鹅掌楸属SRAP分子标记体系优化及遗传多样性分析

doi:10.11707/j.1001-7488.20140706

摘要/Abstract

摘要：

采用L₁₆（4⁵）正交设计，对鹅掌楸属SRAP-PCR反应的5个因素（Mg²⁺、dNTPs、引物、Taq酶和模板DNA浓度）在4个水平上进行优化试验，同时对SRAP反应影响较大的Mg²⁺、Taq酶、模板DNA浓度进行单因素分析。建立鹅掌楸属植物SRAP-PCR最适反应体系（20 μL），反应条件为：Mg²⁺3.0 mmol·L^-1，dNTPs 0.3 mmol·L^-1，引物浓度0.9 μmol·L^-1，Taq酶 2.0 μmol·min^-1，模板DNA 90 ng，10×PCR buffer 2 μL，不足部分以ddH₂O补足。利用优化的SRAP体系对鹅掌楸属10个不同地理种源进行遗传多样性分析。筛选出15对条带清晰、多态性高的引物共扩增出182个位点，其中149个为多态性位点，多态性比例为81.87%。应用POPGEN 32软件通过UPGMA法进行聚类分析，从聚类图可以看出10个地理种源间的遗传距离及亲缘关系，表明SRAP分子标记可以运用于鹅掌楸属的遗传多样性研究。

关键词: 鹅掌楸属, SRAP, 体系优化, 遗传多样性

Abstract:

The SRAP-PCR reaction system of Liriodendron was optimized with an orthogonal design to choose the most proper concentrations of Mg²⁺, dNTPs, primers, Taq polymerase and template DNA at four levels. The concentrations of Mg²⁺, Taq polymerase, template DNA were further fine-adjusted with a single-factor design. The optimized SRAP-PCR system (total 20 μL)was as follows: Mg²⁺3.0 mmol·L^-1, dNTPs 0.3 mmol·L^-1, primer 0.9 μmol·L^-1, Taq polymerase 2.0 μmol·min^-1 and DNA template 90 ng, 2 μL 10×PCR buffer and ddH₂O. With the system, genetic diversity of 10 provenances of Liriodendron was analyzed. The results showed that, 182 loci were generated from the 15 screened primer pairs, of which 149 loci were polymorphic. The percent of polymorphic loci was 81.87%. The dendrogram of Liriodendron was constructed based on cluster analysis (UPGMA) with the Package of POPGEN 32.The genetic distance and relative relationship of the 10 provenances of Liriodendron was shown by the dendrogram distinctly. It indicated that SRAP molecular marker could be widely used in genetic diversity analysis of Liriodendron.

Key words: Liriodendron, SRAP, system optimization, genetic diversity

中图分类号:

S718.46

赵亚琦, 成铁龙, 施季森, 王新民, 徐阳, 刘伟东, 陈金慧. 鹅掌楸属SRAP分子标记体系优化及遗传多样性分析[J]. 林业科学, 2014, 50(7): 37-43.

Zhao Yaqi, Cheng Tielong, Shi Jisen, Wang Xinmin, Xu Yang, Liu Weidong, Chen Jinhui. Optimization of a SRAP-PCR System for Analysis of Genetic Diversity of Liriodendron[J]. Scientia Silvae Sinicae, 2014, 50(7): 37-43.

参考文献

陈海霞, 彭尽晖. 2011. 绣球属植物SRAP-PCR反应体系优化及引物筛选. 中国农学通报, 27(31): 93-98.
(Chen H X, Peng J H. 2011. Optimization of SRAP-PCR system and primers screening in hydrangea. Chinese Agricultural Science Bulletin, 27(31): 93-98. [in Chinese] )
陈万胜, 王元英, 罗成刚, 等.2008. 利用正交设计优化烟草SRAP反应体系. 分子植物育种, 6(1): 177-182.
(Chen W S, Wang Y Y, Luo C G, et al. 2008. Optimization for SRAP-PCR system of tobacoo based on orthogonal design. Molecular Plant Breeding, 6(1): 177-182. [in Chinese] )
郝日明, 贺善安, 汤诗杰, 等. 1995. 鹅掌楸在中国的自然分布及其特点. 植物资源与环境, 4(1):1-6.
(Hao R M, He S A, Tang S J, et al. 1995. Geographical distribution of Liriodendron chinense in China and its significance. Journal of Plant Resources and Environment, 4(1):1-6. [in Chinese] )
何正文, 刘运生, 陈立华, 等. 1998. 正交设计直观分析法优化PCR条件. 湖南医科大学学报, 23(4): 76-77.
(He Z W, Liu Y S, Chen L H, et al. 1998. Orthogonal design-direct analysis for PCR optimization. Bulletin Of Hunan Medical University, 23(4): 76-77. [in Chinese] )
江磊, 李刚, 岳帅, 等. 2013. 11个红花品种遗传多样性与亲缘关系的SRAP分析. 中国油料作物学报, 35(5):546-550.
(Jiang L, Li G, Yue S, et al. 2013. Analysis of genetic diversity and relationship of 11 Carthamus tinctorius L.varieties by SRAP. Chinese Journal of Oil Crop Sciences, 35(5):546-550. [in Chinese] )
李建民, 周志春, 吴开云, 等. 2002. RAPD标记研究马褂木地理种群的遗传分化. 林业科学,38(4):61-66.
(Li J M, Zhou Z C, Wu K Y, et al. 2002. Genetic differentiation of geographic populations in liriodendron chinense using RAPD markers. Scientia Silvae Sinicae, 38(4):61-66. [in Chinese] )
李娟玲, 刘国民, 曹嵩晓, 等.2011. 利用单因子和正交设计双重实验法优化鹧鸪茶RAPD-PCR反应体系. 农学学报, 1(4): 14-21.
(Li J L, Liu G M, Cao S X, et al. 2011. Optimization of rapd-pcr experimental system in mallotus oblongifolius(Miq.)Muell.-Arg. using dual experiments of single factor and orthogonal design. Journal of Agriculture, 1(4): 14-21. [in Chinese] )
李周岐, 王章荣. 2002. RAPD标记在鹅掌楸属中的遗传研究. 林业科学, 1(1): 150-153.
(Li Z Q, Wang Z R. 2002. Genetic study of RAPD markers in liriodendron. Scientia Silvae Sinicae, 1(1): 150-153. [in Chinese] )
梁景霞, 祁建民, 吴为人, 等. 2005. 烟草DNA的提取与SRAP反应体系的建立. 中国烟草学报, 11(4): 33-38.
(Liang J X, Qi J M, Wu W R, et al. 2005. DNA extraction and the establishment of SRAP reaction system in tobacco. Acta Tabacaria Sinica, 11(4): 33-38. [in Chinese] )
林忠旭, 张献龙, 聂以春, 等.2003. 棉花SRAP遗传连锁图构建. 科学通报, 48(15): 1676-1679.
林忠旭, 张献龙, 聂以春. 2004. 新型标记SRAP在棉花F₂分离群体及遗传多样性评价中的适用性分析. 遗传学报, 31(6):622-626.
(Lin Z X, Zhang X L, Nie Y C. 2004. Evaluation of application of a new molecular marker SRAP on analysis of F2 segregation population and genetic diversity in cotton. Acta Genetica Sinica, 31(6):622-626. [in Chinese] )
罗光佐, 施季森, 尹佟明, 等.2000. 利用RAPD标记分析北美鹅掌楸与鹅掌楸种间遗传多样性. 植物资源与环境学报, 9(2): 9-13.
(Luo G Z, Shi J S, Yin T M, et al. 2000. Comparison of genetic diversity between Liriodendron tulipifera Linn. and Liriodendron chinense (Hemsl.) Sarg. by means of RAPD markers. Journal of Plant Resources and Environment, 9(2): 9-13. [in Chinese] )
孙佳琦, 梁建国, 石少川, 等.2010. SRAP标记在观赏植物遗传育种中的应用. 分子植物育种, 8(3): 577-588.
(Sun J Q, Liang J G, Shi S C, et al. 2010. Application of SRAP marker in genetic breeding of ornamental plants. Molecular Plant Breeding, 8(3): 577-588. [in Chinese] )
谭碧玥, 王源秀, 徐立安. 2009. 杨树基因组SRAP扩增体系的建立与优化. 林业科技开发, 23(2): 25-29.
(Tan B Y, Wang Y X, Xu L A. 2009. Establishment and optimization of SRAP-PCR reaction system of Populus. China Forestry Science and Technology, 23(2): 25-29. [in Chinese] )
唐琴, 曾秀丽, 廖明安, 等.2012. 大花黄牡丹遗传多样性的SRAP分析. 林业科学, 48(1): 70-76.
(Tang Q, Zeng X L, Liao M A, et al. 2012. SRAP analysis of genetic diversity of paeonia ludlowii in Tibet. Scientia Silvae Sinicae, 48(1): 70-76. [in Chinese] )
汪成成, 文山, 董健, 等.2010. 正交设计优化日本落叶松SRAP-PCR反应体系. 沈阳农业大学学报, 41(4): 444-448.
(Wang C C, Wen S, Dong J, et al. 2010. Optimization for SRAP-PCR system of Larix kaempferi(Lamb.)Carr.using orthogonal design. Journal of Shenyang Agricultural University, 41(4): 444-448. [in Chinese] )
胥猛, 李火根. 2008.鹅掌楸EST-SSR引物开发及通用性分析. 分子植物育种,6(3):615-618.
(Xu M, Li H G. 2008. Development and characterization of microsatellite markers from expressed sequence tags for liriodendron. Molecular Plant Breeding, 6(3):615-618. [in Chinese] )
占志勇, 徐刚标. 2010. 鹅掌楸ISSR-PCR反应体系的建立及优化. 中南林业科技大学学报, 30(6): 80-84.
(Zhan Z Y, Xu G B. 2010. Establishment and optimization of ISSR-PCR system for Liriodendron. Journal of Central South University of Forestry & Technology, 30(6): 80-84. [in Chinese] )
张红莲, 李火根. 2010. 利用EST-SSR分子标记检测鹅掌楸种间渐渗杂交. 生物多样性, 18(2): 120-128.
(Zhang H L, Li H G. 2010. Detection of interspecific introgressive hybridization in Liriodendron with EST-SSR markers. Biodiversity Science, 18(2): 120-128. [in Chinese] )
赵杨, 李玉璞, 代毅. 2012. 华山松SRAP-PCR反应体系的优化. 西北林学院学报, 27(5): 87-90.
(Zhao Y, Li Y P, Dai Y. 2012. Optimization of SRAP-PCR system for Pinus armandii. Journal of Northwest Forestry University, 27(5): 87-90. [in Chinese] )
朱晓琴, 贺善安, 姚青菊, 等. 1997. 鹅掌楸居群遗传结构及其保护对策.植物资源与环境, 6(4):7-14.
(Zhu X Q, He S A, Yao Q J, et al. 1997. Population genetic structure and conservation strategy of Liriodendron chinense (Hemsl.)Sarg. Journal of Plant Resources and Environment, 6(4):7-14. [in Chinese] )
Aneja B, Yadav N R, Chawla V, et al. 2012. Sequence-related amplified polymorphism (SRAP) molecular marker system and its applications in crop improvement. Molecular Breeding, 30(4): 1635-1648.
Budak H, Shearman R C, Parmaksiz I, et al. 2004. Molecular characterization of Buffalograss germplasm using sequence-related amplified polymorphism markers. Theoretical and Applied Genetics, 108(2): 328-334.
Chen S, Zhou J, Chen Q, et al. 2013. Analysis of the genetic diversity of garlic (Allium sativum L.) germplasm by SRAP. Biochemical Systematics and Ecology, 50(5):139-146.
Esposito M A, Martin E A, Cravero V P, et al. 2007. Characterization of pea accessions by SRAP's markers. Scientia Horticulturae, 113(4): 329-335.
Ferriol M, Pico B, Nuez F. 2003. Genetic diversity of a germplasm collection of Cucurbita pepo using SRAP and AFLP markers. Theoretical and Applied Genetics, 107(2): 271-282.
Li G, Quiros C F. 2001. Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theoretical and Applied Genetics, 103(2/3): 455-461.
Xu J, Li A, Wang X, et al. 2013.Genetic diversity and phylogenetic relationship of kenaf (Hibiscus cannabinu L.) accessions evaluated by SRAP and ISSR. Biochemical Systematics and Ecology, 49(4): 94-100.
Xu M, Li H, Zhang B. 2006. Fifteen polymorphic simple sequence repeat markers from expressed sequence tags of Liriodendron tulipifera. Molecular Ecology Notes, 6(3): 728-730.
Yang A H, Zhang J J, Tian H, et al. 2012. Characterization of 39 novel EST-SSR markers for Liriodendron tulipifera and cross-species amplification in L. chinense (Magnoliaceae). American Journal of Botany, 99(11): e460-e464.

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