| The species of Meretrix meretrix are widely distributed along the south-to-north coast of China.It is an economically important spiecies which belong to the eurythermal and eurysaline benthic in intertidal zone. It is an effective approach to cultivating the good strains with the performances of disease-resistance and fast growth for the sustainable development of Meretrix meretrix aquaculture industry as well as other mariculture species. The marker-assisted selection(MAS) provides the fundamental theories and applied experiences for this task not only in better utilizing the available resources but also increasing production in aquaculture. With the development of MAS in Meretrix meretrix, lots of problems, such as the the genetic diversity maintenance in the subsequent generations, and the genetic analyses of economic characters etc., are raised for further consideration in modern Meretrix meretrix. Genetic variation and diversity of four strains of Meretrix meretrix, Thin Checkered (TC), Black Spot (BS), Dark Fringe (DF) and Red Shell (RS), was conducted using amplified fragment length polymorphism(AFLP),microsatellite markers(SSR) and methylation-sensitive amplified polymorphism (MSAP)analysis in this study, which provid the fundamental theories and applied experiences for this task not only in better utilizing the available resources but also increasing production in aquaculture. Main results were conclusion as following:1. Fifteen AFLP primer combinations were applied to analyze genetic diversities and relationships among strains. Twenty-nine strain-specific bands were found in 789 detected bands, which may be used to distinguish the four strains. The overall polymorphic loci percentage of four strains was 90.75% and the percentage of polymorphic loci ranged from 61.34% to 75.16%. The results of Nei's genetic diversity and Shannon's information index showed that the level of genetic diversity from high to low was BS>RS>DF>TC. AMOVA analysis indicated that the average fixation index (Fst) was 0.2055%, 79.45% of variance was within strains and 20.55% of variance was among strains. The results of cluster analysis indicated that TC strain had higher genetic difference from other strains, second was BS strain, and the genetic variation between DF and RS strain was relatively lower. One AFLP marker was only existed in TC strain and the frequency was 100%, These results suggested that the unique band could be a molecular marker of strain identification.2. The results of SSR showed that every microsatellite locus analyzed were polymorphic in those strains of Meretrix meretrix. A total number of 105 alleles were detected. The most alleles were 21 (WG07) and the least locus were 7(WG11). The distribution of the alleles for the same loci was different in different strains and specific allele type were detected in each strains. The average expected heterozygosity ranged from 0.110 to1.000, while the average observed heterozygosity ranged from 0.486 to 0.867 among strains. PIC value ranged from 0.433 to 0.834. Chi-square tests showed that most of the cases in four strains deviated from Hardy-Weinberg equilibrium. Pairwise Fst ranged from 0.005 to 0.269. Genetic distances among strains ranged from 0.0520 to 0.0980.3. The extent and pattern of cytosine methylation in four strains of Meretrix meretrix genomes were assessed by using the technique of methylation-sensitive amplified polymorphism(MSAP). The result show that 26.7%-30.0% DNA methylation events were detected in the CCGG sequence of Meretrix meretrix genomes, and 9 of the 40 primers showed DNA methylation polymorphisms. In all, 396 fragments were amplified using 9 pairs of selective primers, The average proportion of polymorphism ( P ) was 19.6%. The results demonstrate DNA methylation events occur frequently in M.meretrix genomes, and methylation patterns vary among the strains, which may be related to the appearance of shell color and pattern, This paper is the first time to analyse cytosine methylation in Meretrix meretrix using MSAP, indicating MSAP technique is efficient for detection of cytosine methylation in Meretrix meretrix genomes. |
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