Improved Brassica rapa reference genome by single-molecule sequencing and chromosome conformation capture technologies

Brassica rapa comprises several important cultivated vegetables and oil crops.Current reference genome assemblies of Brassica rapa are quite fragmented and not highly contiguous,thereby limiting extensive genetic and genomic analyses.Here,we report an improved assembly of the B.rapa genome(v3.0)using single-molecule sequencing,optical mapping,and chromosome conformation capture technologies(Hi-C).Relative to the previous reference genomes,our assembly features a contig N50 size of 1.45 Mb,representing a~30-fold improvement.We also identified a new event that occurred in the B.rapa genome~1.2 million years ago,when a long terminal repeat retrotransposon(LTR-RT)expanded.Further analysis refined the relationship of genome blocks and accurately located the centromeres in the B.rapa genome.The B.rapa genome v3.0 will serve as an important community resource for future genetic and genomic studies in B.rapa.This resource will facilitate breeding efforts in B.rapa,as well as comparative genomic analysis with other Brassica species.

Author Correction:Improved Brassica rapa reference genome by single-molecule sequencing and chromosome conformation capture technologies

Since the publication of this article,the authors have noticed that the total gene models(45,985),tandem arrays(2077),tandem genes(4963),redundancy removed(43,099),syntenic genes(39,858),nonsyntenic genes(3241),genes on chromosomes(45,411),genes on scaffolds(574)of B.rapa reference genome v3.0 were mistaken in the article.

A chromosome-level reference genome of nonheading Chinese cabbage[Brassica campestris(syn.Brassica rapa)ssp.chinensis]

Non-heading Chinese cabbage(NHCC)is an important leafy vegetable cultivated worldwide.Here,we report the first high-quality,chromosome-level genome of NHCC001 based on PacBio,Hi-C,and Illumina sequencing data.The assembled NHCC001 genome is 405.33 Mb in size with a contig N50 of 2.83 Mb and a scaffold N50 of 38.13 Mb.Approximately 53%of the assembled genome is composed of repetitive sequences,among which long terminal repeats(LTRs,20.42%of the genome)are the most abundant.Using Hi-C data,97.9%(396.83 Mb)of the sequences were assigned to 10 pseudochromosomes.Genome assessment showed that this B.rapa NHCC001 genome assembly is of better quality than other currently available B.rapa assemblies and that it contains 48,158 protein-coding genes,99.56%of which are annotated in at least one functional database.Comparative genomic analysis confirmed that B.rapa NHCC001 underwent a whole-genome triplication(WGT)event shared with other Brassica species that occurred after the WGD events shared with Arabidopsis.Genes related to ascorbic acid metabolism showed little variation among the three B.rapa subspecies.The numbers of genes involved in glucosinolate biosynthesis and catabolism were higher in NHCC001 than in Chiifu and Z1,due primarily to tandem duplication.The newly assembled genome will provide an important resource for research on B.rapa,especially B.rapa ssp.chinensis.

Combined genomic, transcriptomic, and metabolomic an alyses provide in sights into chayote (Sechium edule) evolution and fruit development

Chayote(Sechium edule)is an agricultural crop in the Cucurbitaceae family that is rich in bioactive components.To enhance genetic research on chayote,we used Nanopore third-generation sequencing combined with Hi-C data to assemble a draft chayote genome.A chromosome-level assembly anchored on 14 chromosomes(N50 contig and scaffold sizes of 8.40 and 46.56 Mb,respectively)estimated the genome size as 606.42 Mb,which is large for the Cucurbitaceae,with 65.94%(401.08 Mb)ofthe genome comprising repetitive sequences;28,237 protein-coding genes were predicted.Comparative genome analysis indicated that chayote and snake gourd diverged from sponge gourd and that a whole-genome duplication(WGD)event occurred in chayote at 25±4 Mya.Transcriptional and metabolic analysis revealed genes involved in fruit texture,pigment,fl avor,fl avonoids,antioxidants,and plant hormones during chayote fruit development.The analysis of the genome,transcriptome,and metabolome provides insights into chayote evolution and lays the groundwork for future research on fruit and tuber development and genetic improvements in chayote.

Zanthoxylum-specific whole genome duplication and recent activity of transposable elements in the highly repetitive paleotetraploid Z.bungeanum genome

Zanthoxylum bungeanum is an important spice and medicinal plant that is unique for its accumulation of abundant secondary metabolites,which create a characteristic aroma and tingling sensation in the mouth.Owing to the high proportion of repetitive sequences,high heterozygosity,and increased chromosome number of Z.bungeanum,the assembly of its chromosomal pseudomolecules is extremely challenging.Here,we present a genome sequence for Z.bungeanum,with a dramatically expanded size of 4.23 Gb,assembled into 68 chromosomes.This genome is approximately tenfold larger than that of its close relative Citrus sinensis.After the divergence of Zanthoxylum and Citrus,the lineage-specific whole-genome duplication event q-WGD approximately 26.8 million years ago(MYA)and the recent transposable element(TE)burst~6.41 MYA account for the substantial genome expansion in Z.bungeanum.The independent Zanthoxylum-specific WGD event was followed by numerous fusion/fission events that shaped the genomic architecture.Integrative genomic and transcriptomic analyses suggested that prominent speciesspecific gene family expansions and changes in gene expression have shaped the biosynthesis of sanshools,terpenoids,and anthocyanins,which contribute to the special flavor and appearance of Z.bungeanum.In summary,the reference genome provides a valuable model for studying the impact of WGDs with recent TE activity on gene gain and loss and genome reconstruction and provides resources to accelerate Zanthoxylum improvement.

De novo assembly of a new Olea europaea genome accession using nano pore seque ncing

Olive(Olea europaea L.)is internationally renowned for its high-end product,extra virgin olive oil.An incomplete genome of O.europaea was previously obtained using shotgun sequencing in 2016.To further explore the genetic and breeding utilization of olive,an updated draft genome of olive was obtained using Oxford Nanopore third-generation sequencing and Hi-C technology.Seven different assembly strategies were used to assemble the fi nal genome of 1.30 Gb,with contig and scaffold N50 sizes of4.67 Mb and 42.60 Mb,respectively.This greatly increased the quality of the olive genome.We assembled 1.1 Gb of sequences of the total olive genome to 23 pseudochromosomes by Hi-C,and 53,518 protein-coding genes were predicted in the current assembly.Comparative genomics analyses,including gene family expansion and contraction,whole-genome replication,phylogenetic analysis,and positive selection,were performed.Based on the obtained high-quality olive genome,a total of nine gene families with 202 genes were identi fi ed in the oleuropein biosynthesis pathway,which is twice the number ofgenes identi fi ed from the previous data.This new accession of the olive genome is of suf fi cient quality for genome-wide studies on gene function in olive and has provided a foundation for the molecular breeding of olive species.

The genome of Chinese flowering cherry(Cerasus serrulata)provides new insights into Cerasus species

Cerasus serrulata is a flowering cherry germplasm resource for ornamental purposes.In this work,we present a de novo chromosome-scale genome assembly of C.serrulata by the use of Nanopore and Hi-C sequencing technologies.The assembled C.serrulata genome is 265.40 Mb across 304 contigs and 67 scaffolds,with a contig N50 of 1.56 Mb and a scaffold N50 of 31.12 Mb.It contains 29,094 coding genes,27,611(94.90%)of which are annotated in at least one functional database.Synteny analysis indicated that C.serrulata and C.avium have 333 syntenic blocks composed of 14,072 genes.Blocks on chromosome 01 of C.serrulata are distributed on all chromosomes of C.avium,implying that chromosome 01 is the most ancient or active of the chromosomes.The comparative genomic analysis confirmed that C.serrulata has 740 expanded gene families,1031 contracted gene families,and 228 rapidly evolving gene families.By the use of 656 single-copy orthologs,a phylogenetic tree composed of 10 species was constructed.The present C.serrulata species diverged from Prunus yedoensis~17.34 million years ago(Mya),while the divergence of C.serrulata and C.avium was estimated to have occurred∼21.44 Mya.In addition,a total of 148 MADS-box family gene members were identified in C.serrulata,accompanying the loss of the AGL32 subfamily and the expansion of the SVP subfamily.The MYB and WRKY gene families comprising 372 and 66 genes could be divided into seven and eight subfamilies in C.serrulata,respectively,based on clustering analysis.Nine hundred forty-one plant disease-resistance genes(R-genes)were detected by searching C.serrulata within the PRGdb.This research provides high-quality genomic information about C.serrulata as well as insights into the evolutionary history of Cerasus species.

VlbZIP30 of grapevine functions in dehydration tolerance via the abscisic acid core signaling pathway

Drought stress limits the growth and development of grapevines,thereby reducing productivity,but the mechanisms by which grapevines respond to drought stress remain largely uncharacterized.Here,we characterized a group A bZIP gene from“Kyoho”grapevine,VlbZIP30,which was shown to be induced by abscisic acid(ABA)and dehydration stress.Overexpression of VlbZIP30 in transgenic Arabidopsis thaliana enhanced dehydration tolerance.Transcriptome analysis revealed that a major proportion of ABA-responsive and/or drought-responsive genes are transcriptionally regulated by VlbZIP30 during ABA or mannitol treatment at the cotyledon greening stage.We identified an A.thaliana G-box motif(CACGTG)and a potential grapevine G-box motif(MCACGTGK)in the promoters of the 39 selected A.thaliana genes upregulated in the transgenic plants and in the 35 grapevine homologs,respectively.Subsequently,using two grapevine-related databases,we found that 74%(23/31)and 84%(21/25)of the detected grapevine genes were significantly upregulated by ABA and drought stress,respectively,suggesting that these genes are involved in ABA or dehydration stress and may be regulated by VlbZIP30 in grapevine.We propose that VlbZIP30 functions as a positive regulator of dehydration-responsive signaling in the ABA core signaling pathway.

The complexity of the Fragaria x ananassa(octoploid)transcriptome by singlemolecule long-read sequencing

Strawberry(Fragaria x ananassa)is an allopolyploid species with diverse and complex transcripts.The regulatory mechanisms of fruit development and maturation have been extensively studied;however,little is known about the signaling mechanisms that direct this process in octoploid strawberry(Fragaria x ananassa).Here,we used long-read sequencing(LRS)technology and RNA-seq analysis to investigate the diversity and complexity of the polyploid transcriptome and differentially expressed transcripts along four successive fruit developmental stages of cultivated strawberry.We obtained a reference transcriptome with 119,897 unique full-length isoforms,including 2017 new isoforms and 2510 long noncoding RNAs.Based on the genome of the plausible progenitor(Fragaria vesca),20,229 alternative splicing(AS)events were identified.Using this transcriptome,we found 17,485 differentially expressed transcripts during strawberry fruit development,including 527 transcription factors(TFs)belonging to 41 families.The expression profiles of all members of the auxin,ABA pathway,and anthocyanin biosynthesis gene families were also examined,and many of them were highly expressed at the ripe fruit stage,strongly indicating that the role of those genes is in the regulation of fruit ripening.We produce a high-quality reference transcriptome for octoploid strawberry,including much of the full-length transcript diversity,to help understand the regulatory mechanisms of fruit development and maturation of polyploid species,particularly via elucidation of the biochemical pathways involved in auxin,ABA,and anthocyanin biosynthesis.

Author Correction:The complexity of the Fragaria x ananassa(octoploid)transcriptome by single-molecule long-read sequencing

Since the publication of this article,the authors have noticed that the part of acknowledgement is missing from article.Here is the acknowledgement.

The chromosome-level Stevia genome provides insights into steviol glycoside biosynthesis

Stevia(Stevia rebaudiana Bertoni)is well known for its very sweet steviol glycosides(SGs)consisting of a common tetracyclic diterpenoid steviol backbone and a variable glycone.Steviol glycosides are 150–300 times sweeter than sucrose and are used as natural zero-calorie sweeteners.However,the most promising compounds are biosynthesized in small amounts.Based on Illumina,PacBio,and Hi-C sequencing,we constructed a chromosome-level assembly of Stevia covering 1416 Mb with a contig N50 value of 616.85 kb and a scaffold N50 value of 106.55 Mb.More than four-fifths of the Stevia genome consisted of repetitive elements.We annotated 44,143 high-confidence protein-coding genes in the high-quality genome.Genome evolution analysis suggested that Stevia and sunflower diverged~29.4 million years ago(Mya),shortly after the whole-genome duplication(WGD)event(WGD-2,~32.1 Mya)that occurred in their common ancestor.Comparative genomic analysis revealed that the expanded genes in Stevia were mainly enriched for biosynthesis of specialized metabolites,especially biosynthesis of terpenoid backbones,and for further oxidation and glycosylation of these compounds.We further identified all candidate genes involved in SG biosynthesis.Collectively,our current findings on the Stevia reference genome will be very helpful for dissecting the evolutionary history of Stevia and for discovering novel genes contributing to SG biosynthesis and other important agronomic traits in future breeding programs.

The genome and transcriptome analysis of snake gourd provide insights into its evolution and fruit development and ripening

Snake gourd(Trichosanthes anguina L.),which belongs to the Cucurbitaceae family,is a popular ornamental and food crop species with medicinal value and is grown in many parts of the world.Although progress has been made in its genetic improvement,the organization,composition,and evolution of the snake gourd genome remain largely unknown.Here,we report a high-quality genome assembly for snake gourd,comprising 202 contigs,with a total size of 919.8 Mb and an N50 size of 20.1 Mb.These findings indicate that snake gourd has one of the largest genomes of Cucurbitaceae species sequenced to date.The snake gourd genome assembly harbors 22,874 protein-coding genes and 80.0%of the genome consists of repetitive sequences.Phylogenetic analysis reveals that snake gourd is closely related to sponge gourd but diverged from their common ancestor~33–47 million years ago.The genome sequence reported here serves as a valuable resource for snake gourd genetic research and comparative genomic studies in Cucurbitaceae and other plant species.In addition,fruit transcriptome analysis reveals the candidate genes related to quality traits during snake gourd fruit development and provides a basis for future research on snake gourd fruit development and ripening at the transcript level.

Genomes reveal selective sweeps in kiang and donkey for high-altitude adaptation

Over the last several hundred years,donkeys have adapted to high-altitude conditions on the Tibetan Plateau.Interestingly,the kiang,a closely related equid species,also inhabits this region.Previous reports have demonstrated the importance of specific genes and adaptive introgression in divergent lineages for adaptation to hypoxic conditions on the Tibetan Plateau.Here,we assessed whether donkeys and kiangs adapted to the Tibetan Plateau via the same or different biological pathways and whether adaptive introgression has occurred.We assembled a de novo genome from a kiang individual and analyzed the genomes of five kiangs and 93 donkeys(including 24 from the Tibetan Plateau).Our analyses suggested the existence of a strong hard selective sweep at the EPAS1 locus in kiangs.In Tibetan donkeys,however,another gene,i.e.,EGLN1,was likely involved in their adaptation to high altitude.In addition,admixture analysis found no evidence for interspecific gene flow between kiangs and Tibetan donkeys.Our findings indicate that despite the short evolutionary time scale since the arrival of donkeys on the Tibetan Plateau,as well as the existence of a closely related species already adapted to hypoxia,Tibetan donkeys did not acquire adaptation via admixture but instead evolved adaptations via a different biological pathway.

Population Genomic Analysis and De Novo Assembly Reveal the Origin of Weedy Rice as an Evolutionary Game

Crop weediness,especially that of weedy rice(Oryza sativa f.spontanea),remains mysterious.Weedy rice possesses robust ecological adaptability;however,how this strain originated and gradually formed proprietary genetic features remains unclear?Here,we demonstrate that weedy rice at Asian high latitudes(WRAH)is phylogenetically well defined and possesses unselected genomic characteristics in many divergence regions between weedy and cultivated rice.We also identified novel quantitative trait loci underlying weedy-specific traits,and revealed that a genome block on the end of chromosome 1 is associated with rice weediness.To identify the genomic modifications underlying weedy rice evolution,we generated the first de novo assembly of a high-quality weedy rice genome(WR04-6),and conducted a comparative genomics study between WR04-6 with other rice reference genomes.Multiple lines of evidence,including the results of demographic scenario comparisons,suggest that differentiation between weedy rice and cultivated rice was initiated by genetic improvement of cultivated rice and that the essence of weediness arose through semi-domestication.A plant height model further implied that the origin of WRAH can be modeled as an evolutionary game and indicated that strategy-based selection driven by fitness shaped its genomic diversity.

A Genomic Variation Map Provides Insights into the Genetic Basis of Spring Chinese Cabbage (Brassica rapa ssp.pekinensis)Selection

Chinese cabbage is the most consumed leafy crop in East Asian countries.However,premature bolting induced by continuous low temperatures severely decreases the yield and quality of the Chinese cabbage, and therefore restricts its planting season and geographic distribution.In the past 40years,spring Chinese cabbage with strong winterness has been selected to meet the market demand.Here,we report a genome variation map of Chinese cabbage generated from the resequencing data of 194 geographically diverse accessions of three ecotypes.In-depth analyses of the selection sweeps and genome-wide patterns revealed that spring Chinese cabbage was selected from a specific population of autumn Chinese cabbage around the area of Shandong peninsula in northern China.We identified 23 genomic loci that underwent intensive selection,and further demonstrated by gene expression and haplotype analyses that the incorporation of elite alleles of VERNALISATION INSENTIVE 3.1(BrVIN3.1)and FLOWER LOCUS C 1(BrFLC1)is a determinant genetic source of variation during selection.Moreover,we showed that the quantitative response of BrVIN3.1 to cold due to the sequence variations in the cis elements of the BrVlN3.1 promoter significantly contributes to bolting-time variation in Chinese cabbage.Collectively, our study provides valuable insights into the genetic basis of spring Chinese cabbage selection and will facilitate the breeding of bolting-resistant Varieties by molecular-marker-assisted selection,transgenic or gene editingapproaches.

Whole-Genome Resequencing of a Worldwide Collection of Rapeseed Accessions Reveals the Genetic Basis of Ecotype Divergence

Rapeseed (Brassica napus),an important oilseed crop,has adapted to diverse climate zones and latitudes by forming three main ecotype groups,namely winter,semiwinter,and spring types. However,genetic variations underlying the divergence of these ecotypes are largely unknown. Here,we report the global pattern of genetic polymorphisms in rapeseed determined by resequencing a worldwide collection of 991 germplasm accessions.A total of 5.56 and 5.53 million singlenucleotide polymorphisms (SNPs)as Well as 1.86 and 1.92 million InDels were identified by mapping reads to the reference genomes of "Darmor-bzh"and "Tapidor,"respectively.We generated a map of allelic drift paths that shows splits and mixtures of the main populations,and revealed an asymmetric evolution of the two subgenomes of B.napus by calculating the genetic diversity and linkage disequilibrium parameters.Selective-sweep analysis revealed genetic changes in genes orthologous to those regulating various aspects of plant development and response to stresses.A genome-wide association study identified SNPs in the promoter regions of FLOWERING LOCUS T and FLOWERING LOCUS C orthologs that corresponded to the different rapeseed ecotype groups. Our study provides important insights into the genomic footprints of rapeseed evolution and flowering-time divergence among three ecotype groups,and will facilitate screening of molecular markers for accelerating rapeseed breeding.

The Coix Genome Provides Insights into Panicoideae Evolution and Papery Hull Domestication

Coix is a grass crop domesticated as early as the Neolithic era.It is still widely cultivated for both highly nutritional food and medicinal use.However,the genetic study and breeding of this crop are hindered by the lack of a sequenced genome.Here,we report de novo sequencing and assembly of the 1619-Mb genome of Coix,and annotation of 75.39%repeats and 39629 protein-coding genes.Comparative genomics analysis showed that Coix is more closely related to sorghum than maize,but intriguingly only Coix and maize had a recent genome duplication event,which was not detected in sorghum.We further constructed a genetic map and mapped several important traits,especially the strength of hull.Selection of papery hull(thin:easy dehulling)from the stony hull(thick:difficult dehulling)in wild progenitors was a key step in Coix domestication.The papery hull makes seed easier to process and germinate.Anatomic and global transcriptome analysis revealed that the papery hull is a result of inhibition of cell division and wall biogenesis.We also successfully demonstrated that seed hull pressure resistance is controlled by two major quantitative trait loci(QTLs),which are associated with hull thickness and color,respectively.The two QTLs were further fine mapped within intervals of 250 kb and 146 kb,respectively.These resources provide a platform for evolutionary studies and will facilitate molecular breeding of this important crop.

Genomic insights into the fast growth of paulownias and the formation of Paulownia witches' broom

Paulownias are among the fastest growing trees in the world,but they often suffer tremendous loss of wood production due to infection by Paulownia witches'broom(PaWB)phytoplasmas.In this study,we have sequenced and assembled a high-quality nuclear genome of Paulownia fortunei,a commonly cultivated paulownia species.The assembled genome of P.fortunei is 511.6 Mb in size,with 93.2%of its sequences anchored to 20 pseudo-chromosomes,and it contains 31985 protein-coding genes.Phylogenomic analyses show that the family Paulowniaceae is sister to a clade composed of Phrymaceae and Orobanchaceae.Higher photosynthetic efficiency is achieved by integrating C3 photosynthesis and the crassulacean acid metabolism pathway,which may contribute to the extremely fast growth habit of paulownia trees.Comparative transcriptome analyses reveal modules related to cambial growth and development,photosynthesis,and defense responses.Additional genome sequencing of PaWB phytoplasma,combined with functional analyses,indicates that the effector PaWB-SAP54 interacts directly with Paulownia PfSPLa,which in turn causes the degradation of PfSPLa by the ubiquitin-mediated pathway and leads to the formation of witches'broom.Taken together,these results provide significant insights into the biology of paulownias and the regulatory mechanism for the formation of PaWB.

Allele-defined genome reveals biallelic differentiation during cassava evolution

Dear Editor,Generation of heterozygous genomes by hybridization between or within species can help maintain plant diversity and serve as a potential source of new species(Baek et al.,2018).Moreover,genomic heterozygosity is associated with genomic coadaptation,developmental stability,and heterosis.Accurate definition of alleles in haplotypes is necessary to precisely characterize allelic variation controlling agriculturally important traits(Shi et al.,2019).Currently,most released genomes have mosaic assembly of haplotypes due to random selection or collapse of alleles during genome assembly(Shi et al.,2019),which masked allelic variation and functional differentiation of divergent alleles in heterozygous species.