甘薯属耐盐植物马鞍藤基因组大小及特征分析
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摘要
马鞍藤(Ipomoea pes-caprae(L.)R. Br.)是甘薯的近缘野生种之一,为热带、亚热带海滩生长的多年生藤蔓植物,作为沿海地区的园林绿化植物之一,具有较强的耐盐性。马鞍藤基因组学的研究可为耐盐甘薯种质创制提供信息参考。本试验调查了马鞍藤基因组大小和特征概况,为后续全基因组精细图谱绘制打下基础。研究以已知基因组大小的三裂叶薯(Ipomoea triloba L.)为对照,运用流式细胞术对马鞍藤基因组大小进行初步预估;使用二代高通量测序技术(Illumina Hiseq2500)对马鞍藤基因组进行survey评估,测序深度30×,利用生物信息学方法估算马鞍藤GC含量(即鸟嘌呤和胞嘧啶所占比例)、杂合率、重复序列等基因组概况。结果表明:流式细胞术估算马鞍藤基因组大小为1012.704±17.37 Mb;经全基因组Survey测定获得马鞍藤有效数据为21.71 Gb,基因组大小经修正后估算为1041.65 Mbp;通过K-mer分布曲线估算马鞍藤基因组中重复序列所占比率为74.52%,杂合率为0.99%;经初步组装后,GC平均深度及含量分布存在异常,出现分层的现象,这可能与马鞍藤基因组杂合率较高有关。本试验首次报导甘薯属耐盐植物马鞍藤基因组大小及特征信息,为马鞍藤进一步的全基因组深度测序和甘薯耐盐基因挖掘打下基础。
Ipomoea pes-caprae(L.)R. Br. is one of the perennial wild species of sweetpotato,a perennial vine plant which grows on tropical and subtropical beaches. It is one of the landscaping plants in coastal areas with strong salt tolerance. The study of Ipomoea pes-caprae(L.)R.Br. genome-wide can provide reference for the improvement of salt-tolerant varieties of Sweetpotato and the innovation of salt-tolerant sweet potato germplasm resources. In order to reduce the blindness of whole-genome deep sequencing,this experiment aims to understand the genome overview and provide insight for the following mapping of the whole genome. We performed the experiments in preliminary estimation of the genome size of Ipomoea pes-caprae(L.)R.Br. by ?ow cytometry,generation of highthroughput sequencing datasets,and the bioinformatics analysis of genomic pro?les such as Ipomoea pes-caprae(L.)R.Br. GC content,heterozygosity,and repeat sequences. The results showed that the genomic size by using the?ow cytometry was 1012.99 Mb,and the clean data of Ipomoea pes-caprae was(L.)R.Br. 21.71 Gb by genomewide Survey. By integrated analysis with 30× genomic dataset,its genome size was re-estimated to be 1041.65 Mb. K-mer analysis suggested 74.52% of repeated sequences of the Ipomoea pes-caprae(L.)R.Br. genome. The heterozygosity rate was 0.99%. The initial depth of GC and the content distribution were abnormal after initial assembly. The strati?cation phenomenon may be related to the high heterozygous rate of the Ipomoea pes-caprae(L.)R.Br. genome. Thus,this work reported the preliminary information of the Ipomoea pes-caprae(L.)R.Br. genome,which lays a foundation for further genome-wide deep sequencing and salt tolerance gene mining.
Ipomoea pes-caprae(L.)R. Br. is one of the perennial wild species of sweetpotato,a perennial vine plant which grows on tropical and subtropical beaches. It is one of the landscaping plants in coastal areas with strong salt tolerance. The study of Ipomoea pes-caprae(L.)R.Br. genome-wide can provide reference for the improvement of salt-tolerant varieties of Sweetpotato and the innovation of salt-tolerant sweet potato germplasm resources. In order to reduce the blindness of whole-genome deep sequencing,this experiment aims to understand the genome overview and provide insight for the following mapping of the whole genome. We performed the experiments in preliminary estimation of the genome size of Ipomoea pes-caprae(L.)R.Br. by ?ow cytometry,generation of highthroughput sequencing datasets,and the bioinformatics analysis of genomic pro?les such as Ipomoea pes-caprae(L.)R.Br. GC content,heterozygosity,and repeat sequences. The results showed that the genomic size by using the?ow cytometry was 1012.99 Mb,and the clean data of Ipomoea pes-caprae was(L.)R.Br. 21.71 Gb by genomewide Survey. By integrated analysis with 30× genomic dataset,its genome size was re-estimated to be 1041.65 Mb. K-mer analysis suggested 74.52% of repeated sequences of the Ipomoea pes-caprae(L.)R.Br. genome. The heterozygosity rate was 0.99%. The initial depth of GC and the content distribution were abnormal after initial assembly. The strati?cation phenomenon may be related to the high heterozygous rate of the Ipomoea pes-caprae(L.)R.Br. genome. Thus,this work reported the preliminary information of the Ipomoea pes-caprae(L.)R.Br. genome,which lays a foundation for further genome-wide deep sequencing and salt tolerance gene mining.
引文
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[27] Sweetpotato Genomics Resource. The Sweetpotato GenomicsResource was released·(2016-06-13)[2018-09-22]·http://sweetpotato.plantbiologry.msu.edu/
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[2]张靖靖·紫草基因组大小的测定研究·南京:南京大学生命科学学院,2013Zhang J J·Study on the measureation of genome size of Lithospermum erythrorhizon. Nanjing:School of Life Sciences,Nanjing University, 2013
[3]Dolezel J, Greilhuber J, Suda J-Estimation of nuclear DNA content in plants using flow cytometry-Nature Protocols, 2007,2(9):2233-2244
[4]李潞滨,武静宇,胡陶,杨学文,彭镇华·毛竹基因组大小测定·植物学通报,2008,25(5):574-578Li L B, Wu J Y, Hu T, Yang X W, Peng Z H. Es-timation of genome size of Moso bamboo(Phyllostachys edulis). ChineseBulletin of Botany, 2008,25(5):574-578
[5]邓果特,刘清波,蒋建雄,陈智勇,艾辛,肖亮,易自力·五节芒基因组大小测定·植物遗传资源学报,2013,14(2):339-341Deng G T,Liu Q B,Jiang J X,Chen Z Y,Ai X,Xiao L,Yi Z L. Estimation of genome size of Miscanthus floridulus.Journal of Plant Genetic Resources, 2013, 14(2):339-341
[6]李秋实,徐江,朱英杰,孙超,宋经元,陈士林·基于流式细胞技术的灵芝基因组大小估测.菌物学报,2013,32(5):899-906Li Q S, Xu J,Zhu Y J, Sun C, Song J Y, Chen S L. Estimation of the genome size of Ganoderma lucidum based on aflow cytometric analysis. Mycosystema, 2013,32(5):899-906
[7]王亚之,李秋实,陈士林,孙超,宋经元·基于流式细胞分析技术的茯苓基因组大小测定.世界科学技术一中医药现代化,2010, 12(3):452-456Wang Y Z, Li Q S, Chen S L, Sun C, Song J Y. Flow cytometric analysis of nuclear DNA content in Poriacocos.Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology, 2010, 12(3):452-456
[8]田新民,周香艳,弓娜·流式细胞术在植物学研究中的应用一检测植物核DNA含量和倍性水平·中国农学通报,2011,27(9):21-27Tian X M, Zhou X Y, Gong N-Applications of flow cytometry in plant research-analysis of nuclear DNA content and ploidy level in plant cells. Chinese Agricultural Science Bulle-tin,2011,27(9):21-27
[9]李彩琴,王泽槐,徐咏珊,张劲霭,李建国·流式细胞术细胞核分离缓冲液的改良及大、小果型荔枝幼果和果皮细胞分裂活性比较·园艺学报,2011,38(9):1781-1790Li C Q, Wang Z H, Xu Y S, Zhang J A, Li J G. Improvment of the cell nuclei suspensions for flow cytometry and comparison on the cell division activity of litchi fruitletand pericarp which having different final fruit size. Acta Horti-culturae Sinica,2011,38(9):1781-1790
[10]施季森,王占军,陈金慧.木本植物全基因组测序研究进展·遗传,2012(2):145-156Shi J S, Wang Z J, Chen J H. Progress on whole genome sequencing in woody plants. Hereditas, 2012(2):145-156
[11]唐其,马小军,莫长明,潘丽梅,韦荣昌,赵欢·罗汉果全基因组Survey分析·广西植物,2015,35(6):786-791Tang Q, Ma X J, Mo C M, Pan L M, Wei R C, Zhao H-Genome survey analysis in Siraitia grosvenorii. Guihaia, 2015,35(6):786-791
[12]张伟,王瑞,杨晓野,祝晓蕊,王新芳,梁萌,杨莲茹,李军燕,罗晓平·捕食性真菌Duddingtonia flagrans基因组DNA的提取及基因组survey分析·中国兽医学报,2017, 37(11):2090-2094Zhang W, Wang R, Yang X Y, Zhu X R, Wang X F, Liang M, Yang L R, Li J Y, Luo X P. Genomic DNA extraction and genome survey of the predatory fungus Duddingtonia flagrans.Chinese Journal of Veterinary Science, 2017,37(11):2090-2094
[13]孔令培·优良海滩植被——厚藤.中国花卉盆景,2001(9):9Kong L P-Excellent beach vegetation---Ipomoea pes-caprae.Chinese Flower Bonsai, 2001(9):9
[14] Kamakura M, Furukawa A. Compensatory function for water transport by adventitious roots of Ipomoea pes-caprae. Journal of Plant Research, 2009(122):327-333
[15]王正加,黄有军,龚宁,夏国华,吕联江,程柯·厚藤耐盐生理指标研究·江西农业大学学报,2006,28(2):264-267Wang Z J, Huang Y J, Gong N, Xia G H, Lv L J, Cheng K-A study on physiological indices of Ipomoea pescaprae under salt stress. Acta Agriculturae Universitatis Jiangxiensis, 2006,28(2):264-267
[16] Dolezel J, Binarova P, Lucretti S. Analysis of nuclear DNA content in plant cells by flow cytometry. Biologia Plantarum,1989,31:113-120
[17] Arumuganathan K, Earle E D-Estimation of nuclear DNA contents of plants by flow cytometry. Plant Molecular Biology Reporter, 1991,9(3):229-241
[18]刘伟,刘争辉,何旭,Dragovich A Yu,杨起简.利用流式细胞术鉴定黑麦草倍性方法的研究·中国农学通报,2011,27(9):212-215Liu W, Liu Z H, He X, Dragovich A Yu, Yang Q J. DNA content and ploidy determination of ryegrass(Lolium perenne)by flow cytometry. Chinese Agricultural Science Bulletin,2011,27(9):212-215
[19]严苏丽,何丽容,陈巧伦·流式细胞仪的应用体会·现代医学仪器与应用,2003,15(1):23-24Yan S L, He L R, Chen Q L. Application experience of flow cytometry. Journal of Molecular Diagnostics and Therapy,2003, 15(1):23-24
[20]赵泓,刘凡·流式细胞仪·安徽农学通报,2006, 12(12):39-41Zhao H, Liu F. Flow cytometry. Anhui Agricultural Science Bulletin, 2006, 12(12):39-41
[21]苏一钧,王娇,戴习彬,唐君,赵冬兰,张安,周志林,曹清河-303份甘薯地方种SSR遗传多样性与群体结构分析·植物资源遗传学报,2018, 19(2):243-251Su Y J, Wang J, Dai X B,Tang J,Zhao D L, Zhang A, Zhou Z L,Cao Q H. Genetic diversity and population structure analysis of303 Sweetpotato landraces using SSR markers. Journal of Plant Genetic Resources, 2018, 19(2):243-251
[22] Huang S S, Li R Q, Zhang Z H, Li L, Gu X F, Fan W, Lucas W J,Wang X W,Xie B Y,Ni P P X,Ren Y Y,Zhu H M,Li J,Lin K, Jin W W, Fei Z J, Li G C, Staub J, Kilian A, van der Vossen EA,Wu Y,Guo J,He J,Jia Z Q,Ren Y,Tian G,Lu Y, Ruan J, Qian W B, Wang M W, Huang Q F, Li B, Xuan Z L, Cao J J, Asan, Wu Z G, Zhang J B, Cai Q L, Bai Y Q, Zhao B W, Han Y H, Li Y, Li X F, Wang S H, Shi Q X, Liu S Q,Cho W K, Kim J Y, Xu Y, Heller-Uszynska K, Miao H, Cheng Z C, Zhang S P, Wu J, Yang Y H, Kang H X, Li M, Liang H Q, Ren X L, Shi Z B, Wen M, Jian M, Yang H L, Zhang G J,Yang Z T, Chen R, Liu S F, Li J W, Ma L J, Liu H, Zhou Y,Zhao J, Fang X D, Li G Q, Fang L, Li Y R, Liu D Y, Zheng H K, Zhang Y, Qin N, Li Z, Yang G H, Yang S, Bolund L,Kristiansen K, Zheng H C, Li S F, Zhang X Q, Yang H M,Wang J, Sun R F, Zhang B, Jiang S Z, Wang J, Du Y C, Li S G-The genome of the cucumber, Cucumis sativus L-Nature Genetics, 2009,41(12):1275-1038
[23]伍艳芳,肖复明,徐海宁,章挺,江香梅·樟树全基因组调查·植物遗传资源学报,2014, 15(1):149-152Wu Y F, Xiao F M, Xu H N, Zhang T, Jiang X M. Genome survey in Cinnamomum camphora L. Presl. Journal of Plant Genetic Resources, 2014, 15(1):149-152
[24] Callan H G. The organization of genetic units in chromosomes.Journal of Molecular Cell Biology,1967,2:1-7
[25]李蔚,刘莉莎,李仁,郭仰东·十字花科蔬菜基因组含量的测定与分析·植物遗传资源学报,2011,12(1):103-106Li W, Liu L S, Li R, Guo Y D. Analysis of genome contents in some cruciferous vegetables. Journal of Plant Genetic Resources, 2011,12(1):103-106
[26] Ipomoea Genome Hub.The haplotype-resovled genome sequence of hexaploid Ipomoea batatas.(2014-05-15)[2018-08-09]. https://ipomoea-genome.org/
[27] Sweetpotato Genomics Resource. The Sweetpotato GenomicsResource was released·(2016-06-13)[2018-09-22]·http://sweetpotato.plantbiologry.msu.edu/
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