Optimization of SSR-PCR Reaction System and Primer Screening of Melia azedarach

WANG Fang; LIAO Bo-yong; LI Pei; LIU Ming-qian; LI Jun-cheng; WU Lin-ying; LIN Wei; CHEN Xiao-yang

Volume 29 Issue 2

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Optimization of SSR-PCR Reaction System and Primer Screening of Melia azedarach

1.
College of Forestry and landscape Architecture, South China Agricultural University/Provincial Key Laboratory of Forest Plant Germplasm Innovation and Utilization/State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangzhou 510642, Guangdong, China
2.
College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China

Received Date: 2015-03-20

Abstract

[Objective] Based on previous achievement on Melia SSR primer to select highly polymorphic, high stability and repeatability Melia azedarach primers, and to lay a foundation for constructing M. azedarach genetic map, QTL mapping and molecular marker-assisted selection and breeding. [Method]The SSR-PCR system was optimized through the single factor experiment and orthogonal test, and by using the genomic DNAs of M. azedarach from 8 provenances as templates, the SSR primers suitable for M. azedarach were screened from 135 pairs of primers. [Result]The optimal SSR-PCR system is as follows: 1.0 μL 50 ngoμL-1 genomic DNA, 1.2 μL 100 μmol·L-1 of each primer, 1.0 μL 10 mmol·L-1 dNTPs, 0.8 μL 25 mmol·L-1 Mg2+, 0.15 μL 5 U·μL-1 Taq polymerase, 1.5 μL 10×Buffer (Mg2+ free), and replenishing ddH2O to 15 μL. And, 15 pairs of SSR primers with high polymorphism, high stability, and good repeatability, were finally screened out. [Conclusion]The SSR-PCR system of M. azedarach was successfully optimized, and 15 pairs of SSR primers applicable to neem were selected.
- Melia azedarach,
- SSR-PCR,
- system optimization,
- primer screening

References

[1]	Peng H, David J M. 16. MELIA Linnaeus, Sp. Pl. 1: 384. 1753[J]. Flora of China. 2008(11): 130-131.
[2]	Evans P T, Rombold J S. Paraiso (Melia azedarach var. "Gigante") woodlots: an agroforestry alternative for the small farmer in Paraguay[J]. Agroforestry Systems, 1984, 2(3): 199-214.
[3]	Vila S, Scocchi A, Mroginski L. Plant regeneration from shoot apical meristems of Melia azedarach L. (Meliaceae)[J]. Acta Physiologiae Plantarum, 2002, 24(2): 195-199.
[4]	Vila S, Gonzalez A, Rey H, et al. Somatic embryogenesis and plant regeneration from immature zygotic embryos of Melia azedarach (Meliaceae)[J]. In Vitro Cellular & Developmental Biology-Plant, 2003, 39(3): 283-287.
[5]	Vila S K, Rey H Y, Mroginski L A. Influence of genotype and explant source on indirect organogenesis by in vitro culture of leaves of Melia azedarach L[J]. Biocell, 2004, 28(1): 35-41.
[6]	Vila S, Gonzalez A, Rey H, et al. Plant regeneration, origin, and development of shoot buds from root segments of Melia azedarach L. (Meliaceae) seedlings[J]. In Vitro Cellular & Developmental Biology-Plant,2005, 41(6): 746-751.
[7]	教忠意,唐凌凌,隋德宗,等. 苦楝的研究现状与展望[J]. 福建林业科技,2009,36(4):269-274.
[8]	张国栓,王少波,刘顺国. 河南省苦楝基因资源及其遗传改良策略研究[J]. 河南林业科技,2009,29(3):41-42.
[9]	程诗明,顾万春. 苦楝中国分布区的物候区划[J]. 林业科学, 2005, 41(3): 186-191.
[10]	程诗明,顾万春. 苦楝遗传资源学研究进展及其展望[J]. 浙江林业科技,2007,27(2): 64-69.
[11]	Chen L, Deng X, Ding M, et al. Geographic variation in traits of fruit stones and seeds of Melia azedarach[J]. Journal of Beijing Forestry University,2014, 36(1): 15-20.
[12]	廖柏勇,陈晓阳,陈丽君,等. 苦楝种源间种子发芽变异的观测[J]. 广东农业科学,2014(11): 43-47,52.
[13]	陈丽君,刘明骞,廖柏勇,等. 苦楝不同种源苗期生长性状和生长节律研究[J]. 西南林业大学学报, 2014,34(4): 1-7.
[14]	陈丽君,邓小梅,丁美美,等. 苦楝种源果核及种子性状地理变异的研究[J]. 北京林业大学学报,2014,36(1): 15-20.
[15]	夏海涛. 药用苦楝遗传多样性ISSR分析和遗传变异规律研究[D]. 2009.
[16]	陈羡德,陈礼光,陈珺,等. 不同来源苦楝种质资源遗传多样性的RAPD分析[J]. 漳州师范学院学报(自然科学版),2010(4):118-122.
[17]	程诗明. 苦楝聚合群体遗传多样性研究与核心种质构建[D]. 中国林业科学研究院, 2005.
[18]	黄映萍. DNA分子标记研究进展[Z]. 2010.
[19]	陈争,姜小凤,童再康. 光皮桦EST-SSR PCR反应体系的优化[J]. 浙江农林大学学报, 2012,29(6):960-965.
[20]	吕学辉,李根前. SSR分子标记及在林木基因组中的应用[J]. 山东林业科技, 2011(5):57-60.
[21]	Varshney R K, Graner A, Sorrells M E. Genic microsatellite markers in plants: features and applications[J]. Trends in Biotechnology, 2005, 23(1): 48-55.
[22]	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.
[23]	Dayanandan S, Dole J, Bawa K, et al. Population structure delineated with microsatellite markers in fragmented populations of a tropical tree, Carapa guianensis (Meliaceae)[J]. Mol Ecol,1999, 8(10): 1585-1592.
[24]	刘军,孙宗修,陈益泰,等. 珍稀濒危树种毛红椿微卫星DNA分离及SSR反应体系优化[J]. 中国生物工程杂志, 2006, 12(26): 50-55.
[25]	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.
[26]	Sexton G J, Frere C H, Dieters M J, et al. Development and characterization of microsatellite loci for Khaya senegalensis (Meliaceae)1[J]. American Journal of Botany, 2010, 97(11): e111-e113.
[27]	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.
[28]	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.
[29]	刘军. 毛红椿天然居群遗传结构研究[D]. 2007.
[30]	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)[J]. Conservation Genetics, 2009, 10(3): 669-671.
[31]	Hernández G, Buonamici A, Walker K, et al. Isolation and characterization of microsatellite markers for Cedrela odorata L. (Meliaceae), a high value neotropical tree[J]. Conservation Genetics,2008, 9(2): 457-459.
[32]	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, 12(2): 344-353.
[33]	White G, Powell W. Cross-species amplification of SSR loci in the Meliaceae family[J]. Molecular Ecology,1997, 6(12): 1195-1197.
[34]	Lemes M R, Brondani R P V, Grattapaglia D. Multiplexed Systems of Microsatellite Markers for Genetic Analysis of Mahogany, Swietenia macrophylla King(Meliaceae), a Threatened Neotropical Timber Species[J]. The Journal of Heredity,2002, 4(93):287-291.
[35]	Hanaoka S, Muturi G M, Watanabe A. Isolation and characterization of microsatellite markers in Melia volkensii Gurke[J]. Conservation Genet Resour. 2012(4): 395-398.
[36]	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.
[37]	张龙,丁西朋,严琳玲,等. 8种柱花草属牧草SSR-PCR反应体系优化及引物筛选[J]. 草业科学, 2014, 31(2): 232-242.
[38]	马洪峥,李珊珊,葛颂,等. 能源作物芒属双药芒组SSR引物的筛选及其评价[J]. 生物多样性, 2011,19(5):535-542.
[39]	李光友,徐建民,吴世军,等. 尾叶桉杂种子代DNA提取和SSR引物筛选[J]. 中南林业科技大学学报,2012,32(2):81-85.

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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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Optimization of SSR-PCR Reaction System and Primer Screening of Melia azedarach

1. College of Forestry and landscape Architecture, South China Agricultural University/Provincial Key Laboratory of Forest Plant Germplasm Innovation and Utilization/State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangzhou 510642, Guangdong, China

2. College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China

Received Date: 2015-03-20

Abstract: [Objective] Based on previous achievement on Melia SSR primer to select highly polymorphic, high stability and repeatability Melia azedarach primers, and to lay a foundation for constructing M. azedarach genetic map, QTL mapping and molecular marker-assisted selection and breeding. [Method]The SSR-PCR system was optimized through the single factor experiment and orthogonal test, and by using the genomic DNAs of M. azedarach from 8 provenances as templates, the SSR primers suitable for M. azedarach were screened from 135 pairs of primers. [Result]The optimal SSR-PCR system is as follows: 1.0 μL 50 ngoμL-1 genomic DNA, 1.2 μL 100 μmol·L-1 of each primer, 1.0 μL 10 mmol·L-1 dNTPs, 0.8 μL 25 mmol·L-1 Mg2+, 0.15 μL 5 U·μL-1 Taq polymerase, 1.5 μL 10×Buffer (Mg2+ free), and replenishing ddH2O to 15 μL. And, 15 pairs of SSR primers with high polymorphism, high stability, and good repeatability, were finally screened out. [Conclusion]The SSR-PCR system of M. azedarach was successfully optimized, and 15 pairs of SSR primers applicable to neem were selected.

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Reference (39)

[1]	Peng H, David J M. 16. MELIA Linnaeus, Sp. Pl. 1: 384. 1753[J]. Flora of China. 2008(11): 130-131.
[2]	Evans P T, Rombold J S. Paraiso (Melia azedarach var. "Gigante") woodlots: an agroforestry alternative for the small farmer in Paraguay[J]. Agroforestry Systems, 1984, 2(3): 199-214.
[3]	Vila S, Scocchi A, Mroginski L. Plant regeneration from shoot apical meristems of Melia azedarach L. (Meliaceae)[J]. Acta Physiologiae Plantarum, 2002, 24(2): 195-199.
[4]	Vila S, Gonzalez A, Rey H, et al. Somatic embryogenesis and plant regeneration from immature zygotic embryos of Melia azedarach (Meliaceae)[J]. In Vitro Cellular & Developmental Biology-Plant, 2003, 39(3): 283-287.
[5]	Vila S K, Rey H Y, Mroginski L A. Influence of genotype and explant source on indirect organogenesis by in vitro culture of leaves of Melia azedarach L[J]. Biocell, 2004, 28(1): 35-41.
[6]	Vila S, Gonzalez A, Rey H, et al. Plant regeneration, origin, and development of shoot buds from root segments of Melia azedarach L. (Meliaceae) seedlings[J]. In Vitro Cellular & Developmental Biology-Plant,2005, 41(6): 746-751.
[7]	教忠意,唐凌凌,隋德宗,等. 苦楝的研究现状与展望[J]. 福建林业科技,2009,36(4):269-274.
[8]	张国栓,王少波,刘顺国. 河南省苦楝基因资源及其遗传改良策略研究[J]. 河南林业科技,2009,29(3):41-42.
[9]	程诗明,顾万春. 苦楝中国分布区的物候区划[J]. 林业科学, 2005, 41(3): 186-191.
[10]	程诗明,顾万春. 苦楝遗传资源学研究进展及其展望[J]. 浙江林业科技,2007,27(2): 64-69.
[11]	Chen L, Deng X, Ding M, et al. Geographic variation in traits of fruit stones and seeds of Melia azedarach[J]. Journal of Beijing Forestry University,2014, 36(1): 15-20.
[12]	廖柏勇,陈晓阳,陈丽君,等. 苦楝种源间种子发芽变异的观测[J]. 广东农业科学,2014(11): 43-47,52.
[13]	陈丽君,刘明骞,廖柏勇,等. 苦楝不同种源苗期生长性状和生长节律研究[J]. 西南林业大学学报, 2014,34(4): 1-7.
[14]	陈丽君,邓小梅,丁美美,等. 苦楝种源果核及种子性状地理变异的研究[J]. 北京林业大学学报,2014,36(1): 15-20.
[15]	夏海涛. 药用苦楝遗传多样性ISSR分析和遗传变异规律研究[D]. 2009.
[16]	陈羡德,陈礼光,陈珺,等. 不同来源苦楝种质资源遗传多样性的RAPD分析[J]. 漳州师范学院学报(自然科学版),2010(4):118-122.
[17]	程诗明. 苦楝聚合群体遗传多样性研究与核心种质构建[D]. 中国林业科学研究院, 2005.
[18]	黄映萍. DNA分子标记研究进展[Z]. 2010.
[19]	陈争,姜小凤,童再康. 光皮桦EST-SSR PCR反应体系的优化[J]. 浙江农林大学学报, 2012,29(6):960-965.
[20]	吕学辉,李根前. SSR分子标记及在林木基因组中的应用[J]. 山东林业科技, 2011(5):57-60.
[21]	Varshney R K, Graner A, Sorrells M E. Genic microsatellite markers in plants: features and applications[J]. Trends in Biotechnology, 2005, 23(1): 48-55.
[22]	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.
[23]	Dayanandan S, Dole J, Bawa K, et al. Population structure delineated with microsatellite markers in fragmented populations of a tropical tree, Carapa guianensis (Meliaceae)[J]. Mol Ecol,1999, 8(10): 1585-1592.
[24]	刘军,孙宗修,陈益泰,等. 珍稀濒危树种毛红椿微卫星DNA分离及SSR反应体系优化[J]. 中国生物工程杂志, 2006, 12(26): 50-55.
[25]	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.
[26]	Sexton G J, Frere C H, Dieters M J, et al. Development and characterization of microsatellite loci for Khaya senegalensis (Meliaceae)1[J]. American Journal of Botany, 2010, 97(11): e111-e113.
[27]	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.
[28]	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.
[29]	刘军. 毛红椿天然居群遗传结构研究[D]. 2007.
[30]	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)[J]. Conservation Genetics, 2009, 10(3): 669-671.
[31]	Hernández G, Buonamici A, Walker K, et al. Isolation and characterization of microsatellite markers for Cedrela odorata L. (Meliaceae), a high value neotropical tree[J]. Conservation Genetics,2008, 9(2): 457-459.
[32]	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, 12(2): 344-353.
[33]	White G, Powell W. Cross-species amplification of SSR loci in the Meliaceae family[J]. Molecular Ecology,1997, 6(12): 1195-1197.
[34]	Lemes M R, Brondani R P V, Grattapaglia D. Multiplexed Systems of Microsatellite Markers for Genetic Analysis of Mahogany, Swietenia macrophylla King(Meliaceae), a Threatened Neotropical Timber Species[J]. The Journal of Heredity,2002, 4(93):287-291.
[35]	Hanaoka S, Muturi G M, Watanabe A. Isolation and characterization of microsatellite markers in Melia volkensii Gurke[J]. Conservation Genet Resour. 2012(4): 395-398.
[36]	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.
[37]	张龙,丁西朋,严琳玲,等. 8种柱花草属牧草SSR-PCR反应体系优化及引物筛选[J]. 草业科学, 2014, 31(2): 232-242.
[38]	马洪峥,李珊珊,葛颂,等. 能源作物芒属双药芒组SSR引物的筛选及其评价[J]. 生物多样性, 2011,19(5):535-542.
[39]	李光友,徐建民,吴世军,等. 尾叶桉杂种子代DNA提取和SSR引物筛选[J]. 中南林业科技大学学报,2012,32(2):81-85.

Optimization of SSR-PCR Reaction System and Primer Screening of Melia azedarach

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通讯作者: 陈斌, bchen63@163.com

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Optimization of SSR-PCR Reaction System and Primer Screening of Melia azedarach

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