Establishment of Genetic Diversity System of Eucommia ulmoides Based on SSR Molecular Markers

HUANG Hai-yan; DU Hong-yan; WUYUN Tana; ZHU Gao-pu

Volume 26 Issue 6

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Establishment of Genetic Diversity System of Eucommia ulmoides Based on SSR Molecular Markers

1.
Non-timber Forestry Research and Development Center, Chinese Academy of Forestry, Zhengzhou 450003, He'nan, China
2.
Key Laboratory of Non-wood Forest Products of State Forestry Administration, Changsha 410004, Hu'nan, China

Received Date: 2012-11-25

Abstract

In order to set up the genetic diversity system of Eucommia ulmoides Oliver based on SSR molecular markers, the establishment of SSR-PCR reaction system and screening out SSR marker primer showing high polymorphism were studied. A L9(34) orthogonal design was performed to optimize the main factors of the SSR-PCR reaction system. The results indicated that the best SSR-PCR reaction system for E.ulmoides was DNA template 1 μL (30~60 ng·μL-1), 2×Taq PCR Master Mix 10 μL, primer 1 μL with the total volume of 25 μL. The PCR reaction system had high stability and repeatability, the pairs of SSR primers with high polymorphism were gotten. The 8 E.ulmoides samples' DNA sequence was amplified with 13 pairs of SSR primers by SSR-PCR technique, 34 alleles were detected, 2.6 alleles were detected from per site on average. Each allele's effective number was 1.751 5, and the h value was 0.379 8, the average I value was 0.643 3. This study is helpful in using SSR molecular marker to analyze genetic diversity and genetic relationship in E. ulmoides.
- Eucommia ulmoides Oliver,
- germplasm resources,
- optimization of SSR-PCR system,
- primer screening

References

[1]	杜红岩. 杜仲优质高产栽培[M]. 北京:中国林业出版社,1996:1-20
[2]	李芳东,杜红岩. 杜仲[M]. 北京:中国中医药出版社, 2001:232-256
[3]	杜红岩. 我国的杜仲胶资源极其开发潜力与产业发展思路[J]. 经济林研究, 2010,28(3):1-6
[4]	杜红岩, 李钦,李福海. 杜仲种仁桃叶珊瑚苷含量的测定及积累规律[J]. 林业科学研究, 2009,22(5):744-746
[5]	周志翔, 王鹏程, 徐永荣, 等. 湖北省杜仲种质资源收集与繁殖保存研究[J]. 华中农业大学学报, 1998,17(5):507-511
[6]	沈作奎, 彭诚. 杜仲不同地区不同种质间生长差异分析[J]. 特产研究, 1999(4):9-11
[7]	唐锡华. 杜仲孢子囊与配子体的发育形态[J]. 植物学报,1962,10(1):29
[8]	田兰馨. 杜仲胚胎学的研究[J]. 西北植物学报, 1993,13 (1):30-35
[9]	张檀, 张康健, 杨吉安, 等. 杜仲酯酶同工酶地理递变及生长预测的研究[J]. 西北林学院学报, 1993,8(4):20-23
[10]	李林初, 徐炳声. 我国八种特有植物的染色体观察[J]. 植物分类学报, 1986,24(2):157
[11]	Kalia R K, Rai M K, Kalia S, et al. Microsatellite markers: an overview of the recent progress in plants[J]. Euphytica,2011,177:309-334
[12]	Agarwal M, Shrivastava N, Padh H. Advances in molecular marker techniques and their applications in plant sciences[J]. Plant Cell Rep,2008,27:617-631
[13]	Xu W J, Wang B W,Cui K M. RAPD and SCAR markers linked to sex determination in Eucommia ulmoides Oliv[J]. Euphytica,2004,136:233-238
[14]	王瑷琦, 黄璐琦, 邵爱娟, 等. 孑遗植物杜仲的遗传多样性RAPD分析和保护策略研究[J]. 中国中药杂志, 2006,31(19):1583-1586
[15]	王宏. 杜仲优良品种(无性系)DNA指纹图谱的构建[D]. 西安:西北农林科技大学, 2007:15-19
[16]	王大玮. 杜仲遗传连锁图谱构建及中药性状的分子标记[D]. 西安:西北农林科技大学, 2011:10-14
[17]	Wu M Q, Chen S L, Wang M X, et al. An analysis of the genetic diversity and genetic structure of Eucommia ulmoides using inter-simple sequence repeat (ISSR) markers[J]. African Journal of Biotechnology, 2011,10(84):19505-19513
[18]	Wang D W, Yu L, Li Z Q. Identification of a Male-Specific Amplified Fragment Length Polymorphism (AFLP) and a Sequence Characterized Amplified Region (SCAR) Marker in Eucommia ulmoides Oliv[J]. Int J Mol Sci, 2011, 12:857-864
[19]	罗冉, 吴委林, 张旸, 等. SSR分子标记在作物遗传育种中的应用[J]. 基因组学与应用生物学, 2010,29(1):137-143
[20]	Morgante M, Olivieri A M. PCR-amplified microsatellites as markers in plant genetics[J]. Plant J, 1993,13(1):175-182
[21]	Dend J Y, Li Y F, Huang H W. Development and characterization of microsatellite markers in Eucommia ulmoides Oliver (Eucommiaceae)[J]. Molecular Ecology Notes, 2006,6:496-498
[22]	邓建云, 李建强, 黄宏文. 一株具有特异AFLP指纹图谱的杜仲古树[J]. 武汉植物学研究, 2006,24(6):509-513
[23]	Yao X H, Deng J Y, Huang H W. Genetic diversity in Eucommia ulmoides (Eucommiaceae), an endangered traditional Chinese medicinal plant[J]. Conserv Genet, 2012, 13:1499-1507
[24]	张龙进, 白成科. 正交优化北重楼ISSR-PCR体系[J]. 植物研究, 2011,31(1):105-108
[25]	罗成, 熊玉婷, 顾蔚, 等. 华中五味子ISSR-PCR反应体系优化及引物筛选[J]. 植物研究, 2010,30(5):588-593
[26]	盖钧镒. 试验统计方法[M]. 北京:中国农业出版社, 2000:286-287
[27]	梁玉琴, 李芳东, 傅建敏, 等. 正交设计优化柿属植物SSR-PCR反应体系[J]. 经济林研究, 2011,29(4):17-22

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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 of Genetic Diversity System of Eucommia ulmoides Based on SSR Molecular Markers

1. Non-timber Forestry Research and Development Center, Chinese Academy of Forestry, Zhengzhou 450003, He'nan, China

2. Key Laboratory of Non-wood Forest Products of State Forestry Administration, Changsha 410004, Hu'nan, China

Received Date: 2012-11-25

Abstract: In order to set up the genetic diversity system of Eucommia ulmoides Oliver based on SSR molecular markers, the establishment of SSR-PCR reaction system and screening out SSR marker primer showing high polymorphism were studied. A L9(34) orthogonal design was performed to optimize the main factors of the SSR-PCR reaction system. The results indicated that the best SSR-PCR reaction system for E.ulmoides was DNA template 1 μL (30~60 ng·μL-1), 2×Taq PCR Master Mix 10 μL, primer 1 μL with the total volume of 25 μL. The PCR reaction system had high stability and repeatability, the pairs of SSR primers with high polymorphism were gotten. The 8 E.ulmoides samples' DNA sequence was amplified with 13 pairs of SSR primers by SSR-PCR technique, 34 alleles were detected, 2.6 alleles were detected from per site on average. Each allele's effective number was 1.751 5, and the h value was 0.379 8, the average I value was 0.643 3. This study is helpful in using SSR molecular marker to analyze genetic diversity and genetic relationship in E. ulmoides.

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Reference (27)

[1]	杜红岩. 杜仲优质高产栽培[M]. 北京:中国林业出版社,1996:1-20
[2]	李芳东,杜红岩. 杜仲[M]. 北京:中国中医药出版社, 2001:232-256
[3]	杜红岩. 我国的杜仲胶资源极其开发潜力与产业发展思路[J]. 经济林研究, 2010,28(3):1-6
[4]	杜红岩, 李钦,李福海. 杜仲种仁桃叶珊瑚苷含量的测定及积累规律[J]. 林业科学研究, 2009,22(5):744-746
[5]	周志翔, 王鹏程, 徐永荣, 等. 湖北省杜仲种质资源收集与繁殖保存研究[J]. 华中农业大学学报, 1998,17(5):507-511
[6]	沈作奎, 彭诚. 杜仲不同地区不同种质间生长差异分析[J]. 特产研究, 1999(4):9-11
[7]	唐锡华. 杜仲孢子囊与配子体的发育形态[J]. 植物学报,1962,10(1):29
[8]	田兰馨. 杜仲胚胎学的研究[J]. 西北植物学报, 1993,13 (1):30-35
[9]	张檀, 张康健, 杨吉安, 等. 杜仲酯酶同工酶地理递变及生长预测的研究[J]. 西北林学院学报, 1993,8(4):20-23
[10]	李林初, 徐炳声. 我国八种特有植物的染色体观察[J]. 植物分类学报, 1986,24(2):157
[11]	Kalia R K, Rai M K, Kalia S, et al. Microsatellite markers: an overview of the recent progress in plants[J]. Euphytica,2011,177:309-334
[12]	Agarwal M, Shrivastava N, Padh H. Advances in molecular marker techniques and their applications in plant sciences[J]. Plant Cell Rep,2008,27:617-631
[13]	Xu W J, Wang B W,Cui K M. RAPD and SCAR markers linked to sex determination in Eucommia ulmoides Oliv[J]. Euphytica,2004,136:233-238
[14]	王瑷琦, 黄璐琦, 邵爱娟, 等. 孑遗植物杜仲的遗传多样性RAPD分析和保护策略研究[J]. 中国中药杂志, 2006,31(19):1583-1586
[15]	王宏. 杜仲优良品种(无性系)DNA指纹图谱的构建[D]. 西安:西北农林科技大学, 2007:15-19
[16]	王大玮. 杜仲遗传连锁图谱构建及中药性状的分子标记[D]. 西安:西北农林科技大学, 2011:10-14
[17]	Wu M Q, Chen S L, Wang M X, et al. An analysis of the genetic diversity and genetic structure of Eucommia ulmoides using inter-simple sequence repeat (ISSR) markers[J]. African Journal of Biotechnology, 2011,10(84):19505-19513
[18]	Wang D W, Yu L, Li Z Q. Identification of a Male-Specific Amplified Fragment Length Polymorphism (AFLP) and a Sequence Characterized Amplified Region (SCAR) Marker in Eucommia ulmoides Oliv[J]. Int J Mol Sci, 2011, 12:857-864
[19]	罗冉, 吴委林, 张旸, 等. SSR分子标记在作物遗传育种中的应用[J]. 基因组学与应用生物学, 2010,29(1):137-143
[20]	Morgante M, Olivieri A M. PCR-amplified microsatellites as markers in plant genetics[J]. Plant J, 1993,13(1):175-182
[21]	Dend J Y, Li Y F, Huang H W. Development and characterization of microsatellite markers in Eucommia ulmoides Oliver (Eucommiaceae)[J]. Molecular Ecology Notes, 2006,6:496-498
[22]	邓建云, 李建强, 黄宏文. 一株具有特异AFLP指纹图谱的杜仲古树[J]. 武汉植物学研究, 2006,24(6):509-513
[23]	Yao X H, Deng J Y, Huang H W. Genetic diversity in Eucommia ulmoides (Eucommiaceae), an endangered traditional Chinese medicinal plant[J]. Conserv Genet, 2012, 13:1499-1507
[24]	张龙进, 白成科. 正交优化北重楼ISSR-PCR体系[J]. 植物研究, 2011,31(1):105-108
[25]	罗成, 熊玉婷, 顾蔚, 等. 华中五味子ISSR-PCR反应体系优化及引物筛选[J]. 植物研究, 2010,30(5):588-593
[26]	盖钧镒. 试验统计方法[M]. 北京:中国农业出版社, 2000:286-287
[27]	梁玉琴, 李芳东, 傅建敏, 等. 正交设计优化柿属植物SSR-PCR反应体系[J]. 经济林研究, 2011,29(4):17-22

Establishment of Genetic Diversity System of Eucommia ulmoides Based on SSR Molecular Markers

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