5 6 2 00 9 1 2 South China Fisheries Science Vol. 5, No. 6 Dec., 2009 doi: 10. 3969/ j. issn. 1673-2227. 2009. 06. 009 16S rrna,,, (, 510300) : PCR ( Pinctada fucata) ( P. chemnitzi) ( Pteria penguin) 16S rrna, PCR,, 439 bp, MEGA 3. 1, 2 ( Transversion), 1 ( Transision), 5 ( Transversion) GenBank 9, 464, 51 / 231 ( 173 53 ) 83. 2%, 55. 4% 59% NJ, : ; 16S rrna ; : Q 755 : A : 1673-2227 - ( 2009) 06-0047 - 07 Phylogenetic relationship of pearl oysters in genus Pinctada based on partial mitochondrial 16S rrna sequence HUANG Guiju, YU Dahui, GUO Yihui, QU Nini ( Key Lab. of Mariculture, Ecology and Quality Control, Ministry of Agriculture; South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China) Abstract: Mitochondrial 16S rrna gene fragments were amplified from Pinctada fucata, P. chemnitzi and Pteria penguin using PCR technique. The PCR products were directly sequenced, and 439 bp nucleotide sequences were obtained after excluding the primers and truncating partial sequences at the end. Sequence variation was analyzed using MEGA 3. 1 software. The results indicated that base substitution was not found among P. fucata, but two transversions were found among P. chemnitzi, one transition and five transversions among P. penguin. Alignment with nine homologous sequences from Pteridae species in GenBank revealed 51 insertion/ deletions and 231 variable sites ( 173 parsimony-informative sites and 53 singletons) among 464 truncated alignment sites. The percentage of the i- dentity of the 16S rrna gene fragments was 83. 2% between P. fucata and P. chemnitzi, 55. 4% between P. fucata and Pteria penguin, and 59% between P. chemnitzi and P. penguin. The NJ tree indicated that P. fucata and P. chemnitzi were assembled together with Pinctada species, yet P. penguin assembled together with the Pteria species, which was consistent with the morphological classification. Key words: pearl oyster; 16S rrna gene; sequence analysis : 2009-07-08; : 2009-09 -18 : ( 2006BAD01A13 ) ; ( 863 ) ( 2006AA10A409 ) ; ( 2006B20201063) : ( 1978 - ),, E-mail: xaxhgj@ 126. com :, E-mail: pearlydh@ 163. com
48 5 ( Pinctada) ( P. fucata ), 3 [ 7 ] ( P. maxima ) ( P. albina), ( P. margaritifera) ( P. nigra) RAPD, ( P. chemnitzi), ( Pteria penguin) [ 1-2 ] [ 8 ] RAPD 7 [ 9],, 6 ITS2,,, ITS2 [ 3], 16S rrna, GenBank 16S rrna, DNA, 1 ( 15. 7 19. 5 kb), [ 4 ] DNA 1. 1 ( ), [ 5] DNA 3, 2 ( mtdna), 95% [ 6] 1 1 Tab. 1 Species and sampling location species Pinctada fucata sampling location GenBank GenBank accession No. P. chemnitzi EF056475EF056476 Pteria penguin EF031242 1. 2 1. 2. 1 DNA DNA 1 [ 1 0] 20 mg NaAc ( 3 mol L - 1, ph 5. 2), - 20 2 h 2, 1. 5 ml, 100 L TEN9 200 L, 4, TEN9 600 L, SDS 2%,, 56 15 min, OD 2 60 OD 28 0 nm, K ( 20 mg ml - 1 ) 10 L, 56 DNA DNA, 20 15 L RNaseA, 37 15 min, ( 25 24 1) ; ; 1. 2. 2 PCR ( 24 1 ) 2 1 /10,, 75% 2 1% EB, ng L - 1
6 : 16S rrna 49 16S rrna, 16S-F:, 3 5 -CGCCTGGTTGATTAAAAACATTGCTGC-3, 16S- R: 5 -CCGGTTTGAACTCAGATCACGTA-3 PCR 20 L, 1 PCR [ 10 mmol L - 1 Tris-HCl ( ph 9. 0, 25 ), 50 mmol L - 1 KCl, 0. 1% Triton X-100], 3 mmol L - 1 MgCl 2, 0. 1 mmol L - 1 dntp, 0. 15 mol L - 1, 1 U TaqDNA ( ), DNA 30 50 ng 93 4 min, 35 : 93 30 s, 55 30 s, 72 30 s, 72 5 min 1. 0% ( Transision) 5 ( Transver-,, ( V-gene) sion) PCR PCR 1. 2. 3, T G C ( 2) Genbank Clustal 1. 81 [ 11 ] 9, 464 MEGA3. 1, KIMURA [ 12] ( twoparameter method) ( 173, 53, 1) Gap, ( maximum parsimony method, MP), Bootstrap 1 000 55. 4% 59. 0%, 2 Clustal 1. 81, 439 bp, ( 3 ) ( : AB214441 AB214448 AB214449 ) ; ( 2 ) 2 ( Transversion), ; ( 2 ) 1,, 3 16S rrna A, 51 / 231 83. 2%, 2. 2 2. 1 16S rrna Kimura 12 DNA 0 0. 490 9 500 bp 16S rrna, PCR 0 0. 288, 2 species 2 16S rrna Tab. 2 Base composition of 16S rrna gene fragments in pearl oysters % base composition A T C G GC GC content Pinctada fucata 28. 0 25. 1 17. 1 29. 8 46. 9 P. radiata 27. 5 25. 3 17. 6 29. 7 47. 3 P. chemnitzi 29. 0 27. 9 15. 8 27. 2 43. 0 P. nigra 29. 7 26. 8 16. 2 27. 3 43. 5 P. maculata 28. 0 25. 6 17. 4 28. 9 46. 3 P. maxima 28. 0 29. 8 15. 2 27. 0 42. 2 P. albina 29. 7 26. 8 16. 2 27. 3 43. 5 P. margaritifera 28. 3 28. 3 15. 4 28. 1 43. 5 Pteria penguin 27. 9 30. 2 14. 8 27. 1 41. 9 P. hirundo 26. 0 26. 9 17. 3 29. 8 47. 1
50 5
6 : 16S rrna 51 1 12 16S rrna Fig. 1 Variable sites in 16S rrna sequences of 12 Pteriidae 3 12 Kimura Tab. 3 Pairwise genetic distances among 12 Pteriidae on the basis of Kimura two-parameter mode species 1 2 3 4 5 6 7 8 9 10 11 12 13 1 P. maxima 2 P. margaritifera 3 P. nigra 4 P. albina 0. 146 0. 288 0. 279 0. 288 0. 279 0. 000 5-0. 279 0. 276 0. 122 0. 122 P. chemnitzi-hk 6-0. 286 0. 283 0. 125 0. 125 0. 007 P. chemnitzi-db 7 0. 245 0. 244 0. 144 0. 144 0. 168 0. 171 P. martensi 8 0. 245 0. 244 0. 144 0. 144 0. 168 0. 171 0. 000 P. fucata 9 0. 274 0. 269 0. 147 0. 147 0. 195 0. 191 0. 070 0. 070 P. imbricata 10 0. 256 0. 248 0. 157 0. 157 0. 188 0. 191 0. 057 0. 057 0. 056 P. radiata 11 P. maculata 12 P. hirundo 0. 270 0. 253 0. 155 0. 155 0. 161 0. 164 0. 103 0. 103 0. 120 0. 097 0. 457 0. 433 0. 490 0. 422 0. 413 0. 436 0. 436 0. 436 0. 474 0. 470 0. 459 13 0. 418 0. 483 0. 468 0. 468 0. 410 0. 401 0. 446 0. 446 0. 464 0. 442 0. 441 0. 163 P. penguin ( P. penguin P. hirundo) 0. 163( 3) ( P. chemnitz) 0. 168,, ( P. fucata) ( P. penguin) ( P. fucata) 0. 446, ( P. chemnitz)
52 5 ( P. penguin) 0. 410 41. 9%,, A T ( P. martensi) ( P. fucata) G C 0, ( P. albina ) 16S rrna ( P. nigra) 0 2. 3 12 16S rrna 0. 446 0. 410, MEGA 3. 1 NJ ( 2), 2 9 0 0. 288,, 2 ( ) 0. 163 ;, 0. 410 0. 490, ( P. hirundo) 3 16S rrna,, 2 (, ), ;, ITS [ 1 4-1 5] RAPD [ 16],, 2, 16S rrna P. fucata P. martensi, [ 13] 16S rrna 0,, ( P. radiata) P. imbricata, DNA 16S rrna 0. 057,, BEAUMONT KHAMDAN [ 17] ( allozyme) 16S rrna BEAUMONT KHAMDAN G C 46. 9% 43. 0% 0. 168,, NJ 2, ( P. radiata) Fig. 2 2 12 16S rrna NJ The NJ tree based on partial mitochondrial 16S rrna gene sequences of 12 species in Pteriidae
6 : 16S rrna 53 P. imbricata P. fucata, [ 18] ITS AFLP P. fucata P. martensi P. imbricata 3, 3 ITS1 ITS2, [ 2 ], 3 P. fucata P. martensi P. imbricata, [ 7],, 0, [ 10],,. [ 2] [ 14 ] ITS1,, 0. 013, 2 HE [ 19 ] 5 ITS 2, [ 16 ] RAPD ( ) ( 3) : 211-217., - - - PCR 16S rrna 439 bp, 3, : [ 1]. [ M] - 2 and its utility in detecting genetic relatedness of pearl oyster.. : 1978, 14: 101-117. [ 2]. [ M] 31. : 2002: 68-98. [ 3],,. [ J]. 2003, 12 ( 1) : 61-64. [ 4] BROWN W M, GEORGE M J, WILSON A C. Rapid evolution of animal mitochondrial DNA [ J]. PNAS, 1979, 76 ( 4) : 1 967-1 971. [ 5] WHITMORE D H, THAI T H, CRAFT C M. The large mouth bass ctyochrome b gene [ J]. J Fish Biol, 1994, 44 ( 4 ) : 637-645. [ 6],. : [ J]. 1983, 2 ( 4) : 321-328.,,. DNA [ J]., 2001, 8 ( 4) : 287-290. [ 8],,. 7 RAPD [ J]., 2006, 2 ( 1) : 18-22. [ 9],. 6 ITS2 [ J]., 2005, 1 ( 4) : 6-12. DNA RAPD [ J]., 2006, 2 ( 4) : 59-64. [ 11] THOMPSON J D, GIBSON T J, PLEWNIAK F J, et al. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools [ J]. Acids Res, 1997, 25 ( 24) : 4 876-4 882. Nucleic [ 12] KUMAR M. A simple method for estimating evolutionary rate of base substitution through comparative studies of nucleotide sequence [ J]. Mol Evol, 1980, 16 ( 2) : 111-120. [ 13]. 3 [ J]., 2001, 25 ( 1 ) : 54-60. [ 14]. ( Pinctada) [ J]., 2006, 37 [ 15]. : rdna ITS [ J]., 2005, 13 ( 4) : 315-323. [ 16],. 5 [ J]. 2009, 28 ( 2) : 293-300. [ 17] BEAUMONT A R, KHAMDAN S A A. Electrophoretic and morphometric characters in population differentiation of the pearl oyster, Pinctada radiata ( Leach), from around Bahrain [ J]. J Molluscan Study, 1991, 57 ( 4 ) : 433-441. [ 18 ],. ITS AFLP 3 [ J]., 2006, 2 ( 5) : 36-44. [ 19 ] HE M, HUANG L, SHI J, et al. Variability of ribosomal DNA ITS [ J]. Mar Biotechnol, 2005, 7 ( 1 ) : 40-45.