鸟巢蕨EST-SSR标记的开发与应用

doi:10.7668/hbnxb.2015.06.020

摘要/Abstract

摘要： 为加速分子标记在鸟巢蕨研究中的应用,利用鸟巢蕨EST序列开展了EST-SSR标记的开发和应用研究.利用MISA软件对鸟巢蕨EST序列进行SSR位点查找,分析EST-SSR的总体特征,并利用Primer 3.0软件设计40对引物,选用10份蕨类材料检测引物的多态性.从42 907条鸟巢蕨EST序列中检测到6 067个SSR位点,其出现频率为1/6.62 kb,包括106种重复基元.在鸟巢蕨EST-SSR中,二核苷酸重复(2 873个,47.35%)是最主要的类型,其次是三核苷酸(1 107个,18.25%)和单核苷酸(972个,16.02%).出现最多的重复基元是AG/TC(1 371个,22.60%),其次是CA/GT(1 066个,17.57%)和A/T(628个,10.35%).设计合成了40对EST-SSR引物对10份蕨类材料进行PCR扩增,26对引物能扩增出明显条带,其中17对引物扩增出了65条多态性条带,平均每对引物多态性带为3.82条.遗传分析表明,10份蕨类材料之间的遗传相似系数为0.338~0.815,可将供试蕨类材料分为2个类群.从鸟巢蕨EST序列中开发SSR标记是有效和可行的,开发的EST-SSR引物可用于蕨类植物遗传分析研究.

关键词: 鸟巢蕨, 分子标记, EST-SSR

Abstract: To accelerate the application of molecular markers in Asplenium nidus ,EST-SSR markers development and utilization form the Asplenium nidus EST sequences were performed.A total of 42 907 EST sequences were obtained from the Asplenium nidus transcriptome,and these sequences were screened by using MISA software to search for SSR motifs.Forty pairs of primers were designed by Primer 3.0 software,and the polymorphism of the primer was tested by PCR amplification of 10 ferns.A total of 6 067 EST-SSR were identified with an average of one SSR per 6.62 kb,and included 106 SSR motif types.Dinucleotide repeats was the dominant type(2 873,47.35%),followed by trinucleotide(1 107,18.25%)and mononucleotide(972,16.02%)repeats.The dominant repeat motif in all EST-SSRs was AG/TC(1 371,22.60%),followed by CA/GT(1 066,17.57%)and A/T(628,10.35%).40 pairs of EST-SSR primers were designed and synthesized based on different motifs types,and verified with 10 fern varieties for polymorphism.Among them,26 primer pairs could produce clear and stable bands,and 17 out of 26 primer pairs could amplify 65 polymorphic bands with an average of 3.82 bands per primer pair.The genetic similarity coefficient and cluster analysis were calculated by NTSYS-pc 2.10e software.The genetic analysis showed that,the genetic similarity coefficients between the 10 cultivars ranged from 0.338 to 0.815,and the cluster analysis showed that all the cultivars could be categorized into two major clusters.These results showed that it is an effective and feasible way to develop EST-SSR markers from Asplenium nidus EST sequences,and the primers designed in this study can be used in genetic analysis of fern.

Key words: Asplenium nidus, Molecular marker, EST-SSR

中图分类号:

S682.03

贾新平, 邓衍明, 孙晓波, 梁丽建. 鸟巢蕨EST-SSR标记的开发与应用[J]. 华北农学报, 2015, 30(6): 134-139. doi: 10.7668/hbnxb.2015.06.020.

JIA Xin-ping, DENG Yan-ming, SUN Xiao-bo, LIANG Li-jian. Development and Application of EST-SSR Marker in Asplenium nidus[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(6): 134-139. doi: 10.7668/hbnxb.2015.06.020.

http://www.hbnxb.net/CN/Y2015/V30/I6/134

参考文献

[1] Morgante M,Hanafey M,Powell W.Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes[J].Nature Genetics,2002,30(2):194-200.
[2] 吕婧,时秋香,任毅,等.黄瓜23对高多态性SSR标记的筛选与评价[J].园艺学报,2011,38(11):2140-2148.
[3] Powell W,Machray G C,Provan J.Polymorphism revealed by simple sequence repeats[J].Trends in Plant Science,1996,1(7):215-222.
[4] Zane L,Bargelloni L,Patarnello T.Strategies for microsatellite isolation:a review[J].Molecular Ecology,2002,11(1):1-16.
[5] Portis E,Nagy I,Sasvári Z,et al.The design of Capsicum spp.SSR assays via analysis of in silico DNA sequence,and their potential utility for genetic mapping[J].Plant Science,2007,172(3):640-648.
[6] 江东,钟广炎,洪棋斌.柑橘EST-SSR分子标记分析[J].遗传学报,2006,33(4):345-353.
[7] 姚利华,滕元文.EST-SSR标记及其在果树研究中的应用[J].果树学报,2008,25(2):219-224.
[8] 董清华,王西成,赵密珍,等.草莓EST-SSR标记开发及在品种遗传多样性分析中的应用[J].中国农业科学,2011,44(17):3603-3612.
[9] Tan L Q,Wang L Y,Wei K,et al.Floral transcriptome sequencing for SSR marker development and linkage map construction in the tea plant(Camellia sinensis)[J].PLoS One,2013,8(11):e81611.
[10] 王娟,陶永焕,宋尚伟.葡萄EST-SSR引物的开发及部分种质聚类分析[J].华北农学报,2014,29(2):121-126.
[11] 陆树刚.蕨类植物学[M].北京:高等教育出版社,2007:34-42.
[12] Cavagnaro P F,Senalik D A,Yang L,et al.Genome-wide characterization of simple sequence repeats in cucumber(Cucumis sativus L.)[J].BMC Genomics,2010,11:569.
[13] 贾新平,叶晓青,梁丽建,等.基于高通量测序的海滨雀稗转录组学研究[J].草业学报,2014,23(6):242-252.
[14] 韩明利,崔娜,于志海,等.番茄果实EST资源SSR信息分析[J].华北农学报,2011,26(4):213-217.
[15] Wei W,Qi X,Wang L,et al.Characterization of the sesame(Sesamum indicum L.)global transcriptome using Illumina paired-end sequencing and development of EST-SSR markers[J].BMC Genomics,2011,12:451.
[16] 贾新平,孙晓波,邓衍明,等.鸟巢蕨转录组高通量测序及分析[J].园艺学报,2014,41(11):2329-2341.
[17] Davey J W,Hohenlohe P A,Etter P D,et al.Genome-wide genetic marker discovery and genotyping using next-generation sequencing[J].Nature Reviews Genetics,2011,12(7):499-510.
[18] 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.
[19] Li D,Deng Z,Qin B,et al.De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree(Hevea brasiliensis Muell.Arg.)[J].BMC Genomics,2012,13:192.
[20] Jia X P,Deng Y M,Sun X B,et al.Characterization of the global transcriptome using Illumina sequencing and novel microsatellite marker information in seashore paspalum[J].Genes & Genomics,2015,37(1):77-86.
[21] 上官凌飞,李晓颖,宁宁,等.杏EST-SSR标记的开发[J].园艺学报,2011,38(1):43-54.
[22] 包文风,王吉明,尚建立,等.基于公共数据库的西瓜EST-SSR信息分析与标记开发[J].华北农学报,2011,26(2):85-89.
[23] 陈琛,庄木,李康宁,等.甘蓝EST-SSR标记的开发与应用[J].园艺学报,2010,37(2):221-228.
[24] Wang S,Wang X,He Q,et al.Transcriptome analysis of the Roots at early and late seedling stages using Illumina paired-end sequencing and development of EST-SSR markers in radish[J].Plant Cell Reports,2012,31(8):1437-1447.
[25] 魏兵强,刘飞云,马宗桓,等.辣椒EST-SSRs的分布特征及在品种多样性研究中的应用[J].园艺学报,2013,40(2):265-274.
[26] Vendramin E,Dettori M T,Giovinazzi J,et al.A set of EST-SSRs isolated from peach fruit transcriptome and their transportability across Prunus species[J].Molecular Ecology Notes,2007,7(2):307-310.
[27] Saha M C,Cooper J D,Mian M A,et al.Tall fescue genomic SSR markers:development and transferability across multiple grass species[J].Theoretical and Applied Genetics,2006,113(8):1449-1458.
[28] 贾新平,叶晓青,梁丽建,等.12份蕨类材料亲缘关系的 SSR分析[J].江西农业学报,2013,25(11):1-5.

选择文件类型/文献管理软件名称

选择包含的内容