瓯江2种的ISSR分析优化和自然种群遗传多样性的研究  

OPTIMIZATION OF ISSR ANALYSIS AND STUDY ON GENETIC DIVERSITY OF TWO NATURE POPULATIONS OF HEMIBARBUS FROM OUJIANG RIVER

作　　者：吕耀平[1] 胡则辉[2] 叶丽平[1] 

机构地区：[1]丽水学院化学与生命科学学院 [2]浙江省海洋水产研究所,舟山316100

出　　处：《水生生物学报》2008年第4期515-521,共7页Acta Hydrobiologica Sinica

基　　金：浙江省自然基金项目(Y307445);浙江省科技厅新苗人才计划项目(2007R40G2260026);浙江省教育厅项目(20070552);浙江省丽水市科技局项目(2007kjh2005)资助

摘　　要：以唇和花为材料,对影响PCR反应的主要因子-模板DNA、引物、dNTPs、Mg2+、Taq酶浓度进行优化,建立了适合唇和花基因组DNA的ISSR-PCR反应体系。25μL的PCR反应液含有的组分和终浓度分别为:约60ng模板DNA、1.2UTaq酶、0.4μmol/L引物、2.0mmol/L MgCl2、0.2mmol/L dNTPs、2%甲酰胺。利用优化反应体系从60个ISSR引物中,筛选出18个稳定性高、重复性好的引物,对25个唇个体和22个花个体的DNA进行正式扩增,分别从两个种群分别扩增到168和156条扩增谱带,扩增片段长度在200—1500bp之间。根据扩增结果计算出两个种群的多态位点比例分别为67.52%和53.21%,Shannon多样性指数分别为0.3450和0.2749,Nei’s指数分别为0.2292和0.1837,平均有效等位基因数分别为1.3875和1.3169。两个种群内样本间的平均遗传距离分别为0.2129和0.1529,结果表明两个种群的遗传多态性较高,其中唇的多样性明显高于花群体。引物UBC845扩增的610bp带和UBC854扩增的1240bp带为唇种的特异性谱带。属鱼类基因组中均含有微卫星DNA序列:(AG)8、(GA)8、(CT)8和(GAA)6。本研究为唇和花的种质保护、合理开发利用以及选择育种提供了一些基础数据。Hemibarbus labeo and H. maculates are the important species for freshwater culture due to their commercial value of fishery industry and their widely distributions over all the river systems except for Xinjiang, Tibet and western plateau in China. Nowadays, several scientific researchers have carried out some works relevant to morphology, taxonomy, nutrition, breeding and culture of H. labeo and H. maculates ,however ,little is known about the genetic diversity of these two natural populations. Hence,it is necessary to detect the genetic diversity of the populations with ISSR （inter-simple sequence repeat） marker in present study for enriching the population genetics data and providing theoretical ground in the aspects of selective breeding and promotion culture. Samples of 25 wild individuals of H. labeo and 22 wild individuals of H. maculates were collected from Dagangtou Stream at Lishui city,Zhejiang Province, Southeast in China,in May,2007. In this paper,we examined the factors affecting the ISSR-PCR reaction of H. labeo and H. maculates using DNA of the caudal fin from kits method as the template,namely concentrations of DNA, Taq DNA polymerase, Mg2＋ and dNTP. The optimal ISSR-PCR reaction system （25μL） in our experiment was as follows： 1 × Taq buffer,60ng template DNA,1.2U Taq DNA polymerase, 0.4μmol/L primes, 2.0mmol/L MgC12,0.2mmol/L dNTPs and 2% formamide. 18 ISSR primers （screened out of 60 primers） produced highly reproducible and polymorphic bands. Using these primers, 168 and 156 bands ranging from 0. 2 to 1.5kb were amplified from the above two populations respectively. The results showed that the percentage of polymorphic bands （PPB） was 67.52% in the H. labeo population ; Shannon＇s diversity index （I）, Nei＇s genetic diversity index （He） and effective number of alleles （Ne） were 0. 3450,0. 2292 and 1. 3875 respectively, which were calculated from ISSR results by software Popgenel. 32; the mean genetic distance （D） was 0. 2129. In the H. maculates populat

关 键 词：唇[鱼骨] 花[鱼骨] ISSR 优化 遗传多样性 分子标记 

分 类 号：Q24[生物学—细胞生物学]