OPTIM IZATI ON OF ISSR ANALYSIS AND STUDY ON GENETIC DIVERSITY OF TWO NATURE POPULATIONS OF HEM IBARBUS FROM OUJI ANG RIVER

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 cuhure.Samples of 25 wild individuals of H.1abeo 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.1abeo 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∞follows:1×Tat/buffer,60ng template DNA,1.2U Taq DNA polymerase,0.4μmol/L primes,2.0mmol/L MgCl2,0.2mmoL/L dNTPs and 2% formamide.18 ISSR primers (screened out of 60 primers)produced higlIly 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’8 diversity index(Ⅰ),Nei’s genetic diversity index (He) and effective nunIber of alleles (Ne) were 0.3450,0.2292 and 1.3875 respectively,which were calculated from ISSR results by software Popgenel.32:the megn genetic distance(D)was 0.2 1 29.In the H.maculates population PPB.1.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 w88 remarkably high existed in the two populations and the level of genetic vailation in the population of H.labeo was hiigher 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 reproduction of H.labeo and H.maculates in Oujiang River is still at the early experimental stage and the natuld populations are not subject to the artificial breeding.The 610bp band amplified by UBC845 and 1240bp band amplified by UBC854 were the character 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)8.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.