高通量RNA-seq技术开发文冠果目的基因SSR标记
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摘要
文冠果(Xanthoceras sorbifolium Bunge)是中国北方重要的集药用、食用、观赏和绿化于一身的多用途经济树种。利用MISA软件从文冠果根、叶、花和果实高通量转录组库的45 335条Unigenes中,搜索挖掘出12 606个SSR标记。在这些SSR标记中,有34.29%的单核苷酸重复、32.82%的二核苷酸重复、29.22%的三核苷酸重复、0.69%的四核苷酸重复、1.59%的五核苷酸重复和1.39%的六核苷酸重复。Unigenes中的408条涉及调控文冠果花色、叶色、果实开裂、成熟期、心室数、种子生长发育、大小和粒重等目的性状,从这些目的基因中共搜索挖掘到64个SSR标记,以辽冠1号叶片DNA为模板从这些SSR标记中共筛选出42对具有清晰扩增条带的SSR标记。以选取的5份表型性状差异明显的文冠果种质叶片为材料,通过对初筛的42对SSR引物的多态性筛选,获得了18对文冠果多态性SSR引物。本研究利用高通量RNA-seq开发的文冠果目的基因SSR标记,可为文冠果指纹图谱构建、遗传多样性分析和标记辅助选择育种等提供分子标记,对文冠果研究和开发利用具有重要意义。
Xanthoceras sorbifolium Bunge is one of important multi-purpose economic trees in the north of China,which includes medicinal, edible, ornamental and greening purpose. Using the MISA software, 12 606 SSR markers were excavated from 45 335 Unigenes of high throughput transcription groups of root, leaf, flower and fruit of X. sorbifolium. In these SSR markers, duplications of mononucleotide, dinucleotide, trinucleotide,tetranucleotide, pentanucleotide and hexanucleotide were 34.29%, 32.82%, 29.22%, 0.69%, 1.59% and 1.39%,respectively. 408 of Unigenes were involved in regulation of target traits of this species, such as flower color, leaf color, fruit cracking, maturity, ventricular number, seed growth and development, size and grain weigh. 64 SSR markers were found in these target genes. 42 pairs of SSR primers with clearly amplified bands were totally screened from these SSR markers using DNA of the line Liao Guan NO.1 as the template. Based on the test materials of five germplasm leaves of Xanthoceras sorbifolia with obviously different phenotypic traits, the polymorphism of 42 SSR markers in the initial sieve was detected, and 18 pairs of polymorphic SSR markers were therefore obtained. SSR markers of Xanthoceras sorbifolia target genes developed by high throughput RNA-seq could provide molecular markers for fingerprinting construction, genetic diversity analysis and marker-assisted selection breeding of X. sorbifolium, which would be important for studies and utilization of Xanthoceras sorbifolia.
Xanthoceras sorbifolium Bunge is one of important multi-purpose economic trees in the north of China,which includes medicinal, edible, ornamental and greening purpose. Using the MISA software, 12 606 SSR markers were excavated from 45 335 Unigenes of high throughput transcription groups of root, leaf, flower and fruit of X. sorbifolium. In these SSR markers, duplications of mononucleotide, dinucleotide, trinucleotide,tetranucleotide, pentanucleotide and hexanucleotide were 34.29%, 32.82%, 29.22%, 0.69%, 1.59% and 1.39%,respectively. 408 of Unigenes were involved in regulation of target traits of this species, such as flower color, leaf color, fruit cracking, maturity, ventricular number, seed growth and development, size and grain weigh. 64 SSR markers were found in these target genes. 42 pairs of SSR primers with clearly amplified bands were totally screened from these SSR markers using DNA of the line Liao Guan NO.1 as the template. Based on the test materials of five germplasm leaves of Xanthoceras sorbifolia with obviously different phenotypic traits, the polymorphism of 42 SSR markers in the initial sieve was detected, and 18 pairs of polymorphic SSR markers were therefore obtained. SSR markers of Xanthoceras sorbifolia target genes developed by high throughput RNA-seq could provide molecular markers for fingerprinting construction, genetic diversity analysis and marker-assisted selection breeding of X. sorbifolium, which would be important for studies and utilization of Xanthoceras sorbifolia.
引文
Bi Q.X.,Mao J.F.,and Guan W.B.,2015,Efficiently developing a large set of polymorphic EST-SSR markers for Xanthoceras sorbifolium by mining raw reads from high-throughput sequencing,Conservation Genet.Resour.,7(2):423-425
Cheng X.M.,and Huang X.X.,2011,Development and application of SSR markers in plants,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),27(5):304-307(程小毛,黄晓霞,2011,SSR标记开发及其在植物中的应用,中国农学通报,27(5):304-307)
Cheng Y.Q.,Wang H.W.,Wan H.X.,Duan J.M.,Zhang P.,and Han W.L.,2013,Construction and characterization of enriched microsatellite library from Xanthoceras sorbifolia,Henan Nongye Daxue Xuebao(Journal of Henan Agricultural University),47(4):440-445(程月琴,王红卫,万惠霞,段景勉,张佩,韩卫丽,2013,文冠果富集SSR文库的构建及特性分析,河南农业大学学报,47(4):440-445)
Li C.T.,Zhang G.H.,Ma C.H.,Meng Z.G.,Chen J.W.,and Yang S.C.,2014,Analysis on SSR loci information in transcriptome of Panax vienamensis var.fuscidiscus and its poly morph,Zhongcaoyao(Chinese Traditional and Herbal Drugs),45(10):1468-1472(李翠婷,张广辉,马春花,孟珍贵,陈军文,杨生超,2014,野三七转录组中SSR位点信息分析及其多态性研究,中草药,45(10):1468-1472)
Li Y.C.,and Meng H.,2009,Analysis of the characteristics and development prospects of Xanthoceras sorbifolia,Neimenggu Linye Diaocha Sheji(Inner Mongolia Forestry Investigation and Design),32(5):86-88(李永春,孟华,2009,文冠果的特性与发展前景浅析,内蒙古林业调查设计,32(5):86-88)
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)
Liu Y.L.,Huang Z.D.,Ao Y.,Li W.,and Zhang Z.,2013,Transcriptome analysis of yellow horn(Xanthoceras sorbifolia Bunge):a potential oil-rich seed tree for biodiesel in China,PLoS One,8(9):e74441
Liu Y.L.,Li W.,Dong S.B.,Mu X.Y.,and Zhang Z.X.,2017,Characterization of SSRs and development of EST-SSRmarkers in transcriptome of Xanthoceras sorbifolia Bunge,Xibei Nonglin Keji(Journal of Northwest A&F University),45(3):89-95(刘玉林,李伟,董树斌,沐先运,张志翔,2017,文冠果转录组SSR特征分析及EST-SSR标记开发,西北农林科技大学学报,45(3):89-95)
Luo D.,Wei W.L.,Zhou B.,and Li Y.H.,2012,Development of simple sequence repeats in Brassica rapa and poly-morphism analysis,Shengwu Jishu Tongxun(Letters in Biotech nology),23(3):402-406(罗丹,吴委林,周波,李玉花,2012,芜菁SSR引物的开发及多态性分析,生物技术通讯,23(3):402-406)
Ruan C.J.,Yan R.,Wang B.X.,Mopper S.,Guan W.K.,and Zhang J.,2017,The importance of yellow horn(Xanthoceras sorbifolia)for restoration of arid habitats and production of bioactive seed oils,Ecological Engineering,99:504-512
Saha M.C.,Mian M.A.R.,Eujayl I.,Zwonitzer J.C.,Wang L.J.,and May G.D.,2004,Tall fescue EST-SSR markers with transferability across several grass species,Theor.Appl.Genet.,109(4):783-791
Shen Z.,Ma L.Y.,Ao Y.,and Duan J.,2017,Analysis of the genetic diversity and construction of core collection of Xanthoceras sorbifolia Bunge using microsatellite marker data,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(8):3341-3350(申展,马履一,敖妍,段劼,2017,基于SSR标记的文冠果遗传多样性分析及核心种质构建,分子植物育种,15(8):3341-3350)
Tang D.H.,Ruan C.J.,Meng T.,and Ding J.,2017,Oil contents and fatty acid composition in different germplasm of Xanthoceras sorbifolia Bunge.,Zhongguo Youzhi(China Oils and Fats),42(3):77-81(唐东慧,阮成江,孟婷,丁健,2017,不同种质文冠果含油量及油中脂肪酸组成分析,中国油脂,42(3):77-81)
Wang L.W.,Wu S.C.,Kou Y.Y.,and Luo G.J.,2012,RAPDanalysis of germplasm resources of Xanthoceras sorbifolia,Liaoning Linye Keji(Journal of Liaoning Forestry Science&Technology),(1):11-12(王丽文,吴松权,寇莹莹,罗广军,2012,文冠果种质资源的RAPD分析,辽宁林业科技,(1):11-12)
Yan R.,Ruan C.J.,Du W.,Ding J.,Wu B.,and Liu L.Y.,2018,Development of SSR markers for target-genes derived from Camellia oleifera by RNA-seq technology,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(8):2540-2548(闫蕊,阮成江,杜维,丁健,吴波,刘祾悦,2018,RNA-seq技术开发油茶目的基因SSR标记,分子植物育种,16(8):2540-2548)
Cheng X.M.,and Huang X.X.,2011,Development and application of SSR markers in plants,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),27(5):304-307(程小毛,黄晓霞,2011,SSR标记开发及其在植物中的应用,中国农学通报,27(5):304-307)
Cheng Y.Q.,Wang H.W.,Wan H.X.,Duan J.M.,Zhang P.,and Han W.L.,2013,Construction and characterization of enriched microsatellite library from Xanthoceras sorbifolia,Henan Nongye Daxue Xuebao(Journal of Henan Agricultural University),47(4):440-445(程月琴,王红卫,万惠霞,段景勉,张佩,韩卫丽,2013,文冠果富集SSR文库的构建及特性分析,河南农业大学学报,47(4):440-445)
Li C.T.,Zhang G.H.,Ma C.H.,Meng Z.G.,Chen J.W.,and Yang S.C.,2014,Analysis on SSR loci information in transcriptome of Panax vienamensis var.fuscidiscus and its poly morph,Zhongcaoyao(Chinese Traditional and Herbal Drugs),45(10):1468-1472(李翠婷,张广辉,马春花,孟珍贵,陈军文,杨生超,2014,野三七转录组中SSR位点信息分析及其多态性研究,中草药,45(10):1468-1472)
Li Y.C.,and Meng H.,2009,Analysis of the characteristics and development prospects of Xanthoceras sorbifolia,Neimenggu Linye Diaocha Sheji(Inner Mongolia Forestry Investigation and Design),32(5):86-88(李永春,孟华,2009,文冠果的特性与发展前景浅析,内蒙古林业调查设计,32(5):86-88)
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)
Liu Y.L.,Huang Z.D.,Ao Y.,Li W.,and Zhang Z.,2013,Transcriptome analysis of yellow horn(Xanthoceras sorbifolia Bunge):a potential oil-rich seed tree for biodiesel in China,PLoS One,8(9):e74441
Liu Y.L.,Li W.,Dong S.B.,Mu X.Y.,and Zhang Z.X.,2017,Characterization of SSRs and development of EST-SSRmarkers in transcriptome of Xanthoceras sorbifolia Bunge,Xibei Nonglin Keji(Journal of Northwest A&F University),45(3):89-95(刘玉林,李伟,董树斌,沐先运,张志翔,2017,文冠果转录组SSR特征分析及EST-SSR标记开发,西北农林科技大学学报,45(3):89-95)
Luo D.,Wei W.L.,Zhou B.,and Li Y.H.,2012,Development of simple sequence repeats in Brassica rapa and poly-morphism analysis,Shengwu Jishu Tongxun(Letters in Biotech nology),23(3):402-406(罗丹,吴委林,周波,李玉花,2012,芜菁SSR引物的开发及多态性分析,生物技术通讯,23(3):402-406)
Ruan C.J.,Yan R.,Wang B.X.,Mopper S.,Guan W.K.,and Zhang J.,2017,The importance of yellow horn(Xanthoceras sorbifolia)for restoration of arid habitats and production of bioactive seed oils,Ecological Engineering,99:504-512
Saha M.C.,Mian M.A.R.,Eujayl I.,Zwonitzer J.C.,Wang L.J.,and May G.D.,2004,Tall fescue EST-SSR markers with transferability across several grass species,Theor.Appl.Genet.,109(4):783-791
Shen Z.,Ma L.Y.,Ao Y.,and Duan J.,2017,Analysis of the genetic diversity and construction of core collection of Xanthoceras sorbifolia Bunge using microsatellite marker data,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(8):3341-3350(申展,马履一,敖妍,段劼,2017,基于SSR标记的文冠果遗传多样性分析及核心种质构建,分子植物育种,15(8):3341-3350)
Tang D.H.,Ruan C.J.,Meng T.,and Ding J.,2017,Oil contents and fatty acid composition in different germplasm of Xanthoceras sorbifolia Bunge.,Zhongguo Youzhi(China Oils and Fats),42(3):77-81(唐东慧,阮成江,孟婷,丁健,2017,不同种质文冠果含油量及油中脂肪酸组成分析,中国油脂,42(3):77-81)
Wang L.W.,Wu S.C.,Kou Y.Y.,and Luo G.J.,2012,RAPDanalysis of germplasm resources of Xanthoceras sorbifolia,Liaoning Linye Keji(Journal of Liaoning Forestry Science&Technology),(1):11-12(王丽文,吴松权,寇莹莹,罗广军,2012,文冠果种质资源的RAPD分析,辽宁林业科技,(1):11-12)
Yan R.,Ruan C.J.,Du W.,Ding J.,Wu B.,and Liu L.Y.,2018,Development of SSR markers for target-genes derived from Camellia oleifera by RNA-seq technology,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(8):2540-2548(闫蕊,阮成江,杜维,丁健,吴波,刘祾悦,2018,RNA-seq技术开发油茶目的基因SSR标记,分子植物育种,16(8):2540-2548)