41 1 Vol.41, No.1 2010 1 OCEANOLOGIA ET LIMNOLOGIA SINICA Jan., 2010 (Sarotherodon melanotheron) 5 * 1, 2 1 2 2 (1. 453007; 2. 201306) 5, 6, : (1) 6 5 22 49, 190 204, 190/190 204/204 270/223 270/243; (2) 5, (N e ) (H e ) (PIC), 1.812 0.331 0.326; (3) 6 2,, 5 ; (4),,, Q959.468 Trewavas(1983), (Tilapias) Tilapia Sarotherodon Oreochroms Oreochroms Tilapia,, 2002 (Sarotherodon melanotheron)(, 2005),,,, (Trewavas, 1983;, 1994;, 2008a) DNA,,, (, 2002) 5 5 1 1.1 6 Sarotherodon 2002, Oreochroms (O. niloticus) 1994 ICLARM, (Israel red tilapia) Oreochroms, 2001, (O. aureus) 1998, Oreochroms (O. niloticus O. mossambica) Tilapia (T. zillii) 30, 95% * 948, 993125 ;, 2005 2007,,, E-mail: xjli67@126.com : 2008-12-23, : 2009-03-15
86 41 1.2 1.2.1 DNA PCR,, / DNA(, 2006) (Kocher et al, 1998; Yue et al, 2002) (PRL) (IGF) (ISP) 5, 1 1.2.2 PCR : 3μl buffer (10mmol/L Tris-HCl, ph 9.0, 50mmol/L KCl, 3.0mmol/L MgCl 2, 0.001% ), 0.1mmol/L dntp, 0.2μmol/L, 50 100ng DNA, 1U Taq DNA (Biostar ), 25μl, 30μl Eppendorf Mastercycler Gradient PCR, : 94 4min, 94 30s, 53.8 (60.5 65.5 67.0 62.5 ) 30s, 72 30s 35, 72 10min 10μl 3.0%, E.B, Syngene GeneTools 1.2.3 (1) (, 2004): (N e ) = 1 m i= 1 r 1 m 2 (H e ) = 1 pi r i = 1 i = 1 n 1 2 n n 2 2 (PIC) = 1 pi 2pi pj i= 1 i= 1 j= i+ 1, p i p j i j, r p 2 i, m (2) Nei (D n )(Nei, 1978): D n = lni, I = J y = 1 r m 2 Y ij j i J xy J J x y, J x =, J xy = 1 r m r r j 1 r m 2 X ij j i r, i X Y ij ij, X ij Y ij X Y j i, r, m 1.2.4, MEGA2 (UPGMA) (NJ) (Nei et al, 2002) 2 2.1 PCR 5 ( 1) PRL1 PRL2, PRL3 IGF, 190 307bp, 197bp 2.2 6 6 5 22 49 5 2 4.2 4.8 2.6 4.4 2; 2.4 4.4 5 2.4 5 2, 3 190 204; 190/190 204/204 270/223 270/243 Tab.1 表 1 5 对微卫星引物序列 重复序列及特异退火温度 Sequence and its repeats of five microsatellite marker primers used in this study, and specific annealing temperature of PCR amplification (5 3 ) ( ) PRL1 PRL2 PRL3 IGF ISP F: GTTAGCCCCCTCCTCACTCT R: ACCTTGCTCGTCACACCTG F: TCGTGTCTTGTGGGGAAACC R: TGAATGGATGCAACAGGATG F: CTTAACATTTTCCACCTTCACG R: CTTGCCTCCATTTTATAGTTCCTT F: ATGCTAGCA AACATCAAAGGTC R: GATATGCTGATGATGCACAGAGTC F: TGAGCTGAGCAGATGGAGCAGAAG R: ATGAACAGCCCTGTGAAGAGAGG TG 53.8 CA 60.5 CA 65.5 ATCT 67.0 CA 62.5
1 : (Sarotherodon melanotheron) 5 87 1 6 5 Fig.1 The partial electrophoresis results of five microsatellite primers in six tilapias species : PRL1 PRL2 PRL3 IGF ISP 1 4: ; 5 8: ; 9 12: ; 13 16: ; 17 20: ; 21 24: ; M: (GeneRuler TM 100bp Ladder) 2.3 6 6 5 2 (N e ) (H e ) (PIC), PRL1 PRL2, H e PIC 0, 3 2.4 6 6 Nei 3 2 : 6 2,, 5,,,, 3 3.1,, 5
88 41 Tab.2 (n=24) 表 2 6 种罗非鱼 5 个微卫星座位的遗传多样性指标平均值 Mean indices of genetic diversity of five microsatellite locus in six tilapias species (n=24) (n=23) (n=21) (n=23) (n=21) N e 1.812 3.211 3.235 1.867 2.747 1.370 H e 0.331 0.667 0.684 0.430 0.576 0.215 PIC 0.326 0.692 0.676 0.366 0.619 0.210 表 3 6 种罗非鱼间的 Nei 氏标准遗传距离 Tab.3 The Nei s standard genetic distances among six tilapias species 0 1.903 0 1.241 0.816 0 2.358 1.071 1.039 0 2.062 1.359 0.961 0.997 0 1.865 1.415 1.339 0.608 1.053 0 Fig.2 2 6 UPGMA NJ Dendrogram of six tilapia species using UPGMA and NJ method for clustering A. UPGMA, B. NJ,,, (Nei et al, 2002), PRL1 204 PRL2 190 IGF 239 ISP 250 0.5, 6,,,, Soduck (1991), 3 15, Tilapia Oreochromis Tilapia Sarotherodon Sarotherodon Oreochromis 0.688 0.150 0.571 0.132 0.366 0.100, Tilapia, Sarotherodon,, Oreochromis, Oreochromis Sarotherodon Tilapia (, 2008a) : 6, 5,, ; 5,, ;, Oreochromis,,
1 : (Sarotherodon melanotheron) 5 89,, (, 2008a), (, 1994),, 3.2, 5 5, Pouyaud (1999) 4 Pouyaud (1995) 24, (2007) AFLP, 16.6%, 0.922 0.995, ( ),, 2002,, 2670,,, :, (, 1995),,, (, 1995),,,, (, 2008b),,,, (, 2008b), ;,,,,,,,,, (, 1996), 500 ( ),, 0.1%,,, 2005.., 12(3): 245 251,,, 2008a. (Sarotherodon melanotheron) 5., 39(6): 683 688,,, 2008b.., 27(5): 777 780, 1996.. :, 131 144,, 1995.., 19(2): 105 111,, 1994.., 29(3): 18 23,,, 2007. AFLP., 16(3): 293 296, 2004.. :, 165 196,, 2002. DNA., 22(3): 714 722 C W, G S,,, 2006. PCR. :, 201 218 Nei M, Kumar S,,,, 2002.. :, 67 128 Kocher T D, Lee W J, Sobolewska H et al, 1998. A genetic linkage map of a cichlid fish, the tilapia (Oreochromis niloticus). Genetics, 148: 1225 1232 Nei M, 1978. Estimation of average heterozygosity and genetic distance from a small number of individual. Genetics, 89: 583 590 Pouyaud L, Agnese J F, 1995. Phylogenetic relationships between 21 species of three tilapiine genera Tilapia, Sarotherodon and Oreochromis using allozyme data. J Fish Biol, 47(1): 26 38 Pouyaud L, Desmarais E, Chenuil A et al, 1999. Kin cohesiveness and possible inbreeding in the mouthbrooding tilapia Sarotherodon melanotheron (Pisces Cichlidae). Molecular Ecology, 8(5): 803 812 Soduck P, McAndrew B J, 1991. Molecular systematics of three tilapiine genera Tilapia, Sarotherodon and Oreochromis using allozyme data. J Fish Biol, 39(supplement A): 301 308 Trewavas E, 1983. Tilapiine Fishes of the Genera Sarotherodon, Oreochromis and Danakilia. London: British Museum (Natural History), Publ Num 878. Comstock Publishing Associates, 583 Yue G H, Orban L, 2002. Microsatellites from genes show polymorphism in two related Oreochromis species. Molecular Ecology Notes, 2: 1 2
90 41 COMPARISON ON GENETIC DIVERSITY BETWEEN SAROTHERODON MELANOTHERON AND OTHER FIVE TILAPIAS SPECIES LI Xue-Jun 1, 2, LI Ai-Jing 1, LI Si-Fa 2, CAI Wan-Qi 2 (1. College of Life Science, Henan Normal University, Xinxiang, 453007; 2. The Key Laboratory of Aquatic Genetic Resources and Aquacultural Ecology Certificated by the Ministry of Agriculture, Shanghai Ocean University, Shanghai, 201306) Abstract Five microsatellite primers were designed for six tilapia species based on the sequence of related gene of known tilapia for PCR amplification and genetic diversity, including blackchin, Nile tilapia, Israel red tilapia, Taiwan red tilapia, blue tilapia and Zilii tilapia. The results indicate that: (1) 22 alleles and 49 genotypes were found in five microsatellite locus of all tilapias. Blackchin tilapia, Taiwan red tilapia and Zilii tilapia have less alleles and genotypes. Specific alleles of blackchin tilapia are 190 and 204, and its specific genotypes are 190/190, 204/204, 270/223 and 270/243, respectively; (2) compared with other five tilapias, blackchin tilapia has lower effective number of alleles (N e ), mean gene heterozygosity (H e ), and polymorphism information content (PIC), which is 1.812, 0.331 and 0.326, respectively; (3) In genetic distance, the six species can be divided into two groups: blackchin tilapia and other five tilapias; (4) inbreeding is considered the primary issue for the genetic resources conservation. Key words Blackchin tilapia, Microsatellite marker, Genetic diversity, Genetic resources conservation