Studies on Genetic Diversity of Selected Population of Hybrid Scallop Chlamys farreri ( ♀ ) × Patinopecten yessoensis ( ♂ ) by Microsatellites Markers

The growth superiority of hybrid scallop Chlamys farreri (♀) × Patinopecten yessoensis (♂), as the following successive generation selection have been reported. However, the data about the genetic diversity in those population remains unexplored. In this study, the genetic structure analysis of F1, F2 and F3 were conducted by PCR with 10 Simple Sequence Repeats (SSR) primers. It showed that a total of 68 alleles were detected, and the number of alleles per locus ranged from 3 to 11, Polymorphism Information Content (PIC) per locus ranged from 0.4729 to 0.8429. And, the average observed heterozygosity (Ho) of the three populations were 0.6100, 0.6975 and 0.7750, while the average expected heterozygosity (He) were 0.7607, 0.7751 and 0.7379 respectively. Fst values among the three populations were also low (Fst<0.05) which suggested low genetic differentiation between each two populations. In all, those data indicated the genetic structure challenge caused by hybridization and selection, supplying a new angle to understand artificial selective breeding.


Introduction
The scallop Chlamys farreri, a native bivalve in China, is one of the most important marine farming species in Northern China.However, massive mortality has caused catastrophic losses to its aquaculture since 1996, which resulted in a sharp production decline.The germplasm quality degeneration have been considered as one major reason for the massive scallop mortality.Therefore, to breed new scallop species with high resistance is an effective method to change the current status quo.
The research on distant hybridization breeding of scallop C. farreri(♀) × Patinopecten yessoensis(♂) have been carried out (Zhou, Yang, & Liu, 2003;Lv, Yang, Wang, Liu, & Zhou, 2006), and surprisingly, the first hybrid generation owned prominent heterosis performance which has been cultivated in large-scale area.The second and third generation individuals with stronger resistance, faster growth than scallop C. farreir have also been selected for farming (Yang, Wang, Liu, & Zhou, 2003).However, during the process of selective breeding, many uncertain factors, such as increasing risk of inbreeding, decreased number of effective groups, may lead to lower genetic diversity, even the genetic effects.Therefore, it is necessary to detect the genetic variation, to understand the changes of genetic structure for developing appropriate scientific measures so that we can smoothly control the progress of selective breeding.
Microsatellite marker (Simple Sequence Repeats, SSR), due to its simple, fast, good stability, higher polymorphism, informative genetic variation and followed Mendelian codominant genetic, has been widely used in various fields as a molecular marker (Li, Park, Endo, & Kijima, 2004;Sakamoto, Danzmann, Okamoto, Ferguson, & Ihssen, 1999;Liu et al., 2004).In terms of hybrid scallop, isoenzyme (He, Yang, Wang, Liu, & Zhou, 2006), RAPD and other labeling techniques were particularly used, however, SSR analysis of different hybrid scallop populations generated from C. farreri (♀) × P. yessoensis (♂) have not been reported.In this study, SSR was employed to analyze the genetic variation of three selective breeding population, to explore the impact of selection process on its genetic structure, which could provide a theoretical basis for molecular marker-assisted breeding.

Sample Collection
Mature female C.farreri and male P. yessoensis were collected as parents from Changdao, Shandong Province.The F 1 hybrid were reproduced from mother C. farreri and father P. yessoensis while F 2 generation from F 1 by self-fertilized, F 3 from F 2 , respectively.30, 40, 40 individuals were selected randomly from F 1 , F 2 , F 3 population, and stored at -80 °C for DNA extracted, respectively.

Preparation of Template DNA
The genome DNA was extracted from adductor muscles of hybrid individual using Phenol-chloroform method.In detail, about 100 mg tissue was sampled into a 1.5 ml centrifuge tube, and then 500 μl homogenization buffer (10 mM Tris-Cl, pH 8.0; 100 mM EDTA, pH 8.0), 50 μl 10% SDS and proteinase K with final concentration of 50 μg/ml were also added.After being mixed adequately, the sample were digested at 55 °C for 3 h, and then the proteins were extracted using phenol, phenol: chloroform (1:1), chloroform, isoamyl:alcohol (24:1), successively.And following, the nucleic acid was precipitated with ethanol, and then dissolved in ddH 2 O.The concentration and quality of extracted DNA were detected by RNA/DNA quantitative analysis using Nanodrop 2000 and agarose gel electrophoresis, respectively.The concentration of genome DNA was diluted to 50 ng/μl and then stored at -20 °C.

PCR Amplification
10 pairs of primers (Table 1) were selected from reported SSR primers of C.farreri and P. yessoensis, to ensure its availability for amplification in hybrid offspring in this study.PCR reactions were carried out in a 10 μl reaction volume on a PCR amplification instrument, including 1 μl 10 × buffer, 0.6 μl Mg 2 + (25 mM), 1 μl dNTP (each 2 mM), 1 μl forward/reverse primer (10 μM) each, 50 ng template DNA, 0.5 U Taq DNA polymerization enzyme (Promega), and PCR-grade water was replenished to 10 μl.PCR reaction program consisted of 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 72 °C for 30 s, and finally 72 °C for5 min.The PCR products were detected by 6 % denaturing polyacrylamide gel electrophoresis, silver staining for detecting polymorphism.The amplified brands were counted by manual mothed for analyzing by software.

Data Analysis
According to the genotype, the value of polymorphic information content (PIC), heterozygosity (Ho), heterozygosity (He), allele number (a), effective number of alleles (a e ), similar coefficient and genetic distances among, and F-statistics of the three groups were calculated using Popgen 32 (Version 1.31).F st value range from 0 to 0.05 was considered as low population genetic differentiation, while 0.05-0.15as middle level, 0.15-0.25 as high level and above 0.25 as significantly high level.

Genetic Diversity of Loci
10 pairs of primers with high polymorphism were used to perform PCR amplification with 110 individuals from F 1 , F 2 and F 3 populations.In all, 68 alleles were obtained, and the numbers of allele for each locus ranged from 3 to 11. PIC value was from 0.4729 to 0.8429.The average observed heterozygosity was F 2 >F 1 >F 3 , with the values of 0.7025, 0.6893, 0.6900, respectively, while the average expected heterozygosity were 0.8315, 0.7751, 0.7379, respectively.
According to P values of genotype, the value of the Hardy-Weinberg equilibrium deviated significantly in three generations (as shown in Table 2), such as CFMSM009 in the F 1 and F 2 populations, CFMSP011 in F 3 population and CFAD213 in F 2 and F 3 .Some representative amplified brands were exampled in Figure 1.

Genetic Similarity Index, Genetic Distance and Cluster Analysis
The genetic distance and genetic similarity in three population was shown in Table 3.It showed that the genetic distance between F 1 and F 3 generation was the largest, while the value between F 2 and F 3 populations was the smallest.According to the genetic distance among groups, UPGMA were used to analyze relationship among three generations populations (Figure 2).It indicated that the F 2 and F 3 clustered firstly and then cluster with F 1.

Population Variation
The F st values between F 1 and F 2 , F 1 and F 3 , F 2 and F 3 were 0.0079, 0.0196, 0.0028, respectively, which indicated that the genetic differentiation among the three generations was weak.The overall genetic differentiation coefficient value was 0.0169, which revealed that only 1.69 % genetic variation was from groups, while 98.31% variation was from individuals.

Discussion
In this study, the data revealed that the genetic diversity among three populations was not significantly different, although the proportion of polymorphic loci and genetic diversity decreased from F 1 to F 3 .This situation was similar to some other reports (Hedgecock, Chow, & Waples, 1992;Mgaya, Gosling, Mercer, & Donlon, 1995;V. Sbordoni, De Matthaeis, M. C. Sbordoni, La Rosa, & Mattoccia, 1986).For example, the genetic diversity in several generations of Chinese shrimp were reported by Zhang et al. (2005) using SSR technology, which showed that the average observed heterozygosity dropped down from the first generation to the sixth.And it was same as the studied in American oysters (Yu and Guo, 2004) and Japanese flounder (Liu et al. 2005).Selective breeding is a complex process, the external environment and artificial selection pressure may cause fluctuations of population genetic variation.And on the other hand, during the artificial breeding process, due to the small effective population the inbreeding rate might increase which caused inbreeding depression and bottleneck effect.And also, high-intensity artificial directional selection might lead to the genetic deterioration and introgression, that these two factors could cause the loss of some particularly alleles, especially some rare gene allele in the breeding population.Therefore, genetic variation of breeding populations should be detected timely in the process of selective breeding.
Coefficient of genetic differentiation is an important parameter reflecting the degree of genetic differentiation among populations.Under controlled conditions, artificial selection, mutagenesis, hybridization could damage balance of genetic, which caused changes of genes and genotype, so the genetic characteristics within a population will also change.In this study, from the first generation to the third generation of breeding populations, the genetic structure within populations changed.Although the genetic structure had a certain differentiation, the degree of differentiation was not significant.The genetic variation analysis showed that genetic differentiation among generations of artificial breeding populations was smaller and differentiated was mainly from individuals.As studies in Chinese shrimp reported by Li et al. (2006), the genetic differentiation coefficient between adjacent groups showed a decreasing trend, and genetic similarity degrees of individuals within a population showed an upward trend with the increase in generation, which indicated the breeding populations tend stability after years of breeding.
Heterozygosity is an important parameter to measure the genetic diversity of populations.In this study, the mean observed heterozygosity were from 0.6893 to 0.7025 among the three generations.High significant deviation phenomena of Hardy-Weinberg equilibrium were observed, and the F is values indicated that seven loci in three populations showed a certain degree loss of heterozygosity, which indicated dumb allele might exist, which was similar to the report of Chinese shrimp (Zhang et al., 2005)).This study enriches the data of hybrid breeding of scallop, opening a new angle to understand genetic diversity change of different generation during artificial selective breeding.

Table 1 .
Microsatellite primers used in this experiment

Table 3 .
Genetic identity and genetic distance in three populations Notes: data below diagonal are genetic distance; data above diagonal are genetic identity.

Table 4 .
F-statistica for three populations of hybrid scallop at ten microsatellite

Table 5 .
F st values of pairwise comparison among different population at 10 microsatellite