40 4 Vol.40, No.4 2009 7 OCEANOLOGIA ET LIMNOLOGIA SINICA July, 2009 (Pelteobagrus eupogon) * 1, 2 1, 3 1, 3 1 1 (1. 150070; 2. 214081; 3. 116023) 40 (YL, 30 ) (SC, 30 ) (SH, 18 ) (YY, 30 ) (QQ, 30 ) 1 (TJ, 30 ) 6 (H o )(H e ) (PIC) (A e ), Nei, x 2 Hardy-Weinberg, (F ST ) (N m ), PHYLIP3.63 Nei UPGMA 6 5042, 150 650bp, 40 1 8, 143, 3.575 (1) 6, PIC 0.00 0.79, 0.40 0.37 0.35 0.39 0.35 0.19, (A e ) 1.00 5.44, 2.05 2.30 1.93 2.07 1.93 1.47, (H e ) 0.00 0.83, 0.45 0.46 0.36 0.46 0.41 0.23; (2) YY QQ (0.9947), TJ SC (0.4846),,,, Q176 (Pelteobagrus eupogon) (Siluriformes) 鲿 (Bagridae) (Pelteobagrus),, (, 2001;, 2007),,,,,,,,,,,,,, (Microsatellite), *, 2004CB117405,, E-mail dayulee2003@yahoo.com.cn :,,, E-mail sunxw2002@163.com : 2008-08-20, : 2009-01-20
4 : (Pelteobagrus eupogon) 461 (, 2003;, 2007) (, 1997;, 2003) (, 2004;, 2007) (, 2006;, 2007) (, 2005;, 2006;, 2005), (, 2001;, 2005;, 2004;, 2006;, 2006) 40 5, PCR,, 1 1.1 1.1.1 (Pelteobagrus eupogon) (YL, 30 ), (SC, 30 ), (SH, 18 ), (QQ, 30 ) (YY, 30 ), 1 (TJ, 30 ) 1.1.2 PCR DNA ABI PE 9700, Eppendorf 5804 R 1.1.3 1( 40 DNA, Primer 5,, 30 ) Promega, 1.2 1.2.1 DNA, 70%,, DNA, DNA (2006) 1.2.2 PCR PCR 25μl, (2006) PCR 94, 3min; PCR 94, 30s; 48 54, 30s; 72, 30s; 40, 72 5min 1.2.3 PCR 2%, 5V/cm 2h, GoldView, Gene Genius Bio imaging system, Gel works (Version 3.0) 1.2.4 PopGene (Version 3.2) (Allele Frequency, P)(Observed number of alleles, N a ) (Effective number of alleles, A e ) (Observed heterozygosity, H o ) (Expected heterozygosity, H e ) (Genetic similarity index, I), (Genetic Distance, D s ) (Polymorphism Information Content, PIC), Botstein (1980) PIC = n n 1 n 2 2 2 Pi Pi Pj i= 1 i+ 1 j= i+ 1, P i P j 1 2 i j, n GenePop(Version 3.4), x 2 Hardy-Weinberg (F ST ) (N m ) PopGene PHYLIP V3.6 (Phylogeny Inference Package) gendist Nei, UPGMA (Unweighted Pair Group Method with Arithmetic means, ) 2 2.1 40 6 5042, 150 650bp, 1 8 1 2.2 (genotypic disequilibrium value), 0.92% P<0.05,,
表 1 部 分 黄 颡 鱼 引 物 序 列 情 况 Tab.1 Part of microsatellite marker sequences of P. eupogon GenBank (5 3 ) ( ) GenBank (5 3 ) ( ) HLJYC009 EU885713 CTGAGGTAGAACAGCACA GACGATCTGTGGAGTTTG (TC) 8 (CA) 6 46 HLJYC059 EU885734 GGCTCGGAGTCAGTTTCT TTGCGTTCTCAAGCTCAT (CA) 26 56 HLJYC012 EU885714 AGACTTGGTTTCAGGCTA CATTCCCATGGTACATATT (CA) 21 56 HLJYC060 EU885735 GATCAACGTCCAACAGAG GGAAAGAAAGATGGCTAG (CA) 28 (TTTG) 7 56 HLJYC013 EU885715 GACCCAGTTCCCACATTG GGCTACCACATCCCTCAT (CA) 23 58 HLJYC061 EU885736 AAGCGATTTATCCATTTT TGACACCCTGATTACACC (GTT) 11 50 HLJYC015 EU885716 CACCTCCACTGAGAATGA CGATAAGCAGGTAAATAA (CA) 23 53 HLJYC066 EU885738 ACACTGACATACACTGGCATAA CTGGCAACGTGTTTCTTACT (TG) 27 56 HLJYC017 EU885717 ATGGTATAAACATGGTGCTA ATGATGCTGATAGGGTGA (TG) 25 58 HLJYC067 EU885739 TACCATTTACAGGCAAGA AACAGCAGGTTTACAATC (TG) 26 49 HLJYC024 EU885718 TCACAATGAGTTTGGAGCCT TCTGGAGAACTGGGTGGG (CA) 27 48 HLJYC068 EU885740 TTTTCTAATCAGCGTTTC CACAGACTCAATCAATCC (CA) 49 53 HLJYC028 EU885720 CCATTTGCTCCATTCCTC TCTCCACCTCCTCACTCC (CA) 36 58 HLJYC074 EU885741 CCTCTGCTGGTCTGTTAG CATCCTTATTCTATCTGAAATG (CA) 50 56 HLJYC031 EU885722 CAGGATGGAGGTGTAAAG ATAAAGCTGTGATGTGCC (CA) 26 56 HLJYC075 EU885742 GTCGACGATCAAATGCTT CTCACCTTTCACCTCCAG (TG) 21 53 HLJYC035 EU885723 ACGGCATGAATAAAGGAA GAGGAGGAGACGAGGTGT (CA) 49 47 HLJYC081 EU885744 AAATATGAGCTGCACAGA AACAAATAACAAGCCCTT (GA) 33 (TG) 4 56 HLJYC037 EU885724 AAAGGTGGAACAGAAGGC ACCAACAAGCGAGAACTA (CA) 35 56 HLJYC084 EU885745 AGATGGTCTGTATGTGCC ATCTTTCAGCGTGTTTCA (CA) 38 48 HLJYC039 EU885725 TCCTACCAGCAGTAACCA GTCCCTCAACCAGAACAC (TG) 42 49 HLJYC086 EU885746 TAAGCCTTGTAAGTATTTTG CTGAAGCTATTTGCTCTT (GA) 39 48 HLJYC040 EU885726 CTGCGTGGTATCAGGTGG AAGGCACTGGATGAGTCAAT (CA) 17 56 HLJYC087 EU885747 TGTGGTGCTATCGGTAAA GAGATTCGCTTCTGTGCC (CA) 29 (GA) 38 56 HLJYC045 EU885729 TGGGTCTCCTCTGGGTTCA GCGGCTTCACTCACTTCC (TG) 29 56 HLJYC089 EU885749 CTACCGCTTATCAGAACTT CGATCTTTGCATCACTCA (CA) 27 48 HLJYC050 EU885731 CCTCCCACTCACCTTCCT GTTGCCACTTCAAACAGC (TG) 43 56 HLJYC090 EU885750 TGAGGCAACATTCAGAGC CCTTGATACAGGAGCAGAG (TG) 49 (GA) 9 48 HLJYC052 EU885732 AGACTTGGCGTTGTTTGG GTGTCAGAGTCGGGTGCT (CA) 118 48 HLJYC093 EU885751 AAAGATGTTGGTTTTGGGTG AGGCCGAAAGATGGCTAA (TC) 30 56
4期 李大宇等: 黄颡鱼(Pelteobagrus eupogon)不同生态地理分布群体遗传多样性的微卫星分析 463-100 图1 六个黄颡鱼群体在基因座 HLJ045 上的遗传差异 Fig.1 The genetic diversity at locus HLJ045 of 6 P. eupogon populations YL 月亮湖黄颡鱼群体; SC 四川黄颡鱼群体; SH 松花江黄颡鱼群体; YY 长湖子代黄颡鱼群体; QQ 长湖亲代黄颡鱼群体; TJ 天津黄颡鱼群体 2.3 群体遗传多样性分析 6 个黄颡鱼群体中, 检测到的有效等位基因数 间, 平均值为 0.46 0.51 0.36 0.63 0.51 和 0.40; 多态信息含量(PIC)在 0.00 0.79 之间, 平均值为 (Ae)在 1.00 5.44 个不等, 各个群体中平均值依次为 0.40 0.37 0.35 0.39 0.35 和 0.19 以上各个群体 2.05 2.30 1.93 2.07 1.93 和 1.47; 无偏期望杂合 多样性指标说明这几个黄颡鱼群体属于中度多态性, 度(He)在 0.00 0.83 之间, 平均值为 0.45 0.46 0.36 遗传多样性水平适中 由表 2 可见, 几种统计参数在 0.46 0.41 和 0.23; 观测杂合度(Ho)在 0.00 1.00 之 6 个群体间具有一定的差异, 但方差分析发现并未达
表 2 六 个 黄 颡 鱼 种 群 在 30 个 微 卫 星 基 因 座 的 多 样 性 指 数 Tab.2 The polymorphic information at 30 microsatellite loci of six P. eupogon populations YL SC SH YY QQ TJ A e H o H e PIC A e H o H e PIC A e H o H e PIC A e H o H e PIC A e H o H e PIC A e H o H e PIC HLJYC009 2.00 1.00 0.51 0.38 1.00 0.00 0.00 0.00 2.00 1.00 0.51 0.38 2.00 1.00 0.51 0.38 HLJYC012 2.90 0.27 0.67 0.58 2.56 0.43 0.62 0.54 2.91 0.72 0.67 0.58 1.00 0.00 0.00 0.00 HLJYC013 3.67 0.70 0.74 0.68 3.33 0.83 0.71 0.65 HLJYC015 1.38 0.13 0.28 0.24 2.14 1.00 0.54 0.42 2.22 0.44 0.57 0.45 HLJYC017 2.60 0.53 0.63 0.54 2.05 0.57 0.52 0.44 HLJYC024 2.23 0.87 0.56 0.46 1.26 0.23 0.21 0.18 1.00 0.00 0.00 0.00 1.00 0.00 0.00 0.00 HLJYC028 4.34 0.87 0.78 0.73 1.56 0.47 0.36 0.29 HLJYC031 3.68 0.80 0.74 0.68 2.94 0.77 0.67 0.59 3.15 0.78 0.70 0.62 2.98 0.63 0.68 0.59 2.77 0.83 0.65 0.56 2.00 1.00 0.51 0.38 HLJYC035 2.00 1.00 0.51 0.38 2.00 1.00 0.51 0.38 1.00 0.00 0.00 0.00 HLJYC037 1.64 0.17 0.40 0.34 HLJYC039 2.00 1.00 0.51 0.38 1.43 0.37 0.30 0.25 1.00 0.00 0.00 0.00 HLJYC040 1.14 0.13 0.13 0.12 1.07 0.07 0.07 0.06 1.25 0.22 0.20 0.18 1.14 0.13 0.13 0.12 1.07 0.07 0.07 0.06 1.76 0.63 0.44 0.34 HLJYC045 2.19 0.27 0.55 0.44 3.77 0.83 0.75 0.69 2.81 0.61 0.66 0.58 2.78 0.67 0.65 0.57 2.98 0.70 0.68 0.61 1.99 0.93 0.51 0.37 HLJYC050 2.45 0.40 0.60 0.55 2.47 0.13 0.61 0.54 3.83 0.39 0.76 0.69 1.00 0.00 0.00 HLJYC052 2.28 0.97 0.57 0.46 1.92 0.80 0.49 0.36 2.00 1.00 0.51 HLJYC059 2.05 0.30 0.52 0.44 1.31 0.27 0.24 0.23 1.25 0.22 0.20 0.18 1.43 0.37 0.30 0.25 1.22 0.20 0.18 0.16 HLJYC060 3.15 0.87 0.69 0.62 3.11 0.37 0.69 0.62 2.62 0.61 0.64 0.55 2.00 1.00 0.51 0.38 HLJYC061 1.00 0.00 0.00 3.56 0.60 0.73 0.67 1.00 0.00 HLJYC066 2.79 0.57 0.65 0.59 3.85 0.93 0.75 0.70 2.99 0.67 0.68 0.60 2.85 0.93 0.66 0.58 2.34 0.87 0.58 0.49 HLJYC067 3.94 0.87 0.76 0.71 1.00 0.00 0.00 0.00 HLJYC068 1.00 0.00 0.00 0.00 1.00 0.00 0.00 0.00 1.33 0.28 0.25 0.23 1.32 0.27 0.24 0.23 1.18 0.17 0.16 0.15 1.46 0.37 0.32 0.29 HLJYC074 5.44 0.87 0.83 0.79 3.27 0.94 0.71 0.64 1.18 0.17 0.16 0.14 HLJYC075 1.72 0.47 0.42 0.37 1.64 0.40 0.40 0.31 1.12 0.11 0.11 0.10 HLJYC081 2.44 0.57 0.60 0.54 3.53 0.77 0.73 0.66 2.00 1.00 0.51 0.38 2.00 1.00 0.51 0.38 2.00 1.00 0.51 0.38 HLJYC084 1.18 0.03 0.16 0.15 1.00 0.00 0.00 0.00 1.77 0.17 0.45 0.39 1.00 0.00 0.00 0.00 HLJYC086 2.60 0.17 0.63 0.54 1.33 0.28 0.26 0.23 1.00 0.00 0.00 0.00 HLJYC087 1.81 0.33 0.45 0.40 2.99 0.30 0.68 0.60 HLJYC089 1.00 0.00 0.00 0.00 2.00 1.00 0.51 0.38 1.00 0.00 0.00 0.00 1.92 0.80 0.49 0.36 HLJYC090 2.00 1.00 0.51 0.38 2.00 1.00 0.51 0.38 2.00 1.00 0.51 0.38 HLJYC093 2.25 0.57 0.56 0.49 1.93 0.63 0.49 0.43 1.53 0.44 0.36 0.29 2.00 1.00 0.51 0.38 1.92 0.80 0.49 0.36 2.05 0.46 0.45 0.40 2.30 0.51 0.46 0.37 1.93 0.36 0.36 0.35 2.07 0.63 0.46 0.39 1.93 0.58 0.41 0.35 1.47 0.40 0.23 0.19 : YL; SC ; SH; YY ; QQ ; TJ
4 : (Pelteobagrus eupogon) 465 (P>0.05, ) 2.4 Hardy-Weinberg (Markov chain method) Hardy-Weinberg P (multi-locus test), 6 ; (multi-group test), 7 ; (HW test for each locus in each population), 6 5.83%(0.01<P <0.05), 8.74% (P<0.01), 85.43% (P>0.05, 3) 2.5 (F ST ) (N m ), 6 0.05, 34 表 3 六 个 黄 颡 鱼 种 群 遗 传 平 衡 分 析 Tab.3 Genotypic equilibrium analysis on six P. eupogon populations YL SC SH YY QQ TJ HLJYC009 1 1 1 1 HLJYC012 0.0000 0.0034 0.7297 0.0001 HLJYC013 0.3163 0.9763 0.8142 HLJYC015 0.0156 1 0.0158 0.1985 HLJYC017 0.3444 0.8404 0.6110 HLJYC024 1 1 1 HLJYC028 0.7229 1 0.9199 HLJYC031 0.8399 0.9133 0.8314 0.3638 0.9591 1 0.9997 HLJYC035 1 1 1 1 1 1 HLJYC037 0.0175 0.0157 HLJYC039 1 1 1 HLJYC040 1 1 1 1 1 1 1 HLJYC045 0.0356 0.8654 0.2202 0.4911 0.2996 1 0.6668 HLJYC050 0.0257 0.0000 0.0006 0.0000 HLJYC052 1 1 1 1 1 1 1 HLJYC059 0.0613 1 1 1 1 0.4674 HLJYC060 0.9699 0.0008 0.4494 1 0.7491 HLJYC061 0.0943 0.0899 HLJYC066 0.2549 0.9986 0.5443 0.9997 1 1 1 HLJYC067 0.8741 0.8864 HLJYC068 1 1 1 1 1 HLJYC074 0.8334 0.9881 1 0.9708 HLJYC075 0.7709 0.6815 1 0.6959 HLJYC081 0.4225 0.7541 1 1 1 1 HLJYC084 0.0009 0.0155 0.0001 HLJYC086 0.0000 1 0.0008 HLJYC087 0.0786 0.0002 0.0001 HLJYC089 1 1 1 HLJYC090 1 1 1 1 1 1 1 HLJYC093 0.3436 1 1 1 1 1 0.3849 0.9571 0.7902 1 1 1 1 YL; SC ; SH; YY ; QQ ; TJ
466 40 (F ST = 0 0.05)(Wright, 1978), 0.4692, 1.5597 2.6 YY QQ, 0.9947( D s = 0.0053); TJ SC, 0.4846( D s = 0.7244) 4 MEGA 3 UPGMA ( 2),, YY QQ, YL SH, SC, TJ 3 3.1, A e H o H e PIC,,, 6 (A e = 1.96, H e = 0.40, H o = 0.49, PIC = 0.34) (2004) RAPD 4 0.0823 0.0926, 5 (2006) 6 SSR 0.5833 PIC 0.488, ; (2006) PIC 0.5889 H o = 0.6427 DeWoody (2000) 78 524 40000, H e = 0.46 A e = 7.50, 6 H e, A e Botstein (1980) PIC 0.50, PIC 0.25, PIC PIC = 0.34,, YL (PIC = 0.40),, TJ, PIC 0.25, 4 (PIC = 0.35 0.39), 6, (, 2004),,,, TJ, PIC,,,, PIC Tab.4 表 4 六 个 黄 颡 鱼 群 体 间 Nei 氏 标 准 遗 传 距 离 ( ) 与 遗 传 相 似 系 数 ( ) Nei s standard distance (below the diagonal) and genetic similarity coefficient (above the diagonal) among 6 P. eupogon populations YL SC SH YY QQ TJ YL 0.6917 0.7812 0.7400 0.7422 0.5807 SC 0.3686 0.6016 0.5766 0.5757 0.4846 SH 0.2469 0.5082 0.7246 0.7187 0.5999 YY 0.3012 0.5506 0.3221 0.9947 0.6062 QQ 0.2982 0.5522 0.3304 0.0053 0.6085 TJ 0.5434 0.7244 0.5110 0.5005 0.4968 YL; SC ; SH; YY ; QQ ; TJ
4 : (Pelteobagrus eupogon) 467 2 UPGMA Fig.2 The UPGMA cluster analysis on 6 P. eupogon populations YL; SC ; SH ; YY ; QQ ; TJ,,,,,,,, 3.2, YY QQ,,, YY, QQ ; TJ SC,, TJ 6, YY QQ, YL SH, SC, TJ Scribner (1986), (geographic proximity), 6, 0.4692, Wright(1978) ; N m = (1 F ST ) / 4F ST, 1.5597, 1, N m <1, ; N m >1, (Slatkin, 1987), 6,, 6 I = 0.4846 0.9947 ( D s = 0.0053 0.7244), ( I = 0.80 0.97, D s = 0.03 0.20) (Thorp, 1982) (2004) RAPD (D s = 0.6720 0.9895), 6,, RAPD,, DNA,,,, 3.3 ( ) 5,,, 5, 5,,,
468 40,,, 2006. ND4., 30(4): 413 419,,, 2006.., 15(2): 136 139,,, 2004. Paralichthys olivaceus 10., 35(6): 530 537,,, 2005. DNA., 12(1): 56 61,,, 2007. (Pelteobagrus fulvidraco, Richardson). ( ), 39(3): 92 97,, 2007.., 26(5): 682 687, 1997.., 32(4): 10 16,, 2007.., 27(5): 23 25,,, 2004.., 26(3): 339 342,,, 2004. DNA RAPD., 24(3): 84 89,,, 2001. RAPD. (), 47(2): 233 237,,, 2003.., 25(5): 615 619,,, 2007. RAPD SCAR., 27(3): 11 12,,, 2006. RAPD., 30(1): 101 106,,, 2005. Sox9., 13(5): 620 623,,, 2006. 17., 28(6): 683 688,,, 2005. P-450arom., 12(5): 541 548,,, 2006.., 28(3): 5 8,,, 2003.., 6: 24 29 Botstein D, White R L, Skolnick M et al, 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human, 32: 314 331 DeWoody J A, Avise J C, 2000. Microsatellite variation in marine, freshwater and anadromous fishes compared with other animals. Journal of Fish Biology, 56: 461 473 Scribner K T, Evans J E, Morreale S J et al, 1986. Genetic divergence among populations of the yellow-bellied slider turtle (Pseudemys scripta) separated by aquatic and terrestrial habitats. Copeia, 3: 691 700 Slatkin M, 1987. Gene flow and the geographic structure of natural populations. Science, 236: 787 792 Thorp J P, 1982. The molecular dock hypothesis: Biochemical evolution, genetic differentiation, and systematic. Annual Review of Ecology Systematic, 13(1): 139 168 Wright, 1978. Evolution and the genetics of populations. University of Chicago Press, 1 30
4 : (Pelteobagrus eupogon) 469 GENETIC DIVERSITY OF DIFFERENT ECOLOGO-GEOGRAPHICAL POPULATIONS OF YELLOW CATFISH PELTEOBAGRUS EUPOGON LI Da-Yu 1, 2, YIN Qian-Qian 1, 3, HOU Ning 1, 3, SUN Xiao-Wen 1, LIANG Li-Qun 1 (1. Key Open Laboratory for Bioengineering Breeding of Northern Fishing, Ministry of Aquaculture, Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin, 150070; 2. Key Open Laboratory for Genetic Breeding of Aquatic Animals and Aquaculture Biology, Ministry of Aquaculture, Freshwater Fishery Research Center, Chinese Academy Fishery Sciences, Wuxi, 214081; 3. College of Aqua-life Science and Technology, Dalian Fisheries University, Dalian, 116023) Abstract Forty microsatellite loci were used for analyzing six populations of yellow catfish Pelteobagrus eupogon: YL [the Yueliang (Moon) Lake, Jilin Province, 30 samples], SC (Sichuan Province, 30 samples), SH (Songhua River at Harbin, Heilongjiang Province, 18 samples), YY (the Changhu Lake, Jingzhou, Hubei Province, 30 samples), QQ (the parent generation of YY, from the Changhu Lake also), and TJ (Huanxin Breeding Farm, Tianjin, 30 samples). The observed (H o ), expected (H e ) heterozygosity, polymorphic information content (PIC), and number of effective alleles (A e ) were determined. Genetic similarity index and genetic distance were computed on the allele frequency. The Hardy-Weinberg Equilibrium was checked by x 2 Test. Genetic differentiation (F ST ) and hierarchical partition of genetic diversity (N m ) were used. A dendrogram was built on the UPGMA results using PHYLIP software package (Ver.3.63). A total of 5042 fragments ranging from 150bp and 650bp in length were obtained. One to eight alleles were amplified in 40 loci and 143 alleles in all six populations. The average number of alleles in each locus is 3.575. The result shows that 1) the level of genetic variability is moderate in the six populations. The polymorphic information contents of the six populations are from 0.00 to 0.79, in average at 0.40, 0.37, 0.35, 0.39, 0.35 and 0.19, respectively. Effective alleles are from 1.00 to 5.44, in average of 2.05, 2.30, 1.93, 2.07, 1.93 and 1.47, respectively. The expected heterozygosity ranges from 0.00 to 0.83, in average at 0.45, 0.46, 0.36, 0.46, 0.41 and 0.23, respectively; 2) the highest genetic similarity index at 0.9947 was found between YY and QQ; and the lowest index at 0.4846 between SC and TJ. Correlation between the clustering result and the geographical distribution may exist. Key words Yellow catfish Pelteobagrus eupogon, Genetic diversity, Molecular marker, Microsatellite