Study On The Change Of AFLP And SSR Markers Polymorphism Induced By Cadmium Chloride In Carassius Auratus Red Variety

Object: On the basis of the study on the standardization and application research ofCarassius auratus red variety, we further research the change of AFLP and SSRmarkers in Carassius auratus red variety induced by cadmium chloride to investigateearly warning role of AFLP and SSR markers for cadmium pollution.Methods: Carassius auratus red variety was used as experimental subject and treatedthem with cadmium chloride for different concentration and different time, andanalyzed the polymorphism of AFLP and SSR markers respectively.According to the96h LC50of cadmium chloride to Carassius auratus redvariety from preliminary experiment, we set up four different concentrations ofcadmium chloride groups treated with cadmium chloride for8d(96h LC50of1/16,1/8,1/4,1/2) and one control group. In the eight days, we collected the blood from livingCarassius auratus red variety every two days, and extracted DNA to analyze thepolymorphism of AFLP and SSR markers.we selected8AFLP primers for selective PCR reaction after digestion-ligationreaction prepared by EcoR I、Trul I and T4DNA, Then we used Quantity one softwareto analyze the PCR electrophoretogram of AFLP markers, and the PopGene softwarewas used to statistics the number of amplification bands (N), the bands range, numberof polymorphic loci (Np) and polymorphism information content (PIC), at last,polymorphism information content was analyzed with Origin8.0software.6SSR primers were selected from the SSR primers of Carp for PCR reactions ofDNA from Carassius auratus red variety, the PCR products were analyzed withQuantity one and PopGene software, and statistics the number of alleles (Na),effective number of alleles (Ne), gene heterozygosis (H), polymorphism information content (PIC), we used Origin8.0software to analyze gene heterozygosis andpolymorphism information content finally.Results:1. Polymorphism of AFLP markers: The numbers of PCR amplificationbands With8pairs of different primers ranged from18to30and the numbers ofpolymorphic bands ranged from14to24. In control group of Carassius auratus redvariety, the numbers of amplified bands with eight pairs of primers was65, the totalnumber of polymorphic bands was30; in the group exposed to cadmium chloride, thenumbers of amplified bands with eight pairs of primers was182, the total number ofpolymorphic bands was147. In the group exposed to1/8LC50concentration ofcadmium chloride, the total number of polymorphic loci increased to36in the secondday after exposed to cadmium chloride, but the number of polymorphic loci will reachto41in the6th day. The number of band amplification with E-ACC/T-CTC primerwas the peak value of10in control group among the8primers, the number ofamplification bands was12in the1/8LC50concentrations of cadmium chlorideexposure group in the4th day. The number of band amplification with E-ACC/T-CTCand E-AAC/T-CAG primer was7in control group,3of them was polymorphism; itwas the lowest among the8primers. With primer E-ACA/T-CTC and1/2LC50concentrations of cadmium chloride exposure group, the numbers of amplificationbands was2in the eighth day and the number of polymorphic bands was1, whichwas the lowest in the8primers.The analysis of PCR products with8primers showed the average value of AFLPmarker polymorphism information content was46.15%and80.77%in control groupand the group exposed to cadmium chloride, respectively. The polymorphisminformation content amplification with E-AAC/T-CAT was37.50%in control group,which is the lowest among the8primers; it was75.00%in the group exposed tocadmium chloride. The polymorphism information content amplification withE-ACT/T-CTC primer was62.50%in control group, which is the highest in the8primers; while in the group exposed to cadmium chloride, the polymorphisminformation content was85.71%, which is the highest in the8primers.2. Polymorphism of SSR markers: The number of polymorphic loci of PCR amplification with6pairs of different SSR primers was0in the control group ofexperimental Carassius auratus red variety, and the number of polymorphic loci willincrease with the increase of the Cadmium chloride concentration and the extension ofexposure time. In the group exposed to cadmium chloride of1/16LC50concentration,the total number of polymorphic loci was8in the2nd day after exposed to cadmiumchloride; But in the1/2LC50concentration group, the number of polymorphic locireached to12in the2nd day; while in the1/4LC50concentration group, the numberof polymorphic loci was the peak value of20in the6th day.The gene polymorphism information content of PCR amplification with6pairsof different SSR primers was the least value of25.00%in the group exposed to thecadmium chloride of1/16LC50concentration in the2nd day; the gene polymorphisminformation content in the1/2LC50concentration group and the1/4LC50concentration group was34.38%and37.50%respectively in the2nd day; in fourdifferent concentration groups, gene polymorphism information content exhibitedmoderate polymorphism in the4th day; the crest value of gene polymorphism contentin1/4the LC50concentration group was62.50%in6th day, reached to highlypolymorphic.The gene heterozygosis of PCR amplification with6pairs of different SSRprimers was0.11±0.20in the1/16LC50concentration group in the2nd day; sincethen, the change of gene heterozygosis in Carassius auratus red variety increases withthe increase of cadmium chloride concentration and the extension of exposure time.Among them, the gene heterozygosis was0.17±0.22in the1/8LC50concentrationgroup in the4th day; the gene heterozygosis was0.28±0.22in the1/4LC50concentration group in the6th day; the gene heterozygosis was0.12±0.20in the1/2LC50concentration group in the8th day.Conclusions:1. The change of SSR and AFLP polymorphism in Carassius auratusred variety will be induced by the Cadmium chloride of certain concentration.2.Polymorphism information content (PIC) of AFLP and SSR will be increased with theincrease of cadmium chloride concentration and the extension of exposure time.3.AFLP and SSR molecular markers in Carassius auratus red variety could be used as biomarkers of early warning for cadmium chloride pollution.