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Acta Agron Sin ›› 2014, Vol. 40 ›› Issue (03): 447-456.doi: 10.3724/SP.J.1006.2014.00447

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2014-03-12 Published: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

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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