尼罗罗非鱼、奥利亚罗非鱼及其杂交后代微卫星标记鉴别和种质纯度分析

Identification and pureness of germplasm analysis of Oreochromis niloticus,Oreochromis aureus,and their hybrids by microsatellite markers

从罗非鱼微卫星遗传图谱前6个连锁群上随机选取43个微卫星位点,对尼罗罗非鱼(Oreochromis niloticus)、尼罗罗非鱼吉富品系(GIFT)、奥利亚罗非鱼(Oreochromis aureus)以及尼罗罗非鱼和奥利亚罗非鱼杂交后代的DNA进行PCR扩增,筛选出7个位点(UNH995、GM066、GM166、UNH162、GM017、GM440、UNH948)在不同罗非鱼中扩增的条带差别明显,具有特异性,可作为罗非鱼鉴定的分子标记,用其中任意一个位点都可以将尼罗罗非鱼或吉富罗非鱼、奥利亚罗非鱼、杂交罗非鱼区别开来。这7个标记位点在尼罗罗非鱼、吉富罗非鱼和奥利亚罗非鱼中平均每个位点检测到等位基因个数分别为3.1、2.3、1.7,等位基因大小在130～316 bp。这7个标记位点上,尼罗罗非鱼与奥利亚罗非鱼PCR扩增的条带大小相差在20 bp以上,对其中2个位点扩增的条带进行测序分析,结果表明是由于微卫星重复次数的差异而造成的。用这7个位点对尼罗罗非鱼、吉富罗非鱼和奥利亚罗非鱼群体的种质纯度进行检测,结果表明,这3个罗非鱼群体中分别有8%、4%、4%的个体在有些位点上的谱带与杂交罗非鱼的相似,可能存在基因污染。

43 microsatellite loci were chosen randomly in the first six linkage groups on the microsatellite genetic map of tilapia,and relevant primers were used to perform PCR amplification from genomic DNA of Oreochromis niloticus,GIFT（Genetically Improved Farmed Tilapia） strain,Oreochromis aureus and hybrids of O.niloticus and O.aureus.And seven microsatellite loci（UNH995,GM066,GM166,UNH162,GM017,GM440,UNH948） were screened in this study.The seven loci could amplify unique bands of variety characters respectively and any loci could be used as a molecular marker to distinguish O.niloticus or GIFT strain,O.aureu,and hybrid tilaplia.The average alleles number detected by these 7 loci in O.niloticus,GIFT strain,and O.aureus was 3.1,2.3 and 1.7 respectively,and the lengths ranged from 124 to 310 bp.The amplification by PCR had an over 20 bp difference between O.niloticus and O.aureus on these 7 loci.The bands amplified by 2 loci were sequenced and then analyzed,and the results showed that the differences were caused by different microsatellite repeats.Germplasm pureness analysis of O.niloticus,GIFT strain and O.aureu populations by these 7 loci indicated that there were 8%,4% and 4% individuals of each population could obtain similar bands to the hybrid tilapia in some loci,which might have some genetic pollution.