Transcriptome analysis to identify candidate genes related to chlorogenic acid biosynthesis during development of Korla fragrant pear in Xinjiang

Korla fragrant pear(KFP)with special fragrance is a unique cultivar in Xinjiang,China.In order to explore the biosynthesis molecular mechanism of chlorogenic acid(CGA)in KFP,the samples at different development periods were collected for transcriptome analysis.High performance liquid chromatography analysis showed that CGA contents of KFP at 88,118 and 163 days after full bloom were(20.96±1.84),(12.01±0.91)and(7.16±0.41)mg/100 g,respectively,and decreased with the fruit development.Pears from these typical 3 periods were selected for de novo transcriptome assemble and 68059 unigenes were assembled from 444037960 clean reads.One‘phenylpropanoid biosynthesis’pathway including 57 unigenes,11 PALs,1 PTAL,64CLs,9 C4Hs,25 HCTs and 5 C3’Hs related to CGA biosynthesis was determined.It was found that the expression levels of 11 differentially expressed genes including 1 PAL,2 C4Hs,34CLs and 5 HCTs were consistent with the change of CGA content.Quantitative polymerase chain reaction analysis further showed that 8 unigenes involved in CGA biosynthesis were consistent with the RNA-seq data.These findings will provide a comprehensive understanding and valuable information on the genetic engineering and molecular breeding in KFP.

Transcriptome profile of Dunaliella salina in Yuncheng Salt Lake reveals salt-stress-related genes under diff erent salinity stresses

Salt stress is an abiotic stress to plants in especially saline lakes.Dunaliella,a halophilic microalga distributed throughout salt lakes and seas,can respond to different salinity stresses by regulating the expression of some genes.However,these genes and their function and biological processes involved remain unclear.Profi ling these salt-stress-related genes in a high-salt-tolerant Dunaliella species will help clarify the salt tolerance machinery of Dunaliella.Three D.salina_YC salt-stress groups were tested under low(0.51 mol/L),moderate(1.03 mol/L),and high(3.42 mol/L)NaCl concentrations and one control group under very low(0.05 mol/L)NaCl concentration and 3 transcriptome results that were deep sequenced and de novo assembled were obtained per group.Twelve high-quality RNA-seq libraries with 46585 upregulated and 47805 downregulated unigenes were found.Relative to the control,188 common differentially expressed genes(DEGs)were screened and divided into four clusters in expression pattern.Fifteen of them annotated in the significant enriched Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were validated via qPCR.Their qPCR-based relative expression patterns were similar to their RNA-seq-based patterns.Two significant DEGs,the geranylgeranyl diphosphate synthase coding gene(1876-bp cDNA)and diacylglycerol O-acyltransferase coding gene(2968-bp cDNA),were cloned and analyzed in silico.The total lipid content,superoxide dismutase specific activity,and betacarotene content of D.salina_YC increased gradually with increasing salinity.In addition,the expression of 11 validated genes involved in fatty acid biosynthesis/degradation,active oxygen or carotenoid metabolisms showed significant changes.In addition,algal photochemical efficiency was diminished with increasing salinity,as well as the expression of 4 photosynthesis-related genes.These results could help clarify the molecular mechanisms underlying D.salina responses to the Yuncheng Salt Lake environment and lay a foundation for further utilization of this algal resource.

De novo assembly of white poplar genome and genetic diversity of white poplar population in Irtysh River basin in China

The white poplar(Populus alba) is widely distributed in Central Asia and Europe. There are natural populations of white poplar in Irtysh River basin in China. It also can be cultivated and grown well in northern China. In this study, we sequenced the genome of P. alba by single-molecule real-time technology. De novo assembly of P. alba had a genome size of 415.99 Mb with a contig N50 of 1.18 Mb. A total of 32,963 protein-coding genes were identified. 45.16% of the genome was annotated as repetitive elements. Genome evolution analysis revealed that divergence between P. alba and Populus trichocarpa(black cottonwood)occurred ～5.0 Mya(3.0, 7.1). Fourfold synonymous third-codon transversion(4 DTV) and synonymous substitution rate(ks)distributions supported the occurrence of the salicoid WGD event(～ 65 Mya). Twelve natural populations of P. alba in the Irtysh River basin in China were sequenced to explore the genetic diversity. Average pooled heterozygosity value of P. alba populations was 0.170±0.014, which was lower than that in Italy(0.271±0.051) and Hungary(0.264±0.054). Tajima's D values showed a negative distribution, which might signify an excess of low frequency polymorphisms and a bottleneck with later expansion of P.alba populations examined.

Oryza pan-genomics: A new foundation for future rice research and improvement

The wild rice species in the genus Oryza harbor a large amount of genetic diversity that has been untapped for rice improvement.Pan-genomics has revolutionized genomic research in plants.However,rice pan-genomic studies so far have been limited mostly to cultivated accessions,with only a few close wild relatives.Advances in sequencing technologies have permitted the assembly of highquality rice genome sequences at low cost,making it possible to construct genus-level pan-genomes across all species.In this review,we summarize progress in current research on genetic and genomic resources in Oryza,and in sequencing and computational technologies used for rice genome and pangenome construction.For future work,we discuss the approaches and challenges in the construction of,and data access to,Oryza pan-genomes based on representative high-quality genome assemblies.The Oryza pan-genomes will provide a basis for the exploration and use of the extensive genetic diversity present in both cultivated and wild rice populations.

Transcriptome analysis reveals dynamic changes in the salt stress response in Salix

Soil salinization is a serious ecological problem worldwide and information regarding the salt tolerance mechanisms of Salix is scarce.To elucidate the dynamic changes in the molecular mechanisms of Salix under salt stress,we generated gene expression profiles and examined changes in the expression of those genes.RNA-Seq was used to produce six cDNA libraries constructed from the leaves of Salix ×jiangsuensis CL‘J2345’treated with NaCl for 0,2,6,12,24 and 48 h.In total,249 million clean reads were assembled into 12,739 unigenes,all of which were clustered into 10 profiles based on their temporal expression patterns.KEGG analysis revealed that as an early defense response,the biosynthesis pathways of cutin,suberin and wax,which are involved in cell wall structure,were activated beginning at 2 h.The expression of secondary metabolism genes,including those involved in the phenylpropanoid,flavonoid,stilbenoid,diarylheptanoid and gingerol pathways,peaked at 6 h and 24 h;the upregulated genes were mainly involved in plant hormone pathways and beta-alanine,galactose and betalain metabolism.We identified roles of key phytohormones and found ETH to be the major signaling molecule activating TFs at 12 h;ETH,ABA,IAA and SA were the key molecules at 24 h.Moreover,we found that the upregulated genes were associated with elevated levels of amino acids,sucrose,inositol,stress proteins and ROS-scavenging enzymes,contributing to the maintenance of water balance.This research constitutes the first detailed analysis of salt stress-related mechanisms in Salix and identifies potential targets for genetic manipulation to improve yields.

A survey on de novo assembly methods for single-molecular sequencing

Background:The single-molecular sequencing(SMS)is under rapid development and generating increasingly long and accurate sequences.De novo assembly of genomes from SMS sequences is a critical step for many genomic studies.To scale well with the developing trends of SMS,many de novo assemblers for SMS have been released.These assembly workflows can be categorized into two different kinds:the correction-and-assembly strategy and the assembly-and-correction strategy,both of which are gaining more and more attentions.Results:In this article we make a discussion on the characteristics of errors in SMS sequences・We then review the currently widely applied de novo assemblers for SMS sequences.We also describe computational methods relevant to de novo assembly,including the alignment methods and the error correction methods.Benchmarks are provided to analyze their performance on different datasets and to provide use guides on applying the computation methods.Conclusion:We make a detailed review on the latest development of de novo assembly and some relevant algorithms for SMS,including their rationales,solutions and results.Besides,we provide use guides on the algorithms based on their benchmark results.Finally we conclude the review by giving some developing trends of third generation sequencing(TGS).

De novo assembly of transcriptome from next-generation sequencing data

Reconstruction of transcriptome by de novo assembly from next generation sequencing （NGS） short-sequence reads provides an essential mean to catalog expressed genes, identify splicing isoforms, and capture the expression detail of transcripts for organisms with no reference genome available. De novo transcriptome assembly faces many unique challenges, including alternative splicing, variable expression level covering a dynamic range of several orders of magnitude, artifacts introduced by reverse transcription, etc. In the current review, we illustrate the grand strategy in applying De Bruijn Graph （DBG） approach in de novo transcriptome assembly. We further analyze many parameters proven critical in transcriptome assembly using DBG. Among them, k-met length, coverage depth of reads, genome complexity, performance of different programs are addressed in greater details. A multi-k-mer strategy balancing efficiency and sensitivity is discussed and highly recommended for de novo transcriptome assembly. Future direction points to the combination of NGS and third generation sequencing technology that would greatly enhance the power of de novo transcriptomics study.

Draft Genome of White-blotched River Stingray Provides Novel Clues for Niche Adaptation and Skeleton Formation

The white-blotched river stingray(Potamotrygon leopoldi)is a cartilaginous fish native to the Xingu River,a tributary of the Amazon River system.As a rare freshwater-dwelling cartilaginous fish in the Potamotrygonidae family in which no member has the genome sequencing information available,P.leopoldi provides the evolutionary details in fish phylogeny,niche adaptation,and skeleton formation.In this study,we present its draft genome of 4.11 Gb comprising 16,227 contigs and 13,238 scaffolds,with contig N50 of 3937 kb and scaffold N50 of 5675 kb in size.Our analysis shows that P.leopoldi is a slow-evolving fish that diverged from elephant sharks about 96 million years ago.Moreover,two gene families related to the immune system(immunoglobulin heavy constant delta genes and T-cell receptor alpha/delta variable genes)exhibit expansion in P.leopoldi only.We also identified the Hox gene clusters in P.leopoldi and discovered that seven Hox genes shared by five representative fish species are missing in P.leopoldi.The RNA sequencing data from P.leopoldi and other three fish species demonstrate that fishes have a more diversified tissue expression spectrum when compared to mammals.Our functional studies suggest that lack of the gc gene encoding vitamin D-binding protein in cartilaginous fishes(both P.leopoldi and Callorhinchus milii)could partly explain the absence of hard bone in their endoskeleton.Overall,this genome resource provides new insights into the niche adaptation,body plan,and skeleton formation of P.leopoldi,as well as the genome evolution in cartilaginous fishes.

The Tartary Buckwheat Genome Provides Insights into Rutin Biosynthesis and Abiotic Stress Tolerance

Tartary buckwheat （Fagopyrum tataricum） is an important pseudocereal crop that is strongly adapted to growth in adverse environments. Its gluten-free grain contains complete proteins with a well-balanced composition of essential amino acids and is a rich source of beneficial phytochemicals that provide significant health benefits. Here, we report a high-quality, chromosome-scale Tartary buckwheat genome sequence of- 489.3 Mb that is assembled by combining whole-genome shotgun sequencing of both Illumina short reads and single-molecule real-time long reads, sequence tags of a large DNA insert fosmid library, Hi-C sequencing data, and BioNano genome maps. We annotated 33 366 high-confidence protein-coding genes based on expression evidence. Comparisons of the intra-genome with the sugar beet genome revealed an independent whole-genome duplication that occurred in the buckwheat lineage after they diverged from the common ancestor, which was not shared with rosids or asterids. The reference genome facilitated the identification of many new genes predicted to be involved in rutin biosynthesis and regulation, aluminum stress resistance, and in drought and cold stress responses. Our data suggest that Tartary buckwheat＇s ability to tolerate high levels of abiotic stress is attributed to the expansion of several gene families involved in signal transduction, gene regulation, and membrane transport. The availability of these genomic resources will facilitate the discovery of agronomically and nutritionally important genes and genetic improvement of Tartary buckwheat.

Chromosome-Level Reference Genome and Population Genomic Analysis Provide Insights into the Evolution and Improvement of Domesticated Mulberry (Morns alba)

Mulberry(Morns spp.)is the sole plant consumed by the domesticated silkworm.However,the genome of domesticated mulberry has not yet been sequenced,and the ploidy level of this species remains unclear.Here,we report a high-quality,chromosome-level domesticated mulberry(Morus alba)genome.Analysis of genomic data and karyotype analyses confirmed that M.alba is a diploid with 28 chromosomes(2/7=2x=28).Population genomic analysis based on resequencing of 134 mulberry accessions classified domesticated mulberry into three geographical groups,namely,Taihu Basin of southeastern China(Hu mulberry),northern and southwestern China,and Japan.Hu mulberry had the lowest nucleotide diversity among these accessions and demonstrated obvious signatures of selection associated with environmental adaptation.Further phylogenetic analysis supports a previous proposal that multiple domesticated mulberry accessions previously classified as different species actually belong to one species.This study expands our understanding of genome evolution of the genus Morus and population structure of domesticated mulberry,which would facilitate mulberry breeding and improvement.

Population transcriptomics reveals a potentially positive role of expression diversity in adaptation

While it is widely accepted that genetic diversity determines the potential of adaptation,the role that gene expression variation plays in adaptation remains poorly known.Here we show that gene expression diversity could have played a positive role in the adaptation of Miscanthus lutarioriparius.RNA-seq was conducted for 80 individuals of the species,with half planted in the energy crop domestication site and the other half planted in the control site near native habitats.A leaf reference transcriptome consisting of 18,503 high-quality transcripts was obtained using a pipeline developed for de novo assembling with population RNA-seq data.The population structure and genetic diversity of M.lutarioriparius were estimated based on 30,609 genic single nucleotide polymorphisms.Population expression（Ep） and expression diversity（Ed）were defined to measure the average level and the magnitude of variation of a gene expression in the population,respectively.It was found that expression diversity increased while genetic Resediversity decreased after the species was transplanted from the native habitats to the harsh domestication site,especially for genes involved in abiotic stress resistance,histone methylation,and biomass synthesis under water limitation.The increased expression diversity could have enriched phenotypic variation directly subject to selections in the new environment.

PacBio Sequencing and Its Applications

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing （SGS） technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation （SV） in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with dis- eases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Addition- ally, PacBio＇s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone.

Oxford Nanopore MinION Sequencing and Genome Assembly

The revolution of genome sequencing is continuing after the successful secondgeneration sequencing （SGS） technology. The third-generation sequencing （TGS） technology, led by Pacific Biosciences （PacBio）, is progressing rapidly, moving from a technology once only capable of providing data for small genome analysis, or for performing targeted screening, to one that promises high quality de novo assembly and structural variation detection for human-sized genomes. In 2014, the MinION, the first commercial sequencer using nanopore technology, was released by Oxford Nanopore Technologies （ONT）. MiniON identifies DNA bases by measuring the changes in electrical conductivity generated as DNA strands pass through a biological pore. Its portability, affordability, and speed in data production makes it suitable for real-time applications, the release of the long read sequencer MiniON has thus generated much excitement and interest in the genomics community. While de novo genome assemblies can be cheaply produced from SGS data, assem- bly continuity is often relatively poor, due to the limited ability of short reads to handle long repeats. Assembly quality can be greatly improved by using TGS long reads, since repetitive regions can be easily expanded into using longer sequencing lengths, despite having higher error rates at the base level. The potential of nanopore sequencing has been demonstrated by various studies in genome surveillance at locations where rapid and reliable sequencing is needed, but where resources are limited.

Review of General Algorithmic Features for Genome Assemblers for Next Generation Sequencers

In the realm of bioinformatics and computational biology, the most rudimentary data upon which all the analysis is built is the sequence data of genes, proteins and RNA. The sequence data of the entire genome is the solution to the genome assembly problem. The scope of this contribution is to provide an overview on the art of problem-solving applied within the domain of genome assembly in the next- generation sequencing （NGS） platforms. This article discusses the major genome assemblers that were proposed in the literature during the past decade by outlining their basic working principles. It is intended to act as a qualitative, not a quantitative, tutorial to all working on genome assemblers pertaining to the next generation of sequencers. We discuss the theoretical aspects of various genome assemblers, identifying their working schemes. We also discuss briefly the direction in which the area is headed towards along with discussing core issues on software simplicity.

High-quality genome assembly of Huazhan and Tianfeng,the parents of an elite rice hybrid Tian-you-hua-zhan

High-quality rice reference genomes have accelerated the comprehensive identification of genome-wide variations and research on functional genomics and breeding.Tian-you-hua-zhan has been a leading hybrid in China over the past decade.Here,de novo genome assembly strategy optimization for the rice indica lines Huazhan(HZ)and Tianfeng(TF),including sequencing platforms,assembly pipelines and sequence depth,was carried out.The PacBio and Nanopore platforms for long-read se-quencing were utilized,with the Canu,wtdbg2,SMARTdenovo,Flye,Canu-wtdbg2,Canu-SMARTdenovo and Canu-Flye assemblers.The combination of PacBio and Canu was optimal,considering the contig N50 length,contig number,assembled genome size and polishing process.The assembled contigs were scaffolded with Hi-C data,resulting in two“golden quality”rice reference genomes,and evaluated using the scaffold N50,BUSCO,and LTR assembly index.Furthermore,42,625 and 41,815 non-transposable element genes were annotated for HZ and TF,respectively.Based on our assembly of HZ and TF,as well as Zhenshan97,Minghui63,Shuhui498 and 9311,comprehensive variations were identified using Nipponbare as a reference.The de novo assembly strategy for rice we optimized and the“golden quality”rice genomes we produced for HZ and TF will benefit rice genomics and breeding research,especially with respect to uncovering the genomic basis of the elite traits of HZ and TF.

A Hu sheep genome with the first ovine Y chromosome reveal introgression history after sheep domestication

The Y chromosome plays key roles in male fertility and reflects the evolutionary history of paternal lineages.Here,we present a de novo genome assembly of the Hu sheep with the first draft assembly of ovine Y chromosome(o MSY),using nanopore sequencing and Hi-C technologies.The o MSY that we generated spans 10.6 Mb from which 775 Y-SNPs were identified by applying a large panel of whole genome sequences from worldwide sheep and wild Iranian mouflons.Three major paternal lineages(HY1a,HY1b and HY2)were defined across domestic sheep,of which HY2 was newly detected.Surprisingly,HY2 forms a monophyletic clade with the Iranian mouflons and is highly divergent from both HY1a and HY1b.Demographic analysis of Y chromosomes,mitochondrial and nuclear genomes confirmed that HY2 and the maternal counterpart of lineage C represented a distinct wild mouflon population in Iran that diverge from the direct ancestor of domestic sheep,the wild mouflons in Southeastern Anatolia.Our results suggest that wild Iranian mouflons had introgressed into domestic sheep and thereby introduced this Iranian mouflon specific lineage carrying HY2 to both East Asian and Africa sheep populations.

High-quality genome assembly and pan-genome studies facilitate genetic discovery in mung bean and its improvement

Mung bean is an economically important legume crop species that is used as a food,consumed as a vegetable,and used as an ingredient and even as a medicine.To explore the genomic diversity of mung bean,we assembled a high-quality reference genome(Vrad_JL7)that was479.35 Mb in size,with a contig N50 length of 10.34 Mb.A total of 40,125 protein-coding genes were annotated,representing96.9%of the genetic region.We also sequenced 217 accessions,mainly landraces and cultivars from China,and identified 2,229,343 high-quality single-nucleotide polymorphisms(SNPs).Population structure revealed that the Chinese accessions diverged into two groups and were distinct from non-Chinese lines.Genetic diversity analysis based on genomic data from 750 accessions in 23 countries supported the hypothesis that mung bean was first domesticated in south Asia and introduced to east Asia probably through the Silk Road.We constructed the first pan-genome of mung bean germplasm and assembled 287.73 Mb of non-reference sequences.Among the genes,83.1%were core genes and 16.9%were variable.Presence/absence variation(PAV)events of nine genes involved in the regulation of the photoperiodic flowering pathway were identified as being under selection during the adaptation process to promote early flowering in the spring.Genomewide association studies(GWASs)revealed 2,912 SNPs and 259 gene PAV events associated with 33 agronomic traits,including a SNP in the coding region of the SWEET10 homolog(jg24043)involved in crude starch content and a PAV event in a large fragment containing 11 genes for color-related traits.This high-quality reference genome and pan-genome will provide insights into mung bean breeding.