基于PacBio三代测序的香瓜茄（人参果）基因组的组装

doi:10.13560/j.cnki.biotech.bull.1985.2022-0344

摘要/Abstract

摘要：

香瓜茄又名人参果,具有抗氧化、抗肿瘤、抗糖尿病等多种生物活性。为丰富茄科作物基因组信息及进化发育历程,获取香瓜茄全基因组序列信息,同时为香瓜茄相关分子研究奠定基础。以香瓜茄植物组织为试验材料,基于Illumina HiSeq构建小片段文库进行基因组特征评估,利用PacBio三代测序技术、Hi-C技术构建及组装香瓜茄全基因组数据库。利用生物信息学方法对获得的基因组序列进行组装、功能注释以及进化分析研究。结果表明,获得54.11 Gb Illumina HiSeq数据;获得55.08 Gb PacBio数据,reads平均长度为14 179 bp;获得Hi-C数据量约143 Gb;拼接得到该基因组contig序列总长为1.16 Gb,Hi-C纠错后contig N50为22.63 Mb;Hi-C挂载染色体,共有1.12 Gb长度的序列可以挂载到12条染色体上,占比97.16%;其中,能够确定顺序和方向的序列长度为1.08 Gb,占定位染色体序列总长度的96.11%,得到基因组大小1.25 Gb;预测有64.22%的重复序列,41 571个基因,99.06%的基因可以注释到NR、GO、KEGG等数据库中;预测得到4 360个tRNA、5 677个rRNA、154个miRNA;得到449个假基因。香瓜茄与马铃薯的进化时间大约在12.82 MYA。

关键词: 香瓜茄, 基因组, PacBio三代测序技术, 基因注释

Abstract:

Pepino（Solanum muricatum）has a variety of biological activities such as antioxidation,antitumor activity,antidiabetic activity. To enrich genomic information and evolutionary development of Solanaceae crops,we obtained the whole genome sequencing information of pepino,which lays the foundation for pepino-related molecular studies. Illumina HiSeq sequencing platform was used to construct a small fragment library for pepino characterization and evaluation while using plant tissues of pepino as experimental material. Then third-generation sequencing technology PacBio's sequencing technology and Hi-C technology were used to construct a whole genome database of the pepino. Different bioinformatics methods were to study assembling the obtained pepino genomes,function annotating and evolutionary analysis. The results showed that a total of 54.11 Gb of Illumina HiSeq data were acquired. First,55.08 Gb of PacBio data were obtained with an average reads length of 14 179 bp. The obtained chromosome conformation capture（Hi-C）was 143 Gb and total length contig sequemce of the assembled genome was 1.16 Gb,with a scaffold N50 of 22.63 Mb,chromosomes with a total of 1.12 Gb length of sequence that can be mapped to 12 chromosomes,accounting for 97.16% of the total genome sequence,respectively. Among the sequences,the length of sequences for which the order and orientation could be determined was 1.08 Gb,96.11% of the total length genes were localized chromosomal sequences. Based on the estimated genome size（1.25 Gb）,the 64.22% repeat sequences were predicted,including 41 571 genes and 99.06% of which could be annotated to NR,GO,KEGG and other databases. Noncoding RNAs included 4 360 tRNAs,5 677 rRNAs,154 miRNAs,and a total of 449 pseudogenes were identified. The evolutionary time between pepino and potato was at approximately 12.82 MYA.

Key words: pepino, genome, PacBio third generation sequencing technology, gene annotation

司诚, 钟启文, 杨世鹏. 基于PacBio三代测序的香瓜茄（人参果）基因组的组装[J]. 生物技术通报, 2022, 38(9): 180-190.

SI Cheng, ZHONG Qi-wen, YANG Shi-peng. Assembly of Pepino Genome Based on PacBio's Third-generation Sequencing Technology[J]. Biotechnology Bulletin, 2022, 38(9): 180-190.

图/表 14

参考文献 49

[1]	Pandey S, Prakash S, Prasad Y, et al. Studies on the effects of different surface sterilization agents under in vitro culture of pepino(Solanum muricatum Ait. )cv[J]. Valentia, 2021, 10(6):371-377.
[2]	Fribourg CE, Gibbs AJ, Adams IP, et al. Biological and molecular properties of wild potato mosaic virus isolates from pepino(Solanum muricatum)[J]. Plant Dis, 2019, 103(7):1746-1756. doi: 10.1094/PDIS-12-18-2164-RE pmid: 31082318
[3]	Wang N, Wang LY, Wang ZH, et al. Solanum muricatum ameliorates the symptoms of osteogenesis imperfecta in vivo[J]. J Food Sci, 2019, 84(6):1646-1650. doi: 10.1111/1750-3841.14637 pmid: 31116433
[4]	岳恒. 香瓜茄多糖结构表征及免疫调节活性的研究[D]. 上海: 上海海洋大学, 2020.
	Yue H. The structure characterization and immunomodulatory activity of polysaccharide from Solanum muricatum[D]. Shanghai: Shanghai Ocean University, 2020.
[5]	Zhao YQ, Zuo JH, Yuan SZ, et al. UV-C treatment maintains the sensory quality, antioxidant activity and flavor of pepino fruit during postharvest storage[J]. Foods, 2021, 10(12):2964. doi: 10.3390/foods10122964 URL
[6]	Pacheco J, Soler S, Figàs MR, et al. Screening of pepino(Solanum muricatum)and wild relatives against four major tomato diseases threatening its expansion in the Mediterranean region[J]. Ann Appl Biol, 2021, 179(3):288-301. doi: 10.1111/aab.12698 URL
[7]	Herraiz FJ, Villaño D, Plazas M, et al. Phenolic profile and biological activities of the pepino(Solanum muricatum)fruit and its wild relative S. caripense[J]. Int J Mol Sci, 2016, 17(3):394. doi: 10.3390/ijms17030394 URL
[8]	Hsu JY, Lin HH, Hsu CC, et al. Aqueous extract of pepino(Solanum muriactum Ait)leaves ameliorate lipid accumulation and oxidative stress in alcoholic fatty liver disease[J]. Nutrients, 2018, 10(7):931. doi: 10.3390/nu10070931 URL
[9]	Herraiz FJ, Blanca J, Ziarsolo P, et al. The first de novo transcriptome of pepino(Solanum muricatum):assembly, comprehensive analysis and comparison with the closely related species S. caripense, potato and tomato[J]. BMC Genomics, 2016, 17:321. doi: 10.1186/s12864-016-2656-8 URL
[10]	Yang SP, Zhu HD, Huang LP, et al. Transcriptome-wide and expression analysis of the NAC gene family in pepino(Solanum muricatum)during drought stress[J]. PeerJ, 2021, 9:e10966. doi: 10.7717/peerj.10966 URL
[11]	Su X, Wang BA, Geng XL, et al. A high-continuity and annotated tomato reference genome[J]. BMC Genomics, 2021, 22(1):898. doi: 10.1186/s12864-021-08212-x pmid: 34911432
[12]	Kim S, Park J, Yeom SI, et al. New reference genome sequences of hot pepper reveal the massive evolution of plant disease-resistance genes by retroduplication[J]. Genome Biol, 2017, 18(1):210. doi: 10.1186/s13059-017-1341-9 pmid: 29089032
[13]	Qin C, Yu CS, Shen YO, et al. Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization[J]. Proc Natl Acad Sci USA, 2014, 111(14):5135-5140. doi: 10.1073/pnas.1400975111 URL
[14]	Kyriakidou M, Anglin NL, Ellis D, et al. Genome assembly of six polyploid potato genomes[J]. Sci Data, 2020, 7(1):88. doi: 10.1038/s41597-020-0428-4 pmid: 32161269
[15]	Hirakawa H, Shirasawa K, Miyatake K, et al. Draft genome sequence of eggplant(Solanum melongena L.):the representative Solanum species indigenous to the old world[J]. DNA Res, 2014, 21(6):649-660. doi: 10.1093/dnares/dsu027 pmid: 25233906
[16]	Sierro N, Battey JND, Ouadi S, et al. The tobacco genome sequence and its comparison with those of tomato and potato[J]. Nat Commun, 2014, 5:3833. doi: 10.1038/ncomms4833 pmid: 24807620
[17]	Takei H, Shirasawa K, Kuwabara K, et al. De novo genome assembly of two tomato ancestors, Solanum pimpinellifolium and Solanum lycopersicum var. cerasiforme, by long-read sequencing[J]. DNA Res, 2021, 28(1):dsaa029. doi: 10.1093/dnares/dsaa029 URL
[18]	Bolger A, Scossa F, Bolger ME, et al. The genome of the stress-tolerant wild tomato species Solanum pennellii[J]. Nat Genet, 2014, 46(9):1034-1038. doi: 10.1038/ng.3046 URL
[19]	Zhou Q, Tang D, Huang W, et al. Haplotype-resolved genome analyses of a heterozygous diploid potato[J]. Nat Genet, 2020, 52(10):1018-1023. doi: 10.1038/s41588-020-0699-x pmid: 32989320
[20]	Sun HQ, Jiao WB, Krause K, et al. Chromosome-scale and haplotype-resolved genome assembly of a tetraploid potato cultivar[J]. Nat Genet, 2022, 54(3):342-348. doi: 10.1038/s41588-022-01015-0 pmid: 35241824
[21]	Rao SSP, Huntley MH, Durand NC, et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[J]. Cell, 2014, 159(7):1665-1680. doi: 10.1016/j.cell.2014.11.021 pmid: 25497547
[22]	Wang MC, Tong SF, Ma T, et al. Chromosome-level genome assembly of Sichuan pepper provides insights into apomixis, drought tolerance, and alkaloid biosynthesis[J]. Mol Ecol Resour, 2021, 21(7):2533-2545. doi: 10.1111/1755-0998.13449 URL
[23]	Yoon YJ, Venkatesh J, Lee JH, et al. Genome editing of eIF4E1 in tomato confers resistance to pepper mottle virus[J]. Front Plant Sci, 2020, 11:1098. doi: 10.3389/fpls.2020.01098 pmid: 32849681
[24]	李宁, 王娟, 王柏柯, 等. 潘那利番茄SpCDPK4基因的克隆、原核表达及表达模式分析[J]. 植物生理学报, 2021, 57(3):672-680.
	Li N, Wang J, Wang BK, et al. Cloning, prokaryotic expression and expression pattern analysis of SpCDPK4 gene in Solanum pennellii[J]. Plant Physiol J, 2021, 57(3):672-680.
[25]	Sato S, Tabata S, Hirakawa H, et al. The tomato genome sequence provides insights into fleshy fruit evolution[J]. Nature, 2012, 485(7400):635-641. doi: 10.1038/nature11119 URL
[26]	王娟, 王柏柯, 李宁, 等. 基因编辑技术在番茄育种中的应用进展[J]. 植物生理学报, 2020, 56(12):2606-2616.
	Wang J, Wang BK, Li N, et al. Advances in the application of genome editing in tomato breeding[J]. Plant Physiol J, 2020, 56(12):2606-2616.
[27]	Salava H, Thula S, Mohan V, et al. Application of genome editing in tomato breeding:mechanisms, advances, and prospects[J]. Int J Mol Sci, 2021, 22(2):682. doi: 10.3390/ijms22020682 URL
[28]	Li RQ, Zhu HM, Ruan J, et al. De novo assembly of human genomes with massively parallel short read sequencing[J]. Genome Res, 2010, 20(2):265-272. doi: 10.1101/gr.097261.109 URL
[29]	Rozen S, Skaletsky H. Primer 3 on the WWW for general users and for biologist programmers[J]. Methods Mol Biol, 2000, 132:365-386. pmid: 10547847
[30]	Flynn JM, Hubley R, Goubert C, et al. RepeatModeler2 for automated genomic discovery of transposable element families[J]. Proc Natl Acad Sci USA, 2020, 117(17):9451-9457. doi: 10.1073/pnas.1921046117 URL
[31]	Ou SJ, Jiang N. LTR_retriever:a highly accurate and sensitive program for identification of long terminal repeat retrotransposons[J]. Plant Physiol, 2018, 176(2):1410-1422. doi: 10.1104/pp.17.01310 URL
[32]	Bao ZR, Eddy SR. Automated de novo identification of repeat sequence families in sequenced genomes[J]. Genome Res, 2002, 12(8):1269-1276. doi: 10.1101/gr.88502 URL
[33]	Price AL, Jones NC, Pevzner PA. De novo identification of repeat families in large genomes[J]. Bioinformatics, 2005, 21(Suppl 1):i351-i358.
[34]	Jurka J, Kapitonov VV, Pavlicek A, et al. Repbase update, a database of eukaryotic repetitive elements[J]. Cytogenet Genome Res, 2005, 110(1/2/3/4):462-467. doi: 10.1159/000084979 URL
[35]	Neumann P, Novák P, Hoštáková N, et al. Systematic survey of plant LTR-retrotransposons elucidates phylogenetic relationships of their polyprotein domains and provides a reference for element classification[J]. Mob DNA, 2019, 10:1. doi: 10.1186/s13100-018-0144-1 URL
[36]	Wheeler TJ, Clements J, Eddy SR, et al. Dfam:a database of repetitive DNA based on profile hidden Markov models[J]. Nucleic Acids Res, 2013, 41(Database issue):D70-D82.
[37]	Chen NS. Using RepeatMasker to identify repetitive elements in genomic sequences[J]. Curr Protoc Bioinform, 2004, 5(1):4. 10.1-4. 10. 14.
[38]	Korf I. Gene finding in novel genomes[J]. BMC Bioinformatics, 2004, 5:59. pmid: 15144565
[39]	Grabherr MG, Haas BJ, Yassour M, et al. Trinity:reconstructing a full-length transcriptome without a genome from RNA-Seq data[J]. Nat Biotechnol, 2011, 29(7):644-652. doi: 10.1038/nbt.1883 pmid: 21572440
[40]	Haas BJ, Salzberg SL, Zhu W, et al. Automated eukaryotic gene structure annotation using evidence modeler and the program to assemble spliced alignments[J]. Genome Biol, 2008, 9(1):R7. doi: 10.1186/gb-2008-9-1-r7 URL
[41]	Griffiths-Jones S, Moxon S, Marshall M, et al. Rfam:annotating non-coding RNAs in complete genomes[J]. Nucleic Acids Res, 2005, 33(Database issue):D121-D124.
[42]	Birney E, Clamp M, Durbin R. GeneWise and genomewise[J]. Genome Res, 2004, 14(5):988-995. pmid: 15123596
[43]	Hu Y, Chen JD, Fang L, et al. Gossypium barbadense and Gossypium hirsutum genomes provide insights into the origin and evolution of allotetraploid cotton[J]. Nat Genet, 2019, 51(4):739-748. doi: 10.1038/s41588-019-0371-5 URL
[44]	Gao ZY, Li ZH, Lin DL, et al. Chromosome-scale genome assembly of the resurrection plant Acanthochlamys bracteata(Velloziaceae)[J]. Genome Biol Evol, 2021, 13(8):evab147. doi: 10.1093/gbe/evab147 URL
[45]	Rabanus-Wallace MT, Hackauf B, Mascher M, et al. Chromosome-scale genome assembly provides insights into rye biology, evolution and agronomic potential[J]. Nat Genet, 2021, 53(4):564-573. doi: 10.1038/s41588-021-00807-0 pmid: 33737754
[46]	Kang MH, Wu HL, Yang Q, et al. A chromosome-scale genome assembly of Isatis indigotica, an important medicinal plant used in traditional Chinese medicine:an Isatis genome[J]. Hortic Res, 2020, 7:18. doi: 10.1038/s41438-020-0240-5 URL
[47]	李育先. 茄科作物基因组的多重序列比对与进化研究[D]. 唐山: 华北理工大学, 2016.
	Li YX. Genome multiple alignment and evolutionary studies of Solanaceae lineages[D]. Tangshan: North China University of Science and Technology, 2016.
[48]	Cao YL, Li YL, Fan YF, et al. Wolfberry genomes and the evolution of Lycium(Solanaceae)[J]. Commun Biol, 2021, 4(1):671. doi: 10.1038/s42003-021-02152-8 URL
[49]	苑克俊, 葛福荣, 牛庆霖. 杏基因组全新组装及杏的进化分析[J]. 植物生理学报, 2020, 56(10):2187-2200.
	Yuan KJ, Ge FR, Niu QL. De novo apricot(Prunus armeniaca)genome assembly and evolutionary analysis[J]. Plant Physiol J, 2020, 56(10):2187-2200.

项目Item	总长度Total length/bp	总数量Total number	最大长度Max length/bp	N50长度N50-length/bp	N90长度N90-length/bp
Contig	1 141 353 553	1 952 498	125 830	2 049	165
Scaffold	1 169 596 440	1 679 956	191 961	3 185	199

项目Item	总长度Total length/bp	总数量Total number	最大长度Max length/bp	N50长度N50-length/bp	N90长度N90-length/bp
Contig	1 141 353 553	1 952 498	125 830	2 049	165
Scaffold	1 169 596 440	1 679 956	191 961	3 185	199

项目Item	基因组信息Genome information
总数TotalNum	1 566
最小长度MinLen	1 000
Scaffold的数量ScfNum	1 566
Scaffold的长度ScfLen/bp	1 156 096 017
Scaffold N50长度ScfN50/bp	87 253 278
Scaffold N90长度ScfN90/bp	74 267 810
最长的scaffold ScfMax/bp	111 830 586
Scaffold数目mScfNum	0
Scaffold的长度mScfLen	0
Contig数目CtgNum	1 813
Ctg长度CtgLen/bp	1 156 071 317
Contig N50的长度CtgN50/bp	22 628 432
Contig N90的长度CtgN90/bp	596 645
最长的contig CtgMax/bp	83 851 337
GC含量GC/%	35.83
Contig数目GapNum	247
Gap长度GapLen/bp	24 700
最大的gap MaxGap	100

项目Item	基因组信息Genome information
总数TotalNum	1 566
最小长度MinLen	1 000
Scaffold的数量ScfNum	1 566
Scaffold的长度ScfLen/bp	1 156 096 017
Scaffold N50长度ScfN50/bp	87 253 278
Scaffold N90长度ScfN90/bp	74 267 810
最长的scaffold ScfMax/bp	111 830 586
Scaffold数目mScfNum	0
Scaffold的长度mScfLen	0
Contig数目CtgNum	1 813
Ctg长度CtgLen/bp	1 156 071 317
Contig N50的长度CtgN50/bp	22 628 432
Contig N90的长度CtgN90/bp	596 645
最长的contig CtgMax/bp	83 851 337
GC含量GC/%	35.83
Contig数目GapNum	247
Gap长度GapLen/bp	24 700
最大的gap MaxGap	100

方法 Method	软件 Software	物种 Species	基因数目 Gene number
Ab initio	Augustus		26 025
	SNAP		47 891
Homology-based	GeMoMa	拟南芥A. thaliana	26 652
		辣椒C. annuum	37 150
		番茄S. lycopersicum	32 312
		潘那利番茄 S. pennellii	33 755
		马铃薯S. tuberosum	51 586
RNAseq	GeneMarkS-T		19 020
	PASA		12 454
Integration	EVM		41 571