Analysis of genetic diversity and population structure of Pleuronectes yokohamae indicated by AFLP markers

a r t i c l e i n f o Article history: Received 30 December 2011 Accepted 28 April 2012 Available online xxx zation of P. yokohamae resource. Genetic diversity is of great importance to the sustainability of populations (Hamrick et al., 1991). Understanding fish stock structure is an important component of successful and sustainable long-term management and has attracted considerable interest; because of a fundamental interest in biotic evolution (Tudela et al., 1999). Determination of population genetic * Corresponding author. Tel./fax: þ86 53285830494. E-mail address: [email protected] (Y. Zhang). Contents lists available at SciVerse ScienceDirect Biochemical Systematics and Ecology Biochemical Systematics and Ecology 44 (2012) 102–108 1. Introduction The righteye flunder, Pleuronectes yokohamae, belongs to Pleuronectiformes, Pleuronectinae, distributes in Northwest Pacific, including southern Hokkaido of Japan to the Yellow Sea, the Gulf of Bohai, the northern part of the East China Sea and Korean island (Li, 1995). It is an important aquaculture species in China. With economic development, fishery activities increased very quickly, the yield of P. yokohamae has declined quickly due to increased efficiency of capture techniques and the leap of catch intensity. There were few studies on P. yokohamae, including its basic biological characteristics and artificial reproduction. As to population genetics studies, there were only Isozyme analysis (Fujio and Kato, 1979) (Zhang et al., 2007), RAPD (Zhang et al., 2008) and mtDNA (Zhang et al., 2009) analysis. No other studies were focused on genetic diversity, which have become an obstacle to further researches on the protection, management, rational exploitation and sustainable utili- Keywords: Pleuronectes yokohamae AFLP Genetic diversity Population structure 0305-1978/$ – see front matter � 2012 Elsevier Ltd doi:10.1016/j.bse.2012.04.024 a b s t r a c t Amplified fragment length polymorphism (AFLP) was used to analyze the genetic diversity and variation of two populations of Pleuronectes yokohamae from China and North Korea. A total of 228 loci ranging in size were detected from 63 individuals, of which 139 were polymorphic. The number of bands per primer combination varied from 37 to 51 and the percentageof polymorphic bands per primer combination ranged from43.14% to72.97%. The proportion of polymorphic loci, the Nei’s genetic diversity and Shannon genetic diversity index of China and North Korea populations were 55.09% and 55.71%, 0.1205 and 0.1917, 0.1370 and 0.2129, respectively. The results showed that the genetic diversity of these two populations was at the same level. Dominant gene frequency revealed that these two pop- ulations have the same genetic population structure. Fst value of the two populations was 0.0986 and AMOVA analysis indicated that 80.75% genetic variation came from individuals withinpopulations, and therewere also 19.25% genetic differentiation occurred between the two populations. Therewas significant genetic difference between two populations. Exact-p test showed there was no random genetic communication between two populations. The UPGMA and NJ trees based on AFLP data supported this result. � 2012 Elsevier Ltd. All rights reserved. Analysis of genetic diversity and population structure of Pleuronectes yokohamae indicated by AFLP markers Hui Zhang a,b, Han Yu b, Tianxiang Gao a, Yan Zhang b,*, Zhiqiang Han c, Yongshuang Xiao d a Fisheries College, Ocean University of China, Qingdao 266003, China bYellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China c Fishery College, Zhejiang Ocean University, Zhoushan 316004, China d Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China journal homepage: www.elsevier .com/locate/biochemsyseco . All rights reserved. structure provides essential information to underpin resource recovery and to aid in delineating and monitoring populations for fishery management. Failure to detect population units can lead to local over-fishing and ultimately to severe declines (Zhang et al., 2006). Molecular genetics techniques offer the ability to identify and delineate fish stock structure where it may not be apparent from phenotypic or behavioral characteristics. Such techniques have been used successfully to understand the structure of marine fish species (Han et al., 2008; Liu et al., 2006). Amplified fragment length polymorphism (AFLP) analysis (Vos et al., 1995) is a PCR-based molecular fingerprinting tech- nique that combines the strengths and overcomes theweaknesses of PCR-RFLP and RAPD-PCR. AFLP analysis has been used for indirect examination of levels of genetic diversity in several species (Liu et al., 2005; Chen et al., 2005; Kim et al., 2007). In this study,we investigated thegeneticdiversityandstructurewithinandamong twowildpopulationsofP. yokohamaeusingAFLP to compare the level of genetic diversity in them. This study provides baseline information on genetic background of this species, and such information should be beneficial to population conservation and fisheries management in this species. 2. Materials and methods 2.1. Samples Thirty-five individuals of P. yokohamae were collected from Penglai sea area north Shandong province in April 2004 and twenty-eight individuals were collected from North Korea sea area in April 2007. The backside muscle of each specimen was sampled and preserved in 95% ethanol for DNA extraction. Sample locations are shown in Fig. 1. H. Zhang et al. / Biochemical Systematics and Ecology 44 (2012) 102–108 103 Fig. 1. The sample locations (PL: PL Population; NK: North Korea Population). genomic DNA was digested with 1 unit of EcoR I and Mse I (NEB) at 37 �C for 6 h. Double-stranded adapters were ligated Table 1 Adapters and primers used in AFLP analysis. Adapter/primer Sequence Adapter EcoR I-1 50-CTCGTAGACTGCGTACC-30 EcoR I-2 30-CATCTGACGCATGGTTAA-50 Mse I-1 50-GACGATGAGTCCTGAG-30 Mse I-2 30-TACTCAGGACTCAT-50 Primer of preamplification EcoR Iþ1 50-GACTGCGTACCAATTCA-30 Mse Iþ1 50-GATGAGTCCTGAGTAAC-30 Primer of selective amplification E-AGA 50-GACTGCGTACCAATTCAGA-30 H. Zhang et al. / Biochemical Systematics and Ecology 44 (2012) 102–108104 to the restriction fragments at 20 �C overnight after adding 1 mL 10 � ligation buffer, 5 pmol EcoR I adapter (EcoR I-1/ EcoR I-2; Tables 1and 2), 50 pmol Mse I adapter (MseI-1/MseI-2; Table 1), 0.3 unit of T4 DNA ligase (Promega) with a final volume of 10 mL. Preamplification PCR reaction was conducted using an Eppendorf Thermocycler (Mastercycler 5334) with a pair of primers containing a single selective nucleotide. Amplification was performed at an annealing temperature of 53 �C for 30 s. The 20 mL PCR product mixture was diluted 10-fold with distilled water and used as 2.2. Genomic DNA extraction Genomic DNA was isolated from muscle tissue by proteinase K digestion followed by a standard phenol–chloroform method. DNA was subsequently resuspended in 100 mL of TE buffer (10 mmol/L Tris-Cl, 1 mmol/L EDTA, PH ¼ 8.0). 2.3. AFLP reaction, gel electrophoresis and silver staining Procedures of AFLP analysis were essentially based on Vos et al. (1995) and Wang et al. (2000). About 100 ng E-AGT 50-GACTGCGTACCAATTCAGT-30 E-AAC 50-GACTGCGTACCAATTCAAC-30 E-ACG 50-GACTGCGTACCAATTCACG-30 E-AGC 50-GACTGCGTACCAATTCAGC-30 M-CAG 50-GATGAGTCCTGAGTAACAG-30 M-CTA 50-GATGAGTCCTGAGTAACTA-30 M-CTC 50-GATGAGTCCTGAGTAACTC-30 M-CTG 50- GATGAGTCCTGAGTAACTG-30 M-CAT 50-GATGAGTCCTGAGTAACAT-30 templates for the subsequent selective PCR amplification. The selective amplifications were carried out in 20 mL PCR reaction volume containing 1 mL productions of preamplifications, 1 � PCR reaction buffer, 150 mM of each dNTP, 30 ng of each selective primer, and 0.5 unit of Taq DNA polymerase on a gradient thermal cycler (Mastercycler 5334) with a touchdown cycling profile of nine cycles of 30 s at 94 �C, 30 s at 65 �C (�1 �C at each cycle), and 30 s at 72 �C followed by the cycling profile of 28 cycles of 30 s at 94 �C, 30 s at 56 �C, and 1 min at 72 �C. The final step was a prolonged extension of 7 min at 72 �C. PCR products were run on 6.0% denaturing polyacrylamide gel electrophoresis (PAGE) for 2.5 h at 50 �C on the Sequi-Gen GT Sequencing Cell (Bio-Rad, USA), and finally detected using the silver staining technique modified from Merril et al. (1979). Five selective primer combinations were used: E-AGA/M-CAG, E-AGT/M- CTA, E-AAC/M-CTC, E-ACG/M-CTG and E-AGC/M-CAT. Adapters and primers used for AFLP preamplification and selective amplification are given in Table 1. Table 2 Number of bands generated by primer combinations. Primer combination No. of total loci No. of total polymorphic loci No. of polymorphic loci Percentage of polymorphism China Korea China Korea E-AGA/M-CAG 45 24 (53.33%) 23 22 51.11% 51.16 E-AGT/M-CTA 44 29 (65.91%) 23 22 56.10% 56.41% E-AAC/M-CTC 51 37 (72.55%) 31 30 67.39% 68.18% E-ACG/M-CTG 37 27 (72.97%) 22 22 64.71% 66.67% E-AGC/M-CAT 51 22 (43.14%) 20 21 39.22% 41.18% Total 228 139 (60.96%) 119 117 55.09% 55.71% Table 3 Parameters of genetic diversity for two populations of Pleuronectes yokohamae. Population Nei genetic diversity Shannon diversity index Average genetic similarity/average genetic distance Genetic similarity/genetic distance between populations Fst value China 0.1205 0.1917 0.8898/0.1167 0.8555/0.1561 0.0986 Korea 0.1370 0.2129 0.8759/0.1325 H. Zhang et al. / Biochemical Systematics and Ecology 44 (2012) 102–108 105 2.4. Data analysis AFLP bands were scored for presence (1) or absence (0), and transformed into 0/1 binary character matrix. Proportion of polymorphic loci, Nei’s genetic diversity and Shannon diversity index were calculated by POPGEN 1.32. Similarity indices were calculated using the formula S ¼ 2Nab/(Na þ Nb) (Nei and Li, 1979), where Na and Nb are the number of bands in individuals a and b, respectively and Nab is the number of sharing bands. Genetic distances between individuals were computed using the formula D ¼ �ln S (Nei and Li, 1979). Genetic relationships among populations were estimated by constructing UPGMA tree and NJ tree based on Nei’s genetic distance and K2P genetic distance respectively in Mega 3.1. Population structure, Fst value and exact-p test were investigated using the molecular variance software package in ARLEQUIN. Fig. 2. Distributions of amplified loci in different frequency intervals. 3. Result A total of 228 putative loci were detected by the five primer combinations, 139 of which were polymorphic (60.96%, Table 2). The number of bands scored per primer combination varied from 37 to 51 and the percentage of polymorphic band per primer combination ranged from 43.14% to 72.97% (Table 2). The proportion of polymorphic loci of the two populations was 54.84% (China) and 55.71% (North Korea). The Nei’s genetic diversity and Shannon diversity index of population China is lower than population North Korea (Table 3). The dominant gene frequency was from 0 to 100%. Fig. 2 showed that in interval 1–9% and 100% they all had peak values; the figure indicated that the two populations had the similar genetic population structure. The average genetic similarity, genetic distance of population Penglai and North Koreawere 0.8898 and 0.8759, 0.1167 and 0.1325, respectively (Table 3). Genetic similarity and genetic distance between the two populations were 0.8555 and 0.1561 (Table 3). The Fst valuewas 0.0986 (Table 3). According to AMOVA analysis, the percentage of variationwithin populations was 80.75%, meanwhile among populations was 19.25% (significant) (Table 4), suggesting a big genetic divergence between the two populations. Two phylogenetic trees of 63 individuals of the two populations were constructed using UPMGA and NJ methods (Fig. 3). Individuals of population Penglai and North Korea were separated into two distinct clusters, but there were also some North Korea individuals insert into Penglai cluster. The exact-p test also indicated that there was no random mating between the two populations (P ¼ 0.0009). Table 4 AMOVA analyses of two populations of Pleuronectes yokohamae. Source of variation d.f. Sum of squares Variance components Percentage of variation Among populations 1 135.877 3.84869 19.25 Within populations 61 984.536 16.13993 80.75 Total 62 1120.413 19.98862 100 Fig. 3. The UPGMA and NJ trees of 63 individuals of P. yokohamae (1–35:China, 36–63:Korea, because both trees are similar to each other, so only one tree was shown). H. Zhang et al. / Biochemical Systematics and Ecology 44 (2012) 102–108106 with the current published data, frequency of polymorphic loci in P. yokohamae is lower than that in Larimichthys crocea, Ictalurus punctatus (18.6%) (Liu et al., 2008;Mickett et al., 2003). These results indicate that genetic diversity of P. yokohamae is at the middle level. H. Zhang et al. / Biochemical Systematics and Ecology 44 (2012) 102–108 107 Generally, genetic diversity of a species was determined by its adaptability, survivability and evolvability. Rich genetic diversity means high survivability and great potential in application of genetic breeding (Hedrick and Miller, 1992). Fst and AMOVA analysis in this study showed the genetic diversity was mostly occurred between the two populations; at the meanwhile there was a little genetic diversity within populations. The UPMGA cluster analysis and NJ tree based on the Nei’s genetic distance and K2P distance indicated the samples from population Penglai were obviously separated from population North Korea, but there were 8 individuals of population North Korea inserted into Penglai’s cluster, indicated there were some genetic differentiation between the two populations, but there was no randommating between the two populations according to the exact-p test. Marine environments are often seen as open habitats in which isolation by distance is the main mechanism that may promote genetic differentiation among populations. The over-wintering ground for China stock is located in 34�450–37�000N, 122�250–123�400E; in March, the adults migrate from the over-wintering ground north to the Bohai strait, they arrived Bohai strait at April, in May there were large shoal of P. yokohamae spawning near Haiyang Island (39�060, 123�100) (Li, 1995). In this study, the North Korea samples were collected in the sea area of North Korea. The North Korea stock may be spawned near Haiyang Island which causing the two populations had some gene exchange, so the UPMGA indicated someNorth Korea samples inserted into Penglai cluster. Penglai population was so far away from North Korea samples, so most of Penglai samples and North Korea samples exited some genetic differentiation. In conclusion, the two populations from China and Korea could not mate randomly and the differentiation between them is significant based on the AFLP marker. Much more work should be addressed to indicate the reason. As the present work provides baseline information on genetic background of this species, it may be beneficial to population conservation and fisheries management to this species and the relative species. Acknowledgment This study was supported by Fishery Germplasm Resource Program of National Science & Technology Infrastructure of China and National High Technology Research and Development Program (No. 2006AA09Z). References Chen, D., Zhang, C., Lu, C., Chang, Y., Chang, J., 2005. Amplified fragment length polymorphism analysis to identify the genetic structure of the Gymnocypris przewalskii (Kessler, 1876) population from the Qinghai Basin. China J. Appl. Ichthyol. 21, 178–183. Fujio, Y., Kato, Y., 1979. Genetic in fish population. Bull. Jap. Soc. Scient. Fish 45, 1169–1178. Hamrick, J.L., Godt, M.J.W., Murawski, D.A., Loveless, M.D., 1991. Correlations between species traits and allozyme diversity: implications for conservation biology. In: Falk, D.A., Holsinger, K.E. (Eds.), Genetics and Conservation of Rare Plants. Oxford University Press, Oxford, pp. 75–86. Hedrick, P.W., Miller, P.S., 1992. Conservation genetics: techniques and fundamentals. Ecol. Appl. 2, 30–46. Han, Z.Q., Li, Y.Z., Chen, G.B., Gao, T.X., 2008. Population genetic structure of coral reef species Plectorhinchus flavomaculatus in South China Sea. Afr. J. Biotechnol. 7, 1774–1781. Keiper, F.J.R., 2000. An analysis of genetic variation in natural populations of Sticherus flabellatus [R. Br. (St John)] using amplified fragment length poly- morphism (AFLP) markers. Mol. Ecol. 9, 571–581. Kim, J.K., Kai, Y., Nakabo, T., 2007. Genetic diversity of Salanx ariakensis (Salangidae) from Korea and Japan inferred from AFLP. Ichthyol. Res. 54, 416–419. Lerceteau, E., Szmidt, A.E., 1999. Properties of AFLP markers in inheritance and genetic diversity studies of Pinus sylvestris. Heredity 82, 252–260. Li, S.Z., 1995. Fauna of China Osteichthyes Pleuronectiformes. Science Press, Beijing, pp. 241–246. Liu, B.Q., Dong, W.Q., Wang, Y.J., 2005. Identification of germ plasm in Pseudosciaena crocea Tai-Chu race by AFLP. Acta Hydrobiol. Sinica 29, 413–416. including wild (76.6%) and cultured (69.2–70.6%) populations (Wang et al., 2002), and Dai-chu race in Zhejiang province (55.80%) (Liu et al., 2005) and is also lower than wild Lutjanus argentimaculatus (57.14%) in China (Zhang and Huang, 2004). But frequency of polymorphic loci in P. yokohamae is higher than that in wild (46.18%) and cultured (40.07%) flounder (Zhang et al., 2004), farmed population of Plectorhinchus cinctus (34.3–41.4%), and partial cultured population of channel catfish 4. Discussion AFLP technique is a highly effective method for examining genetic polymorphism. It does not require any prior sequence information of the species under study and a large number of loci can be generated from a small amount of DNA. Due to its high level of polymorphism, the technique is appropriate to assess organisms with low level of genetic divergence. It has extensively been applied in genetic studies in diverse groups of organisms (Liu et al., 1998; Lerceteau and Szmidt, 1999; Mueller and Wolfenbarger, 1999; Seki et al., 1999; Keiper, 2000; Wang et al., 2000; Nicholas et al., 2001; Ogden and Thorpe, 2002). In the present study, we identified 228 different bands from two P. yokohamae populations by using five primer combinations. Among these bands, about 55.09% bands from population China were polymorphic; about 55.71% bands from population North Korea were polymorphic. Therefore, AFLP can supply rich genetic information for a given species and is good technique to evaluate the genetic diversity. The frequency of polymorphic loci, the Nei’s index, the Shannon’s index and the genetic similarity within the two pop- ulations were very similar, showed that the gene diversity between the two populations was at the same level. Compared Liu, W.B., Zeng, Z.N., Wang, D.X., 2008. Genetic variation in three farmed Pledtorhinchus cinctus stocks with various body color using AFLP analysis. J. Fish. Sci. China 15, 38–46. Liu, J.X., Gao, T.X., Zhuang, Z.M., Jin, X.S., Yokogawa, K., Zhang, Y.P., 2006. Late Pleistocene divergence and subsequent population expansion of two closely related fish species, Japanese anchovy (Engraulis japonicus) and Australian anchovy (Engraulis australis). Mol. Phylogenet. Evol. 40, 712–723. Liu, Z., Nichols, A., Li, P., Dunham, R.A., 1998. Inheritance and usefulness of AFLP markers in channel catfish (Ictalurus punctatus), blue catfish (I. furcatus), and their F1, F2 and backcross hybrids. Mol. Gen. Genet. 258, 260–268. Merril, C.R., Switzer, R.C., van Keuren, M.L., 1979. Trace polypeptides in cellular extracts and human body fluid detected by two-dimensional electrophoresis and a highly sensitive silver stain. Proc. Natl. Acad. Sci. U S A 76, 4335–4339. Mickett, K., Morton, C., Feng, J., Li, P., 2003. Assessing genetic diversity of domestic populations of channel catfish (Ictalurus punctatus) in Alabama using AFLP markers. Aquaculture 228, 91–105. Mueller, U.G., Wolfenbarger, L.L., 1999. AFLP genotyping and fingerprinting. Trends Ecol. Evol. 14, 389–394. Nei, M., Li, W.H., 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. 76, 5269–5273. Nicholas, G., Roger, S.T., Anita, M., 2001. The use of amplified fragment length polymorphism in determining species trees at fine taxonomic levels: analysis of a medically important snake, Trimeresurus albolabris. Mol. Ecol. 10, 419–426. Ogden, R., Thorpe, R.S., 2002. The usefulness of amplified fragment length polymorphism markers for taxon discrimination across graduated fine evolu- tionary levels in Caribbean Anolis lizards. Mol. Ecol. 11, 437–445. Seki, S., Agresti, J.J., Gall, G.A.E., Taniguchi, N., Bernie, M., 1999. AFLP analysis of genetic diversity in three populations of ayu Plecoglossus altivelis. Fish. Sci. 65, 888–892. Tudela, S., Garca-Marn, J.L., Pla, C., 1999. Genetic structure of the European anchovy, Engraulis encrasicolus, in the north-west Mediterranean. J. Exp. Mar. Biol. Ecol. 234, 95–109. Vos, P., Hogers, R., Bleeker, M., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic. Acids Res. 23, 4407–4414. Wang, Z., Jayasankar, P., Khoo, S.K., 2000. AFLP fingerprinting reveals genetic variability in common carp stocks from Indonesia. Asian Fish. Sci. 13, 139–147. Wang, Z.Y., Wang, Y.L., Liu, M.H., 2002. Genetic polymorphisms in wild and cultured large yellow croaker Pseudosciaena crocea using AFLP fingerprinting. J. Fish. Sci. China 9, 198–202. Zhang, J.B., Huang, L.M., 2004. Analyses of genetic diversity of Lutjanus argentimaculatus by AFLP molecular technique. J. Tropical Oceanograph. 23, 50–55. Zhang, J.B., Cai, Z.P., Huang, L.M., 2006. Population genetic structure of crimson snapper Lutjanus erythropterus in East Asia, revealed by analysis of the mitochondrial control region. ICES J. Mar. Sci. 63, 693–704. Zhang, Q.Q., Xu, X.F., Qi, J., 2004. The genetic diversity of wild and farmed Japanese flounder populations. J. Ocean Univ. China 34, 816–820. Zhang, Y., Gao, T.X., Liu, M.H., 2007. Tissual difference and isozyme analysis of Pleuronectes yokohamae Günther. J. Ocean Univ. China 37, 235–242. Zhang, Y., Xiao, Y.S., Gao, T.X., 2008. Analysis of genetic diversity of natural population in Pleuronectes yokohamae. J. Fish. China 32, 492–497. Zhang, Y., Xiao, Y.S., Gao, T.X., 2009. Comparative analysis of mtDNA gene sequences between two species of Pleuronectes. J. Fish. China 33, 201–207. H. Zhang et al. / Biochemical Systematics and Ecology 44 (2012) 102–108108 Analysis of genetic diversity and population structure of Pleuronectes yokohamae indicated by AFLP markers 1. Introduction 2. Materials and methods 2.1. Samples 2.2. Genomic DNA extraction 2.3. AFLP reaction, gel electrophoresis and silver staining 2.4. Data analysis 3. Result 4. Discussion Acknowledgment References