, ,

(1. Key Laboratory of Tropical Disease Control，Sun Yat-sen University, Ministry of Education,Guangzhou 510275,China；2. College of Marine Life Sciences, Ocean University of China, Qingdao 266003,China)

Epinephelusakaarais categorized into Perciformes, Serranidae, Epinephelinae, Epinephelus,there are around 100 species ofEpinephelusakaaratotally in the world. Most ofEpinephelusakaaraare living in the Indian Ocean and tropical and semitropical regions of the Pacific Ocean. Since its rapid growth and high market price,Epinephelusakaara, popularly cultured in the south of China, has become the main encaged culture fish in several provinces, such as Fujian, Guangdong, Zhejiang and Hainan

In recent years, genetic resource ofEpinephelusakaarapopulations have rapidly decreased caused by capturing inordinately. Moreover, the cultural populations also encountered prevalent diseases and highly inbreedingon the reason of the development of fish breeding,. All of these would have effect on the conservation and exploiture ofEpinephelusakaara. At present, the studies ofEpinephelusakaaraare mainly focusing on the regulation of reproduction, growth, and artificial breeding[1-3].However, the study on the genetic diversity and differentiation ofEpinephelusakaara. Has not been reported yet.Generally, genetic diversity of a species was determined by its adaptability, survivability and evolvability. Abundance genetic diversity means high survivability and great potential in application of genetic breeding[4].

Molecular markers have been widely applied in genetic diversity analysis of animals and plants, which is in comparsion to traditional marker techniques, according to the good reproducibility and high polymorphism. Among various molecular techniques, AFLP (amplified fragment length polymorphism) could be used to obtain mass markers without the genomic information by combination of the advantages of RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA). AFLP has been also applied in the study of genetic diversity and differentiation in many aquacultural fishes[5-9].

As one of the main species of Chinese aquiculture,Epinephelusakaarais exporting to many countries with high production. In order to keep the good seeds and avoid the negative effects from artificial breeding on wild resources, it is necessary to investigate the genetic diversity of wild populations. In present study, the AFLP technique was applied to analyze the genetic diversity and differentiation ofE.akaarain 7 different wild populations from East China Sea to South China Sea. Our study would be useful for the conservation and genetic breeding ofE.akaarain the future.

1 Materials and Methods

1.1 Fish samples

The Locations and times of harvestingE.akaarain 7 geographical populations were shown in Table 1.5～16 individuals of each population, which have been conserved at -20 ℃,were used to study.

1.2 DNA extraction and AFLP analysis

Genomic DNA was extracted from blood with phenol/chloroform extraction as described by Sambrook et al[10].

AFLP analysis was conducted essentially as described by Vos et al[10]. Primers and adaptor were designed according to the protocol of Qi and Lindhout[11]were commercially synthesized by Sangon Co. (Shanghai). The DNA samples were digested withEcoR Ⅰ andMseⅠ, and then ligated to restriction site-specific adaptors. Pre-amplification was carried out using adaptor-specific primers with one selective base at 3’ end of each primer. The pre-amplification products were diluted 20-fold and used in selective amplification. The selective amplification used the primers with three selective bases at 3’ end of each primer. Totally 13 primer combinations were selected for AFLP analysis.

Table 1 Sampling time of 7 geographical populations of Epinephelus akaara

Products of the selective amplification were separated by polyacrylamide gel electrophoresis at 60 V for 1.5 hours and were detected by silver staining. The electrophoretic images were scanned and then saved in computer for further analysis.

1.3 Data analysis

The data were scored as dominant markers. Bands present were scored as ‘‘1’’ and absent as ‘‘0’’. The AFLP marker names were referred to the primers used, for example E followed by numbers referred to theEcoR Ⅰ primer and M followed by numbers to theMseⅠ primer. Bands were numbered serially in descending order of fragment length, thus the last numbers of the AFLP marker codes referred to the relative position of the band shown in the gel.

Genetic diversity index and Shannon's index were calculated with POPGEN1.32 software, and AMOVA analysis was used to computeFstwith Arlequin software. UPGMA tree was constructed with MEGA 3.0 software.

2 Results

2.1 AFLP amplification

13 primer combinations were used to analysis 88 samples in 7E.akaarapopulations. Totally, 941 bands ranging from 0.1 kb to 1.5 kb were obtained, and 672 of 941(71.41%) were polymorphic bands. Great differences existed among different primer combinations. Total bands obtained with each primer combination ranged from 41 to 95, on average, 72. Polymorphic bands obtained with each primer combination ranged from 33 to 71 (58.00%～80.49%), on average, 51.7 (Table 2).

2.2 Genetic diversity analysis

Number of bands ranged from 614 to 807 among 7 populations. It showed the great difference existed among 7 populations. The least number of bands have been shown in DYW population, only 614, while that of ZJ population shown the largest number of bands, 807. Frequency of polymorphic loci ranged from 18.40to 47.70%. Nei's index ranged from 0.044 7 to 0.142 9. Shannon's index ranged from 0.066 5 to 0.214 1 (Table 3, 4). Therefore, genetic diversity of DYW population was the lowest, while that of ZJ population was the highest. However, low number of individuals may cause underestimate genetic diversity of DYW population. Genetic similarity within populations ranged from 0.861 5 to 0.947 4. ZJ population was the lowest (genetic distance: 0.149 1), and ZS population was the highest (genetic distance: 0.054 1), indicating that the differentiation within population in ZS was lower than that in ZJ population.

Table 2 Amplification results of different primer combinations

Table 3 Number of loci amplified, ratio of polymorphic loci, index of genetic similarity and genetic distance of 7 Epinephelus akaara populations

Table 4 Nei's index and Shannon's index of 7 Epinephelus akaara populations

2.3 Genetic structure and genetic differentiation

Based on AFLP data, a UPGMA tree was constructed for 88 samples from 7 populations (Fig.2). The 88 samples could be classified into three clades. SY was categorized into single clade A. Part of ZJ population was categorized into single clade B, while the other part of ZJ population and other populations was categorized into clade C. Furthermore, Clade C could be subdivided into 6 subclades subsequently. All these subclades basically clustered according to their geographical location, which implied that the population structure is relative with their geographical location. We also used UPGMA method to construct a tree for 7 populations (Fig.3), which has shown the same result to that abovementioned. Taken together, SY and ZJ populations were different from other 5 populations in genetic structure.

Based on AMOVA analysis, 56.63% variation obtained from among populations (P<0.01) (Table 5), and 55.53% variation ontained from among populations based on Gst estimation. Both of the analysis results indicated great genetic variation and obvious genetic differentiation among populations. Also, Fst showed great genetic difference among 7 populations (Table 6). Genetic distance between populations varied from 0.076 9 to 0.346 7. Genetic distance between ZS and PT population is the lowest (0.076 9), while that SY and ZJ population are the highest (0.346 7) (Table 7). The genetic distance between SY population and other 6 populations ranged from 0.158 2 to 0.346 7, and much more exceeded among 5 populations in clade C, which implied that SY and ZJ population have significant genetic differentiation and might be relatively independent populations.

Fig.1 UPGMA tree of 88 individuals of the 7 Epinephelus akaara populations 1-14：ZS;15-29：PT; 30-39：XM;40-52：ZL;53-57：DYW; 58-73：ZJ;74-88：SY

Fig.2 UPGMA tree of the 7 Epinephelus akaara populations

Table 5 AMOVA analysis of the 7 Epinephelus akaara populations

Table 6 Fst value (above diagonal) and the corresponding P value (below diagonal) among 7 Epinephelus akaara populations

3 Discussion

3.1 Genetic diversity of 7 E. akaara populations

In order to evaluate the genetic diversity in differ ent populations, we sampledE.akaarafrom 7 representative locations. Using 13 primer combinations, 941 bands were obtained from 88 individuals of 7 populations. Among these bands, 672 of 941(about 71.41%) were polymorphic. Therefore, AFLP could be used for obtaining more genetic information for a given species and could be a good technique to evaluate the genetic diversity.

Table 7 Genetic similarity(above diagonal)and genetic distance(below diagonal)among the 7 Epinephelus akaara populations

Results achieved in this study revealed great difference among 7 populations. The frequency of polymorphic loci, the Nei's index, the Shannon's index and the genetic similarity within population varied from 18.40 to 47.70%, from 0.044 7 to 0.142 9, from 0.066 5 to 0.214 1, and from 0.861 5 to 0.947 4, respectively. The genetic variation was the lowest in DYW and ZS populations, while that was the highest in ZJ population. Compared with the current published data, frequency of polymorphic loci inE.akaarawas lower than that inPseudosciaenacrocea, including wild (76.6%) and cultured (69.2%～70.6%) large yellow croaker[6], and Dai-chu race in Zhejiang province (55.80%)[9], it was also lower than that in 3 wild Red sea breamPagrusmajorpopulations (64%～58.4%) and wildLutjanusargentimaculatus(57.14%)in China[5,7]. But frequency of polymorphic loci inE.akaarawas higher than that in wild (46.18%) and cultured (40.07%) flounder[8], partial cultured population of channel catfishLctaluruspunctatus(18.6%) and arowanaScleropagesformosus(12.7%～15.6%)[12-13].These results indicated that genetic diversity ofEpinephelusakaarais below the middle level.

3.2 Genetic differentiation among 7 populaitons of Epinephelus akaara

The 7 populations could be classified into 3 clades. Clade A only contained SY population. But Clade B contained part of individuals of ZJ population and Clade C contained 6 subclades and these subclades basically clustered according to their geographical location. SY population contained 34 specific bands. Significant differentiation could be found among SY population and other populations. But there are no significant differentiation found within individuals of SY population. Individuals of ZJ population was classified into both clade B and C, which implied that significant differentiation was found within individuals of ZJ population. ZS, PT, XM, ZL, SY and part of ZJ population were classified into clade C. Most individuals of the 6 populations in clade C could be further clustered into several subclades. Result of AMOVA and Fst showed that great difference exists among populations. In the 7 populations, ZJ population contained two clades, while individuals from other populations basically were clustered into one clade., We also concluded that gene flow among different populations was weak by AFLP analysis, which may be limited by finite movement and other unknown geographical factors. Another possibility was individuals moving into a population may not be efficient to affect the genetic structure[14-16]. SY population was located in Hainan Island far away from the mainland land. This maight limit the gene flow between SY and other populations. Clade B contains only individuals of ZJ population. But clade C contained individuals of ZJ and other populations, which may indicated that gene flow occur between ZJ and other populations. This process which may be natural or artificial breeding, causes the total number of loci, frequency of polymorphic loci, Nei's index and Shannon's index are higher than other populations. In aquaculture, rich genetic diversity was the basic for seed selection. In this study, we showed that there was significant genetic structure among different populations. Therefore, in order to maintain the development ofEpinephelusakaaraculture, we shall pay more attention to the conservation of wild resources when carrying out artificial breeding. Moreover, we found specific markers to clade A, B and C, respectively. These markers could be applied to population identification and might play important roles in the determination of seed source.

AcknowledgementThis work was funded by ‘863’ Hi-Tech Research and Development Program of China (2006AA09Z418); Natural Science Foundation of China province (40606038 ); Natural Science Foundation of Guangdong province (05003356 ).

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