on reducing awn length and increasing tiller and grain numbers in domesticated rice. Analysis of gene expression patterns suggests that An-1 reg- ulates the formation of awn primordial, and An-2 promotes awn elongation. Nucleotide diversity of the An-2 locus in cultivated rice was found to be significantly reduced compared with that of wild rice, suggesting that the An-2 locus was subjected to artificial selection. We therefore propose that the selection of genetic variation in An-2 was due to reduced awn length and increased grain yield in cultivated rice.

Development and high-throughput genotyping of substitution lines carting the chromosome segments of indica 9311 in the background of japonica Nipponbare

Chromosome segment substitution lines（CSSLs） are useful for the precise mapping of quantitative trait loci（QTLs） and dissection of the genetic basis of complex traits.In this study,two whole-genome sequenced rice cultivars,the japonica Nipponbare and indica 9311 were used as recipient and donor,respectively.A population with 57 CSSLs was developed after crossing and back-crossing assisted by molecular markers, and genotypes were identified using a high-throughput resequencing strategy.Detailed graphical genotypes of 38 lines were constructed based on resequencing data.These CSSLs had a total of 95 substituted segments derived from indica 9311,with an average of about 2.5 segments per CSSL and eight segments per chromosome,and covered about 87.4%of the rice whole genome.A multiple linear regression QTL analysis mapped four QTLs for 1000-grain weight.The largest-effect QTL was located in a region on chromosome 5 that contained a cloned major QTL GW5/qSW5 for grain size in rice.These CSSLs with a background of Nipponbare may provide powerful tools for future whole-genome