利用SSCP技术分析甘蓝型油菜10个功能基因序列差异

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

以甘蓝型油菜SI-1300和Eagle为材料,利用DNA单链构象多态性(single-strand conformation polymorphism,SSCP)技术,对10对功能基因特异性引物进行多态性分析,每对引物均检测到1个多态性位点。随后随机挑选10个多态性片段进行测序,并利用bl2seq软件比较测序序列与基因原始序列。结果显示测序序列与所对应的基因原始序列之间相似程度平均高达98%,差异碱基数平均仅为2.3个。进一步选取5对引物比较分析两个材料间的差异扩增片段序列,发现差异扩增片段在2个材料中高度保守,平均相似度达97%;在所测序的5对引物扩增序列中,共存在39个单核苷酸多态性(single-nucleotide polymorphisms,SNPs)和5个插入/缺失突变(insertion-deletions,INDELs),SNP和INDEL的发生频率分别为1SNP/30bp和1INDEL/233bp。结果表明,SSCP标记能够真实代表原始功能基因,甘蓝型油菜功能基因序列在不同材料间高度保守,其遗传变异类型主要来源于SNP。

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 were 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.