中国50个甘蓝代表品种EST-SSR指纹图谱的构建

doi:10.3864/j.issn.0578-1752.2014.01.012

摘要/Abstract

摘要： 【目的】研究甘蓝DNA指纹鉴定方法,并构建50份中国甘蓝代表品种DNA指纹数据库，为甘蓝品种特异性、真实性、纯度鉴定提供参考依据。【方法】首先，利用6%变性聚丙烯酰胺凝胶电泳技术和来源不同的甘蓝材料对已开发的甘蓝EST-SSR引物进行筛选，获得多态性引物；然后将可用于甘蓝DNA指纹鉴定的核心引物正义链5′末端分别标记TAMRA、HEX、 ROX、6-FAM四种荧光，利用DNA分析仪检测不同等位变异的扩增片段大小，并确定每个等位变异的标准品种。利用核心引物扩增结合标准品种的片段大小，构建中国50个甘蓝代表品种的SSR指纹数据库。通过人工构建模拟群体对‘中甘21’品种真实性进行鉴定，进而对该指纹鉴定技术进行验证。【结果】利用6份来自中国不同生态条件、植物学性状差异较大的甘蓝品种对978对EST-SSR引物进行初步筛选，获得带型清晰、具有多态性的引物128对。进一步根据引物扩增带型清晰、多态性信息指数较高、不同等位变异的带型易于区分、在染色体上分布均匀原则，筛选出20对核心引物。该套引物可以检测甘蓝染色体20个位点的58个等位变异，平均每条染色体上被检测位点2.22个，平均每个位点包含2.9个等位变异，其中引物BoE607、BoE723等位变异数最多为5个，PIC值处于0.34—0.76之间。通过DNA分析仪检测显示等位变异扩增片段长度范围143—296 bp。利用20对核心引物构建50份中国甘蓝代表品种DNA指纹数据库，并阐述了甘蓝SSR-DNA指纹鉴定的应用和技术流程。本研究还对来自甘蓝主产区的31份‘中甘21’品种进行真实性鉴定，结果显示指纹鉴定与田间鉴结果完全一致。【结论】筛选获得20对核心引物，用于构建中国50个甘蓝代表品种DNA指纹数据库，通过构建人工模拟群体对‘中甘21’品种的真实性进行鉴定，准确率达100%。

Abstract: 【Objective】In this study, the method of cabbage DNA fingerprint was drawn, and fifty cabbage representative varieties from China were fingerprinted with EST-SSR primers to provide reference for variety distinctness, authenticity, and purity identification. 【Method】First, EST-SSR primers were screened by using the technologies of 6% denaturing polyacrylamide gel electrophoresis and six cabbage varieties that come from different ecogeography. The length of amplified fragments was detected on DNA Analyzer platform using four fluorescent-labels (TAMRA, HEX, ROX and 6-FAM) in 5′end of forward primer, and then defined the reference variety for every alleles. Total twenty core primers were used to establish fifty cabbage varieties SSR fingerprinting, and for ‘Zhonggan 21’ variety identification. 【Result】 Six cabbage varieties of different resources were used to screen 978 EST-SSR primers, out of 128 polymorphic primers were obtained according to the PCR bands stability, high polymorphism information content (PIC), easy discrimination of different alleles and even distribution of molecular markers on each chromosome, and 20 pairs of primers were selected to detect a total of 58 alleles at 20 loci, with 2.22 loci per chromosome and 2.9 alleles per locus on average. The PIC values varied among the primers ranging from 0.34 to 0.76. The length of amplified fragment varied in the range of 143-296 bp. The maximum number of alleles for each primer pairs of BoE607 and BoE723 was five. Fingerprinting database of 50 cabbage representative varieties from China was established with 20 pairs of core primers. The authenticity of ‘Zhonggan 21’ was identified by artificial simulated population and these results were identical with that made from field investigation.【Conclusion】Twenty pairs of core primers were selected and used to establish DNA fingerprint database of 50 cabbage representative varieties from China, and the authenticity of ‘Zhonggan 21’ was identified by artificial simulated population and the accuracy was 100%.

Key words: cabbage , SSR marker , fingerprinting , varieties identification

王庆彪, 张扬勇, 庄木, 杨丽梅, 刘玉梅, 吕红豪, 方智远. 中国50个甘蓝代表品种EST-SSR指纹图谱的构建[J]. 中国农业科学, 2014, 47(1): 111-121.

WANG Qing-Biao, ZHANG Yang-Yong, ZHUANG Mu, YANG Li-Mei, LIU Yu-Mei, 吕Hong-Hao , FANG Zhi-Yuan. EST-SSR Fingerprinting of Fifty Cabbage Representative Varieties from China[J]. Scientia Agricultura Sinica, 2014, 47(1): 111-121.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.01.012

https://www.chinaagrisci.com/CN/Y2014/V47/I1/111

参考文献

[1]方智远. 我国甘蓝产销变化与育种对策. 中国蔬, 2008(1): 1-2.

Fang Z Y. Changes in production and marketing of cabbage and its breeding counter measures in China. China Vegetables, 2008(1): 1-2. (in Chinese)

[2]杨丽梅, 方智远, 刘玉梅, 庄木, 张扬勇, 孙培田. “十一五”我国甘蓝遗传育种研究进展. 中国蔬菜, 2011(2): 1-10.

Yang L M, Fang Z Y, Liu Y M, Zhuang M, Zhang Y Y, Sun P T. Advances of research on cabbage genetics and breeding during “The Eleventh Five-year Plan” in China. China Vegetables, 2011(2): 1-10. (in Chinese)

[3]王庆彪. 我国甘蓝育成品种系谱及部分骨干亲本的初步分析. 中国园艺学会十字花科分会第九届学术研讨会论文集, 2011: 150-155.

Wang Q B. Pedigree analysis of cabbage cultivars released in China. The Cruciferous Branch Society of Horticulture Ninth Conference Proceedings, 2011: 150-155. (in Chinese)

[4]王庆彪, 方智远, 杨丽梅, 庄木, 张扬勇, 刘玉梅, 孙培田, 吕红豪. 中国甘蓝育成品种系谱分析. 园艺学报, 2013, 40(5): 869-886.

Wang Q B, Fang Z Y, Yang L M, Zhuang M, Zhang Y Y, Liu Y M, Sun P T, Lü H H. Survey and pedigree analysis of cabbage varieties released in China. Acta Horticulturae Sinaca, 2013, 40(5): 869-886. (in Chinese)

[5]戴剑, 李华勇, 丁奎敏, 洪德林. 植物新品种DUS测试技术的现状与展望. 种子, 2007, 26(9): 44-47.

Dai J, Li H Y, Ding K M, Hong D L. Current status and prospects on DUS testing technique system for new plant variety protection. Seed, 26(9): 44-47. (in Chinese)

[6]吴晓玲. 植物新品种保护的技术标准. 种子科技, 1994(4): 43-44.

Wu X L. Technical standards for the protection of new varieties. Seed Science and Technonlogy, 1994(4): 43-44. (in Chinese)

[7]李汝玉, 宋国安, 杨平平. 利用SSR标记进行品种权有争议品种的鉴定. 种子, 2000, 107(1): 66-67.

Li R Y, Song G A, Yang P P. Identification of the rights controversial varieties by SSR markers. Seed, 2000, 107(1): 66-67. (in Chinese)

[8]王立新, 常利芳, 李宏博, 季伟, 刘丽华, 赵昌平. 小麦区试品系DUS测试的分子标记. 作物学报, 2010, 36(7): 1114-1125.

Wang L X, Chang L F, Li H B, Ji W, Liu L H, Zhao C P. Molecular markers for estimating distinctness, uniformity, and stability of wheat lines in regional trials. Acta Agronomica Sinica, 2010, 36(7): 1114-1125. (in Chinese)

[9]Provan J, Powell W, Waugh R. Microsatellite analysis of relationships within cultivated potato(Solanum tuberosum). Theoretical and Applied Genetics, 1996, 92: 1078-1084.

[10]栾雨时, 安利佳, 黄百渠, 张文俊, 何孟元. 用SSR探针进行番茄品种的DNA指纹分析. 园艺学报, 1999, 26(1): 51-53.

Luan Y S, An L J, Huang B Q, Zhang W J, He M Y. Using simple sequence repeat probe for DNA fingerprints in tomato cultivars. Acta Horticulturae Sinica, 1999, 26(1): 51-53. (in Chinese)

[11]许鲲, 张冬晓, 伍晓明, 陆光远, 刘凤兰, 陈碧云, 高桂珍. 国家冬油菜区试新品种SSR指纹图谱构建与遗传关系分析. 中国油料作物学报, 2008, 30(1): 29-34.

Xu K, Zhang D X, Wu X M, Lu G Y, Liu F L, Chen B Y, Gao G Z. SSR analysis of new winter rapeseed cultivars in the national field tests. Chinese Journal of Oil Crop Sciences, 2008, 30(1): 29-34. (in Chinese)

[12]李丽, 何伟明, 马连平, 刘庞源, 徐海明, 徐家柄, 郑晓鹰. 用EST-SSR分子标记技术构建大白菜核心种质及其指纹图谱库. 基因组学与应用生物学, 2009, 28(1): 76-88.

Li L, He W M, Ma L P, Liu P Y, Xu H M, Xu J B, Zheng X Y. Construction Chinese cabbage (Brassica rapa L.) core collection and its EST-SSR fingerprint database by EST-SSR molecular markers. Genomics and Applied Biology, 2009, 28(1): 76-88. (in Chinese)

[13]薄天岳, 刘冲, 葛才林, 任云英, 陈锦秀, 杨晓锋. 甘蓝品种‘争春’和‘寒光2 号’的DNA 指纹图谱构建. 植物生理学通讯, 2006, 42(4): 721-724.

Bo T Y, Liu C, Ge C L, Ren Y Y, Chen J X, Yang X F. Establishment of DNA fingerprinting on cabbage(Brassica oleracea var. capitata) cultivars of ‘Zhengchun’ and ‘Hanguang 2’. Plant Physiology Communications, 2006, 42(4): 721-724. (in Chinese)

[14]宋顺华, 郑晓鹰. 甘蓝品种的AFLP指纹鉴别图谱分析. 分子植物育种, 2006, 4(3): 51-54.

Song S H, Zheng X Y. Analysis of AFLP fingerprinting in cabbage cultivars testing. Molecular Plant Breeding, 2006, 4(3): 51-54. (in Chinese)

[15]田雷, 曹鸣庆, 王辉, 郭晶心, 周乃元, 孙江, 贾希海. AFLP 标记技术在鉴定甘蓝种子真实性及品种纯度中的应用. 生物技术通报, 2001(3): 38-44.

Tian L, Cao M Q, Wang H, Guo J X, Zhou N Y, Sun J, Jia X H. Application of AFLP in the identification of cabbage seed genuineness and purity. Biotechnology Information, 2001(3): 38-44. (in Chinese)

[16]陈琛, 张兴桃, 程斐, 庄木, 刘玉梅, 杨丽梅, 张扬勇, 方智远. 秋甘蓝品种的SSR指纹图谱的构建. 园艺学报, 2011, 38(1): 159-164.

Chen C, Zhang X T, Cheng F, Zhuang M, Liu Y M, Yang L M, Zhang Y Y, Fang Z Y. Establishment of SSR fingerprinting on autumn cabbage hybrids and their parents. Acta Horticulturae Sinica, 2011, 38(1): 159-164. (in Chinese)

[17]司军, 李成琼, 任雪松, 宋洪元, 宋明, 王小佳. 结球甘蓝抗根肿病SSR分子连锁图谱的构建. 植物病理学报, 2011, 41(3): 305-309.

Si J, Li C Q, Ren X S, Song H Y, Song M, Wang X J. Construction of a molecular linkage map for club root resistance in cabbage using SSR markers (Brassica oleracea L. var. capitata). Acta Phytopa Thologica Sinica, 2011, 41(3): 305-309. (in Chinese)

[18]玉米品种鉴定-DNA指纹方法. 中华人民共和国农业行业标准. NY/T 1432-2007.

Maize Variety Identification Molecular Techniques. NY/T 1432-2007.

[19]水稻品种鉴定-DNA指纹方法. 中华人民共和国农业行业标准. NY/T 1433-2007.

Identification of Rice (Oryza sativa L. ) Varieties Using Microsatellite Markers. NY/T 1433-2007. (in Chinesse)

[20]陈琛, 庄木, 李康宁, 刘玉梅, 杨丽梅, 张扬勇, 程斐, 孙培田, 方智远. 甘蓝EST-SSR标记的开发与应用. 园艺学报, 2010, 37(2): 221-228.

Chen C, Zhuang M, Li K N, Liu Y M, Yang L M, Zhang Y Y, Cheng F, Sun P T, Fang Z Y. Development and utility of EST-SSR marker in cabbage. Acta Horticulturae Sinica, 2010, 37(2): 221-228. (in Chinese)

[21]梁宏伟, 王长忠, 李忠, 罗相忠, 邹桂伟. 聚丙烯酰胺凝胶快速、高效银染方法的建立. 遗传, 2008, 30(10): 1379-1382.

Liang H W, Wang C Z, Li Z, Luo X Z, Zou G W. Improvement of the silver-stained technique of polyacrylamide gel electrophoresis. Hereditas, 2008, 30(10): 1379-1382. (in Chinese)

[22]高海娜, 张恩慧, 李殿荣, 王灏, 马英夏, 许忠民. 甘蓝一代杂种纯度SSR鉴定与田间鉴定的一致性研究. 西北农林科技大学学报: 自然科学版, 2012, 40(5): 117-122.

Gao H N, Zhang E H, Li D R, Wang H, Ma Y X, Xu Z M. Research on SSR molecular marker identification technique of hybrids purity in cabbage. Journal of Northwest A＆F University: National Science Edition, 2012, 40(5): 117-122. (in Chinese)

[23]Zhang X, Wu J, Zhang H, Ma Y, Guo A, Wang X. Fine mapping of a male sterility gene MS-cd1 in Brassica oleracea. Theoretical and Applied Genetics, 2011, 123:231-238.