基于花椒转录组序列SSR分子标记开发及花椒种质鉴定

doi:10.7668/hbnxb.2017.05.011

摘要/Abstract

摘要： 为拓展分子标记在花椒种质资源分析中的应用、开发花椒EST-SSR功能性分子标记、分析花椒DNA指纹图谱，利用凤县大红袍花椒的茎尖节点转录组cDNA数据库中45 057条长度大于200 bp的非冗余Unigene序列，使用MIcroSAtellite（MISA）软件搜索SSR位点，分析花椒cDNA序列中SSR位点的频率和密度、SSR重复基元种类及比例、SSR重复次数与数量等分布特征；用Primer 3.0软件在线设计SSR引物并经PCR扩增筛选适合的多态性引物；用Quantity One软件统计多态性条带和分子量大小；NTsys 2.0软件分析12份花椒种质的遗传距离、构建树状聚类图及DNA指纹图谱库。结果表明，45 057条序列中有3 315条Unigene序列包含SSR位点，共3 814个，3 315条序列中SSR位点出现频率为7.07%；在检索出的SSR位点中，二核苷酸、三核苷酸是主要重复类型，分别占29.42%和58.58%，二核苷酸重复中以AG/TC、CT/GA出现频率最高，三核苷酸重复中GAA/CTT、AGA/TCT出现频率最高；二、三、四、五、六核苷酸重复基元总数随着重复次数的增加呈明显下降趋势。利用Primer 3.0设计的64对EST-SSR引物中，55对引物能产生预期片段大小的PCR产物，其中18对引物具有多态性；利用18对引物对12份花椒种质进行PCR扩增，共产生81条扩增条带，其中多态性条带73条，多态率为90.12%；各花椒种质的遗传相似系数为0.552 6~0.894 7，平均为0.725 0；经UPGMA聚类分析在相似系数0.70处12份花椒种质共分为3类，即顶坛花椒为Ⅰ类、竹叶椒为Ⅱ类、其他花椒为Ⅲ类；指纹图谱分析中，8对引物在5份种质中能扩增出特征带型，最少用3对引物进行组合即可将12份花椒种质区分开。成功在凤县大红袍花椒的茎尖节点转录组cDNA序列中开发SSR标记，设计并筛选出8对能扩增出特征带型的引物，最少用3对引物进行组合即可将12份花椒种质区分开，这为今后花椒遗传多样性分析、遗传图谱构建等方面提供新的引物序列并奠定了基础。

关键词: 花椒, EST-SSR, 引物开发, 指纹图谱

Abstract: To improve the application of molecular markers in the Zanthoxylum genus,the stuty developed functional EST-SSR markers and analyzed DNA fingerprint of Zanthoxylum germplasm.Microsatellite software was used to scan the SSR loci from 45 057 non-redundant Unigenes among length above 200 bp derived from the node tissue of stem tip transcriptome sequences of Zanthoxylum bungeanum Maxim Fengxiandahongpao. Then the frequency and density of SSR loci,the type and proportion of SSR motifs and the number of SSR repetitions were also analysed. The SSR primers were then designed by Primer 3.0 online and polymorphic primer were screened by PCR;the polymorphic bands and molecular size were evaluated by using Quantity One software;the genetic distance and clustering map were analyzed by using software NTsys 2.0 and then constructed Fingerprints by Quantity One. The results showed that 3 315 Unigene sequences contained a total of 3 814 SSR loci(7.07%) and the dinucleotide repeat and trinucleotide repeat were the main types and accounted for 29.42% and 58.58% of the total SSRs,respectively. Among dinucleotides,AG/TC and CT/GA were the most frequent repeats;among trinucleotides,GAA/CTT and AGA/TCT appeared high frequency;with the increased of the number of repetition,the total number of repeat motifs showed a clear downward trend. 64 pairs of SSR primers were designed and 55 primer pairs were successfully amplifying DNA fragments. Out of 55 primer pairs,18 pairs of polymorphic primer were used for PCR amplification in 12 Zanthoxylum germplasm. A total of 81 clear bands were amplified and the percentage of polymorphic bands was 90.12%.Genetic similarity coefficients of each Zanthoxylum germplasm among 0.552 6-0.894 7,with an average of 0.725 0. The UPGMA clustering showed that all the Zanthoxylum germplasm was divided into three main groups at the similarity coefficient 0.70:(Ⅰ) Zanthoxylum armatum DC,(Ⅱ) Zanthoxylum bungeanum Maxim,and (Ⅲ) Zanthoxylum planispinum var. dingtanensis;In the fingerprint analysis,8 pairs of primers could amplify characteristic bands on 5 Zanthoxylum germplasms and 3 pairs of primers could be used to separate the 12 Zanthoxylum germplasm at least. We successfully developed SSR markers in the node tissue of stem tip transcriptome sequences of Zanthoxylum bungeanum Maxim Fengxiandahongpao. 8 pairs of primers could amplify characteristic bands were designed and screened,a minimum of 3 pairs of primers could be used to separate 12 pepper cultivars,these new EST-SSR markers from Zanthoxylum germplasm provided a new primer sequence,basis for genetic analysis and fingerprint construction of Zanthoxylum germplasm.

Key words: Zanthoxylum bungeanum Maxim, EST-SSR, Primer development, Fingerprint

中图分类号:

Q78
S573.03

李立新, 司守霞, 魏安智, 刘玉林, 冯世静, 杨途熙. 基于花椒转录组序列SSR分子标记开发及花椒种质鉴定[J]. 华北农学报, 2017, 32(5): 69-77. doi: 10.7668/hbnxb.2017.05.011.

LI Lixin, SI Shouxia, WEI Anzhi, LIU Yulin, FENG Shijing, YANG Tuxi. Study on Development of SSR Molecular Markers Based on Transcriptome Sequencing and Germplasm Identification in Zanthoxylum Germplasm[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2017, 32(5): 69-77. doi: 10.7668/hbnxb.2017.05.011.

http://www.hbnxb.net/CN/Y2017/V32/I5/69

参考文献

[1] 毕君,王春荣,赵京献,等. 北方花椒主产区种质资源考察报告[J]. 河北林果研究,2003,18(2):165-168.
[2] 王双贵,赵京献,毕君,等. 国内外花椒的研究现状及其发展趋势[J]. 内蒙古林业科技,2003(2):32-34.
[3] 白小鸣,王华,曾小峰,等. 气相色谱-质谱法结合保留指数对比初步分析梁平柚麻味物质的组成成分[J]. 食品科学,2015,18(36):103-107.
[4] 杨静,赵镭,史波林,等. 青花椒香气快速气相电子鼻响应特征及GC-MS物质基础分析[J]. 食品科学,2015,36(22):69-74.
[5] 游玉明,任文瑾,刘庆庆,等. 花椒精有效成分对高脂膳食大鼠脂质代谢的影响[J]. 营养学报,2015(3):288-293.
[6] 郑海星,李周岐,薛惠丹,等. 花椒种质资源的RAPD分析[J]. 西北林学院学报,2011,26(2):96-100.
[7] 汉素珍,王有科,李捷,等. 甘肃省主产花椒品种ISSR遗传多样性分析[J]. 甘肃农业大学学报,2011,46(6):46-51.
[8] Feng S J,Yang T X,Liu Z S,et al. Genetic diversity and relationships of wild and cultivated Zanthoxylum germplasms based on sequence-related amplified polymorphism (SRAP) markers[J]. Genetic Resources and Crop Evolution,2015,62(8):1193-1204.
[9] 蒋弘刚,魏安智,杨途熙,等. 花椒茎尖节点转录组测序及基因注释[J]. 西北林学院学报,2014,29(6):94-99.
[10] 潘海涛,汪俊君,王盈盈,等. 小麦EST-SSR标记的开发和遗传作图[J]. 中国农业科学,2010,43(3):452-461.
[11] 孟清照,李仕金,董转年,等. SSR引物开发方法概述[J]. 大众科技,2007(12):116-117,107.
[12] 杨东娟,马瑞君. SSR分子标记在作物遗传多样性研究中的应用现状[J]. 甘肃科技,2007,23(8):99-102.
[13] Zhuang L F,Song L X,Feng Y G,et al. Development and chromosome mapping of new wheat EST-SSR markers and application for characterizing rye chromosomes added in wheat[J]. Acta Agronomica Sinica,2008,34(6):926-933.
[14] Chai L J,Biswas M K,Yi H L,et al. Transferability,polymorphism and effectiveness for genetic mapping of the Pummelo (Citrus grandis Osbeck) EST-SSR markers[J]. Scientia Horticulturae,2013,155(155):85-91.
[15] 曾柏全,邓子牛,熊兴耀,等. 湖南宽皮柑橘EST-SSR反应体系研究[J]. 中国农学通报,2009,25(21):244-247.
[16] 杨春霞,温强,叶金山,等. 枳壳EST-SSR标记的开发[J]. 分子植物育种,2011(1):123-127.
[17] Yao L H,Zheng X Y,Cai D Y,et al. Exploitation of Malus EST-SSRs and the utility in evalution of genetic diversity in Malus and Pyrus[J].Genetic Resources and Crop Evolution,2010,57(6):841-851.
[18] 张俊娥. 苹果EST中微卫星分析[J]. 广西农业生物科学,2008,27(4):378-380.
[19] Celton J M,Tustin D S,Chagné D,et al. Construction of a dense genetic linkage map for apple rootstocks using SSRs developed from Malus ESTs and Pyrus genomic sequences[J]. Tree Genetics & Genomes,2009,5(1):93-107.
[20] 许玉兰,蔡年辉,康向阳,等. EST-SSR标记的开发及其在木本植物中的分布特点[J]. 中国农学通报,2012,28(4):1-7.
[21] 李晓,杨途熙,魏安智,等. 花椒DNA提取方法的研究[J]. 北方园艺,2011(7):130-132.
[22] Mortazavi A,Williams B A,Mccue K,et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq[J]. Nature Methods,2008,5(7):621-628.
[23] 刘博,邵艳卿,滕爽爽,等. 缢蛏(Sinonovacula constricta)EST-SSR分布特征及引物开发利用[J]. 海洋与湖沼,2012,43(1):132-137.
[24] 曾庆国,陈艺燕. 微卫星位点筛选方法综述[J]. 生态科学,2005,24(4):368-372.
[25] 崔海荣,刘金义,佟兆国,等. 砂梨EST-SSR引物开发及其应用[J]. 西北植物学报,2010,30(8):1551-1556.
[26] Rozen S,Skaletsky H. Primer3 on the WWW for general users and for biologist programmers[J]. Methods in Molecular Biology,2000,132(3):365-386.
[27] Rohlf F J. NTSYSpc:numerical taxonomy and multivariate analysis system,version2.02.[M].New York:Exeter software,1998:2010-2015.
[28] Yeh F C,Yang R C,Boyle T B,et al. POPGENE,the user-friendly shareware for population genetic analysis[M]. Edmonton:University of Alberta,1997.
[29] Cardle L,Ramsay L,Milbourne D,et al. Computational and experimental characterization of physically clustered simple sequence repeats in plants[J]. Genetics,2000,156(2):847-854.
[30] 黄海燕,杜红岩,乌云塔娜,等. 基于杜仲转录组序列的SSR分子标记的开发[J]. 林业科学,2013,49(5):176-181.
[31] 樊洪泓,李廷春,李正鹏,等. 银杏EST序列中微卫星的分布特征[J]. 基因组学与应用生物学,2009,28(5):869-873.
[32] Chen C,Zhou P,Choi Y A,et al. Mining and characterizing microsatellites from Citrus ESTs[J]. Theoretical and Applied Genetics,2006,112(7):1248-1257.
[33] 杨华,陈琪,韦朝领,等. 茶树转录组中SSR位点的信息分析[J]. 安徽农业大学学报,2011,38(6):882-886.
[34] 安泽伟,赵彦宏,程汉,等. 橡胶树EST-SSR标记的开发与应用[J]. 遗传,2009,31(3):311-319.
[35] 吴岐奎,邢世岩,王萱,等. 核用银杏品种遗传关系的AFLP分析[J]. 园艺学报,2015,42(5):961-968.
[36] Morgante M,Hanafey M,Powell W. Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes[J]. Nature Genetics,2002,30(2):194-200.
[37] 李小白. 油菜(Brassica napus L.)EST-SSR标记的开发及应用研究[D]. 杭州:浙江大学,2007.
[38] Zhuang L. Development and chromosome mapping of 81 new wheat EST-SSR markers and application for characterizing rye chromosomes added in wheat[J]. Acta Agronomica Sinica,2008,34(6):926-933.
[39] Chen C A. Mining and characterizing microsatellites from citrus ESTs[J].Theoretica and Applied Genetics,2006,7(112):1248-1257.
[40] Fraser L G,Harvey G H,Crowhurst R N. et al. EST-derived microsatellites from Actinidia species and their potential for mapping[J].Theoretical and Applied Genetics,2004,108(6):1010-1016.
[41] 王丽鸳,姜燕华,段云裳,等. 茶树EST-SSRs分布特征及引物开发[J]. 植物遗传资源学报,2009,10(4):511-516.
[42] 忻雅,崔海瑞,张明龙,等. 白菜EST-SSR标记的通用性[J]. 细胞生物学杂志,2006,28(2):248-252.
[43] Kantetyr V,Larotam L,Matthewsd E,et al. Data mining for simple sequence repeats in expressed sequence tags from barley,maize,rice,sorghum and wheat[J]. Plant Molecular Biology,2002,48(5-6):501-510.
[44] Cordeiro G M,Casu R,Mcintyre C L,et al. Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to erianthus and sorghum[J]. Plant Science,2001,160(6):1115-1123.
[45] 杨素丽,明军,刘春,等. 基于EST信息的百合SSR标记的建立[J]. 园艺学报,2008,35(7):1069-1074.
[46] Metzgar D,Bytof J,Wills C. Selection against frameshift mutations limits microsatellite expansion in coding DNA[J]. Genome Research,2000,10(1):72-80.
[47] Gao L F,Jing R L,Huo N X,et al. One hundred and one new microsatellite loci derived from ESTs (EST-SSRs) in bread wheat[J]. Theoretical and Applied Genetics,2004,108(7):1392-1400.
[48] 李淑娴,张新叶,王英亚,等. 桉树EST序列中微卫星含量及相关特征[J]. 植物学报,2010,45(3):363-371.
[49] Eujayl I,Sorrells M E,Baum M,et al. Isolation of EST-derived microsatellite marker for genotying the A and B genomes of wheat[J].Theor Appl Genet,2002,104(2-3):399-407.
[50] Thiel T,Michalek W,Varshney R K,et al. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.[J]. Theoretical and Applied Genetics,2003,106(3):411-422.
[51] 王凤格,赵久然,郭景伦,等. 比较三种DNA指纹分析方法在玉米品种纯度及真伪鉴定中的应用[J]. 分子植物育种,2003,1(5):655-661.

选择文件类型/文献管理软件名称

选择包含的内容