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Acta Agron Sin ›› 2012, Vol. 38 ›› Issue (01): 43-49.doi: 10.3724/SP.J.1006.2012.00043


Polymorphism Analysis of Ten Functional Genes in Brassica napus Using SSCP Method

LI Yuan-Yuan1,2, CHEN Qing-Fang2, FU Ting-Dong2,MA Chao-Zhi2,*   

  1. 1 Department of Bioengineering, Weifang University, Weifang 261061, China; 2 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
  • Received:2011-05-27 Revised:2011-09-13 Online:2012-01-12 Published:2011-11-07
  • Contact: 马朝芝, E-mail: yuanbeauty@hzau.edu.cn

Abstract: A sensitive technology is very necessary to detect the polymorphisms of functional genes in different cultivars, for the coding sequences of functional genes tend to be conserved even between species. Single-strand conformational polymorphism (SSCP) is a desirable method for DNA polymorphism analysis because of its high sensitivity and cost effectiveness. In previous publications, we developed 177 functional markers corresponding to 111 differentially expressed genes between the parents of a Brassica napus hybrid. And, 45 functional markers involved in 39 genes or expressed sequence tags (ESTs) were linked to the QTLs of 12 yield-related traits in the F2 population from SI-1300×Eagle using SSCP analysis. In the present research, we sequenced some polymorphic bands detected by SSCP analysis to confirm the high sensitivity of SSCP analysis. Firstly, a total of ten primer pairs, which were designed according to ten B. napus functional genes or ESTs, were used to survey polymorphisms between SI-1300 and Eagle. All primers showed polymorphisms, resulting ten polymorphic loci. Subsequently, ten polymorphic bands were randomly selected, sequenced and aligned with the gene sequences for primers designed using the bl2seq software. The results indicated that the average identity was 98%, and the average number of different bases was only 2.3 between the sequenced fragments and their functional genes. Furthermore, the sequence comparison of polymorphic fragments amplified by five primer pairs was performed between SI-1300 and Eagle. The polymorphic fragments are highly conserved between SI-1300 and Eagle, and there were 39 single-nucleotide polymorphisms (SNPs) and five insertion-deletions (INDELs) in the DNA fragments amplified by the five primer pairs. The average frequency of sequence polymorphism was estimated to be one SNP every 30 bp and one INDEL every 233 bp. In conclusion, the sequences of functional genes, which could be really amplified by specific primers, are highly conversed among different cultivars in B. napus, and SNP is the most basic genetic variation for functional genes. This study will provide a foundation for investigating the molecular basis of important traits in rapeseed using comparative genomics.

Key words: Brassica napus, Functional genes, SSCP, Sequencing

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