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1 2015 7, 22(4): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 张志伟, 陈爱华, 张志勇, 陆勤勤, 张朝晖, 姚国兴, 蔡永祥, 吴杨平, 曹奕, 张雨, 摘要 : (Solen grandis) (DD) (QHD) (RZ) (LS) (BH) : ~0.853,, 8~22, 199, 14.2, 11.05, (DD), (QHD), (RZ), (LS), (BH), (DD), (QHD), (RZ), (LS), (BH), Hardy-Weinberg, Sg16, 5,, ~ , DD QHD, DD BH ; D a UPGMA, DD QHD, RZ LS, BH,, F st, F st ~ , (P= ),,, : ; ; ; 中图分类号 : S94 文献标志码 : A 文章编号 : (2015) (Solen grandis, Dunker, 1861) (Lamellibranchia), (Heterodonta), (Veneroida), (Solenidae),,,, 30~40 cm [1],,,, 82.7% [2],,,,, [3],, [4], [5], [6] ; [7],, [8] AFLP [9], ; AFLP, 收稿日期 : ; 修订日期 : 基金项目 : (BK ); (2013-NY-013); (J ). 作者简介 : (1977 ),,,,. zhzhwei2005@126.com 通信作者 :, @139.com

2 4 : 5 741,,,,,,,,,, 5,,, 1 mmol/l EDTA), 25 μl 10% SDS, 10 μl K (20 mg/ml), 55, /,, r/min 30 min, 70% 2, 50 μl, 0.8% ( 1), 30 95%, DNA 95% 0.1 g, 1.5 ml, 450 μl STE (150 mmol/l NaCl, 50 mmol/l Tris-HCl, 1 BH: ; DD: ; LS: ; QHD: ; RZ:. Fig. 1 Map of sampling site of Solen grandis BH: Beihai population; DD: Dandong population; LS: Lyusi population; QHD: Qinhuangdao population; RZ: Rizhao population. Tab. 1 表 1 样品采集时间地点数量 Site, date, and the amount of the samples in this study population (BH) Beihai population (LS) Lyusi populaton (RZ) Rizhao population (QHD) Qinhuangdao population (DD) Dandong population sampling site Tieshan Harbor, Beihai, Guangxi Lyusi Harbor, Nantong, Jiangsu Shijiu Harbor, Rizhao, Shandong Dapuhe town, Qinhuangdao, Hebei Donggang Harbor, Dandong, Liaoning latitude and longitude sampling date number N E N E N E N E N E

3 DNA 1.3 SSR [10] 14,, PCR PCR 25 μl, 1 PCR Buffer, Mg mmol/l, 0.2 mmol/l dntps, 1.0 μmol/l, Taq 1.0 U, 100 ng : 94 5 min; s, T m 30 s, 72 1 min, 35, min ABI3730, Tab. 2 表 2 引物名称序列及 PCR 反应条件 Microsatellite loci, primer sequences and conditions of PCR locus (5 3 ) primer sequences (5 3 ) / ann. temp locus (5 3 ) primer sequences (5 3 ) / ann. temp Sg03 Sg04 Sg05 Sg10 Sg11 Sg13 Sg14 F: tttcagtaataaagcgacg R: gactctggtaagaaggattg F: tcatctacaatcccaccc R: ctttattcattttcctacgct F: atcaataagacagcgttcag R: tatcccaatatcatagcct F: acccagaccacaagaagca R: gggcggtgaagtgaagata F: atgaacattccgcccaca R: ccccttcactttaatttcttctg F: ctggatggtaagggttcaa R: ctgcaaatcactaagctaagac F: tcgttagatgtttactttggtt R: cagtatcacgccgcagac 46 Sg15 50 Sg16 46 Sg18 65 Sg22 58 Sg23 58 Sg24 58 Sg26 F: aagggtagtcccaggtct R: ccgtgttttactgaggtcc F: ccaaggaagatacaacgca R: gccgacccagtgatgag F: aaatgtgaaagtgcctac R: taatgctcaaactaccga F: caggataatgtgactgggtg R: tatcagagggtatcaacaaca F: tcaacctatgttacccttttac R: cttcagttagattgggagca F: tgttcttttcttttaccctc R: tatgtaaagcaatggaaag F: tccctggagtatgaacaac R: gatacggacgcagaaaa MICROSATELLITE ANALYSER (MSA, Dieringer and schlotterer, 2003) 5 (A T ) (A M ) (, Allelic richness, A R ) (Fixation index, F is ) (H e ) (H o ) (F st ), GENEPOP V3.3 (Raymond & Rousset 1995), POPULATIONS (http: //bio-informatics. org/~tryphon/populations/) UPGMA STRUCTURE 2.2, Hardy-Weinberg , LS, BH, DD, 8~22, 199, Sg4 Sg23 2 PIC 0.696~0.853,, Sg , Sg F st 0.039~0.098, , Sg15 Sg22 Sg24, 3, 11 F st 0.05~ 0.10, 5 Hardy-Weinberg 14 5, ( 3): Sg16 DD, (14 5 ) 1.11%, Hardy- Weinberg

4 4 : Fig. 2 2 Sg23( ) Sg04( ) Genotype of the microsatellite loci Sg23 (left) and Sg04 (right) in Solen grandis 表 3 14 个微卫星位点在大竹蛏 5 个野生群体扩增等位基因数遗传分化指数多态信息含量及 Hardy-Weinberg 平衡检验 Tab. 3 Allele number, Global F st, PIC and Hardy-Weinberg equilibrium from 14 microsatellite loci among 5 wild populations of Solen grandis Sg03 Sg04 Sg05 Sg10 Sg11 Sg13 Sg14 Sg15 Sg16 Sg18 Sg22 Sg23 Sg24 Sg26 DD ** QHD RZ LS BH total Global F st PIC : ** P<0.01. BH:, DD:, LS:, QHD:, RZ:. Note: ** means P<0.01. BH: Beihai population; DD: Dandong population; LS: Lyusi population; QHD: Qinhuangdao population; RZ: Rizhao population LS, , RZ, , LS 0.815,, , LS 0.007>0, 4 <0, BH, 0.093, DD, 0.088( 4) F st ( 5), ~ , DD BH , LS RZ , 5, (P= ) Nei(1972), POPULATIONS D a ( 5), 5 Tab. 4 表 4 大竹蛏 5 个群体在 14 个微卫星位点的遗传变异情况 Genetic and variation at fourteen microsatellite loci assessed for the 5 Solen grandis populations population A M A R H O H e F is DD QHD RZ LS BH : BH:, DD:, LS:, QHD:, RZ:. Note: BH: Beihai population; DD: Dandong population; LS: Lyusi population; QHD: Qinhuangdao population; RZ: Rizhao population.

5 Tab. 5 表 5 大竹蛏 5 个群体间的遗传分化指数和遗传距离 Genetic differentiation and genetic distance between 5 Solen grandis populations DD QHD RZ LS BH DD QHD ** RZ ** ** LS ** ** ** BH ** ** ** ** :. ** P<0.01. BH: ; DD: ; LS: ; QHD: ; RZ:. Note: The data below diagonal mean genetic differentiation (F st ) and above mean genetic distance (D a ). ** means P<0.01. BH: Beihai population; DD: Dandong population; LS: Lyusi population; QHD: Qinhuangdao population; RZ: Rizhao population ~ , DD QHD, RZ LS, QHD BH 5 UPGMA 3, DD QHD, ; RZ LS ; 4 BH ( 4), K=2, DD QHD, 3 ; K=5,, DD QHD, UPGMA BH: ; DD: ; LS: ; QHD: ; RZ:. Fig. 3 Cluster dendrogram among 5 populations of Solen grandis BH: Beihai population; DD: Dandong population; LS: Lyusi population; QHD: Qinhuangdao population; RZ: Rizhao population. [8], 5 6,,,,,,,, Yuan [11], [12] AFLP (Siliqua minimai) (Sinonovacula constricta) 3 DNA, 3 DNA,, Sun [13] cdna [14] cdna 5, 2~7, [15], 8~22, PCR ~0.853 Botstein [16], PIC>0.5, 0.25<PIC<0.5, PIC< ,

4 : 5 745 4 5 BH: ; DD: ; LS: ; QHD: ; RZ:. Fig.

6 4 : BH: ; DD: ; LS: ; QHD: ; RZ:. Fig. 4 Bayesian clustering analysis of the 5 wild solen grandis populations BH: Beihai population; DD: Dandong population; LS: Lyusi population; QHD: Qinhuangdao population; RZ: Rizhao population 3.2 Hardy-Weinberg, 1.11%, Hardy- Weinberg, 5 Hardy-Weinberg,,, 5,, F is, 1~1 F is, ;,, [17] 5, LS 0.007>0,,,, ; 4 <0, BH 0.093, DD 0.088,, 5 : RZ (0.826) LS (0.815) QHD (0.791) BH (0.785) DD (0.769),, [9], 7 AFLP, 5 141, 4 5 LS ( ), , RZ ( ), DD ( ) QHD ( ) BH ( ), 3.3, 0~0.05, ; 0.05~0.15, ; 0.15~ 0.25, ; 0.25, [18], ~ , DD BH , LS RZ , 5, (P= ),

7 746 22, (Solen strictus) [19] (Portunus trituberculatus) [20],, ~ , DD QHD, ; RZ LS, 4 BH, [21] ISSR 6, [22] 4,, 3.4,,,, 5,,,,, : ( ),,,, ( ),,, 参考文献 : [1] International Bank for Shellfish: Solen grandis. The Taiwan Malacefauna Database [ ]. [ :. [ ].] [2] Dai C J. Evaluation on nutrition components of the soft part in Solen grandis[j]. Journal of Jimei University: Natural Science, 2002, 7(4): [. [J]. :, 2002, 7(4): ] [3] Chen A H, Yao G X, Zhang Z W. Studies on artificial propagation of Solen grandis Dunker in pilot scale[j]. Marine Fisheries, 2009, 31(1): [,,. [J]., 2009, 31(1): ] [4] Chen A H, Zhang Z W, Yao G X, et al. The influence of different environmental factors on the growth and survival rate of juvenile of bamboo clam Solen grandis[j]. Journal of Shanghai Fisheries University, 2008, 17(5): [,,, [J]., 2008, 17(5): ] [5] Chen A H, Yao G X, Zhang Z W, et al. Effects of temperature, salinity and sediment on the growth and survival of solen grandis Dunker juveniles[j]. Journal of Tropical Oceanography, 2010, 29(5): [,,, [J]., 2010, 29(5): ] [6] Yan X W, Zhao S X, Zhang P, et al. Effects of stocking density and alga species on larval growth, survival and metamorphosis in bamboo clam Solen grandis[j]. Journal of Dalian Ocean University, 2010, 25(5): [,,,. [J]., 2010, 25(5): ] [7] Wu Y P, Chen A H, Yao G X, et al. The filtration rate of juvenile Solen grandis Dunker [J]. Marine Science, 2011, 35(1): 6 9. [,,, [J]., 2011, 35(1): 6 9.] [8] Zhang Z W, Yao G X, Chen A H. The tissue specificity and the genetic diversity of the allozymes of Solen grandis Dunker[J]. Marine Science, 2009, 33(3): [,, [J]., 2009, 33(3): ] [9] Zhang T, Liu X Q, Sun Z X, et al. Genetic diversity of five geographical populations in Solen grandis by AFLP markers[j]. Oceanologia et Limnologia Sinica, 2013, 44(2): [,,, (Solen grandis) AFLP [J]., 2013, 44 (2): ] [10] Yuan Y, Li Q, Kong L F, et al. Isolation and characterization

8 4 : of polymorphic microsatellite loci in the grand jackknife clam Solen grandis (Bivalvia: Veneroida)[J]. Genes Genom, 2010, 32(3): [11] Yuan Y, Li Q, Kong L F, et al. The complete mitochondrial genome of the grand jackknife clam, Solen grandis (Bivalvia: Solenidae): a novel gene order and unusual non-coding region[j]. Mol Biol Rep, 2012, 39(2): [12] Shao Y Q, Lu R M, Dong Y H, et al. The genetic structure and diversity analysis of three species of razor clam using AFLP markers[j]. Marine Science, 2009, 33(10): [,,,. [J]., 2009, 33(10): ] [13] Sun G H, Liu X Q, Ren L H, et al Construction of a full-length C DNA Library of Solen grandis Dunker and Identification of Defense- and Immune-Related Genes[J]. J Ocean Univ China, 2014, 13(1): [14] Qiao H J, Liu X D, Sun G H, et al Isolation and characterization of microsatellite markers from cdna library of solen grandis[j]. Oceanologia et Limnologia Sinica, 2012, 43 (6): [,,,. (Solen grandis) cdna [J]., 2012, 43 (6) : ] [15] Zhang T. Study on the genetic diversity of solen grandis populations[d]. Yantai: Ludong University [. [D]. :, 2012.] [16] Botstein D, White R L, Skolnick M, et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms[j]. Am J Hum Genet, 1980, 32(3): [17] Weir B S, Cockerham C C. Estimating F-statistics for the analysis of population structure[j]. Evolution, 1984, 38(6): [18] Wright S. Evolution and the genetics of population variability within and among natural population[m]. Chicago: University of Chicago Press, 1978: 4. [19] Li X P, Liu P, Li J, et al. Genetic diversity among five wild populations of Portunus trituberculatus[j]. Journal of Fishery Sciences of China, 2011, 18(6): [,,,. SSR 5 [J]., 2011, 18(6): ] [20] Chen Y N, Sun Z X, Chang L R. Genetic diversity of different geographical populations in Solen strictus revealed by ISSR markers[j]. Acta Hydrobiologica Sinica, 2010, 34(2): [,,. [J]., 2010, 34(2): ] [21] Niu D H, Li J L, Feng B B, et al. ISSR analysis on genetic structure of six Sinonovacula constricta populations[j]. Chinese Journal of Applied Environmental Biology, 2009, 15(3): [,,,. 6 ISSR [J]., 2009, 15(3): ] [22] Liu B, Shao Y Q, Wang K, et al. Microsatellite analysis of genetic diversity and phylogenetic relationship of four different geographical populations of Sinonovacula constricta[j]. Marine Science, 2013, 37(8): [,,,. 4 [J]., 2013, 37(8): ]

9 Microsatellite analysis of genetic diversity of five wild populations of Solen grandis (Dunker) ZHANG Zhiwei, CHEN Aihua, ZHANG Zhiyong, LU Qinqin, ZHANG Chaohui, YAO Guoxing, CAI Yongxiang, WU Yangping, CAO Yi, ZHANG Yu Jiangsu Institute of Marine Fisheries, Nantong , China Abstract: The grand jackknife clam Solen grandis (Dunker, 1861) is a commercial marine bivalve that is found in coastal waters of China, Japan, and Korea from the shallow subtidal to depths of 20 m. It has high nutritive and medical value according to traditional Chinese medicine. In addition, it is adaptable and has high growth rates and great market potential. Currently, this clam is only available from natural sources. Over the last few decades, however, overfishing and environmental pollution have caused dramatic declines in natural stocks of S. grandis, and production is low and unstable. Fortunately, methods of artificial breeding and cultivation of the jackknife clam are developing. Previous studies on S. grandis focused primarily on morphological and ecological characteristics, allozyme analysis, and evaluation of its nutrition components. Little is known about the molecular ecology of S. grandis or its genetic background. Thus, it is important to evaluate genetic differences among populations along the coast of China. In this study, 14 microsatellite DNA markers were used to examine the genetic diversity and differentiation among five wild populations of the grand jackknife clam. A total of 150 samples were collected from Dandong (DD) in Liaoning Province, Qinhuangdao (QHD) in Hebei Province, Rizhao (RZ) in Shandong Province, Lyusi (LS) in Jiangsu Province, and Beihai (BH) in Guangxi Province. Comparisons of 199 alleles among the five populations showed that, in the fourteen loci, polymorphism information content (PIC)values ranged from to with high polymorphism. The number of alleles ranged from 8 to 22 and the average number (A M ) and richness(a R ) of alleles were 14.2 and 11.05, respectively. Expected (H e ) and observed heterozygosity (H o ) ranged and , respectively, suggesting high levels of genetic diversity in all populations. Only the Sg16 locus in the DD population deviated significantly from Hardy-Weinberg equilibrium, further indicating stability in the genetic diversity of all populations. The genetic distance among the five populations ranged from to , with minimum and maximum distances between DD and QHD and between DD and BH, respectively. The UPGMA tree indicated highest similarity among geographically close populations, clustering DD with QHD and RZ with LS, the structure of which was confirmed by Bayesian cluster analysis. The F st were , suggesting a moderate degree of differentiation among the five wild populations. Overall, the results suggest a high level of genetic diversity and differentiation among five grand jackknife clam populations from geographically distinct locations along the coast of China. Precaution should be taken to protect the germplasm of this species, particularly when designing stock enhancement programs. Key words: Solen grandis; microsatellite; wild population; genetic diversity Corresponding author: ZHANG Zhiyong @139.com

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L 2012 13 2 201-206 Journal of Plant Genetic Resources / 071001 41 SSR 21 SSR 41 52 2 ~ 4 2. 5 Shannon 0. 21 ~ 1. 40 0. 73 5. 54 3 7 9 13. 16% ~ 186. 49% 5. 48 6 11 SSR Genetic Diversity of Peanut Arachis