甘蓝显性雄性不育基因的延长随机引物扩增DNA(ERPAD)标记

An Extended Random Primer Amplified DNA(ERPAD)Marker Linked to a Dominant Male Sterile Gene in Cabbage

获得了一个与甘蓝显性雄性不育基因连锁的延长随机引物扩增ＤＮＡ（ＥｘｔｅｎｄｅｄＲａｎｄｏｍＰｒｉｍｅｒＡｍｐｌｉｆｉｅｄＤＮＡ，ＥＲＰＡＤ）标记ＥＰＴ１１９００。ＥＰＴ１１９００是在与甘蓝显性雄性不育基因连锁的ＲＡＰＤ标记ＯＴ１１９００的基础上，通过逐步筛选３’端延长的随机引物的方法转化而成的稳定性和特异性都得到增强的新标记。这种标记的稳定性和特异性接近ＳＣＡＲ标记，但不需要克隆和测序。该方法首先根据ＯＰＴ１１的序列合成３’端添加Ａ、Ｔ、Ｃ、Ｇ４种碱基的４个引物，组成１０个引物对。以不育池为模板筛选出ＯＴ１１Ｃ＋ＯＴ１１Ｇ为唯一能够扩增长度与ＯＴ１１９００相同的一个片段的引物对。在此引物对基础上，又添加４种碱基得到８个新的引物，相互组合成１６个引物对。进一步以不育池为模板筛选出ＯＴ１１ＣＴ＋ＯＴ１１ＧＡ，在严格条件下，它可特异扩增长度与ＯＴ１１９００相同的一个片段ＥＰＴ１１９００。通过分离群体分析证实这一片段与ＯＴ１１９００处于同一位点。

An extended random primer amplified DNA(ERPAD)marker EPT11900 linked to a dominant male sterile gene was identified in cabbage(Brassica oleracea var.capitata).EPT11900 was developed from OT11900,an RAPD marker linked to the same gene,by successive screening of 3F endextended random primers.This new marker was more stable and specific than the original RAPD marker.It was comparable to SCAR in stability and specificity,but its development needed no cloning and sequencing.According to the sequence of OPT11,A,T,C or G was added to 3end of the primenr and four extended primers were obtained.From the ten combinations made by using the four primers OT11C+OT11G was identified as the only primer pair that had the ability to amplify a segment with the same size as OT11900,when using MSbulk as template.Based on OT11C+OT11G,another eight primers were synthesized by adding each of A,T,C and G.Sixteen primer pairs were then formed by combining the eight primers.After stringent amplification,OT11CT+OT11GA was screened as the specific primer pair to produce single segment EPT11900 with the same size as OT11900.Segregating analysis proved that EPT11900 and OT11900 located at the same locus.