厚朴转录组特征分析及EST-SSR标记的开发
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摘要
为探明厚朴的基因组信息,开发合适的生物学工具以促进厚朴的遗传育种,本研究利用Illumina-Solexa测序技术对厚朴根、茎皮、叶不同组织的9个样本进行转录组分析,获得大量的序列信息,并以转录组序列为基础进行厚朴SSR分子标记的大规模发掘。结果表明,共获得了109 526条厚朴转录组序列,平均序列长度为1 023 bp,通过与蛋白数据库NR、Swiss-Prot、GO、KOG和KEGG五个数据库比对,分别有6 0361、39 942、65 535、51 351和27 310条厚朴转录组的Unigene被注释;厚朴转录组表达信息进行大通量SSR位点的发掘,发现了含SSR位点的序列25 925条,共27 721个SSR位点,SSR出现的频率为23.67%。厚朴转录组共发现了374种碱基重复模式,且CT/GA和AG/TC是主要的双碱基重复序列,AGA/TCT和AAG/TTC是主要的三碱基重复序列;随机挑选的180对SSR引物中有104对产生清晰可重复的条带,21对引物具有多态性,厚朴SSR引物多态性较高。本研究结果为厚朴及近缘种的遗传多样性分析、遗传图谱构建及比较基因组研究奠定了一定的理论基础。
In order to investigate genomic information and develop molecular markers of Houpo?a officinalis, Illumina-Solexa Genome sequencing platform was used to analyze different tissues of velamen, stem bark and leaf of 9 individuals to get a large number of Unigenes, and to develop abundant SSR markers on the basis of the transcription sequencing data. The results showed that a total of 109 526 Unigenes with an average length of 1 023 bp were produced, among which 60 361, 39 942, 65 535, 51 351 and 27 310 were annotated in NR, Swiss-Prot, GO, KEGG and KOG database, respectively. A total of 25 949 sequences with 27 721 SSRs were detected through transcription of H. officinalis, SSR sites occurred with a frequency of 23.67%. Among the total of 374 identified EST-SSRs, CT/GA(31.8%) and AG/TC(27.90%) were the dominant di-nucleotide repeat motifs, AGA/TCT and AAG/TTC were the dominant tri-nucleotide repeat motifs. A total of 180 SSR markers were randomly selected for validation, among these, 104 produced reproducible amplicons and 21 were polymorphic which showed that EST-SSRs of H. officinalis were polymorphic. The results of this study laid a good foundation for genetic analysis, map construction and comparative genome of H. officinalis and related species.
In order to investigate genomic information and develop molecular markers of Houpo?a officinalis, Illumina-Solexa Genome sequencing platform was used to analyze different tissues of velamen, stem bark and leaf of 9 individuals to get a large number of Unigenes, and to develop abundant SSR markers on the basis of the transcription sequencing data. The results showed that a total of 109 526 Unigenes with an average length of 1 023 bp were produced, among which 60 361, 39 942, 65 535, 51 351 and 27 310 were annotated in NR, Swiss-Prot, GO, KEGG and KOG database, respectively. A total of 25 949 sequences with 27 721 SSRs were detected through transcription of H. officinalis, SSR sites occurred with a frequency of 23.67%. Among the total of 374 identified EST-SSRs, CT/GA(31.8%) and AG/TC(27.90%) were the dominant di-nucleotide repeat motifs, AGA/TCT and AAG/TTC were the dominant tri-nucleotide repeat motifs. A total of 180 SSR markers were randomly selected for validation, among these, 104 produced reproducible amplicons and 21 were polymorphic which showed that EST-SSRs of H. officinalis were polymorphic. The results of this study laid a good foundation for genetic analysis, map construction and comparative genome of H. officinalis and related species.
引文
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[15] 李娜,史彩红,车晶玉.厚朴的药理活性与临床应用研究进展[J].中国医药指南,2011,9(9):191-193
[16] Xiong X,Xiao Y,Wei X P,Xie R,Hou Z Y.Research progress on comprehensive utilization of Magnolia officinalis resource [J].Forest Inventory & Planning,2009,34(4):88-92
[17] 周玉亮.分子标记技术及其在植物遗传育种中的应用 [J].生物技术通讯,2005,16(3):350-352
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[19] Faville M J,Vecchies A C,Schreiber M.Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.) [J].Theoretical & Applied Genetics,2004,110(1):12-32
[20] Choi H K,Luckow M A,Doyle J,Cook D R.Development of nuclear gene-derived molecular markers linked to legume genetic maps [J].Molecular Genetics & Genomics,2006,276(1):56-70
[21] Ramu P,Deshpande S P,Senthilvel S,Jayashree B,Billot C,Deu M.In silico mapping of important genes and markers available in the public domain for efficient sorghum breeding[J].Molecular Breeding,2010,26(3):409-418
[22] Bordes J,Goudemand E,Duchalais L,Chevarin L,Oury F X,Heumez E.Genome-wide association mapping of three important traits using bread wheat elite breeding populations [J].Molecular Breeding,2014,33(4):755-768
[23] Santos F H C D,Cavalcanti J J Ⅴ,Silva F P D.Detection of quantitative trait loci for physical traits of cashew apple [J].Crop Breeding & Applied Biotechnology,2010,10(2):101-109
[24] 孔玲.厚朴良种选择及其RAPD种源鉴别研究[D].福州:福建农林大学,2010
[25] 蒋燕锋.厚朴遗传多样性层次变异规律研究[D].杭州:浙江农林大学,2010
[26] 于华会.厚朴遗传多样性和遗传关系研究及ITS序列分析[D].杭州:中国林业科学研究院,2010
[27] Yu H H,Yang Z L,Sun B,Liu R N,Yang X.Genetic diversity and relationship of the endangered plant Magnolia officinalis (Magnoliaceae) assessed with ISSR polymorphisms [J].Biochemical Systematics & Ecology,2011,39(2):71-78
[28] 郑志雷.厚朴遗传多样性研究及指纹图谱的构建[D].福州:福建农林大学,2010
[29] 翁剑.不同种源凹叶厚朴TRAP分析及苗期种源初选[D].福州:福建农林大学,2014
[30] 麦静,杨志玲,杨旭,陈慧,刘起胜,廖海军.濒危植物厚朴SSR引物筛选及反应体系优化[J].生态与农村环境学报,2015,31(4):600-607
[31] Chen H,Wang L,Wang S,Liu C,Blair M W,Cheng X.Transcriptome sequencing of mung bean (Vigna radiate) genes and the identification of EST-SSR markers [J].PLoS One,2015,10(4):221-229
[32] Garg R,Patel R K,Tyagi A K,Jain M.De novo assembly of chickpea transcriptome using short reads for gene discovery and marker identification [J].DNA Research,2011,18(1):53-63
[33] Initiative A G.Analysis of the genome sequence of the flowering plant Arabidopsis thaliana [J].Nature,2000,408(6814):796-815
[34] Arumugam V,Riju A,Arunachalam V,Kumar N,Soorianathasundaram K,Jeyakumar P.Mining of Expressed Sequence Tag (EST) libraries and core nucleotide sequences for Simple Sequence Repeats (SSR) in papaya [J].Acta Horticulturae,2010,851:197-200
[35] Sahu J,Sen P,Choudhury M D,Barooah M,Modi M K,Talukdar A D.Towards an efficient computational mining approach to identify EST-SSR markers [J].Bioinformation,2012,8(4):201-202
[36] Wang L,Yang Y,Zhao Y,Yang S,Udikeri S,Liu T.De novo characterization of the root transcriptome and development of EST-SSR markers in Paris polyphylla smith var.yunnanensis,an endangered medical plant [J].Journal of Agricultural Science & Technology,2016,18(2):437-452
[37] Lu J J,Gao L,Kang J Y,Feng S G,He R F,Wang H Z.Thirteen novel polymorphic microsatellite markers for endangered Chinese endemic herb Dendrobium officinale [J].Conservation Genetics Resources,2013,5(2):359-361
[38] Schlautman B,Fajardo D,Bougie T,Wiesman E,Polashock J,Vorsa N.Development and validation of 697 novel polymorphic genomic and EST-SSR markers in the american cranberry (Vaccinium macrocarpon ait.) [J].Molecules,2015,20(2):2001-2013
[39] 王学勇,周晓丽,高伟,崔光红,黄璐琦,刘春生.丹参新的EST-SSR分布规律及分子标记的建立[J].中国中药杂志,2011,36(3):289-293
[40] Cameron M,Williams H E,Cannane A.Improved gapped alignment in BLAST[J].IEEE/ACM Transactions on Computational Biology & Bioinformatics,2004,1(3):116-129
[41] Wrzodek C,Dr?ger A,Zell A.KEGG translator:visualizing and converting the KEGG PATHWAY database to various formats [J].Bioinformatics,2011,27(16):2314-2315
[42] Lewontin R C.The apportionment of human diversity[J].BMC Evolutionary Biology,1972,6:381-398
[43] Yang Y,Xu M,Luo Q,Wang J,Li H.De novo transcriptome analysis of Liriodendron chinense petals and leaves by Illumina sequencing[J].Gene,2014,534(2):155-162
[44] Shi S G,Yang M,Zhang M,Wang P,Kang Y X,Liu J J.Genome-wide transcriptome analysis of genes involved in flavonoid biosynthesis between red and white strains of Magnolia sprengeri,pamp[J].BMC Genomics,2014,15(1):706
[45] 石文芳.基于RNA-seq测序的梅花转录组分析[D].北京:北京林业大学,2012
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