Analysis Of Genetic Diversity In Five Populations Of Grass Carp (Ctenopharyngodon Idella) And Screening Of SSR Markers Related To Growth Traits

Grass carp(Ctenopharyngodon idella) is one of most important cultured freshwater fishes which belongs to Cypriniformes, Cyprinida, Leuciscus, Grass carp. Because of fast growth and low cost farming, it has been the main breeding species in the ponds, lakes and reservoirs. It distributes in major river systems and lakes in Asia except for Xinjiang and Qinghai Tibet Plateau without the natural distribution, forming rich germplasm resources. In recent years, the unclear genetic background of broodstock and inbreeding led to germplasm degradation; executive arrest and pollution of the natural environment makes natural grass carp populations decreased significantly. Therefore, in order to protect and use the freshwater fish resources, the genome of different groups in grass carp should be researched. We need to develop and use a variety of molecular genetic markers to find out their genetic polymorphism and relationship and study their genetic background for the basis of grass carp breeding population. On the other hand, due to long period of sexual maturity of grass carp, big broodstock individuals and other reasons, so it is difficult to breed superior strains. The current has not yet acquired the genetic improvement of grass carp strains. Marker assistant selection(MAS) has become an important method to improve breed. Selection and use of grass carp economic traits associated molecular markers can effectively improve the selection efficiency and shorten the breeding period. It is important significantly to accelerate the development of superior economic traits of grass carp breeding strains. In this paper, microsatellite markers were selected from the EST database of grass carp. They were employed to detect the genetic diversity of three groups of grass carp populations from the Yangtze River System(SS, JL, CS) and two groups from the Pearl River System(QY, ZQ). Meanwhile, we identified microsatellite loci associated with growth traits in grass carp. The detailed content was as follows:1. Development of EST derived Microsatellites in grass carp. 5556 microsatellite sequences were found from ESTs database (760000 items) which were constructed from brain, muscle,liver tissues of grass carp. According to these microsatellite sequences, 118 pairs of primers were designed with Primer Premier 5.0. Nineteen pairs of primers can amplified clear and highly polymorphic products by method of PCR.2. Development of EST derived Microsatellites and Analysis of Genetic Diversity in Five Populations of Grass Carp. Nineteen pairs of EST SSRs primers were employed to detect the genetic diversity of three groups of grass carp populations from the Yangtze River System(SS, JL, CS) and two groups from the Pearl River System(QY, ZQ) . The result displayed that 93 polymorphic loci were amplified and each primer were detected 2 to 8 alleles or 4.89 alleles by average. Genetic diversity data showed that: the average polymorphism information content(PIC) ranged from 0.4154 to 0.4604 displayed that grass carp population had lower genetic diversity; the average observed heterozygosity was from 0.4158 to 0.5013, the average expected heterozygosity was 0.4506 and 0.5028 , CS had the highest mean expected heterozygosity(0.5028). JL had the lowest mean expected heterozygosity(0.4506) and mean observed heterozygosity(0.4158). SS, QY and ZQ had the middle mean observed heterozygosity and mean expected heterozygosity. These observation indicated that CS has the highest genetic diversity, whereas JL the lowest; Fst value indicated that the populations were lowly differentiated. Chi square test was used to analyze the genotypes based on Hardy Weinberg equilibrium, the P value denoted that the five populations deviated equilibrium partially. The dendrogram based on genetic distance showed two major clusters according with basin distribution of grass carp populations: the stock from CS , SS and JL were in one cluster, which were sampled from the Yangtze River. The other cluster consists of ZQ and QY from sampling localities distributed in the Pearl River.3. Screening of EST SSRs markers related to growth traits in grass carp. Eighteen pairs of EST SSRs primers selected from EST base of grass carp were employed to examine the associations between their genotypes and growth traints and detect the genetic diversity of random Grass carp populations. Microsatellite loci of 13118, 13305, 24017, 25085, 35939 and 40698 were significantly associated with body weight, body length., and body height (P<0.05 or P <0.01). The most favorable genotypes for growth traits were AA at 13118, AD at 13305, AC at 24017, BE at 25085, BB at 35939 and BB at 40698. In addition, a total of alleles for the loci were detected (2~9 alleles for each locus). The average effective number of alleles(Ne), observed heterozygosity(Ho), expected heterozygosity(He) and mean polymorphic information content(PIC) was 4.556, 0.4529, 0.4571 and 0.4017, respectively, both of which indicated that the population genetic diversity was low. The homology identity of the 6 correlative ESTs was determined by GenBank of NCBI blast programmer on the amino acid levels. It was found, through BLAST analysis, that 2 ESTs with significant similarity to the known functional sequences in GenBank. 24017 was highly homologous to the known natural killer enhancing factor(NKEF) with an identity of 86%. 25085 was highly homologous to the known cAMP response element binding protein with an identity of 80%. The other 4 ESTs were not significantly homologous to any of the known functional genes from GenBank.