Using the SSR with fluorescent labeling to establish SSR molecular ID code for cultivars of the Chinese herbaceous peony
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摘要: 为了开发芍药品种分子鉴定方法,本研究采用SSR标记技术,在目前芍药SSR引物开发较少的情况下,采用文献相关引物84对,并结合自行设计的27对引物,将6%非变性聚丙烯酰胺凝胶与四重荧光毛细管电泳技术相结合,从111对引物中筛选出29对扩增稳定、多态性高的核心引物;共检测到223个SSR等位位点,平均每对引物为7.69个,有效等位基因数(Ne)为3.658±0.328,Shannon多样性指数(I)为1.371±0.104,观测杂合度(Ho)为0.532±0.045,期望杂合度(He)为0.645±0.036,多态性信息含量PIC值介于0.20~0.84,平均0.60。基于最少引物鉴定最多种质的原则,按照PIC值由高到低顺序串联排序的方式,最终筛选出4对SSR引物(Pmg153、Knu73、Pmg209、J38)。结果表明:筛选出的4对SSR引物组合可以有效鉴定66个芍药品种;采用个位数字和大写英文字母结合对不同带型进行编码,成功绘制了66个中国传统芍药品种的分子身份证。本研究为芍药品种鉴定提供了一种快速、准确与高效的分子检测方法,也有助于中国传统芍药品种的遗传改良与芍药新品种保护。Abstract: Herbaceous peony(Paeonia lactiflora Pall.)is a perennial herbaceous plant which belongs to Sect. Paeonia in family Paeoniaceae. It is a traditional flower with 3000-year history and has hundreds of cultivars in China. However, it is difficult to distinguish cultivars accurately only by the traditional morphological identification method. Establishing the multi-level molecular identification techniques can improve the accuracy of cultivar identification. In order to develop a molecular identification method,we used the SSR technique to establish the molecular ID code of 66 cultivars of Chinese traditional herbaceous peony. In this research, 111 pairs of SSR primers with 84 from literatures and 27 self-designed, were labeled with 6% none-denaturing polyacrylamide gel electrophoresis and quadruple fluorescence capillary electrophoresis. Then 29 pairs of primers with high polymorphism and good repeatability were selected. The results showed that 223 polymorphic alleles were revealed by the 29 primer pairs,with an average of 7.69 alleles per pair. The effective number of alleles (Ne) was 3.658±0.328, Shannon diversity index (I) was 1.371±0.104, observed heterozygosity (Ho) was 0.532±0.045, expected heterozygosity (He) was 0.645±0.036, and the range of PIC at 29 SSR loci of 66 cultivars was 0.20-0.84, 0.60 on average. Based on the principle of distinguishing the most varieties using the minimum pairs of primers, 4 pairs of primers (Pmg153, Knu73, Pmg209, J38) were selected out. This study indicates that the 4 pairs of primers can identify the 66 varieties of Chinese traditional herbaceous peony and can successfully establish 66 molecular ID codes by different single digits and uppercase letters. It not only provides a rapid, accurate and efficient molecular identification method for herbaceous peony cultivars, but also contributes to genetic improvement of cultivars of Chinese traditional herbaceous peony and is significant for protection of the new varieties.
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[1] TENG H B,LÜ B,ZHAO J R, et al. DNA fingerprint profile involved in plant variety protection practice [J]. Biotechnology Bulletin, 2009(1): 1-6. [2] 滕海涛,吕波,赵久然,等. 利用DNA指纹图谱辅助植物新品种保护的可能性[J]. 生物技术通报, 2009(1): 1-6. [3] 张辕. 基于三种标记的中国传统菊花品种鉴定及分类研究[D]. 北京:北京林业大学, 2014. [4] ZHANG Y.Studies on the identification and classification of Chinese traditional chrysanthemum cultivars based on three types of markers [D]. Beijing:Beijing Forestry University, 2014. [5] GE L.Development of SSR markers based on transcriptome and their application in germplasm identification of Lilium [D]. Beijing:Chinese Academy of Agricultural Sciences, 2012. [6] 葛亮. 基于转录组信息的百合SSR标记开发及种质分子鉴定研究[D]. 北京:中国农业科学院, 2012. [7] 徐雷锋. 利用荧光标记SSR构建百合种质资源分子身份证[J]. 园艺学报, 2014, 41(10): 2055-2064. [8] XU L F. Using the fluorescent labeled SSR markers to establish molecular identity of Lily germplasms [J]. Acta Horticulturae Sinica,2014, 41(10): 2055-2064. [9] HUANG P,CUI P J,ZHENG Y Q,et al.SSR-Based molecular identification and analysis of genetic relationships among Rose varieties[J]. Scientia Silvae Sinicae,2012,48(10): 55-62. [10] 黄平,崔鹏娇,郑勇奇,等. 基于SSR标记月季品种鉴定及遗传关系分析[J]. 林业科学,2012,48(10): 55-62. [11] 季丽静. 观赏芍药部分新种质SSR遗传多样性分析及DNA指纹图谱构建[D]. 北京:北京林业大学, 2013. [12] JI L J.Study of genetic diversity on partial new germplasm of Paeonia and establishment of DNA fingerprint by SSR markers[D]. Beijing:Beijing Forestry University, 2013. [13] 段一凡,王贤荣,梁丽丽,等. 桂花品种SSR荧光指纹图谱的构建[J]. 南京林业大学学报(自然科学版),2014,38(增刊): 1-6. [14] DUAN Y F,WANG X R,LIANG L L, et al. Fingerprinting and identification of Osmanthus fragrans cultivars using fluorescence-labeled SSR markers[J].Journal of Nanjing Forestry University (Natural Sciences Edition),2014,38(Suppl.): 1-6. [15] HU C L, ZHANG C,LIU C L,et al. Establishment of molecular ID for Fraxinus germplasms based on SSR markers[J]. Shandong Agricultural Sciences, 2015,47(5): 6-9,22. [16] 胡春龙,张晨,刘翠兰,等. 基于SSR标记构建白蜡种质资源分子身份证[J]. 山东农业科学, 2015,47(5): 6-9,22. [17] ZHANG J J, JI L J,LIU Z H,et al.Constructing SSR fingerprinting for introduced new germplasm of Paeonia L.[J]. Journal of Northeast Forestry University, 2015,43(3): 70-74. [18] CHENG Y, KIM C, SHIN D, et al. Development of simple sequence repeat (SSR) markers to study diversity in the herbaceous peony ( Paeonia lactiflora )[J].Journal of Medicinal Plants Research, 2011, 5(31): 6744-6751. [19] SUN J, YUAN J, WANG B, et al. Development and characterization of 10 microsatellite loci in Paeonia lactiflora (Paeoniaceae)[J]. American Journal of Botany, 2011, 98(9): 242-243. [20] LI J W, LI N. Establishment of rapid silver-stained method of denaturing polyacrylamide gel of microsatellite markers[J].Chinese Potato Journal, 2015,29(3): 136-140. [21] LI L, CHENG F, ZHANG Q. Microsatellite markers for the Chinese herbaceous peony Paeonia lactiflora (Paeoniaceae)[J]. American Journal of Botany, 2011, 98(2): 16-18. [22] WANG R, TIAN H L, WANG F G, et al.The application comparison of two SSR data analysis softwares GeneMapper and GeneMarker used in the identification of Maize variety[J/OL]. Fenzi Zhiwu Yuzhong(Online),2012,10:1171-1178. [23] CHENG B Y, XIA J H, GONG J Y, et al.Application of capillary electrophoresis detection with fluorescent SSR markers in rice DNA fingerprint identification[J]. Chinese Journal Rice Science, 2011,26(6): 672-676. [24] 张建军,季丽静,刘仲赫,等. 引进观赏芍药新种质的SSR指纹图谱构建[J]. 东北林业大学学报, 2015,43(3): 70-74. [25] ZHANG J G,TIAN R,CHEN Q L, et al.Establishment of molecular ID for Pyrus based on SSR markers [J]. Journal of Huazhong Agricultural University, 2014,33(1): 12-17. [26] 李建武,李宁. SSR标记变性聚丙烯酰胺凝胶快速银染方法的建立[J]. 中国马铃薯, 2015,29(3): 136-140. [27] GAO Y,TIAN L M, LIU F Z, et al.Using the SSR fluorescent labeling to establish SSR fingerprints for 92 cultivars in Pyrus [J]. Acta Horticulturae Sinica, 2012,39(8): 1437-1446. [28] 王蕊,田红丽,王凤格,等. 玉米DNA指纹鉴定中两种SSR数据分析软件GeneMapper和GeneMarker的应用比较[J/OL]. 分子植物育种(网络版),2012,10:1171-1178. [29] QIU Y, LI X X, LI Q X,et al.Establishment of the molecular identification for radishgermplasm using SSR markers[J]. Journal of Plant Genetic Resources, 2014,15(3): 648-654. [30] BOTSTEIN D, WHITE R L, SKOLNICK M, et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms.[J]. American Journal of Human Genetics, 1980, 32(3): 314-331. [31] 程本义,夏俊辉,龚俊义,等. SSR荧光标记毛细管电泳检测法在水稻DNA指纹鉴定中的应用[J]. 中国水稻科学, 2011,25(6): 672-676. [32] MA L Y,KONG D C,LIU H B, et al.Construction of SSR fingerprint on 36 Chinese Jujube cultivars[J].Acta Horticulturae Sinica, 2012,39(4): 647-654. [33] 张靖国,田瑞,陈启亮,等. 基于SSR标记的梨栽培品种分子身份证的构建[J]. 华中农业大学学报,2014,33(1): 12-17. [34] 高源,田路明,刘凤之,等. 利用SSR荧光标记构建92个梨品种指纹图谱[J]. 园艺学报, 2012,39(8): 1437-1446. [35] 邱杨,李锡香,李清霞,等. 利用SSR标记构建萝卜种质资源分子身份证[J]. 植物遗传资源学报, 2014,15(3): 648-654. [36] 麻丽颖,孔德仓,刘华波,等. 36份枣品种SSR指纹图谱的构建[J]. 园艺学报, 2012,39(4): 647-654. -
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