Microsatellite analysis of genetic diversity and population structure of Chinese mitten crab (Eriocheir sinensis)

Microsatellite analysis of genetic diversity and population structure of Chinese mitten crab (Eriocheir sinensis)

JOURNAL OF GENETICS AND GENOMICS J. Genet. Genomics 35 (2008) 171176 www.jgenetgenomics.org Microsatellite analysis of genetic diversity and popul...

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JOURNAL OF

GENETICS AND GENOMICS J. Genet. Genomics 35 (2008) 171176

www.jgenetgenomics.org

Microsatellite analysis of genetic diversity and population structure of Chinese mitten crab (Eriocheir sinensis) Yumei Chang a, b, Liqun Liang a, Haitao Ma a, c, Jianguo He b, Xiaowen Sun a, * a

Key Laboratory of Finfish Bioengineering & Breeding in North China, Ministry of Agriculture, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070,China b College of Life Sciences, Zhongshan University, Guangzhou 510275, China c Life Science of Technology Institute, Dalian Fisheries University, Dalian 116023, China Received for publication 19 April 2007; revised 12 July 2007; accepted 12 July 2007

Abstract Chinese mitten crab (Eriocheir sinensis) has higher commercial value as food source than any other species of Eriocheir in China. To evaluate the germplasm resources and characterize the genetic diversity and population structure of the crabs in different water systems, two stocks and two farming populations were assessed with 25 polymorphic microsallite loci available in public GenBank. Basic statistics showed that the average observed heterozygosity (Ho) amongst populations ranged from 0.5789 to 0.6824. However, a remarkable presence of inbreeding and heterozygote deficiencies were observed. To analyze population structure, pairwise FST coefficients explained only ~10.3% variability from the subdivision of mitten crab populations, the remaining variability stems from the subdivision within subpopulations. Although the four populations had slight differentiation, different allelic frequencies resulted in distinct population structures. Two stocks and one farming population were clustered together to the phylogenetic branch of Yangtze crab, with an approximate membership of 95%. Whereas, another farming population was clustered singly to the phylogenetic branch of the Liaohe crab, with a membership of 97.1%. The tests for individual admixture showed that Yangtze crab had probably been contaminated with individuals from other water systems. Genetic relationships between populations also supported the conclusion that Yangtze crab and Liaohe crab had different gene pools in spite of the origins of the same species. Keywords: Chinese mitten crab; genetic diversity; population structure; admixture

Introduction Chinese mitten crab (Eriocheir sinensis) is popular for its taste, nutrition, and economic value, which facilitate the aquaculture of the mitten crab in China. Although mitten crabs have a wide distribution in northeast and south of China, the population in the Yangtze River has the best reputation for its big size and specific taste. However, crab populations in the Yangtze River have diminished for several years due to dam construction, over fishing, and water pollution. For example, the number of crab fry has decreased significantly from 70 tons in the 1960s to less than * Corresponding author. E-mail address: [email protected]

10 tons in 1980, and to less than 0.2 tons in 1998 (Tang et al., 2000). Due to these impacts, numerous cultured crab fry are released annually into the river for recruitments. In this context, two viewpoints have emerged amongst experts regarding the potential for genetic mixing and degradation of the preferred (i.e., size and taste) Yangtze River crab. The first view is that crab populations from the Liaohe River (referred to as the “Liaohe crab”) in northeast of China and the Oujiang River (referred to as the “Oujiang crab”) in south of China could interbreed with Yangtze River crab populations (referred to as the “Yangtze crab”) and lead to degradation and dilution of this preferred germplasm. The second viewpoint is that crab populations from the three large rivers are not interbreeding due to propagation differences, such as time for spawning (Liaohe

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crab spawns earlier than Yantze crab) (Tang et al., 2000). In general, most experts support the former probability, given the phenotypic traits of Yangtze crab, such as small body and early maturity. Genetic research on Chinese mitten crab has developed slowly in comparison with other aquacultured species in China. Early researches focused on two fields including taxa confusion and different populations’ identification using isozymes, random amplified polymorphic DNAs (RAPDs) and mtDNAs, as well as predicted by morphologic judges (Wang and Yu, 1995; Xu and Li, 1996; Qiu et al., 1997; Li and Li, 1999; Li and Zhou, 1999; Zhao and Li, 1999; Zhou and Gao, 1999; Tand et al., 2003; Zhang and Ma, 2004). In population identification, the findings are inadequate and inaccurate due to the limitations of methods, such as isozyme (lower polymorphisms) and RAPDs (unstable and inaccurate results). Microsatellite markers, also known as short sequence tandom repeats (SSRs), are very popular in genome researches, such as genetic linkage maps, population genetics and many other fields. In addition, it is easy to obtain the results in combination with the PCR method. Therefore, more microsatellite markers need to be developed and applied in genome research for the Chinese mitten crab. At present, there are a total of 35 microsatellite markers that are available in the public GenBank (Hänfling and Weetman, 2003; Zhu et al., 2006; Chang et al., 2006). Given the current status of Chinese mitten crab industry in China, 18 polymorphic markers were developed and eight markers available from the public GenBank were utilized to (1) investigate genetic diversities between crab stocks and farming crabs, and (2) identify specific markers for geographically different populations, especially referred to as Yangtze and Liaohe crab, (3) estimate the differentiation within and amongst populations and their genetic relationships, and (4) evaluate the admixture estimates for individual crabs.

Materials and methods Samples collection Samples were collected from four sites. Two of them were the stock sanctuaries, located in the Gaochun County, Jiangsu Province and Mingguang County, Anhui Province, south of China, respectively. The samples hereafter were abbreviated as CJS and CAH. Samples collected from a private farming site, Tianjin, were referred to as CTJ (in fact, the crab fry originated from the Yangtze River). Another group of samples, also collected from a private farming site, Panjin County, Liaoning Province, northeast of

China, were called CLN (the crab fry originated from the Liaohe River). All samples were the adult individuals and each population was sampled 30 individuals separately, consisting of 15 females and 15 males. Sampled leg muscle of each individual was preserved in 100% ethanol. DNA extraction and microsatellite genotyping Ethanol-preserved leg muscle of each individual was first immersed into non-ionic water for about 1.5 h thrice. Then, the leg muscle was dried and ground. DNA isolation of each individual was conducted as described by Chang et al. (2004). As a result, one male individual of CLN failed to isolate DNA and was abandoned. A set of 26 polymorphic microsatellites were subjected to PCR amplification in a 25 PL volume. The composition of PCR reaction mixture and the procedures of PCR were referenced to Chang et al. (2006). As a result, marker ESC57 was not chosen for further research due to its confusing stuttering bands. PCR products were separated by 8% non-denaturing polyacrylamide gel electrophoresis and visualized following silver stain. The allelic determination was made manually with the software package of GEL-PRO analyzer (http: //www.mediacy.com/index.aspx?page=GelPro). Diversity analysis The following data statistics were calculated using the computer program PowerMarker (Liu, 2002). Basic statistics, including the number of alleles, observed heterozygosity (Ho), gene diversity or expected heterozygosity (He), and inbreeding coefficient (f) were calculated across 25 loci and populations with 1,000 bootstrap samplings. Tests for deviations from Hardy-Weinberg equilibrium (HWE, P<0.05) were performed across 25 loci and populations by applying the unbiased estimates of the exact P-value with default settings of the Monte Carlo Markov Chain method (MCMC). Tests for genotypic linkage disequilibrium for all pairs of loci within each population were also conducted using the MCMC method. Fixation coefficient (Fis ) as a measure of heterozygote deficiency was evaluated amongst populations using computer program POPGEN (Yeh and Yang, 1999). Population structure analysis Population structure performed the hierarchical Wright’s F-statistics using the estimators of Weir and Cockerham (1984) as implemented in the computer program POPGEN. Pair-wise FST estimates amongst all population combinations were computed through the construction of coancestry matrix using the software PowerMarker. The assign-

Yumei Chang et al. / Journal of Genetics and Genomics 35 (2008) 171176

ment of Individual crabs to their most likely populations was performed by the computer program STRUCTURE (Pritchard et al., 2000). In this analysis, three simulations were conducted for each model (k =15) assuming admixture and correlated allele frequencies between populations with a 10,000 replications burn-in period and 10,000 MCMC replicates. Running this procedure demonstrates that sampled crabs have obvious population structure.

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strated the presence of linkage disequilibrium between locus pairs, with the number of one for CAH, two for CJS, three for CTJ and seven for CLN, respectively.

Population relationship analysis The shared allele distance DSA was used to calculate genetic distance between populations (Chakraborty and Jin, 1993). The phylogenetic tree was constructed on the basis of the DSA genetic distance matrix, which was calculated by the unweighted pair-group method using arithmetic average (UPGMA) and corroborated by 1,000 bootstrap procedures (Sneath and Sokal, 1973). Given the presence of subsequent migrants between populations, UPGMA algorithm was considered to be superior to the Neighbor-Joining algorithm (Achmann et al., 2004). The output of a list of trees obtained was integrated to a consensus tree by the software package PHYLIP (Felsenstein, 1993).

Results Genetic diversity across 25 loci and populations The total number of alleles for the 25 microsatellite loci in the sampled crab was 183, and the mean number of alleles (MNE) per locus was 7.32 with the range of 310. The values of Ho ranged from 0.3277 to 0.8739 with the mean of 0.6453. The values of He ranged from 0.5607 to 0.8688 with the mean of 0.7857. Obviously, the above-mentioned data showed a relatively higher genetic diversity in Chinese mitten crab, and Fig. 1 gives a good example for this points view. The Fis pronounced most loci present heterozygote deficiencies. Two loci Es06 and ESC29 had over 40% heterozygote deficiencies in all samples (Table 1). Although MNE for each population was almost identical, Ho, He, and f expressed slight differences between populations. CAH and CJS had relatively higher Ho (0.6784 and 0.6824) and lower f (0.1188 and 1.1227). CLN had the lowest Ho (0.5789) and the highest f (0.2009) amongst populations (Table 2). An exact P-value test indicated that most loci significantly deviated from HWE in the CAH and CLN with the number of 12 and 14 loci, respectively (P< 0.05). The deviations of HWE were also detected in CJS and CTJ with the number of 8 and 9 loci, separately. In addition, an exact P-value test for linkage disequilibrium (Pİ0.05) demon-

Fig. 1. Microsatellite locus Es74 showed a high genetic diversity in the four crab populations.

Genetic structure of populations Population differentiation was presented by pair-wise FST coefficients through the construction of coancestry matrix. According to the values of matrix, the FST coefficients ranged from 0.0275 (between CAH and CJS) to 0.103 (between CJS and CLN). Thus, microsatellite variability from 2.75% to 10.3% was explained by the subdivision of crab populations. The remaining variability was explained by the variation within the subpopulation. The levels of differentiation for separate locus (FST) were relatively low and ranged from 0.0129 to 0.1725, except for locus ESC29 (0.4368) and locus Es87 (0.3378). FST estimates were significantly different from zero for all loci (P < 0.01) (data not shown). To test admixture of individual crabs amongst populations, especially the Yangtze crab and the Liaohe crab, the computer program STRUCTURE was used to evaluate the assignments of individuals to 15 assumed populations with three simulations. As a result, it was found that the output of program parameters set for two clusters assumedwere more stable. Populations CAH, CJS, and CTJ converged one cluster together and showed average membership of 95.6%, 97.8%, and 94.6%, respectively. As expected, population CLN was clustered singly with an aver age membership in its expected cluster of 97.1%. This result indicated that the Yangtze crab and the Liaohe crab were of remarkably different gene pools despite belonging to the same species with only 10.3% genetic variability. Examinations for individual admixture showed that two

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174 Table 1

Summary statistics of 25 loci for crab subpopulations Loci

No. of samples

No. of alleles

Size range

He

Ho

Fis

Es06

119

6

315349

0.7778

0.4622

0.4093

Es18

118

6

213309

0.7747

0.5508

0.2929

Es36

119

10

182247

0.8600

0.7983

0.0759

Es38

119

8

211280

0.8259

0.7143

0.1390

Es42

119

6

237422

0.7284

0.7731

0.057

Es67

119

5

213270

0.7272

0.5210

0.2874

Es74

119

6

257393

0.7768

0.7647

0.0198

Es75

116

6

193257

0.7785

0.8103

0.037

ESA42

119

7

245287

0.7776

0.6555

0.1612

ESA67

119

9

134189

0.8546

0.5798

0.3253

ESB25

119

7

193236

0.8168

0.6891

0.1605

ESB72

119

10

321341

0.8391

0.6639

0.2129

ESB88

118

9

328444

0.8292

0.7881

0.0538

ESC11

118

9

125176

0.8688

0.6780

0.2237

ESC20

118

8

186290

0.7752

0.8136

0.045

ESC29

119

5

53171

0.5607

0.3277

0.4190

ESC34

119

8

105180

0.8029

0.8739

0.084

ESC56

119

6

170225

0.7257

0.5294

0.2744

ESC65

119

8

227430

0.8365

0.7395

0.1201

ESC86

118

8

90185

0.8340

0.5932

0.2926

ESD02

119

6

235275

0.7359

0.5378

0.2731

ESD11

119

10

105170

0.8677

0.7059

0.1896

ESD52

119

9

105195

0.8577

0.5294

0.3864

ESA76

112

8

165260

0.8393

0.6339

0.2490

Es87

118

3

156182

0.5714

0.3983

0.3068

Mean

118

7.32

0.7857

0.6453

Table 2 Summary statistics amongst populations across 25 loci

CAH

MNE

Ho

He

f

6.56

0.6784 (0.0243)

0.7380 (0.0210)

0.1188 (0.0274)

CJS

6.64

0.6824 (0.0299)

0.7450 (0.0240)

0.1227 (0.0238)

CTJ

6.48

0.6395 (0.0371)

0.7226 (0.0261)

0.1531 (0.0345)

CLN

6.40

0.5789 (0.0330)

0.7052 (0.0341)

0.2009 (0.0292)

individuals from the Yangtze cluster had over 50% membership in the Liaohe cluster, demonstrating the probable existence of the Yangtze-Liaohe hybrids or introgressants.

struction of phylogenetic tree with a robust 1,000 samplings (Fig. 2). As this phylogenetic tree shows, the crab populations CJS, CAH, and CTJ converged together, which

Population genetic relationships The data of genetic distance matrix (DSA) showed that populations CJS and CAH had the nearest genetic distance of 0.26, the farthest genetic distance was 0.33 between populations CJS and CLN. The genetic relationships between populations were further confirmed by the recon-

Fig. 2. Reconstruction of phylogenetic tree for crab populations based on the shared allele distance DSA using UPGMA algorithm method.

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indicated the same ancestor that was derived, corresponding to the background knowledge acquired.

Discussion Chinese mitten crab has a wide range of distributions in China. The geographically characterized populations are the Yangtze crab, the Liaohe crab and the Oujiang crab named after their living river systems. However, it is difficult to distinguish these populations due to the limitations of detective methods. Despite genetic differences having been tested between populations using RAPD and mtDNA, population specific markers are still undefined successfully (Qiu et al., 1997; Li and Zhou, 2002). In this study, population specific markers for the Yangtze and the Liaohe crabs were not identified even using 25 highly polymorphic microsatellite markers, except that several individuals of each population displayed certain private alleles. According to previous and the present findings, it was inferred that the Yangtze and the Liaohe crabs had a weak divergence not only confirmed by molecular markers but by sequence comparisons of 16S rDNA (Qiu et al., 1997; Li and Zhou, 2002; Sun et al., 2002). The dynamics of allelic frequencies causes the genetic differences between populations. According to the knowledge acquired, the direct factors for the dynamics of allelic frequencies includes mating system, stresses stemmed from fragmented habitat, impeded migration route and pollutant water quality, which likely incur genetic drifts and/or gene mutations. Thus, it is quite normal that no specific markers were detected presently for crab populations from different water systems. However, with the increased number of microsatellite markers and the development of new markers like single nucleotide polymorphisms (SNPs) and/or genes, specific markers for each crab population will be detected in future. Although the Yangtze and the Liaohe crabs are the same species, computer program STRUCTURE with three simulations showed that they had different gene pools and were embodied by different allelic frequencies in their genomes. The three Yangtze crab populations sampled from different areas were always clustered together, whereas the Liaohe crab was clustered singly, demonstrating that the two populations were independent and there was no occurrence of gene exchanges except for two skeptical individuals from the Yangtze crab studied. Sun et al. (2002) proved that there were few differences except for one base substitution in part of 16S rDNA sequences between the Yangtze and the Liaohe crabs. In contrast, a few individuals sampled in the estuary of the Yangtze River are of ‘AA’ mutations through sequence comparisons (‘GG’ are specific for the Yangtze crab). Nevertheless, ‘AA’ mutations in 16S rDNA characterize to Eriocheir japonica hepuensis, considered to be a kind of subspecies of Eriocheir japonica,

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mainly distributed in the Ming River, the Nanliu River and the Jiulong River, south of China. Thus, it was inferred that these two individuals are likely to be E. j. hepuensis, since Liaohe crab individuals are not expected to migrate to the Yangtze region in this study. Unlike the morphological discrimination and isozymic analysis, molecular markers, especially microsatellite markers, are very sensitive and polymorphic. Due to its higher variability, microsatellite markers offer more genetic information, such as a wide range of heterozygosity, genetic distance and population structure. Twenty-five microsatellite markers were used for disclosing genetic information of Chinese mitten crab including stocks and farming crabs to assess the current status of crab industry and germplasm enhancements precisely. In line with the findings, the mean Ho and He observed amongst all samples were 0.6453 and 0.7857, respectively (Table 1), demonstrating a relatively higher genetic diversity within crab individuals. This was in correspondence with those reported by other documents no matter where the samples collected (Hänfling and Weetman, 2003; Zhu et al., 2006; Chang et al., 2006). The unique mating system of Chinese mitten crab may correspond to its higher genetic diversity. One female mitten crab often attracts several males to donate their sperms to its sperm storage chamber when they achieve sex maturity (Cheng YX, personal communication). Fitzsimmons (1998) considered multiple paternities provide a good mechanism to counteract inbreeding depression by increasing the genetic diversity (heterozygosity). However, most microsatellite loci exhibited drastic departures from HWE, as shown by the fact that He was apparently higher than Ho (Table 1). If a population in HWE, the value of He is close to Ho, otherwise, the population deviates HWE due to the excess and/or deficit of heterozygotes (Quan et al., 2006). In this study, heterozygote deficiency at most loci seems to be strong evidence for the deviations of HWE. Sampling (i.e., sample size and representative) is likely to be a case resulting in a biased allelic frequency. However, small populations and nonrandom mating also lead to significant heterozygote deficits, which might be a potential risk for populations to lose genetic diversity rapidly and suffer further inbreeding depression. Therefore, it is extremely urgent and necessary to conserve Chinese mitten crab. Since there are certain traits or genes unique to the Yangtze and the Liaohe crabs, they should be conserved as different units of management and conservation, even though they have a weak differentiation. Two tentative and constructive plans or measures are suggestive of the following: (1) the preservation of genetic diversity. More healthy individuals should be introduced into the stock sanctuaries to increase the observed heterozygosity, a key valuable index for the estimates of genetic diversity; (2) the recruitment of crab resources in rivers. To date, the exhaustions of natural crab resources in

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different water systems are severe, and the crab fry produced artificially are highly needed to restore and complement crab populations in different rivers. However, the crab fry must be originated from rivers locally before releasing on a large scale.

Acknowledgements The authors thank Dr. Dale Bruns, College of Science and Engineering, Wilkes University for re-editing the English language of this manuscript. This work is supported by National Basic Research Program of China (No. 2004CB117405).

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