基于转录组测序的花椒属物种EST-SSR标记开发
|
摘要
【目的】开发适用于花椒(Zanthoxylum bungeanum)和竹叶花椒(Zanthoxylum armatum)的EST-SSR引物,为花椒属物种的鉴定及遗传多样性探究提供分子标记。【方法】对花椒和竹叶花椒分别进行转录组测序(RNA-seq),基于得到的EST序列开发SSR引物。在2种花椒的EST-SSR引物中分别随机挑选60对引物,用12份材料(4份花椒和8份竹叶花椒)为样本,利用琼脂糖凝胶电泳验证其特异性。从有特异性条带的引物中,再随机选取花椒和竹叶花椒SSR引物各15对,选取6个(2份花椒和4份竹叶花椒)样本,利用聚丙烯酰胺银染电泳进行多态性检测。【结果】花椒共有64 944条Unigene,碱基对长度共54 073 890bp,含有12 746个SSR位点,分布在10 595条Unigene上,SSR位点出现频率为19.63%,平均分布距离4.24kb。竹叶花椒Unigene共75 669条,碱基对长度58 975 053bp,共15 096个SSR位点,分布在12 612条Unigene上。SSR位点出现频率为19.95%,平均分布距离为3.91kb。60对花椒引物中,有46对成功扩增出特异性条带,扩增效率76.67%;60对竹叶花椒引物中,有41对扩增出特异性条带,扩增效率68.33%。花椒和竹叶花椒的特异性条带大小在100～300bp,主要集中在150～250bp。多态性验证试验中,15对花椒引物中有12对产生了多态性条带,多态率80.00%;15对竹叶花椒引物中则有14对产生了多态性条带,多态率93.33%。【结论】基于花椒和竹叶花椒转录组高通量测序结果开发的EST-SSR分子标记引物,具有较明显的特异性和多态性。
【Objective】To develope suitable primers for Zanthoxylum bungeanumand Zanthoxylum armatum,which is aim to provide molecular markers for genetic diversity and species identity of Zanthoxylum.【Method】Sequences of Z.bungeanumand Z.armatum were determined by RNA-seq and SSR primers were developed based on obtained EST sequences.Randomly 60 pairs primers were selected from the EST-SSR primers of two species and 12 samples(4 Z.bungeanumand 8 Z.armatum)were used to detect specificity by agarose gel electrophoresis.Then,15 pairs of primers with specificity strands were randomly selected from the two species and polymorphism of 6 samples of Z.bungeanumand Z.armatum was detected by polyacrylamide gel electrophoresis(PAGE).【Result】In Z.bungeanum,there were 64 944 Unigene in total and length of base pair was 54 073 890 bp including 12 746 SSR loci on 10 595 Unigene.The occurrence frequency of SSR was 19.63% with average distance of 4.24 kb.In Z.armatum,here were 75 669 Unigene including 15 096 SSR loci on 12 612 Unigene.The length of base was 58 975 053 bp,occurrence frequency of SSR was 19.95%,and average distance was 3.91 kb.There were 46 pairs of primers of Z.bungeanumand 41 pairs of primers of Z.armatum with specific bands and the ratio was 76.67%for Z.bungeanumand 68.33%for Z.armatum.The lengths of bands were 100-300 bp and mainly distributed in 150-250 bp.A total of 12 pairs of primers of Z.bungeanumproduced polymorphic bands with polymorphism ratio of 80.00%,and 14 pairs of Z.armatum produced polymorphic bands with polymorphism ratio of 93.33%.【Conclusion】The screened EST-SSR primers for Z.bungeanumand Z.armatum based on RNA-seq showed significant specificity and polymorphism.
【Objective】To develope suitable primers for Zanthoxylum bungeanumand Zanthoxylum armatum,which is aim to provide molecular markers for genetic diversity and species identity of Zanthoxylum.【Method】Sequences of Z.bungeanumand Z.armatum were determined by RNA-seq and SSR primers were developed based on obtained EST sequences.Randomly 60 pairs primers were selected from the EST-SSR primers of two species and 12 samples(4 Z.bungeanumand 8 Z.armatum)were used to detect specificity by agarose gel electrophoresis.Then,15 pairs of primers with specificity strands were randomly selected from the two species and polymorphism of 6 samples of Z.bungeanumand Z.armatum was detected by polyacrylamide gel electrophoresis(PAGE).【Result】In Z.bungeanum,there were 64 944 Unigene in total and length of base pair was 54 073 890 bp including 12 746 SSR loci on 10 595 Unigene.The occurrence frequency of SSR was 19.63% with average distance of 4.24 kb.In Z.armatum,here were 75 669 Unigene including 15 096 SSR loci on 12 612 Unigene.The length of base was 58 975 053 bp,occurrence frequency of SSR was 19.95%,and average distance was 3.91 kb.There were 46 pairs of primers of Z.bungeanumand 41 pairs of primers of Z.armatum with specific bands and the ratio was 76.67%for Z.bungeanumand 68.33%for Z.armatum.The lengths of bands were 100-300 bp and mainly distributed in 150-250 bp.A total of 12 pairs of primers of Z.bungeanumproduced polymorphic bands with polymorphism ratio of 80.00%,and 14 pairs of Z.armatum produced polymorphic bands with polymorphism ratio of 93.33%.【Conclusion】The screened EST-SSR primers for Z.bungeanumand Z.armatum based on RNA-seq showed significant specificity and polymorphism.
引文
[1]Zhang D X,Thomas G,Hartley.Flora of China[M].Beijing:Science Press,2008:11.
[2]张华,叶萌.青花椒的分类地位及成分研究现状[J].北方园艺,2010(14):199-203.Zhang H,Ye M.Research status on the taxonomic and component of green Zanthoxylum bungeanum Maxim[J].Northern Horticulture,2010(14):199-203.
[3]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].甘肃农大学报,2011,46(6):46-51.Han S Z,Wang Y K,Li J,et al.ISSR genetic diversity analysis of Zanthoxylum bungeanumin Gansu province[J].Journal of Gansu Agricultural University,2011,46(6):46-51.
[4]李猛,王平,孙吉康,等.蚬壳花椒ISSR-PCR反应体系的建立及优化[J].广西植物,2013,33(2):185-190.Li M,Wang P,Sun J K,et al.Establishment and optimization of ISSR-PCR reaction system on Zanthoxylum dissitum[J].Guihaia,2013,33(2):185-190.
[5]邓洪平,徐洁,陈锋,等.九叶青花椒遗传多样性的形态与分子鉴定[J].西北植物学报,2008,28(10):2103-2109.Deng H P,Xu J,Chen F,et al.Morphological and molecular identification on genetic diversity of Zanthoxylum armatum var.Novemfolius[J].Acta Botanica Boreali-Occidentalia Sinica,2008,28(10):2103-2109.
[6]Medhi K,Sarmah D K,Deka M,et al.High gene flow and genetic diversity in three economically important Zanthoxylum spp.of upper Brahmaputra Valley Zone of NE India using molecular markers[J].Meta Gene,2014,2:706-721.
[7]Chen J,Li R,Xia Y,et al.Development of EST-SSR markers in flowering Chinese cabbage(Brassica campestris L.ssp.chinensis var.utilis Tsen et Lee)based on de novo transcriptomic assemblies[J].PLoS ONE,2017,12(9):e0184736.
[8]Guo R,Landis J B,Moore M J,et al.Development and application of transcriptome-derived microsatellites in Actinidia eriantha(Actinidiaceae)[J].Frontiers in Plant Science,2017,8:1383.
[9]Zhang M Y,Fan L,Liu Q Z,et al.A novel set of EST-Derived SSR markers for pear and cross-species transferability in Rosaceae[J].Plant Molecular Biology Reporter,2014,32(1):1-13.
[10]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.
[11]Chen C,Ping Z,Choi Y A,et al.Mining and characterizing microsatellites from Citrus ESTs[J].Theoretical&Applied Genetics,2006,112(7):1248-1257.
[12]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.Huang H Y,Du H Y,Wu Y T N,et al.Development of SSRmolecular markers based on transcriptome sequencing of Eucommia ulmoides[J].Scientia Silvae Sinicae,2013,49(5):176-181.
[13]陈琪,杨华,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.Chen Q,Yang H,Wei C L,et al.Analysis on SSR information in Camellia sinensis transcriptome[J].Journal of Anhui Agricultural University,2011,38(6):882-886.
[14]Bassam B J,Caetano-Anollés G,Gresshoff P M.Fast and sensitive silver staining of DNA in polyacrylamide gels[J].Analytical Biochemistry,1991,196(1):80-83.
[15]Feng S,Zhao L,Liu Z,et al.De novo transcriptome assembly of Zanthoxylum bungeanum using illumina sequencing for evolutionary analysis and simple sequence repeat marker development[J].Scientific Reports,2017,7(1):16754.
[16]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.
[17]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.Fan H H,Li T C,Li Z P,et al.Characteristics of EST-SSRdistribution in ginkgo ESTs[J].Genomics and Applied Biology,2009,28(5):869-873.
[18]Kota R,Varshney R K,Thiel T,et al.Generation and comparison of EST-derived SSRs and SNPs in barley(Hordeum vulgare L.)[J].Hereditas,2001,135(2/3):145-151.
[19]Tóth G,Gáspári Z,Jurka J.Microsatellites in different eukaryotic genomes:survey and analysis[J].Genome Research,2000,10(7):967.
[20]潜宗伟,陈海丽,崔彦玲.菠菜转录组SSR位点分析及其分子标记的开发[J].农业生物技术学报,2016(11):1688-1697.Qian Z W,Chen H L,Cui Y L.Analysis of the SSR loci and development of molecular markers in Spinacia oleracea transcriptome[J].Journal of Agricultural Biotechnology,2016(11):1688-1697.
[21]Agarwal M,Shrivastava N,Padh H.Advances in molecular marker techniques and their applications in plant sciences[J].Plant Cell Reports,2008,27(4):617-631.
[22]Dreisigacker S,Zhang P,Warburton M L,et al.SSR and pedigree analyses of genetic diversity among CIMMYT wheat lines targeted to different mega environments[J].Crop Science,2004,44(2):381-388.
[23]Simon S A,Zhai J,Nandety R S,et al.Short-read sequencing technologies for transcriptional analyses[J].Annual Review of Plant Biology,2009,60(1):305.
[24]刘峰,王运生,田雪亮,等.辣椒转录组SSR挖掘及其多态性分析[J].园艺学报,2012,39(1):168-174.Liu F,Wang Y S,Tian X L,et al.SSR mining in pepper(Capsicum annuum L.)transcriptome and the polymorphism analysis[J].Acta Horticulturae Sinica,2012,39(1):168-174.
[25]Saha M C,Mian M A,Eujayl I,et al.Tall fescue EST-SSRmarkers with transferability across several grass species[J].Theoretical&Applied Genetics,2004,109(4):783-791.
[2]张华,叶萌.青花椒的分类地位及成分研究现状[J].北方园艺,2010(14):199-203.Zhang H,Ye M.Research status on the taxonomic and component of green Zanthoxylum bungeanum Maxim[J].Northern Horticulture,2010(14):199-203.
[3]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].甘肃农大学报,2011,46(6):46-51.Han S Z,Wang Y K,Li J,et al.ISSR genetic diversity analysis of Zanthoxylum bungeanumin Gansu province[J].Journal of Gansu Agricultural University,2011,46(6):46-51.
[4]李猛,王平,孙吉康,等.蚬壳花椒ISSR-PCR反应体系的建立及优化[J].广西植物,2013,33(2):185-190.Li M,Wang P,Sun J K,et al.Establishment and optimization of ISSR-PCR reaction system on Zanthoxylum dissitum[J].Guihaia,2013,33(2):185-190.
[5]邓洪平,徐洁,陈锋,等.九叶青花椒遗传多样性的形态与分子鉴定[J].西北植物学报,2008,28(10):2103-2109.Deng H P,Xu J,Chen F,et al.Morphological and molecular identification on genetic diversity of Zanthoxylum armatum var.Novemfolius[J].Acta Botanica Boreali-Occidentalia Sinica,2008,28(10):2103-2109.
[6]Medhi K,Sarmah D K,Deka M,et al.High gene flow and genetic diversity in three economically important Zanthoxylum spp.of upper Brahmaputra Valley Zone of NE India using molecular markers[J].Meta Gene,2014,2:706-721.
[7]Chen J,Li R,Xia Y,et al.Development of EST-SSR markers in flowering Chinese cabbage(Brassica campestris L.ssp.chinensis var.utilis Tsen et Lee)based on de novo transcriptomic assemblies[J].PLoS ONE,2017,12(9):e0184736.
[8]Guo R,Landis J B,Moore M J,et al.Development and application of transcriptome-derived microsatellites in Actinidia eriantha(Actinidiaceae)[J].Frontiers in Plant Science,2017,8:1383.
[9]Zhang M Y,Fan L,Liu Q Z,et al.A novel set of EST-Derived SSR markers for pear and cross-species transferability in Rosaceae[J].Plant Molecular Biology Reporter,2014,32(1):1-13.
[10]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.
[11]Chen C,Ping Z,Choi Y A,et al.Mining and characterizing microsatellites from Citrus ESTs[J].Theoretical&Applied Genetics,2006,112(7):1248-1257.
[12]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.Huang H Y,Du H Y,Wu Y T N,et al.Development of SSRmolecular markers based on transcriptome sequencing of Eucommia ulmoides[J].Scientia Silvae Sinicae,2013,49(5):176-181.
[13]陈琪,杨华,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.Chen Q,Yang H,Wei C L,et al.Analysis on SSR information in Camellia sinensis transcriptome[J].Journal of Anhui Agricultural University,2011,38(6):882-886.
[14]Bassam B J,Caetano-Anollés G,Gresshoff P M.Fast and sensitive silver staining of DNA in polyacrylamide gels[J].Analytical Biochemistry,1991,196(1):80-83.
[15]Feng S,Zhao L,Liu Z,et al.De novo transcriptome assembly of Zanthoxylum bungeanum using illumina sequencing for evolutionary analysis and simple sequence repeat marker development[J].Scientific Reports,2017,7(1):16754.
[16]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.
[17]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.Fan H H,Li T C,Li Z P,et al.Characteristics of EST-SSRdistribution in ginkgo ESTs[J].Genomics and Applied Biology,2009,28(5):869-873.
[18]Kota R,Varshney R K,Thiel T,et al.Generation and comparison of EST-derived SSRs and SNPs in barley(Hordeum vulgare L.)[J].Hereditas,2001,135(2/3):145-151.
[19]Tóth G,Gáspári Z,Jurka J.Microsatellites in different eukaryotic genomes:survey and analysis[J].Genome Research,2000,10(7):967.
[20]潜宗伟,陈海丽,崔彦玲.菠菜转录组SSR位点分析及其分子标记的开发[J].农业生物技术学报,2016(11):1688-1697.Qian Z W,Chen H L,Cui Y L.Analysis of the SSR loci and development of molecular markers in Spinacia oleracea transcriptome[J].Journal of Agricultural Biotechnology,2016(11):1688-1697.
[21]Agarwal M,Shrivastava N,Padh H.Advances in molecular marker techniques and their applications in plant sciences[J].Plant Cell Reports,2008,27(4):617-631.
[22]Dreisigacker S,Zhang P,Warburton M L,et al.SSR and pedigree analyses of genetic diversity among CIMMYT wheat lines targeted to different mega environments[J].Crop Science,2004,44(2):381-388.
[23]Simon S A,Zhai J,Nandety R S,et al.Short-read sequencing technologies for transcriptional analyses[J].Annual Review of Plant Biology,2009,60(1):305.
[24]刘峰,王运生,田雪亮,等.辣椒转录组SSR挖掘及其多态性分析[J].园艺学报,2012,39(1):168-174.Liu F,Wang Y S,Tian X L,et al.SSR mining in pepper(Capsicum annuum L.)transcriptome and the polymorphism analysis[J].Acta Horticulturae Sinica,2012,39(1):168-174.
[25]Saha M C,Mian M A,Eujayl I,et al.Tall fescue EST-SSRmarkers with transferability across several grass species[J].Theoretical&Applied Genetics,2004,109(4):783-791.