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1 HEREDITAS (Beijing) , 30(11): ISSN 研究报告 DOI: /SP.J 贾智英 1, 石连玉 1, 刘晓峰 2, 孙效文 1., ; 2., : 利用 12 对微卫星标记对黑龙江水系 6 个野生鲫鱼不同倍性群体进行遗传结构分析结果表明 : 6 个采集群体的平均等位基因数为 5.8~6.8, 平均有效等位基因数为 2.8~4.6, 平均期望杂合度为 ~0.6962, 平均多态信息含量为 ~0.6481, 说明这几个群体遗传多样性水平较高根据 d 值, 各群体均有不同程度的偏离 Hardy-Weinberg 平衡的现象, 均表现为杂合度过度 Kruskal-Wallis 检验表明两种倍性 6 个采集群体鲫鱼遗传多样性差异不显著, 没有发现三倍体鲫由于倍性增加而出现额外的等位基因片段群体间基因分化系数 (G ST ) 为 , 表明群体间存在轻度遗传分化聚类分析表明, 同水体两种鲫鱼亲缘关系最近, 不同采集群体间松花江与乌苏里江分化最小, 月亮湾与新荒泡其次, 双凤水库群体分化最大 : 鲫鱼 ; 遗传多样性 ; 微卫星标记 The genetic diversity of diploid and triploid crucian carp from six populations in Heilongjiang River System JIA Zhi-Ying 1, SHI Lian-Yu 1, LIU Xiao-Feng 2, SUN Xiao-Wen 1 1. Heilongjiang River Fisheries Research Institute, Chinese Academy of Fisheries Science, Harbin , China; 2. Life Science and Technology, Dalian Fishery University, Dalian , China Abstract: Twelve microsatellite markers from silver crucian carp were used to investigate the genetic structuring of the diploid and triploid crucian carp from six natural populations in Heilongjiang River System. In the six populations, the number of average allele (A) is from 5.8 to 6.8, the number of effective allele (Ne) is from 2.8 to 4.6, the expected heterozygosity value (H e ) is from to and the average PIC value is from to 0.648, which indicated that the genetic diversity of the populations investigated is rich. According to genetic deviation index (d), deviation from Hardy-Weinberg equilibrium was found in these populations and all of them showed heterozygosity excess. Kruskal-Wallis test indicated that there was no significant variance in different ploid level and populations. No extra alleles that present with the increase of ploid level were found. The coefficient of gene differentiation between populations(g ST )was which indicated low values of genetic differentiation between these populations. Genetic distance was calculated and cluster analysis was also carried out. The results showed that distance between polyploid and diploid in same water was the nearest. Among populations, Songhua River and Ussuri River were the nearest, Xinhuangpao Lake and were nearer, and Shuangfeng reservoir has the largest genetic distance with others. Keywords: crucian carp; genetic diversity; microsatellite marker 收稿日期 : ; 修回日期 : 基金项目 : ( )[Supported by the National Natural Sciences Foundation of China(No )] 作者简介 : (1976 ),,,,, xiaojiade_41@163.com 通讯作者 : (1955 ),,,,, Tel: ; sunxw2002@163.com

2 1460 HEREDITAS (Beijing) , (Carassius auratus gibelio)(carassius auratus L.),, , 100, [1],, 20 90,,,,,, [2] [3],,,, QTL, , 3, 3,, 75% Cell Lab Quanta SC ( Beckman ), 1 表 1 样本采集时间和数目 Table 1 Collecting time and number of samples Population Time Number Ploidy Diploid Shuangfeng Reseviour Triploid Hulun Lake Songhua River Ussuri River Xinhuangpao Lake DNA DNA /, [4] , J4, J9, J12, J58, J69, HLJY- 016, HLJY-018, HLJY-019, HLJY-025, HLJY-030, HLJY-034, HLJY-035, [5, 6] PCR 25 μl, DNA 50 ng, PCR buffer 18 μl(10 mmol/l Tris-Cl (ph 8.3), 50 mmo l/l KCl, 1.5 mmol/l MgCl 2, 0.001%, 0.1% Tweven- 100, datp dgtp dttp dctp 2 mmol/l,), 0.2 mmol/l, Taq 1 U, 25 μl 5% (Average number of allele, A) (Average number of effective allele, N e ) (Average observed heterozygosity, H o ) (Average expected heterozygosity, H e ) Hardy-Weinberg (Genetic deviation index, d), (Genetic identity index, I)(Genetic distance, D) (Gene differentiation, G ST ) [7] : : A = (1/n)Σχ i ; : N e = 1/Σƒ 2 i ;

3 11 : 1461 : H o = / ; : H e = 1 Σƒ 2 i ; : PIC = 1 Σƒ 2 i ΣΣ2 ƒ 2 i ƒ 2 j ; Hardy-Weinberg : d = (H o H e )/H e ; : I =Sp i q i /(Σp 2 i Σq 2 i ) 1/2 ; : D = lni; : G ST = (H T H S ) / H T χ i i, ƒ i i, j=i+1 n, H T, H S p i q i i SPSS 12.0 (H e ) Kruskal-Wallis, UPGMA (Unweighted Pair Group Method with Arithmetic means, ) DNA ( 1), DNA 200, 300, DNA PCR 12 6,, 100%, A Ne Ho He d PIC ~12.8, 5.8~6.8 ; 1.3~8.2, 2.8~4.6, 3 4, 12 6, Botstein, J4 HLJY-016, 10, 6 PIC ( 2 3) 6 (He=0.6915, PIC=0.6481),, ~ ~0.6962, Ho He 6 12 (He) Kruskal-Wallis 图 1 流式细胞仪测定 DNA 相对含量图 A: ; B: Fig. 1 Histogram picture of nuclear DNA content of the crucian carp taken by flow cytometry A: Diploid; B: Triploid.

4 1462 HEREDITAS (Beijing) 表 2 6 个鲫鱼群体在 12 个微卫星位点的遗传多态性 Table 2 Allelic variability for six populations at twelve microsatellite loci Locus A Ne Ho He d PIC J J J J J HLJY HLJY HLJY HLJY HLJY HLJY HLJY 表 3 6 个鲫鱼群体的遗传多样性参数 Table 3 Genetic diversity of six crucian carp populations Parameter A Ne Ho He d PIC Ploidy Xinhuangpao Lake 6 Six crucian carp populations Ussuri River Shuangfeng resevoir Hulun Lake Songhua River 2n n total n n total n n total n n total n n total n n total , (H= , d f =5, P>0.05; H= , d f =11, P>0.05) d, Hardy-Weinberg,, Hardy-Weinberg, (Multi-group test), 12 6 Hardy-Weinberg, J9 2.4 Nei [8] 6 (D)(I), 0.5, 0.5 (D ), (I ); (D ), (I ) 6, (I ), (D ), 4 5, MEGA4.0, UPGMA, 2,

5 表 4 不同倍性鲫鱼群体遗传一致度与遗传距离 Table 4 Genetic identity (I) and distance (D) in different ploidy populations Population Ploidy Xinhuangpao Lake Ussuri River Shuangfeng resevoir Hulun Lake Songhua River 2N 3N 2N 3N 2N 3N 2N 3N 2N 3N 2N 3N Xinhuangpao Lake Ussuri River Shuangfeng resevoir Hulun Lake Songhua River 2n n n n n n n n n n n n , Notes: Numbers above the diagonal are genetic distance and numbers below are genetic identity.

6 11 : 1463

1464 HEREDITAS (Beijing) 2008 30 表 5 6 个采集群体的遗传一致度与遗传距离 Table 5 Genetic identity (I) and distance (D) in six populations collected Xinhuangpao Lake Ussuri River Shuangfeng resevoir Hulun Lake Songhua

7 1464 HEREDITAS (Beijing) 表 5 6 个采集群体的遗传一致度与遗传距离 Table 5 Genetic identity (I) and distance (D) in six populations collected Xinhuangpao Lake Ussuri River Shuangfeng resevoir Hulun Lake Songhua River Xinhuangpao Lake Ussuri River Shuangfeng resevoir Hulun Lake Songhua River , Notes: Numbers above the diagonal are genetic distance and numbers below are genetic identity. 图 2 Fig.2 群体的 UPGMA 分子系统树 UPGMA molecular trees of populations, ;,,,, 6 (G ST ) PIC Ne Ho He,,,, Ne Ho He 3.5~ ~ ~ , Ne Ho He, Ne Ho He, [3] ~0.6091,, [9] 0.607~0.721, DeWoody

8 11 : 1465 [10] , He=0.46, A=7.5, He Ne, Hardy-Weinberg, Kruskal-Wallis,,,,,, (G ST ),, G ST , ; G ST , ; G ST , ; G ST 0.25, [11], 6 (G ST ) ,, 3.2,,, ;, PCR DNA,, [12] ; Kruskal-Wallis,,, (References): [1] SHEN Jun-Bao, LIU Ming-Hua, FAN Zhao-Ting. Silver crucian carp (Carassius auratus gibelio (Bloch)) in Heilongjiang Province, China. Harbin: The Scientific and Technological Press of Heilongjiang, 1997.,,.. :, [2] SHEN Jun-Bao, WANG Guo-Rui, FAN Zhao-Ting. The ploidy of crucian carp and its geographical distribution in Heilongjiang main river. Journal of Fisheries of China, 1983, 7(2): ,,.., 1983, 7(2): [3] LU Cui-Yun, YANG Yan-Hao, TONG Guang-Xiang, HAO Jun, SUN Xiao-Wen, LIANG Li-Qun, LEI Qing-Quan. Comparative studies of genetic diversity between C. auratus gibelio (Bloch) and C. auratus auratus (L.) in the same reservoir. Chinese Journal of Fisheries, 2006, 19(2): ,,,,,,.., 2006, 19(2): [4] LIANG Li-Qun, SUN Xiao-Wen, WANG Peng, YAN Xue-Chun, SHEN Jun-Bao. The research on the DNA extraction from the fish finny. Biotechnology, 1994, 4(1): ,,,,. DNA, 1994, 4(1): [5] Jia ZY, Sun XW, Liang LQ, Lu CY, Lei QQ. Isolation and characterization of microsatellite markers from Fangzheng silver crucian carp, Carassius auratus gibelio (Bloch), and cross-amplification in the closely related species crucian carp, Carassius auratus auratus (Linnaeus). Molecular Ecology Notes, 2006, 6: [DOI] [6] Yue GH, Orban L. Polymorphic microsatellites from silver crucian carp (Carassius auratus gibelio Bloch) and crossamplification in common carp (Cyprinus carpio L.). Molecular Ecological Notes, 2002, 2: [7] Nei M. Molecular Population Genetics and Evolution. Amsterdam: North-Holland Publishing Company, 1975, [8] Nei M. Genetic distance between populations. American Naturalist, 1972, 106: [DOI] [9] LU ShuangQing, LIU Zhen, LIU Hong-Yu, SHAO Tiao-Yi, SU Jian-Ming. Microsatellite DNA analysis of genetic diversity and the phylogenetic relationships of four breed varieties of Carassius sp. Journal of Fisheries Sciences of China, 2005, 12(4): ,,,,. 4 DNA., 2005, 12(4): [10] DeWoody JA, Avise JC. Microsatellite variation in marine, freshwater and anadromous fishes compared with other animals. J Fish Biol, 2000, 56(3): [DOI] [11] Balloux F, Lugon-Moulin N. The estimation of population differentiation with microsatellite markers. Mol Ecol, 2002, 11: [12] LI Yin-Chuan. Comparative investigation of the genetic diversity between C.a. gibelio and C.a. auratus[ Dissertation]. Northeast Agricultural University, [ ]., 2001.

Microsoft Word - 李学军.doc

Microsoft Word - 李学军.doc 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)