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

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 population PPB,I,He,Ne and D were 53.21% ,0. 2749,0. 1837,1. 3169 and 0. 1529 respectively. The ISSR analysis revealed that the genetic diversity was remarkably high existed in the two populations and the level of genetic variation in the population of H. labeo was higher than that in the population of H. maculates. One reason is that H. labeo and H. maculates have lived in Zhejiang streams for a long time and adapted to the different stream environments. Long time natural selection gives rise to stronger adaptability so as to enrich their genetic diversity. The other reason is that the repro-duction of H. labeo and H. maculates in Oujiang River is still at the early experimental stage and the natural populations are not subject to the artificial breeding. The 610bp band amplified by UBC845 and 1240bp band amplified by UBC854 were the character of H. labeo,which the sequence of two specific bands can be designed by the molecular probe, specific primer,and other application in molecular genetics after validation, recovery and clone. The genome of Hemibarbus contained these microsatellite DNA sequences： （AG）8, （GA）8, （CT）8 and （GAA）6. These results supply information for protection and utilization of H. labeo and H. maculates and also provide data for the further study of genetic variation and selective breeding of Hemibarbus.