Abstract: The Asian arowana, Scleropages formosus (Osteoglossidae) is one of the most valuable species of the ornamental fish which has an important role on academic study and economic development. The present study assessed the genetic variation of three S. formosus stocks cultured in ponds using 39 polymorphic microsatellite primer combinations. The results showed the middle level of genetic diversity among all three stocks. The average values of observed Heterozygosity (Ho) expected Heterozygosity (He) for the red stocks are the highest which were 0.509 and 0.552, respectively. Genetic diversity of the green stocks was higher than the golden group. The inbreeding coeffcients within the subpopulation (Fis) and the differentiation index of population (Fst) analyses showed low genetic differentiation among three cultured stocks. The cluster analysis based on Nei’s standard genetic distance showed that the golden and the green stocks clustered together and the red stock was in another clade. These results would have important implications for future breeding programs, conservation and production, suitable management guideline projects for S. formosus in mainland China.

INTRODUCTION

Scleropages formosus commonly known as Asian arowana, the Dragonfish, bony-tongue belongs to the order Osteoglossiformes, one of the ancestral teleost clade with extant representatives restricted to freshwater habitats in Southeast Asia. Three basic colour (green, golden and red) varieties were identified for S. formosus which were probably related to freshwater habitats during the Pleistocene glacial ages. Depending on lovely elegance shape and a long-life, S. formosus acquired a special status in some Asian countries, a very popular but extremely valuable ornamental fish. An adult red or golden arowana individual might cost over $20,000 at the ornamental fish market (Yue et al., 2004). Due to its popularity and great demand, S. formosus have been fiercely hunted in its native habitat, leading to its including species threatened with extinction fish in the wild listed by the Convention on International Trades in Endangered Species of Wild Fauna and Flora (CITES) as an endangered species since 1980. From then on, their commercial export, import and sales were normally prohibited in all members of CITES. On the other hand, S. formosus is a primitive fish retaining anatomical characteristics from the Jurassic era and of great value on scientific research to explore genetic evolution.

Given their great evolutionary and economic importance, more information should be required on genetic variation of the inter or intra-population which has important implication in fishery management and development of aquaculture technology. Previous studies have demonstrated that genetic diversity among S. formosus isolates in Southeast Asian countries including wild caught stocks (Shafiqur et al., 2008) and cultured stocks (Yue et al., 2000, 2006a; Mu et al., 2009) using several DNA-based molecular markers and techniques such as RAPD (Yue et al., 2002), AFLP (Yue et al., 2004), STS (Yue et al., 2003), mtDNA cytochrome b (cytb) gene (Hu et al., 2009), D-loop regions (Pan et al., 2005), ATP synthase gene (Tang et al., 2004) of mitochondrial (mt) DNA. Generally, the choice of an appropriate genetic marker contributed to a population genetics survey (Sunnucks, 2000). Above studies focused on approaching different questions such as identification of different stocks whether wild or cultured, conservation of different sampling locations using different mtDNA markers. In addition compared with RAPDs that results thought not to promise (Fernando, 1997), microsatellite is proved to be a powerful tool emerged as those with finest resolution for labeling of populations and individuals and have been widely used accurate genetic assessment of population differentiation of aquaculture fish species such as grass carp (Zhang et al., 2005; Liu et al., 2009), tilapia (Rutten et al., 2004) and Asian seabass (Zhu et al., 2006), due to their high variation, abundance, neutrality, co-dominance and unambiguously scoring of alleles (Tautz, 1989). Although, relatively few scientific studies using microsatellite have been published about different stocks in peer-reviewed, in order to avoid the loss of genetic variability posing a major stumbling block to aquaculture, the objectives of the present study was to examine genetic diversity for future commercial production within three stocks of S. formosus cultured in mainland China using microsatellite.

MATERIALS AND METHODS

Fish samples and DNA extraction: Three stocks of S. formosus were collected from Guangzhou tiny-lake aquatic organism technology co., ltd, including green (17), golden (21), red (15) arowana individuals. Fin clip of each fish was collected and kept in absolute ethanol. DNA was extracted according to the instruction of kit (Tianwei Co. Ltd, China) and dissolved in deionized distilled water. The integrity and purity of the DNA samples were examined on a 0.8% agarose gel by electrophoresis. All DNA samples were stored at -20°C until use.

Microsatellite analysis: A total of forty four microsatellite primer combinations described by previous studies (Yue et al., 2000, 2006a; Tang et al., 2004) were used. Detail information of all microsatellite primers were shown in Table 1. PCR reactions were carried out in a volume of 20 μL containing 2 mM of each MgCl₂, 0.2 mM of each dNTPs, 0.5 mM primer, 2.0 μL 10xrTaq buffer, 0.15 U Taq DNA polymerase (Takara) and 1 μL DNA in a thermocycler (Bio-Rad) under the following conditions: after an initial denaturation at 94°C for 4 min, then 94°C for 30 sec (denaturation); 45°C for 57 sec (Table 1) for 30 sec (annealing); 72°C for 30 sec (extension) for 30 cycles, followed by a final extension at 72°C for 7 min.

Table 1:	Microsatellites DNA primer
F: Forward primer; R: Reverse primer

Data analysis: Amplified products were manually scored as 1 for the presence and 0 for the absence of a band as binary data using each of the primer. The effective number of alleles each primer (Ae) observed (Ho) and expected (He) Heterozygosities and Hardy-Weinberg Equilibrium (HWE) were analyzed using program POPGENE (Version 1.31) (Yeh and Boyle, 1997). Nei’s genetic distances (d) among stocks were calculated using MEGA4.0 (Tamura et al., 2007) and used to draw phylogenetic trees by an Unweighted Pair-Group Method using an Arithmetic mean method (UPGMA). The reliability of the dendrogram was evaluated with 1,000 bootstraps.

RESULTS AND DISCUSSION

The current study analyzed DNA polymorphism for forty-four microsatellite primer and assessed the genetic variability of three cultured stocks of S. formosus in mainland China. Although, each primer was used to amplify in all stocks, the 39 polymorphic primers except for D16, D31, D32, D95, K16 were included in the analyses.

Table 2:	Genetic diversity of three stocks of Scleropages formosus

Table 3:	Chi-square test for Hardy-Weinberg equilibrium of three stocks of Scleropages formosus
*significant deviation from equilibaium;**terribly significant deviation from equilibaium; there is only one allele at the primer

A total of 144 alleles were detected across polymorphic primer ranging from 2-7, corresponding to an average of 3.7 alleles per primer. The genentic diversity at the population level was determined using the effective number of alleles (Ae), observed (Ho) and expected (He) heterozygosities. Value of Ho ranged from 0.493 (Red) to 0.509 (Green and Golden) whereas He ranged from 0.552 (Golden) to 0.549 (Green) with the number of effective alleles ranging from 2.53 (Red) to 2.60 (Golden) (Table 2) showing middle diversity across each stocks. Probability of deviation from Hardy-Weinberg equilibrium per primer combination was shown in Table 3. The F_ST estimating within stocks ranged from 0.0026-0.01 with an average of 0.0097 (p<0.01), (Table 4) showing significant genetic differences between different stocks.

Fig. 1:	Cluster analysis of three Seleropages formosus based on Ds using UPGMA method (Mu et al., 2009)

Table 4:	Genetic Distances (Ds)\genetic similarity Indices (I) (above diagonal), gene flow (Nm)\genetic differentiation coefficient (FST) (below diagonal) among three stocks of Scleropages formosus

Table 5:	Heterozygosity of different stocks of Scleropages formosus

Low level of genetic distances were observed between the golden and green arowana ranging from 0.006-0.0245, suggested that S. formosus perhaps did not evolve to the level of specie. However, a little high level of distances were found between red and green arowana. Genetic relationships of three stocks were calculated using Nei’s genetic distances to infer phylogenies by the Unweighted Pair-Group Method with Arithmetic mean (UPGMA) (Fig. 1). The phylogenetic relationships among three stocks showed that golden and green arowana clustered together, sistered to the red stock.

The average allele number in the study was 3.7 per primer, lower than that of the S. formosus previously reported by Yue et al. (2000, 2006a) and Tang et al. (2004). The difference in allele number could be affected by sample size of the individuals used for the characterization of primers. Heterozygosity, gene diversity was an important measurement of population diversity at genetic level and has drawn much attention from ecologists and aquaculturists (Xu et al., 2001).

Heterozygosity of three stocks ranged from 0.543-0.552 which was similar to the statistical data of approximately 40000 individuals, 524 microsatellite primers of 78 species (DeWoody and Avise, 2000) suggested that the genetic diversity of three S. formosus stocks was relatively rich. However, compared with those of other wild or cultured S. formosus stocks, the results were higher than that between wild green and captive golden arowana stocks (Shafiqur et al., 2008) but far below to that between wild green and wild red arowana stocks (Yue et al., 2000, 2006a) (Table 5). The possible explaination could be the inheritance of single or multiple genes affected by factors in the environment. Intra-specific relationship relating to S. formosus among different geographical populations isolates in Southeast Asian countries has different results using different types of nuclear genome markers and techniques in earlier studies (Kumazawa and Mutsumi, 2000; Tang et al., 2004). The study showed that golden arowana and green arowana clearly fall into different clades from red arowana, other than the former studies (Tang et al., 2004; Yue et al., 2006b) which was relevant to different geographical population and size.

Traditionally, cluster analysis maybe solve the problem of compositor of classifications in one collectivity. Yue et al. (2004) monitoring phylogenetic relationship of three Asian arowana stocks by AFLP showed that red and green varieties clustered. It is the same with the results that of Tang et al. (2004) supported red arowana was closely related with Malaysian red-tail gold arowana. Something else, Hu et al. (2009) anaylzing mt cytb gene showed that red arowana clustered with golden arowana after clustering with green arowana.

This study render different results that gold arowana and green arowana clearly fall into different clades from red arowana but Tang et al. (2004) estimated the time of divergence between the green and red arowana (1.5-2.6 MYA) close to the probable time of the fluctuation of sea level during the late Pliocene to early Pleistocene era by mt ATPase subunit 6 and 8 assuming that the sequence divergence rate for ATPase determined for fish is 1.3% per million years (Bermingham et al., 1997). In view of the present case to be a primitive fish possessing of academic and economical value, it is necessary to make more researches to clarify the evolution and breeding of different color stocks for a fuller understanding using more molecular markers.

CONCLUSION

The present study assessed genetic diversity among different stocks of S. formosus cultured in mainland China using microsatellite markers. The results have important implication for studying genetic structure of S. formosus as well as for the effective breeding management and development.

ACKNOWLEDGEMENTS

Project support was provided by the People's Republic of China, Ministry of Agriculture Affairs State (2009-Z13) to YC Hu, Science and Technology Developing Fisheries Program of liwan (20082109029) to YC Hu and the Open Fund of Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture (BZ2009-10) to XD Mu.