Isolation Of Microsatellite Markers In Grass Carp (Ctenopharyngodon Idella) And Genetic Diversity Analysis For Nine Cultured Populations

Grass Carp (Ctenopharyngodon idella) is native fish of China, widelydistributed in the Yangtze River, Pearl River and Heilongjiang area, whichis the most important cultured freshwater fish in China. In this study, weused magnetic beads enrichment method, isolated more than400polymorphic microsatellite markers, lay the foundation for genetic linkagemap encryption work of grass carp; established five different groups ofmicrosatellite multiplex PCR system, for the detecting genetic diversity indifferent populations of grass carp; studied genetic variation for a total ofnine cultured grass carp populations from China and abroad, in order toprovide a theoretical basis of monitoring, evaluation and utilization forgrass germplasm. The main contents are as follows:1. Screening and analysis of polymorphic microsatellite markers forgrass carpBy improving the magnetic beads enrichment method, we used biotin-labeled (CA)10and (GACA)6as probe, build two microsatellite libraries fortwo and four bases of repeating unit, received a total of more than3000microsatellite sequences. Primers were designed and followed by AGE andPAGE electrophoresis, successfully screened polymorphic microsatelliteprimer465pairs, from which randomly selected25pairs, amplification andSTR for polymorphisms level analysis through60genomic DNA fromHeilongjiang grass carp. The results show: These25microsatellite locitotally showed polymorphism, the number of alleles per locus between3-15, with an average of6.52; observed heterozygosity between0.350-0.950;expected heterozygosity between0.439-0.843;3loci showed deviationfrom Hardy population-Weinberg equilibrium (P<0.05);18loci showedhigh levels of polymorphism information content (PIC>0.5), can be used as backup markers for genetic linkage map encryption work of grass carp, butalso can be used as an ideal microsatellite markers for genetic diversity,genetic structure and any other research.2. Establishment of microsatellite multiplex PCR system for detectinggenetic diversity in different populations of grass carpWe picked out53microsatellite markers from genetic linkage mapand the library which have high heterozygosity and polymorphism,fluorescent primers were synthesized, by secondary screening andcombination through four different wild grass genomes DNA, finallyestablished five groups multiplex PCR system (a total of20microsatellitemarkers), with polymorphisms detection by a total of80genomic DNAfrom Yuanjiang, Chengdu, Honghu and Anxiang groups. The resultsshowed that: the number of alleles (Na) between8-28, the effective numberof alleles (Ne) between3.09-19.66, observed heterozygosity (Ho) rangedfrom0.713to0.975, expected heterozygosity heterozygosity (He) rangedfrom0.681to0.955, polymorphic information content (PIC) ranged from0.652to0.947,20microsatellite loci are all highly polymorphic loci(PIC>0.5). The alleles, heterozygosity and polymorphism informationcontent of these20polymorphic microsatellite loci belong to higher level,which can provide a wealth of genetic information for detection andanalysis of genetic diversity in grass carp populations.3. Analysis of genetic diversity for nine cultured grass carppopulations from domestic and abroadIn this research, microsatellite multiplex PCR systems were used toanalyze the genetic diversity and genetic structure of nine culturedpopulations of grass carp, which were collected from domestic and abroadaquaculture farms. The genetic diversity analysis shows, all of the twentymicrosatellite loci were highly polymorphic level (PIC from0.542to0.950), all of the populations showed high genetic diversity level (Ho from0.552to0.972, and Na from13to35), India and Nepal populations showedrelatively low level for genetic diversity. Bottleneck effect shows that nine groups heterozygous serious excess, except Chengdu groups, other groupshave varying degrees of deviation from the mutation-drift equilibrium,indicating that due to the human factor, the number of grass carp breedingpopulation have sudden drop recently, normal generation transmission andchange of genetic genes (genetic drift) is seriously threatened. The index ofpopulation genetic differentiation FSTand AMOVA analysis shows, thereare highly significant genetic differentiation among and within populations(P<0.01), the overall level of differentiation was higher (53%FST>0.05).The analysis of genetic distance and UPGMA tree shows,6domesticpopulations were clustered together first with closer genetic distance;Vietnam population was closer to domestic populations, the populations ofIndia and Nepal were relatively independent. The analysis of geneticstructure shows, nine cultured populations were divided into six groups intheory, Anxiang and Shaoguan populations were relatively independent,the rest of the Yangtze river populations showed mixed genetic informationwith Tianjin population, India and Nepal populations showed higherindependence of genetic structure.