638 



Genetic diversity of yellow grouper 

 iEpinephelus awoara) 

 determined by random amplified 

 polymorphic DNA (RAPD) analysis 



Satyendra K. Upadhyay (contact author) 



Wang Jun 



Su Yong-Quan 



Ding Shao-Xiong 



Sonal Chaturvedi 



Laboratory of Fish Genetics 



Diseases and Breeding 



College of Oceanograpfiy and Environmental Science 



Xiamen University 



Xiamen 361005 



Fujian, Peoples Republic of China 



Present address (for S K Upadhyay): A-37, Satya Vi|aya Society 



J, M. Road 



Bhandup (W) 



Mumbai 400078 (Maharashtra), India 

 Email address for S K. Upadhyay satyaxmu yahoo.com 



The development of molecular tech- 

 niques has enhanced our ability to 

 identify fish species. A few molecular 

 markers, such as mitochondrial DNA 

 and ribosomal DNA, have been used 

 to assist in the identification of fish 

 species. One such technique now rou- 

 tinely used is the random amplified 

 polymorphic DNA polymerase chain 

 reaction (RAPD-PCR; Williams et al.. 

 1990). Genetic analysis with RAPD 

 markers is relatively easy, fast, and 

 efficient. RAPD analysis, however, 

 may not be practical for identifing 

 species that interbreed (Martinez et 

 al., 1997). Although the major limi- 

 tation of RAPD technique for iden- 

 tification of intraspecific specimens 

 is its repeatability, adherence to 

 protocol and standardized reactions 

 can improve the method (Jones et 

 al., 1997). This technique has been 

 used for identification and detection 

 of genetic diversity in various fish 

 species (tilapia; Naish et al., 1995; 

 striped bass; Bielawski and Pumo, 

 1997; grouper; Asensio et al., 2002). 

 Most other DNA-based methods are 

 more laborious and time consuming 

 than RAPD, making them less suit- 



able for large population or genetic 

 diversity studies. Suitable loci with 

 high reproducibility allow the identi- 

 fication of unequivocally distinct spe- 

 cies (i.e., where there is large genetic 

 differentiation between species, com- 

 pared to within species; Greig et al., 

 2005). 



Groupers (Epinephelinae: Serrani- 

 dae) are among the most important 

 and highly valued demersal species 

 of tropical and subtropical coastal ar- 

 eas worldwide. In general, grouper 

 species lack distinct morphological 

 specializations, but color patterns 

 and geographic location are used in 

 the field. Some confusion exists over 

 the names of some important Indo- 

 Pacific grouper species (Heemstra 

 and Randall, 1993; Sadovy, 1997) 

 and little work has been done to es- 

 tablish species identities and their 

 genetic diversity. One alternative to 

 gathering information on these spe- 

 cies is the use of molecular genetic 

 markers. In the present study, RAPD 

 analysis has been used to investigate 

 the genetic variation in two popula- 

 tions of yellow grouper iEpinephelus 

 awoara) from the South China Sea. 



Materials and methods 



Sample preparation and 

 DNA extraction 



Yellow grouper were obtained from 

 two populations (30 individuals from 

 Xiamen and 26 individuals from 

 Guangdong) of the South China 

 Sea. Muscle samples from fish were 

 taken, placed in 95% ethanol, trans- 

 ported to the laboratory, and stored at 

 -20"C until analysis. Genomic DNA 

 was extracted according to the DNA 

 extraction method of DeSalle et al. 

 (1993). 



Primer selection and 

 RAPD reaction profile 



Twenty primers (Table 1) were selected 

 on the basis of presence of intense, 

 well-distinguished, and reproducible 

 bands for further analysis. PCRs for 

 each population were performed in 

 2-,i(L volumes containing 100 mM 

 Tris-HCl (pH 8.3), 500 niM KCl, 15 

 mM MgCU, 1 f(L dNTPs. 0.4 unit 

 rTaq polymerase and approximately 

 20 ng of template DNA and 1 ^L of 

 each primer. Thermocycling condi- 

 tions were as follows; initial dena- 

 turation step of 95°C for 5 min., 30 

 cycles of 45 s at 93°C, annealing tem- 

 perature of 45 s, primary extension of 

 72'C for 2 min., and a final extension 

 of 72°C for 5 minutes. 



Detection of amplified DNA 



Electrophoresis of a lO-j/L portion 

 of the amplification reaction was 

 performed for 45 minutes at 100 

 V in a 1.2 % agarose gel contain- 

 ing ethidium bromide (1 f(g/mL) in 

 Tris-acetate buffer (0.004 M Tris 

 acetate, 0.001 M EDTA, pH 8.0). 

 DNA fragments were viewed with 

 UV transillumination and analyzed 

 with Genescan (Gene-Geneius Bio- 

 imaging System, Cambridge, Eng- 

 land). Their sizes were estimated 

 by comparison with a commercial 

 1-kb plus DNA ladder. 



Manuscript submitted 10 August 2004 

 to the Scientific Editor's Office. 



Manuscript approved for publication 



19 December 2006 by the Scientific Editor. 



Fish. Bull. 104:638-642(2006). 



