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作物学报 ›› 2014, Vol. 40 ›› Issue (03): 447-456.doi: 10.3724/SP.J.1006.2014.00447

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甜叶菊微卫星富集文库的构建与多态性标记的筛选

秦海峰1,龙宁1,吴建国1,2,*,石春海1,*   

  1. 1浙江大学农业与生物技术学院农学系, 浙江杭州 310058；2 浙江农林大学农业与食品科学学院, 浙江省农业产品品质改良技术研究重点实验室, 浙江临安 311300
  • 收稿日期:2013-06-07 修回日期:2013-08-30 出版日期:2014-03-12 网络出版日期:2013-11-14
  • 通讯作者: 吴建国, E-mail: jianguowu@zafu.edu.cn, Tel: 0571-88982691; 石春海, E-mail: chhshi@zju.edu.cn, Tel: 0571-88982691
  • 基金资助:

    本研究由浙江省科技攻关项目(2009C32030)资助。

Construction of Microsatellite-Enriched Library and Isolation of Icrosatellite Markers in Stevia rebaudiana

QIN Hai-Feng1,LONG Ning1,WU Jian-Guo1,2,*,SHI Chun-Hai1,*   

  1. 1 College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China; 2 The Key Laboratory for Qulaity Improvement of Agricultural Products of Zhejiang University, College of Agriculture and Food Science, Zhejiang A&F University, Lin’an 311300, China
  • Received:2013-06-07 Revised:2013-08-30 Published:2014-03-12 Published online:2013-11-14
  • Contact: 吴建国, E-mail: jianguowu@zafu.edu.cn, Tel: 0571-88982691; 石春海, E-mail: chhshi@zju.edu.cn, Tel: 0571-88982691

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摘要：

甜叶菊是我国一种重要特种经济作物, 其分子标记相关遗传背景研究甚少。本研究基于生物素与链霉亲和素的强亲和性原理, 用链霉亲和素顺磁颗粒捕捉人工合成的标记有生物素的寡核苷酸探针(AG)15, 间接筛选出含有甜叶菊基因组微卫星序列的DNA酶切片段, 将筛选得到的片段连接到pUC-T载体中, 构建甜叶菊微卫星序列的富集文库。挑取354个克隆进行菌落PCR检验, 从中筛选出158个阳性克隆进行测序。结果表明, 134个(84.81%)克隆中含有微卫星序列, 其中完美型85个(63.43%)、非完美型15个(11.19%)、复合型34个(25.38%)。根据微卫星序列共设计出71对微卫星引物, 其中62对能扩增出稳定的条带。利用24个甜叶菊品系对这62对引物的遗传多样性的分析表明, 有16个位点表现出多态性, 等位基因数为2~8个, 平均每个位点扩增得到4.5个等位基因, 多态性信息含量在0.3163~0.7595之间, 观测杂合度(Ho)与期望杂合度(He)的范围分别为0.2174~0.9167与0.3555~0.8076。通过聚类分析, 将甜叶菊分为大小叶两大类。本研究开发出的微卫星标记可为甜叶菊的分子遗传育种提供有效的遗传标记。

关键词: 甜叶菊, 富集文库, 微卫星, 磁珠, 遗传多样性

Abstract:

Stevia is one of important special crops in China, but its genetic background about molcular markers is known a little so far.Based on the principle of the strong affinity between biotin and streptavidin, we used streptavidin paramagnetic particles to catch a synthetic oligonucletide probe (AG)15 during an indirect selection from the genomic microsatellite sequence of restriction fragments of DNA in Stevia rebaudiana. The enriched DNA fragments were ligated to the pUC-T vector to construct a S. rebaudiana microsatellite sequence enriched library. From a library of 354 cloned colonies, 158 were screened by PCR examination and the positive clones were sequenced, resulting in 134 (84.81%) clones containing microsatellite sequences. Among these microsatellites, 85 (64.34%) belonged to the perfect type, 15 (11.19%) to the imperfect type and 34 (25.38%) to the compound type. From the primers designed for the 71 microsatellite loci, 62 could amplify stable PCR products. An analysis of individual genetic diversity of the 62 pairs primers using 24 S. rebaudiana varietiesshowed 16 loci had polymorphism and the number of alleles at each locus ranged from two to eight. The average site amplified 4.5 alleles and the polymorphic information content ranged from 0.3163 to 0.7595. The observed heterozygosity (Ho) varied from 0.2174 to 0.9167 and the expected heterozygosity (He) varied from 0.3555 to 0.8076. By clustering analysis, S. rebaudiana were divided into two categories. The microsatellite markers developed at this study could provide useful genetic markers for molecular breeding of S. rebaudiana.

Key words: Stevia rebaudiana, Enrichement library, Microsatellite, Magnetic-beads, Genetic diversity

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