薄壳山核桃转录组中的SSR位点信息分析
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摘要
通过对薄壳山核桃转录组数据的分析,开发新的薄壳山核桃分子标记,为研究薄壳山核桃的遗传多样性、分子标记辅助育种提供科学依据。利用ESTtrimmer和cross-match对转录组测序获得的Unigene进行过滤,除去冗杂序列;利用MISA对无冗余Unigene进行SSR搜索,并应用Primer3进行SSR引物设计。从搜索序列中发现了10 303个SSR,分布于9 871条Unigenes中,出现频率为18.31%,分布密度为1/4.06 kb。二核苷酸和单核苷酸是主要重复类型,分别占SSR总数的44.27%和40.64%;优势重复基元为A/T、AG/CT和AAC/GTT,分别占单核苷酸的39.66%、二核苷酸的35.22%和三核苷酸的4.90%。88.86%的基序长度集中在10～20 bp。成功设计了5 547对SSR引物。通过对薄壳山核桃高通量转录组序列的SSR信息的研究,表明薄壳山核桃转录组SSR位点出现频率高、分布密度大,具有较高的多态性潜能,能提供丰富的重复类型,可以为研究薄壳山核桃的遗传多样性、分子标记辅助育种、遗传图谱绘制等方面提供有效的理论依据。
In order to develop new molecular markers of Carya illinoinensis, SSR markers were developed based on transcriptome data. This study lay foundations for studies of genetic diversity and molecular marker assisted breeding of C. illinoinensis. The Unigenes obtained from transcriptome sequencing were removed miscellaneous sequences by ESTtrimmer and cross-match. MISA was used to search SSR without redundant Unigene, and the SSR primer was designed by using primer3. We found that 10 303 SSRs distributed in 9 871 Unigenes were detected, accounting for 18.31% of all the Unigenes. The average distribution distance of the transcriptomic SSRs is about 1/4.06 kb. The dinucleotide and mononucleotide are the major repetitive types, accounting for 44.27% and40.64% of all the SSRs, respectively. The dominating motifs were A/T in mononucleotide repeats, AG/CT in dinucleotide repeats, and AAC/GTT in trinucleotide repeats, accounting for 39.66%, 35.22% and 4.90%,respectively. 88.86 percent of the motif length is centered in 10～20 bp. 5 547 pairs of SSR primers were successfully designed. The results showed that the SSR loci of the pecan transcriptome were higher in frequency,rich in type, large in density and higher in polymorphism potential. There is great potential for genetic diversity,molecular marker assisted breeding and genetic mapping.
In order to develop new molecular markers of Carya illinoinensis, SSR markers were developed based on transcriptome data. This study lay foundations for studies of genetic diversity and molecular marker assisted breeding of C. illinoinensis. The Unigenes obtained from transcriptome sequencing were removed miscellaneous sequences by ESTtrimmer and cross-match. MISA was used to search SSR without redundant Unigene, and the SSR primer was designed by using primer3. We found that 10 303 SSRs distributed in 9 871 Unigenes were detected, accounting for 18.31% of all the Unigenes. The average distribution distance of the transcriptomic SSRs is about 1/4.06 kb. The dinucleotide and mononucleotide are the major repetitive types, accounting for 44.27% and40.64% of all the SSRs, respectively. The dominating motifs were A/T in mononucleotide repeats, AG/CT in dinucleotide repeats, and AAC/GTT in trinucleotide repeats, accounting for 39.66%, 35.22% and 4.90%,respectively. 88.86 percent of the motif length is centered in 10～20 bp. 5 547 pairs of SSR primers were successfully designed. The results showed that the SSR loci of the pecan transcriptome were higher in frequency,rich in type, large in density and higher in polymorphism potential. There is great potential for genetic diversity,molecular marker assisted breeding and genetic mapping.
引文
Agarwal M.,Shrivastava N.,and Padh H.,2008,Advances in molecular marker techniques and their applications in plant sciences,Plant Cell Reports,27(4):617-631
Beedanagari S.R.,Dove S.K.,Wood B.W.,and Conner P.J.,2005,A first linkage map of pecan cultivars based on RAPDand AFLP markers,Theor.Appl.Genet.,110(6):1127-1137
Cai N.H.,Xu Y.L.,Xu Y.,Deng L.L.,Zhou L.,Wang D.W.,He C.Z.,and Duan A.A.,2015,The distribution and characteristics of SSR in transcriptome of Pinus yunnanensis,Yunnan Daxue Xuebao(Journal of Yunnan University),37(5):770-778(蔡年辉,许玉兰,徐杨,邓丽丽,周丽,王大玮,何承忠,段安安,2015,云南松转录组SSR的分布及其序列特征,云南大学学报,37(5):770-778)
Chen L.,Li L.N.,Yang G.D.,and Dai Y.P.,2016,Microsatellites characteristics of transcriptomic sequences from Osmanthus serrulatus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(4):959-965(陈林,李龙娜,杨国栋,戴亚平,2016,特有植物短丝木犀(Osmanthus serrulatus)转录组微卫星特征分析,分子植物育种,14(4):959-965)
Chen X.D.,Sun D.F.,Rong D.F.,Peng J.H.,and Li C.D.,2011,A recessive gene controlling male sterility sensitive to short day length/low temperature in wheat(Triticum aestivum L.),J.Zhejiang Univ.Sci.B,12(11):943-950
Du F.K.,Xu F.,Qu H.,Feng S.S.,Tang J.J.,and Wu R.L.,2013,Exploiting the transcriptome of Euphrates poplar,Populus euphratica(Salicaceae)to develop and characterize new E-ST-SSR markers and construct an EST-SSR database,PLoSOne,8(4):e61337
Fang Z.Z.,Ye X.F.,Zhou D.R.,Jiang C.C.,and Pan S.L.,2016,Analysis on SSR information in'Furongli'plum transcriptome and development of molecular markers in Prunus salicina Lindl.,Guoshu Xuebao(Journal of Fruit Science),33(4):416-424(方智振,叶新福,周丹蓉,姜翠翠,潘少霖,2016,'芙蓉李'转录组SSR信息分析与分子标记开发,果树学报,33(4):416-424)
Huang H.Y.,Du H.Y.,Wuyun T.N.,and Liu P.F.,2013,Development of SSR molecular markers based on transcrip tome sequencing of Eucommia ulmoides,Linye Kexue(Scientia Silvae Sinicae),49(5):176-181(黄海燕,杜红岩,乌云塔娜,刘攀峰,2013,基于杜仲转录组序列的SSR分子标记的开发,林业科学,49(5):176-181)
Jia X.D.,Wang T.,Zhai M.,Li Y.R.,and Guo Z.R.,2011,Genetic diversity and identification of Chinese-grown pecan using ISSR and SSR markers,Molecules,16(12):10078-10092
Jiang D.,Zhong G.Y.,and Qi-Bing H.,2006,Analysis of microsatellites in citrus unigenes,Acta Genetica Sinica,33(4):345-353
Jin J.Q.,Cui H.R.,Chen W.Y.,Lu M.Z.,Yao Y.L.,Xin Y.,and Gong X.C.,2006,Data mining for SSRs in ESTs and development of EST-SSR marker in tea plant(Camellia sinensis),Chaye Kexue(Journal of Tea Science),26(1):17-23(金基强,崔海瑞,陈文岳,卢美贞,姚艳玲,忻雅,龚晓春,2006,茶树EST-SSR的信息分析与标记建立,茶叶科学,26(1):17-23)
Kalia R.K.,Rai M.K.,Kalia S.,Singh R.,and Dhawan A.K.,2011,Microsatellite markers:an overview of the recent progress in plants,Euphytica,177(3):309-334
Kantety R.V.,Rota M.L.,Matthews D.E.,and Sorrells M.E.,2002,Data mining for simple sequence repeats in expressed sequence tags from barley,maize,rice,sorghum and wheat,Plant Molecular Biology,48(5-6):501-510
Li S.X.,Zhang X.Y.,Wang Y.Y.,and Yin T.M.,2010,Content and characteristics of microsatellites detected in expressed sequence tag sequences in Eucalyptus,Zhiwuxue Tongbao(Chinese Bulletin of Botany),45(3):363-371(李淑娴,张新叶,王英亚,尹佟明,2010,桉树EST序列中微卫星含量及相关特征,植物学通报,45(3):363-371)
Li X.,Yang N.,Zhao K.G.,Chen Y.X.,Tang R.J.,and Chen L.Q.,2013,Development and primer selection of EST-SSRmolecular markers based on transcriptome sequencing of Chimonanthus praecox,Beijing Linye Daxue Xuebao(Jour nal of Beijing Forestry University),35(S1):25-32(李响,杨楠,赵凯歌,陈玉星,唐锐君,陈龙清,2013,蜡梅转录组EST-SSR标记开发与引物筛选,北京林业大学学报,35(S1):25-32)
Li X.Y.,Xu H.X.,Feng J.J.,and Chen J.W.,2014,Development and application of genic simple sequence repeat markers from the transcriptome of loquat,Journal of the American Society for Horticultural Science,139(5):507-517
Li Y.L.,Yang X.X.,Zhang J.Y.,Huang S.W.,and Xiong X.Y.,2014,Studies on SSR molecular markers based on transcriptome of Taxus chinensis var.mairei,Yuanyi Xuebao(Acta Horticulturae Sinica),41(4):735-745(李炎林,杨星星,张家银,黄三文,熊兴耀,2014,南方红豆杉转录组SSR挖掘及分子标记的研究,园艺学报,41(4):735-745)
Ma Q.Y.,Liao Z.Y.,Zhang D.F.,Dai X.G.,Chen Y.N.,and Li S.X.,2015,Deep sequenced-based transcriptome analysis of microsatellites in peach(Prunus persica cv.duplex)flowers,Nanjing Linye Daxue Xuebao(Journal of Nanjing Forestry University(Natural Sciences Edition)),39(3):34-38(马秋月,廖卓毅,张得芳,戴晓港,陈赢男,李淑娴,2015,碧桃花瓣转录组微卫星特征分析,南京林业大学学报(自然科学版),39(3):34-38)
Sui Y.,2005,Mutation mechanisms and evolutionary patterns of microsatellite sequences in common almond(Prunus Amygdalus L.)and other Prunus species,Thesis for M.S.,Capi tal Normal University,Supervisor:Ma R.C.,and Xie H.,pp.28-30(隋毅,2005,扁桃及其它李属物种微卫星序列(SSR)突变机制和进化模式分析,硕士学位论文,首都师范大学,导师:马荣才,谢华,pp.28-30)
Thao D.V.,Yamashita M.,Watanabe A.,and Shiraishi S.,2013,Development of tetranucleotide microsatellite markers in Pinus kesiya Royle ex Gordon,Conservation Genetics Resources,5(2):405-407
Villarreal-Lozoya J.E.,Lombardini L.,and Cisneros-Zevallos L.,2007,Phytochemical constituents and antioxidant capacity of different pecan(Carya illinoinensis(Wangenh.)K.Koch)cultivars,Food Chemistry,102(4):1241-1249
Wang Y.S.,Nie F.,and Lin S.Q.,2015,The latest research progress in high-throughput sequencing of fruit tree,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(9):2034-2043(王云生,聂飞,林顺权,2015,果树高通量测序的最新研究进展,基因组学与应用生物学,34(9):2034-2043)
Wen Y.F.,Han W.J.,Zhou H.,and Xu G.B.,2015,SSR mining and development of EST-SSR markers for Cunninghamia lanceolata based on transcriptome sequences,Linye Kexue(Scientia Silvae Sinicae),51(11):40-49(文亚峰,韩文军,周宏,徐刚标,2015,杉木转录组SSR挖掘及EST-SSR标记规模化开发,林业科学,51(11):40-49)
Wood B.W.,2000,Pollination characteristics of pecan trees and orchards,Horttechnology,10(1):120-126
Xu M.,and Li H.G.,2008,Development and characterization of microsatellite markers from expressed sequence tags for liriodendron,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),6(3):615-618(胥猛,李火根,2008,鹅掌楸EST-SSR引物开发及通用性分析,分子植物育种,6(3):615-618)
Xu X.,Peng M.,Fang Z.,and Xu X.P.,2000,The direction of microsatellite mutations is dependent upon allele length,Nature Genetics,24(4):396-399
Yang X.D.,Tan H.W.,and Zhu W.M.,2016,SpinachDB:Awell-characterized genomic database for gene family classification and snp information of spinach,PLoS One,11(5):e0152706
Zhang G.W.,Xu S.C.,Mao W.H.,Hu Q.Z.,and Gong Y.M.,2013,Determination of the genetic diversity of vegetable soybean(Glycine max(L.)Merr.)using EST-SSR markers,J.Zhejiang Univ.Sci.B,14(4):279-288
Zhang Q.T.,Li X.Y.,Yang Y.M.,Fan S.T.,and Ai J.,2016,Analysis on SSR information in transcriptome and development of molecular markers in Lonicera caerulea,Yuanyi Xuebao(Acta Horticulturae Sinica),43(3):557-563(张庆田,李晓艳,杨义明,范书田,艾军,2016,蓝靛果忍冬转录组SSR信息分析及其分子标记开发,园艺学报,43(3):557-563)
Zhang T.F.,Qi W.C.,Gu M.F.,Zhang X.L.,Li T.,and Zhao H.,2016,Exploration and transferability evaluation of EST-SS-Rs in quinoa,Zuowu Xuebao(Acta Agronomica Sinica),42(4):492-500(张体付,戚维聪,顾闽峰,张晓林,李坦,赵涵,2016,藜麦EST-SSR的开发及通用性分析,作物学报,42(4):492-500)
Beedanagari S.R.,Dove S.K.,Wood B.W.,and Conner P.J.,2005,A first linkage map of pecan cultivars based on RAPDand AFLP markers,Theor.Appl.Genet.,110(6):1127-1137
Cai N.H.,Xu Y.L.,Xu Y.,Deng L.L.,Zhou L.,Wang D.W.,He C.Z.,and Duan A.A.,2015,The distribution and characteristics of SSR in transcriptome of Pinus yunnanensis,Yunnan Daxue Xuebao(Journal of Yunnan University),37(5):770-778(蔡年辉,许玉兰,徐杨,邓丽丽,周丽,王大玮,何承忠,段安安,2015,云南松转录组SSR的分布及其序列特征,云南大学学报,37(5):770-778)
Chen L.,Li L.N.,Yang G.D.,and Dai Y.P.,2016,Microsatellites characteristics of transcriptomic sequences from Osmanthus serrulatus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(4):959-965(陈林,李龙娜,杨国栋,戴亚平,2016,特有植物短丝木犀(Osmanthus serrulatus)转录组微卫星特征分析,分子植物育种,14(4):959-965)
Chen X.D.,Sun D.F.,Rong D.F.,Peng J.H.,and Li C.D.,2011,A recessive gene controlling male sterility sensitive to short day length/low temperature in wheat(Triticum aestivum L.),J.Zhejiang Univ.Sci.B,12(11):943-950
Du F.K.,Xu F.,Qu H.,Feng S.S.,Tang J.J.,and Wu R.L.,2013,Exploiting the transcriptome of Euphrates poplar,Populus euphratica(Salicaceae)to develop and characterize new E-ST-SSR markers and construct an EST-SSR database,PLoSOne,8(4):e61337
Fang Z.Z.,Ye X.F.,Zhou D.R.,Jiang C.C.,and Pan S.L.,2016,Analysis on SSR information in'Furongli'plum transcriptome and development of molecular markers in Prunus salicina Lindl.,Guoshu Xuebao(Journal of Fruit Science),33(4):416-424(方智振,叶新福,周丹蓉,姜翠翠,潘少霖,2016,'芙蓉李'转录组SSR信息分析与分子标记开发,果树学报,33(4):416-424)
Huang H.Y.,Du H.Y.,Wuyun T.N.,and Liu P.F.,2013,Development of SSR molecular markers based on transcrip tome sequencing of Eucommia ulmoides,Linye Kexue(Scientia Silvae Sinicae),49(5):176-181(黄海燕,杜红岩,乌云塔娜,刘攀峰,2013,基于杜仲转录组序列的SSR分子标记的开发,林业科学,49(5):176-181)
Jia X.D.,Wang T.,Zhai M.,Li Y.R.,and Guo Z.R.,2011,Genetic diversity and identification of Chinese-grown pecan using ISSR and SSR markers,Molecules,16(12):10078-10092
Jiang D.,Zhong G.Y.,and Qi-Bing H.,2006,Analysis of microsatellites in citrus unigenes,Acta Genetica Sinica,33(4):345-353
Jin J.Q.,Cui H.R.,Chen W.Y.,Lu M.Z.,Yao Y.L.,Xin Y.,and Gong X.C.,2006,Data mining for SSRs in ESTs and development of EST-SSR marker in tea plant(Camellia sinensis),Chaye Kexue(Journal of Tea Science),26(1):17-23(金基强,崔海瑞,陈文岳,卢美贞,姚艳玲,忻雅,龚晓春,2006,茶树EST-SSR的信息分析与标记建立,茶叶科学,26(1):17-23)
Kalia R.K.,Rai M.K.,Kalia S.,Singh R.,and Dhawan A.K.,2011,Microsatellite markers:an overview of the recent progress in plants,Euphytica,177(3):309-334
Kantety R.V.,Rota M.L.,Matthews D.E.,and Sorrells M.E.,2002,Data mining for simple sequence repeats in expressed sequence tags from barley,maize,rice,sorghum and wheat,Plant Molecular Biology,48(5-6):501-510
Li S.X.,Zhang X.Y.,Wang Y.Y.,and Yin T.M.,2010,Content and characteristics of microsatellites detected in expressed sequence tag sequences in Eucalyptus,Zhiwuxue Tongbao(Chinese Bulletin of Botany),45(3):363-371(李淑娴,张新叶,王英亚,尹佟明,2010,桉树EST序列中微卫星含量及相关特征,植物学通报,45(3):363-371)
Li X.,Yang N.,Zhao K.G.,Chen Y.X.,Tang R.J.,and Chen L.Q.,2013,Development and primer selection of EST-SSRmolecular markers based on transcriptome sequencing of Chimonanthus praecox,Beijing Linye Daxue Xuebao(Jour nal of Beijing Forestry University),35(S1):25-32(李响,杨楠,赵凯歌,陈玉星,唐锐君,陈龙清,2013,蜡梅转录组EST-SSR标记开发与引物筛选,北京林业大学学报,35(S1):25-32)
Li X.Y.,Xu H.X.,Feng J.J.,and Chen J.W.,2014,Development and application of genic simple sequence repeat markers from the transcriptome of loquat,Journal of the American Society for Horticultural Science,139(5):507-517
Li Y.L.,Yang X.X.,Zhang J.Y.,Huang S.W.,and Xiong X.Y.,2014,Studies on SSR molecular markers based on transcriptome of Taxus chinensis var.mairei,Yuanyi Xuebao(Acta Horticulturae Sinica),41(4):735-745(李炎林,杨星星,张家银,黄三文,熊兴耀,2014,南方红豆杉转录组SSR挖掘及分子标记的研究,园艺学报,41(4):735-745)
Ma Q.Y.,Liao Z.Y.,Zhang D.F.,Dai X.G.,Chen Y.N.,and Li S.X.,2015,Deep sequenced-based transcriptome analysis of microsatellites in peach(Prunus persica cv.duplex)flowers,Nanjing Linye Daxue Xuebao(Journal of Nanjing Forestry University(Natural Sciences Edition)),39(3):34-38(马秋月,廖卓毅,张得芳,戴晓港,陈赢男,李淑娴,2015,碧桃花瓣转录组微卫星特征分析,南京林业大学学报(自然科学版),39(3):34-38)
Sui Y.,2005,Mutation mechanisms and evolutionary patterns of microsatellite sequences in common almond(Prunus Amygdalus L.)and other Prunus species,Thesis for M.S.,Capi tal Normal University,Supervisor:Ma R.C.,and Xie H.,pp.28-30(隋毅,2005,扁桃及其它李属物种微卫星序列(SSR)突变机制和进化模式分析,硕士学位论文,首都师范大学,导师:马荣才,谢华,pp.28-30)
Thao D.V.,Yamashita M.,Watanabe A.,and Shiraishi S.,2013,Development of tetranucleotide microsatellite markers in Pinus kesiya Royle ex Gordon,Conservation Genetics Resources,5(2):405-407
Villarreal-Lozoya J.E.,Lombardini L.,and Cisneros-Zevallos L.,2007,Phytochemical constituents and antioxidant capacity of different pecan(Carya illinoinensis(Wangenh.)K.Koch)cultivars,Food Chemistry,102(4):1241-1249
Wang Y.S.,Nie F.,and Lin S.Q.,2015,The latest research progress in high-throughput sequencing of fruit tree,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(9):2034-2043(王云生,聂飞,林顺权,2015,果树高通量测序的最新研究进展,基因组学与应用生物学,34(9):2034-2043)
Wen Y.F.,Han W.J.,Zhou H.,and Xu G.B.,2015,SSR mining and development of EST-SSR markers for Cunninghamia lanceolata based on transcriptome sequences,Linye Kexue(Scientia Silvae Sinicae),51(11):40-49(文亚峰,韩文军,周宏,徐刚标,2015,杉木转录组SSR挖掘及EST-SSR标记规模化开发,林业科学,51(11):40-49)
Wood B.W.,2000,Pollination characteristics of pecan trees and orchards,Horttechnology,10(1):120-126
Xu M.,and Li H.G.,2008,Development and characterization of microsatellite markers from expressed sequence tags for liriodendron,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),6(3):615-618(胥猛,李火根,2008,鹅掌楸EST-SSR引物开发及通用性分析,分子植物育种,6(3):615-618)
Xu X.,Peng M.,Fang Z.,and Xu X.P.,2000,The direction of microsatellite mutations is dependent upon allele length,Nature Genetics,24(4):396-399
Yang X.D.,Tan H.W.,and Zhu W.M.,2016,SpinachDB:Awell-characterized genomic database for gene family classification and snp information of spinach,PLoS One,11(5):e0152706
Zhang G.W.,Xu S.C.,Mao W.H.,Hu Q.Z.,and Gong Y.M.,2013,Determination of the genetic diversity of vegetable soybean(Glycine max(L.)Merr.)using EST-SSR markers,J.Zhejiang Univ.Sci.B,14(4):279-288
Zhang Q.T.,Li X.Y.,Yang Y.M.,Fan S.T.,and Ai J.,2016,Analysis on SSR information in transcriptome and development of molecular markers in Lonicera caerulea,Yuanyi Xuebao(Acta Horticulturae Sinica),43(3):557-563(张庆田,李晓艳,杨义明,范书田,艾军,2016,蓝靛果忍冬转录组SSR信息分析及其分子标记开发,园艺学报,43(3):557-563)
Zhang T.F.,Qi W.C.,Gu M.F.,Zhang X.L.,Li T.,and Zhao H.,2016,Exploration and transferability evaluation of EST-SS-Rs in quinoa,Zuowu Xuebao(Acta Agronomica Sinica),42(4):492-500(张体付,戚维聪,顾闽峰,张晓林,李坦,赵涵,2016,藜麦EST-SSR的开发及通用性分析,作物学报,42(4):492-500)