Development of SSR Molecular Markers Based on Transcriptome Sequences of Alnus

RAO Long-bing; YANG Han-bo; GUO Hong-ying; DUAN Hong-ping; CHEN Yi-tai

Volume 29 Issue 6

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Article Navigation > Forest Research > 2016 > 29(6): 875-882

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Development of SSR Molecular Markers Based on Transcriptome Sequences of Alnus

1.
Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, Zhejiang, China
2.
Sichuan Academy of Forestry, Chengdu 610081, Sichuan, China
3.
College of Resources and Environment, Yunnan Agricultural University, Kunming 650201, Yunnan, China

Received Date: 2016-03-25

Abstract

[Objective] To develop SSR markers of gene transcriptional areas from Alnus based on the results of transcriptome sequencing. [Methods] Distribution patterns of the markers in the transcriptome sequences and their characteristics were analyzed, in order to provide more powerful tools for molecular marker-assisted breeding Alnus. The SSR locus from transcriptome sequences were searched by MicroSAtellite (MISA), and statistical analyses were conducted for the amount, distribution and characteristics of SSR loci. 100 pairs of SSR primers were designed and synthesized. Agarose electrophoresis was used for initial check and capillary electrophoresis for separation and detection of the polymorphism of the primers. [Results] A total of 8 298 Unigenes containing 8 678 SSR locus were searched from 85 769 Unigenes by MicroSAtellite (MISA) sequence analysis software, accounting for 9.67% of the transcriptome sequences, with an average of one SSR per 14.04kb. The dinucleotide repeat is the most abundant repeat type, accounting for 65.87% of the total number of SSRs. Additionally, a small amount of GC repeats was found. A total of 4 531 pairs of primers were designed by Primer 3 software according to the Unigene sequences of transcriptional. 18 pairs of primers produced bands with expected sizes were selected from 100 pairs of primers. [Conclusion] The amplified primers of the polymorphism loci were mainly dinucleotide and trinucleotide repeats. This study has an important value to develop SSR molecular markers based on the transcriptome sequencing analysis. This study is also important for analyzing genetic diversity, marker assisted selection, genetic linkage mapping and functional gene mining of Alnus by using SSR molecular markers.
- Alnus,
- transcriptome,
- SSR,
- primer

References

[1]	于东阳, 梅芳, 王军辉, 等. 不同种源桤木制浆造纸性能的遗传变异[J]. 河南农业大学学报, 2013, 47(6):703-709.
[2]	饶龙兵, 杨汉波, 郭洪英, 等. 17种桤木属植物的亲缘关系研究及模糊种鉴定[J]. 植物研究, 2015(4):528-534.
[3]	杨汉波, 饶龙兵, 郭洪英, 等. 5种桤木属植物的核型分析[J]. 植物遗传资源学报, 2013, 14(6):1203-1207.
[4]	刘军. 桤木和四川桂花的形态、细胞及分子分类学研究. 雅安:四川农业大学, 2006.
[5]	饶龙兵, 杨汉波, 郭洪英, 等. 桤木属7种植物的核型分析[J]. 西北植物学报, 2013, 33(7):1333-1338.
[6]	李秀媛, 刘西平, Hang DUONG, 等. 美国海滨桤木和薄叶桤木水分生理特性的比较[J]. 植物生态学报, 2011, 35(1):73-81.
[7]	饶龙兵, 杨汉波, 郭洪英,等. 桤木属植物AFLP反应体系的建立与优化[J]. 分子植物育种, 2014(3):547-553.
[8]	饶龙兵, 杨汉波, 郭洪英, 等. 不同倍性桤木属植物遗传差异的AFLP分析[J]. 植物研究, 2014(6):803-809.
[9]	夏勇. 江南桤木遗传多样性研究. 南京:南京林业大学, 2012.
[10]	Kalia R K, Rai M K, Kalia S, et al. Microsatellite markers: An overview of the recent progress in plants [J].Euphytica, 2011, 177(3):309-334.
[11]	李翠婷, 张广辉, 马春花, 等. 野三七转录组中SSR位点信息分析及其多态性研究[J]. 中草药, 2014, 45(10):1468-1472.
[12]	Morgante M, Olivieri A M. PCR-amplified microsatellites as markers in plant genetics [J]. Plant Journal for Cell & Molecular Biology, 1993, 3(1):175-182.
[13]	Milee A, Neeta S, Harish P. Advances in molecular marker techniques and their applications in plant sciences[J]. Plant Cell Reports, 2008, 27(4):617-631.
[14]	程小毛, 黄晓霞. SSR标记开发及其在植物中的应用[J]. 中国农学通报, 2011, 27(5):304-307.
[15]	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):305-333.
[16]	李炎林, 杨星星, 张家银, 等. 南方红豆杉转录组SSR挖掘及分子标记的研究[J]. 园艺学报, 2014, 41(4):735-745.
[17]	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.
[18]	赵凯歌, 陈玉星, 唐锐君, 等. 蜡梅转录组 EST-SSR 标记开发与引物筛选[J]. 北京林业大学学报, 2013(S1):25-32.
[19]	张振, 张含国, 莫迟, 等. 红松转录组SSR分析及EST-SSR标记开发[J]. 林业科学, 2015(8):114-120.
[20]	Drašnarová A, Krak K, Vít P, et al. Cross-amplification and multiplexing of SSR markers for Alnus glutinosa and A. incana[J]. Tree Genetics & Genomes, 2014, 10(4):865-873.
[21]	Lepais O. De novo discovery and multiplexed amplification of microsatellite markers for black alder(Alnus glutinosa)and related species using SSR-enriched shotgun pyrosequencing [J]. Journal of Heredity, 2011, 102(5):627-632.
[22]	Kalinowski S T, Taper M L, Marshall T C. Revising how the conputer program CERVUS accommondates genotyping error increases success in paternity assignment [J]. Molecular Breeding, 2007, 16(5): 1099-1106.
[23]	Bradbury P J, Zhang Z, Kroon D E, et al. TASSEL: software for association mapping of complex traits in diverse samples [J]. Bioinform, 2007, 23(19): 2633-2635.
[24]	Peakall R, Smouse P E. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research-an update [J]. Bioinformatics, 28(19): 2537-2539.
[25]	Rohlf F J. NYSYS pc, numerical taxonomy and multivariate analysis system, version 2.1. Setauket, NY: Exeter Publishing, Ltd.,2000
[26]	李小白, 向林, 罗洁, 等. 转录组测序(RNA-seq)策略及其数据在分子标记开发上的应用[J]. 中国细胞生物学学报, 2013(5):720-726.
[27]	Jiang D, Zhong G Y, Hong Q B. Analysis of Microsatellites in Citrus Unigenes[J]. Investigación Agraria Sistemas Y Recursos Forestales, 2000, 9(1):233-254.
[28]	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.
[29]	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.
[30]	鄢秀芹, 鲁敏, 安华明. 刺梨转录组SSR信息分析及其分子标记开发[J]. 园艺学报, 2015, 42(2):341-349.
[31]	姚刘慧, 唐翠明, 戴凡炜, 等. 基于桑树转录组测序的SSR标记开发和引物筛选[J]. 蚕业科学, 2015(1):18-27.
[32]	郁永明, 田丹青, 潘晓韵, 等. 基于红掌转录组序列的SSR标记分析与开发[J]. 分子植物育种, 2015(6):1349-1354.
[33]	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.
[34]	Saha M C, Mian M A R, Imad E, et al. Tall fescue EST-SSR markers with transferability across several grass species [J]. Tagtheoretical & Applied Geneticstheoretische Und Angewandte Genetik, 2004, 109(4):783-791.
[35]	Dreisigacker S, Zhang P, Warburton M L, et al. SSR and pedigree analyses of genetic diversity among CIMMYT wheat lines targeted to different megaenvironments [J]. Crop Science, 2004, 44(2):381-388.
[36]	文亚峰,韩文军,周宏,等. 杉木转录组SSR挖掘及EST-SSR标记规模化开发[J]. 林业科学, 2015, 51(11): 40-49.
[37]	Duran C, Singhania R, Raman H, et al. Predicting polymorphic EST-SSRs in silico[J]. Molecular Ecology Resources, 2013, 13(3): 538-545.
[38]	Blacket M J, Robin C, Good R T, et al. Universal primers for fluorescent labelling of PCR fragments-an efficient and cost-effective approach to genotyping by fluorescence[J]. Molecular Ecology Resources, 2012, 12(3): 456-463.

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Development of SSR Molecular Markers Based on Transcriptome Sequences of Alnus

1. Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, Zhejiang, China

2. Sichuan Academy of Forestry, Chengdu 610081, Sichuan, China

3. College of Resources and Environment, Yunnan Agricultural University, Kunming 650201, Yunnan, China

Received Date: 2016-03-25

Abstract: [Objective] To develop SSR markers of gene transcriptional areas from Alnus based on the results of transcriptome sequencing. [Methods] Distribution patterns of the markers in the transcriptome sequences and their characteristics were analyzed, in order to provide more powerful tools for molecular marker-assisted breeding Alnus. The SSR locus from transcriptome sequences were searched by MicroSAtellite (MISA), and statistical analyses were conducted for the amount, distribution and characteristics of SSR loci. 100 pairs of SSR primers were designed and synthesized. Agarose electrophoresis was used for initial check and capillary electrophoresis for separation and detection of the polymorphism of the primers. [Results] A total of 8 298 Unigenes containing 8 678 SSR locus were searched from 85 769 Unigenes by MicroSAtellite (MISA) sequence analysis software, accounting for 9.67% of the transcriptome sequences, with an average of one SSR per 14.04kb. The dinucleotide repeat is the most abundant repeat type, accounting for 65.87% of the total number of SSRs. Additionally, a small amount of GC repeats was found. A total of 4 531 pairs of primers were designed by Primer 3 software according to the Unigene sequences of transcriptional. 18 pairs of primers produced bands with expected sizes were selected from 100 pairs of primers. [Conclusion] The amplified primers of the polymorphism loci were mainly dinucleotide and trinucleotide repeats. This study has an important value to develop SSR molecular markers based on the transcriptome sequencing analysis. This study is also important for analyzing genetic diversity, marker assisted selection, genetic linkage mapping and functional gene mining of Alnus by using SSR molecular markers.

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Reference (38)

[1]	于东阳, 梅芳, 王军辉, 等. 不同种源桤木制浆造纸性能的遗传变异[J]. 河南农业大学学报, 2013, 47(6):703-709.
[2]	饶龙兵, 杨汉波, 郭洪英, 等. 17种桤木属植物的亲缘关系研究及模糊种鉴定[J]. 植物研究, 2015(4):528-534.
[3]	杨汉波, 饶龙兵, 郭洪英, 等. 5种桤木属植物的核型分析[J]. 植物遗传资源学报, 2013, 14(6):1203-1207.
[4]	刘军. 桤木和四川桂花的形态、细胞及分子分类学研究. 雅安:四川农业大学, 2006.
[5]	饶龙兵, 杨汉波, 郭洪英, 等. 桤木属7种植物的核型分析[J]. 西北植物学报, 2013, 33(7):1333-1338.
[6]	李秀媛, 刘西平, Hang DUONG, 等. 美国海滨桤木和薄叶桤木水分生理特性的比较[J]. 植物生态学报, 2011, 35(1):73-81.
[7]	饶龙兵, 杨汉波, 郭洪英,等. 桤木属植物AFLP反应体系的建立与优化[J]. 分子植物育种, 2014(3):547-553.
[8]	饶龙兵, 杨汉波, 郭洪英, 等. 不同倍性桤木属植物遗传差异的AFLP分析[J]. 植物研究, 2014(6):803-809.
[9]	夏勇. 江南桤木遗传多样性研究. 南京:南京林业大学, 2012.
[10]	Kalia R K, Rai M K, Kalia S, et al. Microsatellite markers: An overview of the recent progress in plants [J].Euphytica, 2011, 177(3):309-334.
[11]	李翠婷, 张广辉, 马春花, 等. 野三七转录组中SSR位点信息分析及其多态性研究[J]. 中草药, 2014, 45(10):1468-1472.
[12]	Morgante M, Olivieri A M. PCR-amplified microsatellites as markers in plant genetics [J]. Plant Journal for Cell & Molecular Biology, 1993, 3(1):175-182.
[13]	Milee A, Neeta S, Harish P. Advances in molecular marker techniques and their applications in plant sciences[J]. Plant Cell Reports, 2008, 27(4):617-631.
[14]	程小毛, 黄晓霞. SSR标记开发及其在植物中的应用[J]. 中国农学通报, 2011, 27(5):304-307.
[15]	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):305-333.
[16]	李炎林, 杨星星, 张家银, 等. 南方红豆杉转录组SSR挖掘及分子标记的研究[J]. 园艺学报, 2014, 41(4):735-745.
[17]	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.
[18]	赵凯歌, 陈玉星, 唐锐君, 等. 蜡梅转录组 EST-SSR 标记开发与引物筛选[J]. 北京林业大学学报, 2013(S1):25-32.
[19]	张振, 张含国, 莫迟, 等. 红松转录组SSR分析及EST-SSR标记开发[J]. 林业科学, 2015(8):114-120.
[20]	Drašnarová A, Krak K, Vít P, et al. Cross-amplification and multiplexing of SSR markers for Alnus glutinosa and A. incana[J]. Tree Genetics & Genomes, 2014, 10(4):865-873.
[21]	Lepais O. De novo discovery and multiplexed amplification of microsatellite markers for black alder(Alnus glutinosa)and related species using SSR-enriched shotgun pyrosequencing [J]. Journal of Heredity, 2011, 102(5):627-632.
[22]	Kalinowski S T, Taper M L, Marshall T C. Revising how the conputer program CERVUS accommondates genotyping error increases success in paternity assignment [J]. Molecular Breeding, 2007, 16(5): 1099-1106.
[23]	Bradbury P J, Zhang Z, Kroon D E, et al. TASSEL: software for association mapping of complex traits in diverse samples [J]. Bioinform, 2007, 23(19): 2633-2635.
[24]	Peakall R, Smouse P E. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research-an update [J]. Bioinformatics, 28(19): 2537-2539.
[25]	Rohlf F J. NYSYS pc, numerical taxonomy and multivariate analysis system, version 2.1. Setauket, NY: Exeter Publishing, Ltd.,2000
[26]	李小白, 向林, 罗洁, 等. 转录组测序(RNA-seq)策略及其数据在分子标记开发上的应用[J]. 中国细胞生物学学报, 2013(5):720-726.
[27]	Jiang D, Zhong G Y, Hong Q B. Analysis of Microsatellites in Citrus Unigenes[J]. Investigación Agraria Sistemas Y Recursos Forestales, 2000, 9(1):233-254.
[28]	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.
[29]	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.
[30]	鄢秀芹, 鲁敏, 安华明. 刺梨转录组SSR信息分析及其分子标记开发[J]. 园艺学报, 2015, 42(2):341-349.
[31]	姚刘慧, 唐翠明, 戴凡炜, 等. 基于桑树转录组测序的SSR标记开发和引物筛选[J]. 蚕业科学, 2015(1):18-27.
[32]	郁永明, 田丹青, 潘晓韵, 等. 基于红掌转录组序列的SSR标记分析与开发[J]. 分子植物育种, 2015(6):1349-1354.
[33]	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.
[34]	Saha M C, Mian M A R, Imad E, et al. Tall fescue EST-SSR markers with transferability across several grass species [J]. Tagtheoretical & Applied Geneticstheoretische Und Angewandte Genetik, 2004, 109(4):783-791.
[35]	Dreisigacker S, Zhang P, Warburton M L, et al. SSR and pedigree analyses of genetic diversity among CIMMYT wheat lines targeted to different megaenvironments [J]. Crop Science, 2004, 44(2):381-388.
[36]	文亚峰,韩文军,周宏,等. 杉木转录组SSR挖掘及EST-SSR标记规模化开发[J]. 林业科学, 2015, 51(11): 40-49.
[37]	Duran C, Singhania R, Raman H, et al. Predicting polymorphic EST-SSRs in silico[J]. Molecular Ecology Resources, 2013, 13(3): 538-545.
[38]	Blacket M J, Robin C, Good R T, et al. Universal primers for fluorescent labelling of PCR fragments-an efficient and cost-effective approach to genotyping by fluorescence[J]. Molecular Ecology Resources, 2012, 12(3): 456-463.

Development of SSR Molecular Markers Based on Transcriptome Sequences of Alnus

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通讯作者: 陈斌, bchen63@163.com

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Development of SSR Molecular Markers Based on Transcriptome Sequences of Alnus

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