Char Plant:A De Novo Open Chromatin Region Prediction Tool for Plant Genomes

Chromatin accessibility is a highly informative structural feature for understanding gene transcription regulation,because it indicates the degree to which nuclear macromolecules such as proteins and RNAs can access chromosomal DNA.Studies have shown that chromatin accessibility is highly dynamic during stress response,stimulus response,and developmental transition.Moreover,physical access to chromosomal DNA in eukaryotes is highly cell-specific.Therefore,current technologies such as DNase-seq,ATAC-seq,and FAIRE-seq reveal only a portion of the open chromatin regions(OCRs)present in a given species.Thus,the genome-wide distribution of OCRs remains unknown.In this study,we developed a bioinformatics tool called Char Plant for the de novo prediction of OCRs in plant genomes.To develop this tool,we constructed a three-layer convolutional neural network(CNN)and subsequently trained the CNN using DNase-seq and ATACseq datasets of four plant species.The model simultaneously learns the sequence motifs and regulatory logics,which are jointly used to determine DNA accessibility.All of these steps are integrated into Char Plant,which can be run using a simple command line.The results of data analysis using Char Plant in this study demonstrate its prediction power and computational efficiency.To our knowledge,Char Plant is the first de novo prediction tool that can identify potential OCRs in the whole genome.The source code of Char Plant and supporting files are freely available from https://github.com/Yin-Shen/Char Plant.

Recent Advances in Assembly of Complex Plant Genomes

Over the past 20 years,tremendous advances in sequencing technologies and computational algorithms have spurred plant genomic research into a thriving era with hundreds of genomes decoded already,ranging from those of nonvascular plants to those of flowering plants.However,complex plant genome assembly is still challenging and remains difficult to fully resolve with conventional sequencing and assembly methods due to high heterozygosity,highly repetitive sequences,or high ploidy characteristics of complex genomes.Herein,we summarize the challenges of and advances in complex plant genome assembly,including feasible experimental strategies,upgrades to sequencing technology,existing assembly methods,and different phasing algorithms.Moreover,we list actual cases of complex genome projects for readers to refer to and draw upon to solve future problems related to complex genomes.Finally,we expect that the accurate,gapless,telomere-totelomere,and fully phased assembly of complex plant genomes could soon become routine.

The Distribution of Repetitive DNAs Along Chromosomes in Plants Revealed by Self-genomic in situ Hybridization

The distribution of repetitive DNAs along chromosomes is one of the crucial elements for understanding the organization and the evolution of plant genomes. Using a modified genomic in situ hybridization （GISH） procedure, fluorescence in situ hybridization （FISH） with genomic DNA to their own chromosomes （called self-genomic in situ hybridization, self-GISH） was carried out in six selected plant species with different genome size and amount of repetitive DNA. Nonuniform distribution of the fluorescent labeled probe DNA was observed on the chromosomes of all the species that were tested. The signal patterns varied among species and were related to the genome size. The chromosomes of the small Arabidopsis genome were labeled almost only in the pericentromeric regions and the nucleolus organizer regions （NORs）. The signals in the relatively small genomes, rice, sorghum, and Brassica oleracea var. capitata L., were dispersed along the chromosome lengths, with a predominant distribution in the pericentromeric or proximal regions and some heterochromatic arms. All chromosomes of the large genomes, maize and barley, were densely labeled with strongly labeled regions and weakly labeled or unlabeled regions being arranged alternatively throughout the lengths. In addition, enhanced signal bands were shown in all pericentromeres and the NORs in B. oleracea var. capitata, and in all pericentromeric regions and certain intercalary sites in barley. The enhanced signal band pattern in barley was found consistent with the N-banding pattern of this species. The GISH with self-genomic DNA was compared with FISH with Cot-1 DNA in rice, and their signal patterns are found to be basically consistent. Our results showed that the self-GISH signals actually reflected the hybridization of genomic repetitive DNAs to the chromosomes, thus the self-GISH technique would be useful for revealing the distribution of the regions where repetitive DNAs concentrate along chromosomes and some chromatin differentiation associated with repetitive DNAs in plants.

The design of aided software for osmotic stress responding genes mining in plant genome

A software and algorithm which based on random sequence model uses osmotic stress responding cis elements from existing information sources of biology was designed. It can infer the genic downstream function of Arabidopsis thaliana through analyzing its promoter region, and can offer effective aided analysis to mine osmotic stress responding genes in Arabidopsis thatiana genome. The practical application proves that this software can aid to analyze vast genic data and offer important data evidence.

A Widely Applicable Method for Plant Genomic DNA Extraction

[Objective] This study aimed to explore a method for plant genomic DNA extraction, to provide guidance for rapid extraction of genomic DNA from plant tissues. [ Method] Based on the published genomic DNA extraction methods, operation steps, reagent amount and processing time of SDS extraction method were optimized. [Result] A widely applicable method was established initially for genomic DNA extraction from various varieties of plants and various kinds of plant tissues. Quality of the extracted genomic DNA was relatively good, which meets the requirements for further operation. [ Conclusion] This study provided guidance for rapid plant genomic DNA extraction.

New Methods in Genomic Research of Plants

Association mapping(as opposed to population mapping) is becoming more important in establishing associations between a phenotype and a genotype.The major advantage of association mapping,

Genome editing in plants using the TnpB transposase system

The widely used clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease(Cas)system is thought to have evolved from IS200/IS605 transposons.TnpB proteins,encoded by one type of IS200/IS605 transposon,are considered to be the evolutionary ancestors of Cas12 nucleases,which have been engineered to function as RNA-guided DNA endonucleases for genome editing in bacteria and human cells.TnpB nucleases,which are smaller than Cas nucleases,have been engineered for use in genome editing in animal systems,but the feasibility of this approach in plants remained unknown.Here,we obtained stably transformed genome-edited mutants in rice(Oryza sativa)by adapting three recently identified TnpB genome editing vectors,encoding distinct TnpB nucleases(ISAam1,ISDra2,and ISYmu1),for use in plants,demonstrating that the hypercompact TnpB proteins can effectively edit plant genomes.ISDra2 and ISYmu1 precisely edited their target sequences,with no off-target mutations detected,showing that TnpB transposon nucleases are suitable for development into a new genome editing tool for plants.Future modifications improving the genome-editing efficiency of the TnpB system will facilitate plant functional studies and breeding programs.

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.

Exploiting viral vectors to deliver genome editing reagents in plants

Genome editing holds great promise for the molecular breeding of plants,yet its application is hindered by the shortage of simple and effective means of delivering genome editing reagents into plants.Conventional plant transformation-based methods for delivery of genome editing reagents into plants often involve prolonged tissue culture,a labor-intensive and technically challenging process for many elite crop cultivars.In this review,we describe various virus-based methods that have been employed to deliver genome editing reagents,including components of the CRISPR/Cas machinery and donor DNA for precision editing in plants.We update the progress in these methods with recent successful examples of genome editing achieved through virus-based delivery in different plant species,highlight the advantages and limitations of these delivery approaches,and discuss the remaining challenges.

Plant genomic resources at National Genomics Data Center:assisting in data-driven breeding applications

Genomic data serve as an invaluable resource for unraveling the intricacies of the higher plant systems,including the constituent elements within and among species.Through various efforts in genomic data archiving,integrative analysis and value-added curation,the National Genomics Data Center(NGDC),which is a part of the China National Center for Bioinformation(CNCB),has successfully established and currently maintains a vast amount of database resources.This dedicated initiative of the NGDC facilitates a data-rich ecosystem that greatly strengthens and supports genomic research efforts.Here,we present a comprehensive overview of central repositories dedicated to archiving,presenting,and sharing plant omics data,introduce knowledgebases focused on variants or gene-based functional insights,highlight species-specific multiple omics database resources,and briefly review the online application tools.We intend that this review can be used as a guide map for plant researchers wishing to select effective data resources from the NGDC for their specific areas of study.

Over 100 Million Years of Enzyme Evolution Underpinning the Production of Morphine in the Papaveraceae Family of Flowering Plants

Phylogenomic analysis of whole genome sequences of five benzylisoquinoline alkaloid(BIA)-producing species from the Ranunculales and Proteales orders of flowering plants revealed the sequence and timing of evolutionary events leading to the diversification of these compounds.(S)-Reticuline is a pivotal intermediate in the synthesis of many BIAs and our analyses revealed parallel evolution between the two orders,which diverged122 million years ago(MYA).Berberine is present in species across the entire Ranunculales,and we found co-evolution of genes essential for production of the protoberberine class.The benzophenanthridine class,which includes the antimicrobial compound sanguinarine,is specific to the Papaveraceae family of Ranunculales,and biosynthetic genes emerged after the split with the Ranunculaceae family110 MYA but before the split of the three Papaveraceae species used in this study at77 MYA.The phthalideisoquinoline noscapine and morphinan class of BIAs are exclusive to the opium poppy lineage.Ks estimation of paralogous pairs indicates that morphine biosynthesis evolved more recently than 18 MYA in the Papaver genus.In the preceding 100 million years gene duplication,neofunctionalization and recruitment of additional enzyme classes,combined with gene clustering,gene fusion,and gene amplification,resulted in emergence of medicinally valuable BIAs including morphine and noscapine.

Genome editing in plants with MAD7 nuclease

MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.

Current and future editing reagent delivery systems for plant genome editing

Many genome editing tools have been developed and new ones are anticipated; some have been extensively applied in plant genetics, biotechnology and breeding, especially the CRISPR/Cas9 system. These technologies have opened up a new era for crop improvement due to their precise editing of user-specified sequences related to agronomic traits. In this review, we will focus on an update of recent developments in the methodologies of editing reagent delivery, and consider the pros and cons of current delivery systems. Finally, we will reflect on possible future directions.

Unraveling the 3D Genome Architecture in Plants:Present and Future

The eukaryotic genome has a hierarchicalthree-dimensional(3D)organization with functional implications for DNA replication,DNA repair,and transcriptional regulation.Over the past decade,scientists have endeavored to elucidate the spatial characteristics and functions of plant genome architecture using high-throughput chromatin conformation capturing technologies such as Hi-C,ChlA-PET,and HiChIP.Here,we systematically review current understanding of chromatin organization in plants at multiple scales.We also discuss the emerging opinions and concepts in 3D genome research,focusing on state-of-the-art 3D genome techniques,RNA-chromatin interactions,liquid-liquid phase separation,and dynamic chromatin alterations.We propose the application of single-cell/single-molecule multi-omics,multiway(DNA-DNA,DNA-RNA,and RNA-RNA interactions)chromatin conformation capturing methods,and proximity ligation-independent 3D genome-mapping technologies to explore chromatin organization structure and function in plants.Such methods could reveal the spatial interactions between trait-related SNPs and their target genes at various spatiotemporal resolutions,and elucidate the molecular mecha-nisms of the interactions among DNA elements,RNA molecules,and protein factors during the formation of key traits in plants.

Expanding plant genome-editing scope by an engineered iSpyMacCas9 system that targets A-rich PAM sequences

The most popular CRISPR-SpCas9 systemrecognizes canonical NGG protospacer adjacent motifs(PAMs).Previously engineered SpCas9 variants,such as Cas9-NG,favor G-rich PAMs in genome editing.In this manuscript,we describe a new plant genome-editing system based on a hybrid iSpyMacCas9 platform that allows for targeted mutagenesis,C to T base editing,and A to G base editing at A-rich PAMs.This study fills amajor technology gap in the CRISPR-Cas9 system for editing NAAR PAMs in plants,which greatly expands the targeting scope of CRISPR-Cas9.Finally,our vector systems are fully compatible with Gateway cloning and will work with all existing single-guide RNA expression systems,facilitating easy adoption of the systems by others.We anticipate that more tools,such as prime editing,homology-directed repair,CRISPR interference,and CRISPR activation,will be further developed based on our promising iSpyMac-Cas9 platform.

TALENs:Customizable Molecular DNA Scissors for Genome Engineering of Plants

Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases（TALENs）,consisting of an engineered specific（TALE） DNA binding domain and a Fok I cleavage domain,are newly developed versatile reagents for genome engineering in different organisms.Because of the simplicity of the DNA recognition code and their modular assembly,TALENs can act as customizable molecular DNA scissors inducing double-strand breaks（DSBs） at given genomic location.Thus,they provide a valuable approach to targeted genome modifications such as mutations, insertions,replacements or chromosome rearrangements.In this article,we review the development of TALENs,and summarize the principles and tools for TALEN-mediated gene targeting in plant cells,as well as current and potential strategies for use in plant research and crop improvement.

Shortened snRNA promoters for efficient CRISPR/Cas-based multiplex genome editing in monocot plants

Dear Editor,CRISPR(clustered regularly interspaced short palindromic repeats)/Cas genome editing is a powerful tool for introducing specific mutations in organisms including plants.The system is composed of a nuclease such as Cas9 or Cas12a and an engineered single-guide RNA(sgRNA)incorporating a target sequence(Li et al.,2019).A Cas9/sgRNA complex recognizes its target site in the genome,resulting in a mutation at that site.

Targeted Gene Manipulation in Plants Using the CRISPR/Cas Technology

The CRISPR/Cas technology is emerging as a revolutionary genome editing tool in diverse organisms including plants,and has quickly evolved into a suite of versatile tools for sequence-specific gene manipulations beyond genome editing.Here,we review the most recent applications of the CRISPR/Cas toolkit in plants and also discuss key factors for improving CRISPR/Cas performance and strategies for reducing the off-target effects.Novel technical breakthroughs in mammalian research regarding the CRISPR/Cas toolkit will also be incorporated into this review in hope to stimulate prospective users from the plant research community to fully explore the potential of these technologies.

Decoding the plant genome: From epigenome to 3D organization

The linear genome of eukaryotes is partitioned into diverse chromatin states and packaged into a threedimensional(3D)structure,which has functional implications in DNA replication,DNA repair,and transcriptional regulation.Over the past decades,research on plant functional genomics and epigenomics has made great progress,with thousands of genes cloned and molecular mechanisms of diverse biological processes elucidated.Recently,3D genome research has gradually attracted great attention of many plant researchers.Herein,we briefly review the progress in genomic and epigenomic research in plants,with a focus on Arabidopsis and rice,and summarize the currently used technologies and advances in plant 3D genome organization studies.We also discuss the relationships between onedimensional linear genome sequences,epigenomic states,and the 3D chromatin architecture.This review provides basis for future research on plant 3D genomics.

Call for Papers Special Issue on"Plant Genomics"

We are very pleased to announce a special issue, to be published in the fall of 2018, on ＂Plant Genomics＂ in the journal Genomics, Proteomics ＆ Bioinformatics （GPB）.