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1、 Applied Basic Research Programs of the Science and Technology Commission Foundation of Tianjin, P.R.China (Key Program 09JCZDJC19300; General Program 06YFJMJC11800) An Analysis of genetic diversity in Cyclina sinensis (Mollusca Bivalvia) of four Geographical Populations using AFLP Technique Pan Bao
2、ping, Zhang Liyan, Song Xin, Gao Weiwei, Yuan Yuan School of Chemistry and Life Science Tianjin Key Laboratory of Cyto-Genetical and Molecular Regulation Tianjin Normal University Tianjin, P. R. China AbstractThis article preliminarily studied the genetic diversity in 109 individuals of Cyclina sin
3、ensis (Mollusca Bivalvia) of four geographical populations distributed around Chinas Bohai region based on amplified fragment length polymorphism, AFLP technique. Using 6 primer pairs including E-ACT/M-CGT，a total of 90 bands were detected of which 82 bands (91.11%) were polymorphic. The diversity i
4、ndex of each wild population was: TG 66.67%，DG 70%，HG 76.67% and BDH 83.33% while the Neis genetic diversity index followed 0.2149，0.2162，0.2520 and 0.2895. Analysis suggested similar genetic diversity levels among the four populations in Bohai area and there was no significant difference (P0.05). A
5、MOVA analysis and UPGMA analysis conducted to the genetic differentiation degree of the four groups revealed an unnoticeable level of genetic diversity, and there was a certain degree of inter-population gene flow phenomenon. The genetic variations among the individuals within one population make up
6、 the main part of the total, which accounted for 86.02% total variance. The result above agreed with the conclusion of Y. Yuan et al. (2008) on the genetic structures of different geographical populations of Cyclina sinensis distributed in Yellow Sea and Bohai region based on ITS sequencing methods.
7、 Keywords-Cyclina sinensis, Amplified Fragment Length Polymorphism (AFLP), genetic diversity, AMOVA I. INTRODUCTION Cyclina sinensis (Gmelin, 1791) is naturally distributed along the coastal waters of China, from Liaoning Province in the North to Hainan Province in the South (Q. Q. Zhuang 1, 2001).
8、Since it has a couple of advantages conducive to industrialization and commercialization, such as a wide range of temperature and salinity tolerance, stronger resistibility, high-output ratio and a long survey time without water etc, In China, C.sinensis is of considerable economic importance (X. Q.
9、 Liu 2 et al, 2003) and the artificial breeding of C.sinensis has already been placed emphasis in the National High Technology Research and Development Program 863, which is carried out by the Ministry of Science and Technology of the PRC. In recent years, with the rapid rise of domestic C.sinensis
10、farming, disordered genetic exchanges and blind selection of parent-shells appeared frequently. As a result, geographical germ-plasm intermixed seriously. This situation is of great disadvantage to the healthy development of the C.sinensis industry. Therefore, understanding of genetic diversity in C
11、yclina sinensis and features of germ-plasm resources distribution have abundance empirical significance for both reasonable introduction and culture of seedling. Whats more, considering its wide distribution around Asian countries, exploration of the genetic structure of C.sinensis among populations
12、 has directive significance to reveal the distribution of bivalves in China, pathways of population changes and the reasons for habitat sere. To support the increasing industry of C.sinensis, researches have been carried out, but mainly focus on the techniques of artificial breeding, growth and habi
13、tat (Y. S. Yu 3 et al., 1995; L. Zhou 4 et al., 2000; Y. K. Mo 5, 2007). To date, available research literatures about genetic structure of C.sinensis among geographical populations are relatively the less (Z. L. Mo 6 et al., 2005；B. P. Pan 7 et al., 2005; Y. Yuan 8 et al., 2008). The study of genet
14、ic diversity in C.sinensis using AFLP Technique is rarely reported. II. MATERIALS AND METHODS A. Collection of Samples Samples were collected from four different geographical populations distributed around Beidaihe in Hebei Province (BDH, 30 individuals), Hangu in Tianjin (HG, 25 individuals), Tangt
15、u in Tianjin (TG, 28 individuals) and Dagang in Tianjin (DG, 26 individuals) in March and April 2007 (Table I). TABLE I. THE SAMPLING LOCATIONS Population Location Individuals Longitudes(E) Latitudes(N) BDH Beidaihe 30 119.57E 39.28N HG Hangu 25 117.47E 39.13N TG Tanggu 28 117.50E 39.21N DG Dagang 2
16、6 117.37E 38.49N 978-1-4244-4713-8/10/$25.00 2010 IEEE All the wild samples were collected randomly and had been identified as C.sinensis by morphological classification. The adductor muscle were collected from living swatches and immediately fixed in 70% alcohol until DNA preparation. All the sampl
17、e tissues were stored at -20 after brought back to the laboratory till DNA extraction. B. DNA extraction DNA was extracted according to Grewe 9 (l993) et al. 150mg tissue from each sample was digested in 600l buffer (50mmol/L Tris-HCl pH8.0, 0.7mol/L NaCl, 10mmol/L ETDA, 1% CTAB, 0.1% 2-mercaptoetha
18、nol, 100g/ml proteinase K) at 60 for 2.5 hours. The DNA was extracted twice by the mixture made up of hydroxybenzene/chloroform/isoamylalcohol followed by ethanol precipitation. After wash twice with 70% alcohol, each DNA sample was dissolved in 50l ddH2O and kept at -70. DNA concentration and quali
19、ty were measured by UV spectrophotometry and agarose gel electrophoresis. C. AFLP analysis AFLP analysis was altered from P. Vos 10 et al. High quality genomic DNA was digested with a pair of restriction enzymes (EcoR I /Mse I) in a solution whose total volume was 25l (20ng/l DNA 3l, 10U/l EcoR I 0.
20、25l, 10U/l Mse I 0.25l, 10Buffer Tango 5l, PCR H2O 16.5l). After centrifugal mixing for a few seconds, we kept the mixture at constant temperature of 37 for 4 hours. Then the enzyme was inactivated at 80 for 20 minutes. DNA ligation was performed in a 20l volume containing 2PM/l EcoR I adepter1l, 2P
21、M/l Mse I adepter 1l, 3U/l T4 DNA ligase 0.1l, 10Buffer 2l and PCR H2O 11l. After centrifugal mixing, 5l from each solution above was kept at 18-20 overnight to finish the ligation reaction. The mixtures were kept at 65 for 10 minutes to inactivate the ligase before use. The total volume of pre-ampl
22、ification system was 20l, containing 10 x PCR Buffer 2l, MgCl2 (25mM) 1.6l, dNTPs (5mM) 0.8l, EcoR I pre-amplification primer (50ng/l) 1.2l, Mse I pre-amplification primer (50 ng/l) 1.2l, Taq polymerase (5U/l) 0.2l, genomic DNA solution 2l and PCR H2O11l. After centrifuge for a few seconds, the ampl
23、ification started with initial denaturation at 94 for 2min followed by 30 cycles contained denaturation at 94 for 30s, annealing at 35 for 45s and elongation at 72 for 1 min. 2l of the products was diluted with ddH2O (1:50) and used as the templates of the selective amplification. (Fig. 1) Figure 1.
24、 Electrophoresis Result of Cyclina sinensis AFLP Pre-amplification As selective amplification primer, E+3/M+3 were used. The selective amplification system (20ul) contained 10x PCR Buffer 2l, MgCl2 (25mM)1.6l, dNTPs (5mM) 0.8l, EcoR I pre-amplification primer (50ng/l) 1.2l , Mse I pre-amplification
25、primer (50 ng/l) 1.2l, Taq polymerase (5U/l) 0.2l, PCR H2O12l, diluted product of pre-amplification 1l. After a few seconds centrifugal mixing, gradient PCR was performed as follow. In the first cycle 94 30s, 65 30s, 72 80s, then 12 cycles decreased by 0.7 each cycle, and 23 cycles 94 30s, K55 30s,
26、72 80s at last. Amplified products were measured and analyzed by using SequiGen GT Nucleic Acid Electrophoresis Cell system (BIO-RAD Company, U.S.A.). Add 10ul into 10ul denaturing agent (98% deionized formamide, 10mmol/L EDTA, 0.25% bromophenol blue and xylene Dracocephalum). After 95 5 minutes den
27、aturation, immediately put the mixture in ice bath, then 4% denaturing polyacrylamide gel electrophoresis with 5.5ul each specimen. III. RESULTS A. AFLP genetic Diversity 6 primer pairs were used in this research including E-AAC/ M-CCT, E-AAC/ M-CAG, M-CAC/ E-ACG etc. Totally 90 bands were detected,
28、 of which fragment size were between 100bp and 1000bp. 82 of the 90 were polymorphic (91.11%). The diversity index of each wild population was: TG 66.67%，DG 70%，HG 76.67% and BDH 83.33%. The Neis genetic diversity index followed 0.2149，0.2162，0.2520 and 0.2895 while the Shannon genetic diversity ind
29、ex followed 0.3274, 0.3341, 0.3848 and 0.4367. The average diversity index was 0.4516. Result of T-test suggested similar genetic diversity levels among the four populations (P0.05). (Fig. 2) B. Genetic distance We made an analysis of the genetic distance and the genetic similarity index according t
30、o the Neis genetic diversity index (Table II). The genetic distance among HG, DG and BDH were discovered to be relatively nearer while the genetic distance is the fastest between TG and BDH (0.1662). Figure 2. Electrophisis of the amplified product TABLE II. GENETIC DISTANCE AND SIMILARITY BY NEIS I
31、NDEX TG DG HG BDH TG 0.9324 0.8935 0.8469 DG 0.0700 0.9730 0.9336 HG 0.1126 0.0273 0.9705 BDH 0.1662 0.0687 0.0300 The figure under and above the diagonal line are genetic distance and similarity, respectively Figure 3. Phylogenetic tree based on UPGMA of four populations According to genetic distan
32、ces among populations, we made a clustering analysis by using UPGMA method in the software of TFPGA. (Fig. 3) To acquaint the genetic diversity among populations of C. sinensis, we used ARLEQUIN software package to reveal genetic variation. The results of both Reynolds and Slatkin showed it was furt
33、her between TG and BDH and nearer among HG, BDH and DG. In order to further analyze the genetic diversity levels among populations, we set BDH as the out group and evaluate the 3 groups distributed around Tianjin. The genetic variation levels and subdivision both within and among populations were te
34、sted by analysis of molecular variance (AMOVA) using the ARLEQUIN software package. The result showed the variation among regions makes up 7.26% while it among populations makes 6.72%. The variation between individuals within populations accounted for 86.02%, which is corresponding to Shnanon index
35、calculated proportion (82.09%). (Table III) TABLE III. GENETIC DISTANCE AND SIMILARITY BY NEIS INDEX Source of Variation Degree of Freedom Sum. of Square Estimated Variance Percent of Variation of Components Among Regions 1 48.725 1.18500 7.26% Among Populations 2 50.050 1.09778 6.72% Within Populat
36、ions 36 505.700 14.04722 86.02% Total 39 604.475 16.33000 IV. DISCUSSION AFLP (Amplified fragment length polymorphism) technology as a new type of molecular markers, with informative, high sensitivity and rich in polymorphism, etc., are now widely used in plant and animal genetic diversity analysis,
37、 gene mapping, construction of genetic mapping and diagnosis of diseases 11-14 etc. This paper studied the genetic variation and genetic structure among wild C.sinnensis populations naturally distributed around Chinas Bohai Region. Preliminary analysis showed that in the 90 bands detected, 82 were p
38、olymorphic (91.11%), which is similar to the result (94.2%) got by H. M. J. L. T. Bai 15 (2008) on researching the C.sinnensis populations in the north of China based on RAPD technique. It is indicating that in Chinas Bohai region, C.sinnensis has a higher genetic diversity and a better condition of
39、 germ-plasm resources. According to the initial results, the polymorphism in TG population is lower than the other three ones. This may because of artificial disturbance in that area recent years, such as reclamation projects, introduction restoration, decline of habitats etc. The amount of C.sinnen
40、sis production in this region is decreasing year after year. The diversity index of each wild population was: TG 66.67%，DG 70%，HG 76.67% and BDH 83.33%. The Neis genetic diversity index was 0.2149，0.2162 ， 0.2520 and 0.2895 while the Shannon genetic diversity index were 0.3274, 0.3341, 0.3848 and 0.
41、4367. Results of statistical calculation showed P were greater than 0.05, indicating similar genetic diversity levels among the four populations in Bohai area. The genetic similarity coefficients of the groups were all above 84%, revealing no significant population differentiation and none different
42、 geographical species formed. This result agreed with Z. L. Mo 6 et al. (2005) on the analysis of genetic variation in 5 C.sinnensis geographical populations in Liaoning Province, Tianjin, Jiangsu Province and Fujian Province. From the genetic structure of the four populations, the genetic distance
43、based on Neis genetic diversity index was basically the same as that based on both Reynolds genetic distance and genetic distance matrix of Slatkin, indicating a farther distance between TG and BDH. In Neis analysis, the distance between BDH and HG is the nearest (0.0273), meanwhile it between BDH a
44、nd HG is 0.0300. But in Reynolds and matrix of Slatkin, the nearest distance appeared between BDH and HG (0.0016). These slightly different results might be caused by the nuances of the way the different data processed in. Neis index considered each band as an independent locus and accepted the assu
45、mption of one missing site as a homozygous recessive. Therefore, in AFLP, this hypothesis might cause overestimations of the number of analyzed loci. 16 Thus, Z. Q. Yue 17 (2008) considered in the application of AFLP analysis of population genetic structure, AMOVA should be selected as the preferred
46、 statistical method. Molecular variance AMOVA analysis showed a variation percentage among individuals within groups of 86.02%, which roughly agreed to the statistics based on Shannon genetic diversity index (82.09%). This figure indicated a certain degree of inter-population gene flow phenomenon, w
47、hich agreed with Y. Yuan 8 et al. (2008) on the genetic structures of different geographical populations of Cyclina sinensis distributed in Yellow Sea and Bohai region based on ITS technique. And also, this study was in accordance with the view points of Q. S. Tang 18 (2006) written in his book “Mar
48、ine Living Resources and Habitats” that the bio-communitie in Bohai Sea region is a geographical one. The analysis of genetic diversity in Cyclina sinensis (Mollusca Bivalvia) of four geographical populations provided considerable experimental data for many applications including reasonable species
49、introduction, culture of seedling, protection of resources and biological repair. ACKNOWLEDGMENT This work is supported by the Applied Basic Research Programs of the Science and Technology Commission Foundation of Tianjin P.R.China (Key Program 09JCZDJC19300; General Program 06YFJMJC11800). REFERENC
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