草鱼Ⅰ型微卫星标记的发掘及其多态性检测

IDENTIFICATION OF TYPEⅠMICROSATELLITE MARKERS AND THEIR POLYMORPHISM IN GRASS CARP (CTENOPHARYNGODON IDELLUS)

表达序列标签(EST)是发掘Ⅰ型微卫星标记的重要资源。研究运用生物信息学方法,从草鱼头肾组织3027条EST序列中搜索到322个微卫星位点,占整个EST数据库的10.6%。其中,二核苷酸重复位点151个(46.9%),三核苷酸重复位点137个(42.5%),四、五、六核苷酸重复位点较少;在二核苷酸重复位点中,AC/GT重复位点最为丰富,占二核苷酸重复位点总数的50.3%,AG/CT重复次之,占二核苷酸重复位点总数的40.4%,AT和GC重复较少。10个微卫星位点的多态性检测结果显示,4个位点在草鱼测试群体中呈多态性,多态性位点的平均多态信息含量(PIC)和平均遗传杂合度(H)分别为0.5236和0.5441,其中,2个多态性位点的PIC值大于0.5,呈现高度多态性特征。Ⅰ型微卫星标记将为草鱼遗传连锁图谱构建和QTL分析提供有效的基因分子标记。

Expressed sequence tags（ESTs） may serve as an effective source for identifying type Ⅰ microsatellite markers.In the present study,the database containing 3027 ESTs from head kidney of grass carp was screened for type Ⅰmicrosatellite sequences using Tandem repeats finder v4.0 software.A total of 322 type Ⅰ microsatellites were iden-tified,among which 151 were the type of dinucleotide repeat being the most abundant microsatellite and accounting for 46.9% of the identified microsatellite markers,and 137 were the type of trinucleotide accounting for 42.5%,while hexa-,penta-and tetra-nucleotide repeats were rare.Among the dinucleotide repeats,AC/GT was the most abundant type with percentage of 50.3%,followed by the AG/CT type with 40.4%,while AT and GC were rare.The ESTs were used to design primers to amplify grass carp genomic DNA,and polymerase chain reaction products of the ESTs were analyzed to determine the length of polymorphic sequences,and then 27 microsatellites were selected for polymorphism analyses.10 of the 27 primer pairs resulted in PCR products of expected size,of which 4 loci showed polymorphism in a wild population of grass carp,with 2 loci having high polymorphism.The allele number of all the loci was 23,and the mean PIC and heterozygosity values were 0.5236 and 0.5441,respectively.It is concluded that these type Ⅰ microsatellite markers can be used for further study in genetic mapping and quantitative trait loci（QTLs） identification in grass carp.