铁皮石斛EST-SSR引物的评价、甄选与应用研究

doi:10.7525/j.issn.1673-5102.2017.01.011

摘要/Abstract

摘要： 开发、评价已筛选的铁皮石斛EST-SSR引物，并探讨其在遗传多样性、物种鉴定、遗传图谱构建、亲缘关系鉴定等方面应用。利用前期筛选的20对多态性较好的引物用于16份材料（15份铁皮石斛与1份串珠石斛）的聚丙烯酰氨凝胶电泳，统计扩增条带。采用PopGen32软件进行Nei's基因多样度和Simpson指数等多态性指标的统计，分析单个引物和组合引物的区分率，之后采用NTSYSpc2.1软件计算遗传相似系数聚类图的构建。结果表明，引物DN4等16对引物多态性较高，而DN13、DN23、DN60和DN67多态性较低。单个引物均不能将所有供试样品区分开来，其中DN4、DN10、DN105、DN81、DN39和DN71区分率相对较高；利用组合引物能够将所有样品进行有效区分，其中DN4+DN10+DN105+DN39引物组合可作为核心引物予以使用，可用于铁皮石斛指纹图谱的构建和遗传多样性分析。聚类分析结果表明，该20对引物可以较好地应用于铁皮石斛种内、种间亲缘关系的鉴定。此外，引物DN4、DN13、DN39、DN58、DN65、DN67、DN10和DN99可用于铁皮石斛与串珠石斛的鉴定。本研究开发的20对引物可较好地应用于铁皮石斛遗传多样性分析、种质鉴定、亲缘关系鉴定和遗传图谱构建等方面，具有广泛的应用性。

关键词: 铁皮石斛, 分子标记, EST-SSR, 引物评价, 应用研究

Abstract: By developing and evaluating EST-SSR primers, we studied their application in genetic diversity, species identification, genetic map construction, genetic relationship identification of Dendrobium officinale. The early developed 20 pairs of primers with good polymorphism were used for the analysis of 16 samples(15 samples of D.officinale and 1 sample of D.falconeri) by polyacrylamide gel electrophoresis, and the amplified bands were calculated. Nei's gene diversity and Simpson index were tested using PopGen32 to analysis the differentiation rate of single primer and combination primers. Finally, the construction of genetic similarity coefficient clustering map was calculated by using NTSYSpc2.1. The 16 pairs of primers, such as DN4, were highly polymorphic, while lower in DN13, DN23, DN60 and DN67. Although single primer could not distinguish all the samples, DN4, DN10, DN105, DN81, DN39 and DN71 displayed relatively higher distinguish rate. All the samples could be distinguished effectively by combined primers, and the DN4+DN10+DN105+DN39 primer set could be used as the core primer for fingerprint construction and genetic diversity analysis of D.officinale. By cluster analysis, the 20 pairs of primers could be used for the identification of both intraspecific and interspecific phylogenetic relationships of D.officinale. The primers DN13, DN39, DN4, DN58, DN65, DN67, DN10 and DN99 could be used for the identification of D.officinale and D.falconeri. The 20 pairs of primers could be wildly used in the application of genetic diversity analysis, germplasm identification, genetic relationship identification and genetic map construction of D.officinale.

Key words: Dendrobium officinale, molecular markers, EST-SSR, primer evaluation, applied research

中图分类号:

S567.9

胡仲义, 付涛, 何月秋, 李文, 林立. 铁皮石斛EST-SSR引物的评价、甄选与应用研究[J]. 植物研究, 2017, 37(1): 78-87.

HU Zhong-Yi, FU Tao, HE Yue-Qiu, LI Wen, LIN Li. Evaluation,Selection and Application of Dendrobium officinale EST-SSR Primers[J]. Bulletin of Botanical Research, 2017, 37(1): 78-87.

参考文献

1. 高正华,杨兵勋,陈立钻. 铁皮石斛的研究进展[J]. 中国现代应用药学,2008,25(8):692-695.Gao Z H,Yang B X,Chen L Z. Progress of research on the Dendrobium officinale[J]. Chinese Journal of Modern Applied Pharmacy,2008,25(8):692-695.
2. 管惠娟,张雪,屠风娟,等. 铁皮石斛化学成分的研究[J]. 中草药,2009,40(12):1873-1876.Guan H J,Zhang X,Tu F J,et al. Study on the chemical constituents of the Dendrobium officinale[J]. Chinese Traditional and Herbal Drugs,2009,40(12):1873-1876.
3. 李燕,王春兰,王芳菲,等. 铁皮石斛化学成分的研究[J]. 中国中药杂志,2010,35(13):1715-1719.Li Y,Wang C L,Wang F F,et al. Chemical constituents of Dendrobium candidum[J]. China Journal of Chinese Materia Medica,2010,35(13):1715-1719.
4. Hokanson S C,Szewc-mcfadden A K,Lamboy W F,et al. Microsatellite(SSR) markers reveal genetic identities,genetic diversity and relationships in a Malus×domestica borkh. core subset collection[J]. Theoretical and Applied Genetics,1998,97(5-6):671-683.
5. BérubéY,Zhuang J,Rungis D,et al. Characterization of EST-SSRs in loblolly pine and spruce[J]. Tree Genetics & Genomes,2007,3(3):251-259.
6. Varshney R K,Sigmund R,B rner A,et al. Interspecific transferability and comparative mapping of barley EST-SSR markers in wheat,rye and rice[J]. Plant Science,2005,168(1):195-202.
7. 苑鹤,林二培,朱波,等. 铁皮石斛人工栽培居群的遗传多样性研究[J]. 中草药,2011,42(3):566-569.Yuan H,Lin E P,Zhu B,et al. Genetic diversity in cultivated populations of Dendrobium officinale[J]. Chinese Traditional and Herbal Drugs,2011,42(3):566-569.
8. 王慧中,卢江杰,施农农,等. 13种石斛属植物遗传多样性的AFLP分析[J]. 分子细胞生物学报,2007,40(3):205-210.Wang H Z,Lu J J,SHI N N,et al. Analysis of genetic diversity among 13 Chinese species of Dendrobium based on AFLP[J]. Journal of Molecular Cell Biology,2007,40(3):205-210.
9. 李明焱,谢小波,朱惠照,等. 铁皮石斛新品种"仙斛1号"的选育及其特征特性研究[J]. 中国现代应用药学,2011,28(4):281-284.Li M Y,Xie X B,Zhu H Z,et al. Breeding of Dendrobium officinale cv. Xianhu 1 and its characteristics[J]. Chinese Journal of Modern Applied Pharmacy,2011,28(4):281-284.
10. 樊洪泓,李廷春,邱婧,等. 药用石斛遗传多样性的SRAP标记研究[J]. 中国中药杂志,2008,33(1):6-10.Fan H H,Li T C,Qiu J,et al. Studies on genetic diversity of medicinal Dendrobium by SRAP[J]. China Journal of Chinese Materia Medica,2008,33(1):6-10.
11. 邱道寿,郑希龙,蔡时可,等. 石斛SSR标记的开发及可转移性分析[J]. 植物科学学报,2013,31(5):500-509.Qiu D S,Zheng X L,Cai S K,et al. Development and transfer analysis of SSR in Dendrobium[J]. Plant Science Journal,2013,31(5):500-509.
12. Silfverberg-dilworth E,Matasci C L,van de Weg W E,et al. Microsatellite markers spanning the apple(Malus×domestica Borkh.) genome[J]. Tree Genetics & Genomes,2006,2(4):202-224.
13. 王西成,姜淑苓,上官凌飞,等. 梨EST-SSR标记的开发及其在梨品种遗传多样性分析中的应用评价[J]. 中国农业科学,2010,43(24):5079-5087.Wang X C,Jiang S L,Shangguan L F,et al. Development of EST-derived SSR markers for pear and evaluation of their application in pear genetic diversity analysis[J]. Scientia Agricultura Sinica,2010,43(24):5079-5087.
14. 上官凌飞,李晓颖,宁宁,等. 杏EST-SSR标记的开发[J]. 园艺学报,2011,38(1):43-54.Shangguan L F,Li X Y,Ning N,et al. Development of EST-SSR markers in apricot[J]. Acta Horticulturae Sinica,2011,38(1):43-54.
15. 宋海斌,崔喜波,马鸿艳,等. 基于SSR标记的甜瓜品种(系)DNA指纹图谱库的构建[J]. 中国农业科学,2012,45(13):2676-2689.Song H B,Cui X B,Ma H Y,et al. Construction of DNA fingerprint database based on SSR marker for varieties(Lines) of Cucumis melo L. [J]. Scientia Agricultura Sinica,2012,45(13):2676-2689.
16. 董清华,王西成,赵密珍,等. 草莓EST-SSR标记开发及在品种遗传多样性分析中的应用[J]. 中国农业科学,2011,44(17):3603-3612.Dong Q H,Wang X C,Zhao M Z,et al. Development of EST-derived SSR markers and their application in strawberry genetic diversity analysis[J]. Scientia Agricultura Sinica,2011,44(17):3603-3612.
17. 王佳卉. 软枣猕猴桃EST-SSR分子标记的开发及遗传多样性分析[D]. 长春:吉林农业大学,2014.Wang J H. Development of EST-derived SSR markers in Actinidia arguta and genetic diversity analysis[D]. Changchun:Jilin Agricultural University,2014.
18. 向旭,欧良喜,陈厚彬,等. 中国96个荔枝种质资源的EST-SSR遗传多样性分析[J]. 基因组学与应用生物学,2010,29(6):1082-1092.Xiang X,Ou L X,Chen H B,et al. EST-SSR analysis of genetic diversity in 96 litchi(Litchi chinensis Sonn.) Germplasm resources in China[J]. Genomics and Applied Biology,2010,29(6):1082-1092.
19. 赖勇,王鹏喜,范贵强,等. 大麦SSR标记遗传多样性及其与农艺性状关联分析[J]. 中国农业科学,2013,46(2):233-242.Lai Y,Wang P X,Fan G Q,et al. Genetic diversity and association analysis using SSR markers in barley[J]. Scientia Agricultura Sinica,2013,46(2):233-242.
20. 潘海涛. 小麦EST-SSR标记的开发和苗期养分吸收QTL定位[D]. 泰安:山东农业大学,2009.Pan H T. Development of EST-SSR markers and QTL mapping for absorption of elements in wheat[D]. Tai'an:Shandong Agricultural University,2009.
21. Wei W L,Qi X Q,Wang L H,et al. Characterization of the sesame(Sesamum indicum L.) global transcriptome using Illumina paired-end sequencing and development of EST-SSR markers[J]. BMC Genomics,2011,12(1):451.
22. Han Z G,Wang C B,Song X L,et al. Characteristics,development and mapping of Gossypium hirsutum derived EST-SSRs in allotetraploid cotton[J]. Theoretical and Applied Genetics,2006,112(3):430-439.
23. 张曙光,董丽娟,杨阳,等. 茶树品种"玉绿"和"玉笋"父本的EST-SSR的标记鉴定[J]. 茶叶科学,2009,29(6):430-435.Zhang S G,Dong L J,Yang Y,et al. Identification of male parents for "Yulv" and "Yusun" tea variety based on EST-SSR markers[J]. Journal of Tea Science,2009,29(6):430-435.
24. 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.Huang H Y,Du H Y,Wuyun T N,et al. Development of SSR molecular markers based on transcriptome sequencing of Eucommia ulmoides[J]. Scientia Silvae Sinicae,2013,49(5):176-181.
25. 鲍露,张东,滕元文,等. DNA标记技术在梨属植物研究中的应用[J]. 果树学报,2006,23(2):270-275.Bao L,Zhang D,Teng Y W,et al. DNA marker technologies in Pyrus research[J]. Journal of Fruit Science,2006,23(2):270-275.
26. 鲁敏. 苹果SSR标记在梨属植物亲缘关系研究中的应用[D]. 雅安:四川农业大学,2011.Lu M. Application of apple SSRs in genetic relationship of pyrus[D]. Ya'an:Sichuan Agricultural University,2011.
27. 陈媛媛. 猪苓EST-SSR标记的开发及遗传多样性研究[D]. 杨凌:西北农林科技大学,2014.Chen Y Y. Development of EST-SSR markers and genetic diversity for Polyporus umbellatus[D]. Yangling:Northwest A& F University,2014.

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