Study On Individual Identification Of Grass Carp(Ctenopharyngodon Idellus) Through The Use Of Molecular Marker Technology

In order to provide the theoretical foundation for the breeding of grass carp,in this research,DNA barcode technology which based on the COI gene and microsatellite molecular marker technology are applied to the individual identification about grass carp.As a common method,traditional phenol-chloroform method extracts the DNA which has many advantages such as large output.However it will cause a great damage to the experiment samples since it often selects muscular tissue or blood as the extract material.By using the Chelex100 method that costs less time,the research which use fish scale as a raw material got the improved DNA extraction technology.The results revealed that the improved phenol-chloroform method was obviously superior to the traditional method and PCR results showed that the extractions of the nuclear and mitochondrial genes could be used for the subsequent research.COI gene(cytochrome oxidase I)which is about 600 bp is used to encode the cytochrome c oxidase subunit I from the mitochondrial.Scholars often apply it to the species identification or biological system development.To further explore the feasibility that DNA barcode technology based on the COI gene was applied to the individual identification about grass carp,the research compared with the COI gene sequence from 9 grass carp and analysis the interrelation about 9 COI gene sequences by using some software.Results revealed that the length of COI gene is 1440 bp.The average contents of T,A,C,G show the decreasing trend about 30.2%,27.1%,25.0%,17.7%.The content of G which obviously blows the others shows the strong base composition bias.There were 24 base mutations in the COI gene sequences of 9 grass carp.There were 7 and 6 mutation sites between 280bp-440 bp and 1300bp-1410 bp.They respectively belonged to the high mutation frequency section and could be considered as a miniature DNA barcode.Genetic distances between grass carp were ranging from 0.1% to 0.6% and average genetic distance was 0.39%.It is less than the 2% critical value within the same species.Therefore the DNA barcode technology based on the COI gene doesn’t apply to individual identification of grass carp.Microsatellite also named simple sequence repeat(SSR)which exists in eukaryotic genomes.Because it possess the higher mutation rate,the neutral DNA,the widespread and the better conservatism at the pole of the sequence,microsatellite marker technique is widely used in a variety of biological field.By arranged and combined a small amount of rich polymorphism of microsatellite loci,the research eventually choose 8 pairs(HLJC115 HLJC104 HLJC107,13118,HLJC2,HLJC111,HLJC9 and HLJC126)from the 85 pairs of microsatellite primers which used to the individual identification of 11 and 63 grass carps.11 grass carp amount to amplify 19 alleles,the average loci containing 4.75 alleles,allele numbers range from 2 to7.63 grass carp amount to amplify 38 alleles,the average loci containing 6.33 alleles,allele numbers range from 4 to 9.The results showed that microsatellite molecular marker technology can reach 100% for small-scale samples(11)or large-scale samples(63).