Establishment and Primer Screening of SSR-PCR Reaction System for Toona ciliata

ZHAN Xin; LU Hao-jun; ZHAO Shuai; CHEN Xiao-yang; DENG Xiao-mei

Volume 29 Issue 4

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Establishment and Primer Screening of SSR-PCR Reaction System for Toona ciliata

1.
College of Forestry and Landscape Architecture/Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, South China Agricultural University, Guangzhou 510642, Guangdong, China

Received Date: 2015-07-06

Abstract

[Objective] To establish SSR-PCR reaction system, and screen high polymorphism primers for SSR analysis of Toona ciliata. [Method] The orthogonal design was employed to obtain the optimal SSR-PCR system of T. ciliate. Using optimized system of SSR-PCR, 135 pairs of SSR primers from Meliaceae plants were amplificated in six populations. [Result] The optimal 10 μL reaction system contained 1.5 μL 30 ng·μL-1Template DNA, 0.8 μL 10 umol·L-1 primers,0.025 μL 10 mol·L-1 dNTPs, 0.8 μL 25mol·L-1 Mg2+, 0.1 μL 5 U·μL-1 taq DNA polymerase, 0.01μL 1 mol·L-1 F-dUTP, 1μL 10× PCR buffer (Mg2+free) and 5.765 μL ddH2O. 12 primers with high polymorphism were selected among 29 primer combinations. [Conclusion] The optimal reaction system of SSR-PCR were established and high polymorphism primers were selected, which could be used in analysis of fingerprinting genetic diversity, molecular breeding and variety identification in T. ciliate.
- Toona ciliate,
- SSR-PCR,
- system optimization,
- primers screening

References

[1]	李志辉,李柏海,祁承经,等. 我国南方珍贵用材树种资源的重要性及其发展策略[J]. 中南林业科技大学学报. 2012, 32(11): 1-8.
[2]	甘文峰,余林,叶金山,等. 珍贵用材树种毛红椿研究进展综述[J]. 江西林业科技. 2014(05): 33-37.
[3]	黄红兰,张露,郭晓燕,等. 九连山毛红椿种群的结实特性及其生殖力[J]. 林业科学. 2013(07): 170-174.
[4]	刘军,陈益泰,罗阳富,等. 毛红椿天然林群落结构特征研究[J]. 林业科学研究. 2010, 23(1): 93-97.
[5]	李明芳,郑学勤. 开发SSR引物方法之研究动态[J]. 遗传. 2004, 26(5): 769-776.
[6]	刘军,陈益泰,姜景民,等. 毛红椿群体遗传结构的SSR分析[J]. 林业科学研究. 2009(01): 37-41.
[7]	Li F, Gan S. An Optimised Protocol for Fluorescent-dUTP Based SSR Genotyping and its Application to Genetic Mapping in Eucalyptus[J]. SILVAE GENETICA. 2011, 60(1): 18-25.
[8]	Lemes M R, Esashika T, Gaoue O G. Microsatellites for mahoganies: Twelve new loci for Swietenia macrophylla and its high transferability to Khaya senegalensis[J]. American Journal of Botany. 2011, 98(8): e207-e209.
[9]	Dayanandan S, Dole J, Bawa K. Population structure delineated with microsatellite markers in fragmented populations of a tropical tree, Carapa guianensis(Meliaceae)[J]. Molecular Ecology. 1999, 8(10): 1585-1592.
[10]	Liu J, Jiang J M, Chen Y T. Genetic diversity of central and peripheral populations of Toona ciliata var. pubescens, an endangered tree species endemic to China[J]. Genet Mol Res. 2014, 13(2): 4579-4590.
[11]	Cespedes M, Gutierrez M V, Holbrook N M, et al. Restoration of genetic diversity in the dry forest tree Swietenia macrophylla(Meliaceae) after pasture abandonment in Costa Rica[J]. Molecular Ecology. 2003, 12(12): 3201-3212.
[12]	Sexton G J, Frere C H, Dieters M J, et al. Development and characterization of microsatellite loci for Khaya senegalensis (Meliaceae)[J]. American Journal of Botany. 2010, 97(11): e111-e113.
[13]	Pereira M F, Bandeira L F, Blanco A J V, et al. Isolation and characterization of microsatellite loci in Cabralea canjerana (Meliaceae)[J]. American Journal of Botany. 2010, 98(1): e10-e12.
[14]	White G, Powell W. Isolation and characterization of microsatellite loci in Swietenia humilis (Meliaceae): an endangered tropical hardwood species[J]. Molecular Ecology. 1997, 6(9): 851-860.
[15]	Boontong C, Pandey M, Changtragoon S. Isolation and characterization of microsatellite markers in Indian neem (Azadirachta indica var. indica A. Juss) and cross-amplification in Thai neem (A-indica var. siamensis Valenton)[N]. 03/08/2013(669-671).
[16]	Hernandez G, Buonamici A, Walker K. Isolation and characterization of microsatellite markers for Cedrela odorata. (Meliaceae), a high value neotropical tree[J]. Conservation Genetics. 2008, 9(2): 457-459.
[17]	Karan M, Evans D S, Reilly D, et al. Rapid microsatellite marker development for African mahogany (Khaya senegalensis, Meliaceae) using next-generation sequencing and assessment of its intra-specific genetic diversity[J]. Molecular Ecology Resources. 2012, 1(2): 344-353.
[18]	White G, Powell W. Cross-species amplification of SSR loci in the Meliaceae family[J]. Molecular Ecology. 1997, 6(12): 1195-1197.
[19]	Lemes M, Brondani R, Grattapaglia D. Multiplexed Systems of Microsatellite Markers for Genetic Analysis of Mahogany, Swietenia macrophylla King (Meliaceae), a Threatened Neotropical Timber Species[J]. Journal Of Heredity. 2002, 93(4): 287-291.
[20]	Hanaoka S, Muturi G M, Watanabe A. Isolation and characterization of microsatellite markers in Melia volkensii Gurke[J]. Conservation Genetics Resources. 2012, 4(2): 395-398.
[21]	De Lima P F, Ramos F N, Zucchi M I, et al. Development and characterization of microsatellite markers from Guarea guidonia (Meliaceae), a tree species from different habitats within the Brazilian Atlantic forest[J]. Conservation Genetics Resources. 2009, 1(1): 171-173.
[22]	Magnusson P K E, Wilander E, Gyllensten U. Analysis of loss of heterozygosity in microdissected tumor cells from cervical carcinoma using fluorescent dUTP labeling of PCR products[J]. Biotechniques. 1996, 21(5): 844-847.
[23]	Woolbright S A, Difazio S P, Yin T, et al.. A dense linkage map of hybrid cottonwood (Populus fremontii x P-angustifolia) contributes to long-term ecological research and comparison mapping in a model forest tree[J]. Heredity. 2008, 100(1): 59-70.
[24]	Busch J D, Benford R, Pearson T, et al. Development of polymorphic tetranucleotide microsatellites for pinyon jays (Gymnorhinus cyanocephalus)[J]. Conservation Genetics. 2009, 10(3): 689-691.
[25]	Macavoy E S, Mcgibbon L M, Sainsbury J P, et al. Genetic variation in island populations of tuatara (Sphenodon spp) inferred from microsatellite markers. [J]. Conservation Genetics., 8(2): 305-318.
[26]	徐阳,陈金慧,王颖,等. 杉木SSR-PCR体系优化[J]. 林业科技开发. 2014(01): 15-20.
[27]	陈怀琼,隋春,魏建和.植物SSR引物开发策略简述[J]. 分子植物育种. 2009, 7(4): 845-851.
[28]	曾庆国,陈艺燕. 微卫星位点筛选方法综述[J]. 生态科学. 2005(04): 368-372.
[29]	王丽,赵桂仿. 植物不同种属间共用微卫星引物的研究[J]. 西北植物学报,2005(08): 1540-1546.

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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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Establishment and Primer Screening of SSR-PCR Reaction System for Toona ciliata

1. College of Forestry and Landscape Architecture/Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, South China Agricultural University, Guangzhou 510642, Guangdong, China

Received Date: 2015-07-06

Abstract: [Objective] To establish SSR-PCR reaction system, and screen high polymorphism primers for SSR analysis of Toona ciliata. [Method] The orthogonal design was employed to obtain the optimal SSR-PCR system of T. ciliate. Using optimized system of SSR-PCR, 135 pairs of SSR primers from Meliaceae plants were amplificated in six populations. [Result] The optimal 10 μL reaction system contained 1.5 μL 30 ng·μL-1Template DNA, 0.8 μL 10 umol·L-1 primers,0.025 μL 10 mol·L-1 dNTPs, 0.8 μL 25mol·L-1 Mg2+, 0.1 μL 5 U·μL-1 taq DNA polymerase, 0.01μL 1 mol·L-1 F-dUTP, 1μL 10× PCR buffer (Mg2+free) and 5.765 μL ddH2O. 12 primers with high polymorphism were selected among 29 primer combinations. [Conclusion] The optimal reaction system of SSR-PCR were established and high polymorphism primers were selected, which could be used in analysis of fingerprinting genetic diversity, molecular breeding and variety identification in T. ciliate.

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Reference (29)

[1]	李志辉,李柏海,祁承经,等. 我国南方珍贵用材树种资源的重要性及其发展策略[J]. 中南林业科技大学学报. 2012, 32(11): 1-8.
[2]	甘文峰,余林,叶金山,等. 珍贵用材树种毛红椿研究进展综述[J]. 江西林业科技. 2014(05): 33-37.
[3]	黄红兰,张露,郭晓燕,等. 九连山毛红椿种群的结实特性及其生殖力[J]. 林业科学. 2013(07): 170-174.
[4]	刘军,陈益泰,罗阳富,等. 毛红椿天然林群落结构特征研究[J]. 林业科学研究. 2010, 23(1): 93-97.
[5]	李明芳,郑学勤. 开发SSR引物方法之研究动态[J]. 遗传. 2004, 26(5): 769-776.
[6]	刘军,陈益泰,姜景民,等. 毛红椿群体遗传结构的SSR分析[J]. 林业科学研究. 2009(01): 37-41.
[7]	Li F, Gan S. An Optimised Protocol for Fluorescent-dUTP Based SSR Genotyping and its Application to Genetic Mapping in Eucalyptus[J]. SILVAE GENETICA. 2011, 60(1): 18-25.
[8]	Lemes M R, Esashika T, Gaoue O G. Microsatellites for mahoganies: Twelve new loci for Swietenia macrophylla and its high transferability to Khaya senegalensis[J]. American Journal of Botany. 2011, 98(8): e207-e209.
[9]	Dayanandan S, Dole J, Bawa K. Population structure delineated with microsatellite markers in fragmented populations of a tropical tree, Carapa guianensis(Meliaceae)[J]. Molecular Ecology. 1999, 8(10): 1585-1592.
[10]	Liu J, Jiang J M, Chen Y T. Genetic diversity of central and peripheral populations of Toona ciliata var. pubescens, an endangered tree species endemic to China[J]. Genet Mol Res. 2014, 13(2): 4579-4590.
[11]	Cespedes M, Gutierrez M V, Holbrook N M, et al. Restoration of genetic diversity in the dry forest tree Swietenia macrophylla(Meliaceae) after pasture abandonment in Costa Rica[J]. Molecular Ecology. 2003, 12(12): 3201-3212.
[12]	Sexton G J, Frere C H, Dieters M J, et al. Development and characterization of microsatellite loci for Khaya senegalensis (Meliaceae)[J]. American Journal of Botany. 2010, 97(11): e111-e113.
[13]	Pereira M F, Bandeira L F, Blanco A J V, et al. Isolation and characterization of microsatellite loci in Cabralea canjerana (Meliaceae)[J]. American Journal of Botany. 2010, 98(1): e10-e12.
[14]	White G, Powell W. Isolation and characterization of microsatellite loci in Swietenia humilis (Meliaceae): an endangered tropical hardwood species[J]. Molecular Ecology. 1997, 6(9): 851-860.
[15]	Boontong C, Pandey M, Changtragoon S. Isolation and characterization of microsatellite markers in Indian neem (Azadirachta indica var. indica A. Juss) and cross-amplification in Thai neem (A-indica var. siamensis Valenton)[N]. 03/08/2013(669-671).
[16]	Hernandez G, Buonamici A, Walker K. Isolation and characterization of microsatellite markers for Cedrela odorata. (Meliaceae), a high value neotropical tree[J]. Conservation Genetics. 2008, 9(2): 457-459.
[17]	Karan M, Evans D S, Reilly D, et al. Rapid microsatellite marker development for African mahogany (Khaya senegalensis, Meliaceae) using next-generation sequencing and assessment of its intra-specific genetic diversity[J]. Molecular Ecology Resources. 2012, 1(2): 344-353.
[18]	White G, Powell W. Cross-species amplification of SSR loci in the Meliaceae family[J]. Molecular Ecology. 1997, 6(12): 1195-1197.
[19]	Lemes M, Brondani R, Grattapaglia D. Multiplexed Systems of Microsatellite Markers for Genetic Analysis of Mahogany, Swietenia macrophylla King (Meliaceae), a Threatened Neotropical Timber Species[J]. Journal Of Heredity. 2002, 93(4): 287-291.
[20]	Hanaoka S, Muturi G M, Watanabe A. Isolation and characterization of microsatellite markers in Melia volkensii Gurke[J]. Conservation Genetics Resources. 2012, 4(2): 395-398.
[21]	De Lima P F, Ramos F N, Zucchi M I, et al. Development and characterization of microsatellite markers from Guarea guidonia (Meliaceae), a tree species from different habitats within the Brazilian Atlantic forest[J]. Conservation Genetics Resources. 2009, 1(1): 171-173.
[22]	Magnusson P K E, Wilander E, Gyllensten U. Analysis of loss of heterozygosity in microdissected tumor cells from cervical carcinoma using fluorescent dUTP labeling of PCR products[J]. Biotechniques. 1996, 21(5): 844-847.
[23]	Woolbright S A, Difazio S P, Yin T, et al.. A dense linkage map of hybrid cottonwood (Populus fremontii x P-angustifolia) contributes to long-term ecological research and comparison mapping in a model forest tree[J]. Heredity. 2008, 100(1): 59-70.
[24]	Busch J D, Benford R, Pearson T, et al. Development of polymorphic tetranucleotide microsatellites for pinyon jays (Gymnorhinus cyanocephalus)[J]. Conservation Genetics. 2009, 10(3): 689-691.
[25]	Macavoy E S, Mcgibbon L M, Sainsbury J P, et al. Genetic variation in island populations of tuatara (Sphenodon spp) inferred from microsatellite markers. [J]. Conservation Genetics., 8(2): 305-318.
[26]	徐阳,陈金慧,王颖,等. 杉木SSR-PCR体系优化[J]. 林业科技开发. 2014(01): 15-20.
[27]	陈怀琼,隋春,魏建和.植物SSR引物开发策略简述[J]. 分子植物育种. 2009, 7(4): 845-851.
[28]	曾庆国,陈艺燕. 微卫星位点筛选方法综述[J]. 生态科学. 2005(04): 368-372.
[29]	王丽,赵桂仿. 植物不同种属间共用微卫星引物的研究[J]. 西北植物学报,2005(08): 1540-1546.

Establishment and Primer Screening of SSR-PCR Reaction System for Toona ciliata

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通讯作者: 陈斌, bchen63@163.com

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Establishment and Primer Screening of SSR-PCR Reaction System for Toona ciliata

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