Genetic structure of cultured Haliotis diversicolor supertexta populations

Genetic structure of cultured Haliotis diversicolor supertexta populations

Zhongbao Li

ABSTRACT Genetic structure of four cultured Haliotis diversicolor supertexta populations were investigated using the assay of vertical slab polyacrylamide gel electrophoresis. Low levels of genetic diversity of populations was found: mean numbers of alleles per locus ranged from 1.4 to 1.5: proportions of polymorphic loci ([P.sub.o.99%]) ranged from 27.78 to 33.33; observed heterozygosities ranged from 0.120 to 0.150; expected heterozygosities ranged from 0.123 to 0.131. The results showed that the coefficient of gene differentiation among populations was low ([F.sub.ST] = 0.04), gene flow among populations was large (Nm = 6). Genetic structure was very similar among four cultured populations. The genetic distances between any two of the four populations were 0.002-0.009, with an average of 0.0047.

KEY WORDS: Haliotis diversicolor supertexta, allozyme, genetic structure, genetic diversity, genetic differentiation

INTRODUCTION

Haliotis diversicolor supertexta (Reeve) is widely distributed in China and Japan (Nie & Wang 2000). In recent years, H. diversicolor supertexta has been playing an economically important role in the fishing and farming industries of China. Numerous studies on the culture and ecology have been undertaken on H. diversicolor supertexta, however, few are concerned with genetic structure. Knowledge of genetic structure among populations is particularly important for managing exploited species. This knowledge is used in aquaculture to assess the potential for adaptive divergence available from different sources of broodstock. To protect this valuable marine resource and supply genetic information for the study of genetics and breeding, it is necessary to investigate and assess the genetic structure of H. diversicolor supertexta population.

Allozyme electrophoresis is considered to be an extremely useful technique in population genetics (Allendorf & Utter 1979, Lavery & Shaklee 1991). Within the genus Haliotis this kind of study has only been carried out in H. fulgens (Zuniga et al. 2000), H. roei Grey (Hancock 2000), H. rubra Leach (Brown 1991, Brown & Murray 1992) and H. laevigata Donovan (Brown & Murray 1992). This technique is used in the present study to examine the genetic structure of cultured H. diversicolor supertext populations. In this study we describe the pattern of population genetic structure in four cultured H. diversicolor supertext populations from one commercial abalone farm of Dongshan (population 1) and three commercial abalone farms of Zhangpu (population 2, population 3, and population 4) in Fujian Province, using allozyme analysis. The results can be useful for protecting and improving the resources of this species.

MATERIALS AND METHODS

Samples

Vertical slab polyacrylamide gel electrophoresis was used to estimate genetic structures and genetic diversities of the four cultured H. diversicolor supertexta populations. One hundred and twenty eight individuals were collected in 2002 from one commercial abalone farm in Dongshan (population 1, 32 individuals) and three commercial abalone farms in Zhangpu (population 2, 32 individuals; population 3, 32 individuals; and population 4, 32 individuals) in Fujian Province. They were alive on the way to the laboratory. A muscle tissues was dissected from each individual and preserved in a deep freezer at -80[degrees]C. The muscle sample (0.5 g) was homogenized with Tris-HC1 extract solution (pH = 7.0). The homogenates were centrifuged at 12000 rpm for 15 min at 4[degrees]C. The supernatant was then collected for electrophoresis.

Electrophoresis

The allozymes were detected using polyacrylamide gel electrophoresis (PAGE). The buffer systems and staining procedure followed those described by Taniguchi and Sugama (1990), Zen and Xiang (1989), and Wang (1996). A total of 10 enzymes at 18 loci were analyzed: lactate dehydrogenase (LDH, E.C.1.1.1.27), superoxide dismutase (SOD, E.C.1.15.1.1), esterase (EST, E.C.3.1.1-), aspartate aminotransferase (AAT, E.C.2.6.1.1), alcohol dehydrogenase (ADH, E.C.1.1.1.), malic enzyme (M E, E.C.1.1.1.40), malate dehydrogenase (MDH, E.C.1.1.1.37), sorbital dehydrogenase (SDH, E.C.1.1.1.14), isocitrate dehydrogenase (IDH, E.C. 1.1.1.42), and amylase (AMY,3.2.1.1). The names, abbreviations and numbers of the enzymes were described by Shaklee et al. (1990), and the description of the loci and alleles by Wang (1996).

Statistical Analysis

To assess the genetic diversities of the four populations, the allele frequencies, the mean numbers of alleles per locus (A), the proportions of polymorphic loci (P), observed heterozygosities (Ho), and expected heterozygosities (He) at Hardy-Weinberg equilibrium were calculated (Wang 1996). A locus was considered to be polymorphic ([P.sub.0.9]%) if the frequency of most common allele was equal or less than 0.99 at one or more localities. To estimate the degree of genetic differentiation among populations, the genetic distances between any two of the four populations were calculated using Nei’s formulas (Nei 1978), the coefficient of gene differentiation ([F.sub.ST) among populations was calculated following Wright (1977), and gene flow among populations (Nm) was calculated using Wright’s formulas (Wright 1969). Dendrogram from the matrix of genetic distances was constructed using unweighted pair group method with arithmetic averaging (UPGMA, Sheath & Sokal 1973). Data were analyzed using BIOSYS-1 software (Swofford & Selander 1989).

RESULTS

Ten enzymes coded by 18 loci were clearly resolved in all populations (Li et al. 2004a). Six loci (Est-2, Est-3, Mdh-1, Sdh-2, Aat-1, Amy-1) were polymorphic in at least one population among populations. Allele frequencies at polymorphic loci in four cultured H. diversicolor supertexta populations are presented in Table 1 and showed little variability among them.

Low levels of genetic diversity were estimated in the four cultured H. diversicolor supertexta populations (Table 2). The mean numbers of alleles per locus ranged from 1.4 to 1.5. The proportions of polymorphic loci ([P.sub.0.99]%) ranged from 27.78 to 33.33. Observed heterozygosities ranged from 0.120 to 0.150. Expected heterozygosities ranged from 0.123 to 0.131. The genetic diversity was very similar among populations.

To estimate the degree of genetic difference among the four populations, the coefficient of gene differentiation ([F.sub.ST]) and the genetic distance (D) were calculated. The coefficient of gene differentiation among populations was 0.04 (Table 3). The genetic distances between any two of the four populations were 0.0020.009, with the average of 0.0047, the genetic identities between any two of the four populations were 0.991-0.998, with the average of 0.9953 (Table 4). Gene flow was large (Nm = 6) (Table 3). The UPGMA dendrogrom of four cultured H. diversicolor supertexta populations was shown in Figure 1. The first formed by population 1 and population 2 at a distance of 0.002. The second formed by population 3 and population 4 (D = 0.002), which was linked to the first cluster at a distance of 0.006.

DISCUSSION

Genetic diversity is fundamental for the maintenance of species, population, and ecosystem diversities. Many studies have demonstrated that a loss of genetic diversity leads to a reduction in the adaptive fitness of a population, increasing the risk of extinction (Vrijenhoek 1994). In the present study, low level of genetic diversity was estimated in the four cultured H. diversicolor supertexta populations (Table 2). The proportions of polymorphic loci of H. diversicolor supertexta populations ranged from 27.78 to 33.33, which was lower than that of 48 kinds of seashells (P = 0.471 [+ or -] 0.159) (Singh 1984). The proportion of polymorphic loci is influenced by the number of loci examined. The average observed heterozygosity we found among H. diversicolor supertexa populations ([H.sub.0] = 0.135) was similar to those reported for H. rubra ([H.sub.0] = 0.136; Brown 1991), H. fulgens ([H.sub.0] = 0.119; Zuniga et al. 2000), H. discus hannai Ino ([H.sub.0] = 0.123; Fujio et al. 1983) and 48 kinds of seashells ([H.sub.0] = 0.147; Singh & Green 1984), however, lower than that of H. laevigata ([H.sub.0] = 0.195; Brown & Murray 1992) and higher than that of 10 kinds of invertebrates ([H.sub.0] = 0.098; Powell 1975). It is probably a reasonable assumption that the amount of isozyme variation reflects the relative amount of genetic variation found at other loci in the genome (McAndrew & Majumder 1983). Six loci (Est-2, Est-3, Mdh-1, Sdh-2, Aat-1 and Amy-1) in 18 gene loci were polymorphic in at least one population among populations. From the genetic point of view, Est-2, Est-3, Mdh-1, Sdh-2, Aat-1 and Amy-1 can be used as the biochemical genetic markers of H. diversicolor supertexta for genetic diversity assessment and selective breeding program. Similar studies have been carried out in H. diversicolor diversicolor (Li et al. 2004a), H. discus discus, and H. discus hannai (Li et al. 2004b).

Genetic structure was very similar among four cultured populations. The coefficient of gene differentiation among populations was low ([F.sub.sr] = 0.04), with 4% gene differentiation of the total diversity coming from interpopulation and 96% from intrapopulation. The result showed that gene flow among populations in our study was large (Nm = 6) (Table 3). The estimate obtained in this study for [F.sub.ST] values in H. diversicolor supertexta is in general agreement with those reported for H. cracherodii ([F.sub.ST] = 0.039; Harem & Burton 2000), H. fulgens ([F.sub.ST] = 0.036; Zuniga et al. 2000), H. rubra ([F.sub.ST] = 0.022; Brown 1991), H. laevigata ([F.sub.ST] = 0.014; Brown & Murray 1992), H. rufescens ([F.sub.ST] = 0.012; Burton & Tegner 2000) and H. roei ([F.sub.ST] = 0.009; Hancock 2000) within the genus Haliotis.

The mean genetic distance and mean genetic identity among populations were 0.0047 and 0.9953 respectively, which means that there were no significant differences in the biochemical genetic character among populations. Nei (1987) considers the threshold genetic distance between populations, between subspecies, and between species at about 0.01, 0.1, and 1.0. They belong to population level of H. diversicolor supertexta.

Modern fisheries science demands a holistic management of fisheries; this concept includes the maintenance of genetic diversity and population structure, which are critical for ensuring the long-term survival of any fishery (Shepherd & Brown 1993). The results in this study are likely to be indicative of population genetics of cultured H. diversicolor supertexta in China and the factors influencing it, which should be considered in the development of H. diversicolor supertexta conservation and future management plans.

TABLE 1.

Allele frequencies atpolymorphic loci in cultured Haliotis

diversicolor superlexta populations.

Population Population Population Population

Locus Allele 1 2 3 4

Est-2 A 0.333 0.438 0.708 0.646

B 0.667 0.563 0.292 0.354

Est-3 A 0.854 0.792 0.875 0.708

B 0.125 0.208 0.125 0.229

C 0.021 0.000 0.000 0.063

Mdh-1 A 0.188 0.188 0.188 0.167

B 0.813 0.813 0.813 0.833

Sdh-2 A 0.979 1.000 1.000 1.000

B 0.021 0.000 0.000 0.000

Aat-1 A 0.042 0.021 0.167 0.042

B 0.188 0.146 0.292 0.146

C 0.313 0.354 0.208 0.354

D 0.458 0.479 0.333 0.458

Amy-1 A 0.438 0.688 0.563 0.417

B 0.563 0.313 0.438 0.583

TABLE 2.

Genetic diversities of cultured Haliotis diversicolor

supertexta populations.

Population Population Population

Index 1 2 3

A 1.5 (0.2) 1.4 (0.2) 1.4 (0.2)

[P.sub.0.99]% 33.33 27.78 27.78

Ho 0.134 (0.068) 0.137 (0.063) 0.120 (0.060)

He 0.124 (0.051) 0.124 (0.051) 0.123 (0.053)

Population

Index 4

A 1.4 (0.2)

[P.sub.0.99]% 27.78

Ho 0.150 (0.069)

He 0.131 (0.053)

TABLE 3.

F-statistics and gene flow at polymorphic loci in cultured Haliotis

diversicolor supertexta populations.

Locus [F.sub.IS] [F.sub.IT] [F.sub.ST] Nm

Est-2 0.493 0.540 0.093

Est-3 0.029 0.051 0.022

Mdh-1 -0.084 -0.083 0.001

Sdh-2 -0.021 -0.005 0.016

Aat-1 -0.508 -0.477 0.020

Amy-1 -0.206 -0.149 0.047

Mean -0.130 -0.060 0.040 6

TABLE 4.

Genetic distances (above diagonal) and genetic identities (below

diagonal) among cultured Haliotis diversicolor

supertexta populations.

Population 1 2 3 4

1 ***** 0.002 0.009 0.004

2 0.998 ***** 0.006 0.005

3 0.991 0.994 ***** 0.002

4 0.996 0.995 0.998 *****

ACKNOWLEDGMENTS

The project was supported by National Natural Science Foundation of China (No: 30271013), Natural Science Foundation of Fujian province (No: B0110036), and The project of the Committee of Science and Technology of Fujian Province (No: 2001Z073).

LITERATURE CITED

Allendorf, F. W. & F. M. Utter. 1979. population genetics. In: W. S. Hoar, D. J. Randall & J. R. Brett, editors. Fish physiology, vol. 8. New York: Academic Press. pp. 407454.

Brown, L. D. 1991. Genetic variation and population structure in the black-lip abalone, Haliotis rubra. Aust. J. Mar. Freshwater Res. 42:77-90.

Brown, L. D. & N. D. Murray. 1992. Population genetics, gene flow and stock structure in Haliotis rubra and Haliotis laevigata. In: S. A. Shepherd, M. J. Tegner & S. A. Guzman del Proo, editors. Abalone of the world: biology, fisheries and culture. Oxford: Blackwell Scientific: pp. 24-33.

Burton, R. S. & M. J. Tegner. 2000. Enhancement of red abalone Haliotis rufescens stocks at San Miguel Island: reassessing a success story. Mar. Ecol. Prog. Ser. 202:303-308.

Fujio, Y., R. Yamanaka & P. J. Smith. 1983. Genetic variation in marine mollusks. Bull. Jpn. Sac. Sci. Fish. 49:1809-1817.

Hancock, B. 2000. Genetic subdivision of Roe’s abalone, Haliotis roei Grey (mollusca Gastropoda), in south-western Australia. Mar. Freshwater Res. 51:679-687.

Hamm, D. E. & R. S. Burton. 2000. Population genetics of the black abalone, Haliotis cracherodii, along the central Californian coast. J. Exp. Mar Biol. Ecol. 254:235-247.

Lavery, S. & J. B. Shaklee. 1991. Genetic evidence for separation of two sharks, Carcharhinus limbatus and C.tilstoni, from Northern Australia. Marine Biology 108:1-4.

Li, Z. B., Z. Tian, D. R. Zhu & C. Y. Ye. 2004a. Biochemical genetic analysis of allozymes of Haliotis diversicolor supertexta and Haliotis diversicolor diversicolor. Marine Science 28(2):27-31. (In Chinese).

Li, Z. B., S. L. Deng, Y. Ding & X. Q. Xu. 2004b, Biochemical genetic analysis of allozymes of Haliotis discus discus and Haliotis discus hannai, Marine Science 28(4):43-9. (In Chinese).

McAndrew, B. J. & K. C. Majumder. 1983. Tilapia stock identification using electrophoretic markers. Aquaculture 30:249-261.

Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583-590.

Nei, M. 1987. Molecular evolutionary genetics. New York: Columbia University Press. 512 pp.

Nie, Z. Q. & S. P. Wang. 2000. Practice cultured technique in abalone. Chinese Agricultural Press, Beijing. (In Chinese). pp. 18-29. Powell, J. R. 1975. Protein variation in natural populations of animal. Evolutianary boil. 8:79 119.

Shepherd, S. A. & L. D. Brown. 1993. What an abalone stock: implications for the role of refuge in conservation. Can. J. fish. Aquat. Sci. 50:2001-2009.

Shaklee, J. B., F. W. Allendorf & D. C. Morizot. 1990. Gene nomenclature for protein-coding loci in fish. Trans. Am. Fish. Sac. 119:2-15.

Singh, S. M. & R. H. Green. 1984. Excess of allozyme homozygosity in marine mollusks and its possible biological significance. Malaeologica 25(2):569-581.

Sneath, P. H. A. & R. S. Sokal. 1973. Numerical taxonomy–the principle and practice of numerical classifications. San Francisco: W. H. Freeman.

Swofford, D. L. & P. K. Selander. 1989. A computer program for the analysis of allelic variation in population genetics and biochemical systematics. J. Hered. 72:281-283.

Taniguchi, N. & K. Sugama. 1990. Genetic variation and population structure of red sea bream in the coastal waters of Japan and the East China Sea. Nippon Suisan Gakkaishi. 56(7): 1069-1077.

Vrijenhoek, R. C. 1994. Genetic diversity and fitness in small populations. In: V. Loescheke, J. Tomiuk & S. K. Jain, editors. Conservation genetics. Boston: Birkauser. pp. 37-53.

Wang, Z. R. 1996. Allozyme analysis of plants. Beijing: Science Press. pp. 77-119. (In Chinese).

Wright, S. 1969. Evolution and the genetics of populations, vol. 2. The theory of gene frequencies. Chicago: University of Chicago Press.

Wright, S. 1977. Evolution and the Genetics of populations, vol. 3. Experimental results and evolutionary deductions. Chicago: University of Chicago Press.

Zen, C. K. & J. H. Xiang. 1989. Marine biotechnology. Jinan, China: Shan Dang Science Technology Press. (In Chinese).

Zuniga, G., S. A, Guzman Del Proo, R. Cisneros & G. Rodriguez. 2000. Population genetic analysis of the abalone Haliotis Jidgens (mollusca Gastropoda) in Baja California, Mexico. J. Shellfish Res. 19:853-859.

ZHONGBAO LI (1,2) * AND CHANGSHENG CHEN (1)

(1) Fisheries College, Jimei University, Xiamen, Fujian 361021, People’s Republic of China; (2) School of Marine and Environmental Studies, Xiamen University, Xiamen, Fujian 361005, People’s Republic of China

* Corresponding author. Fax: +86-592-618-1420; E-mail: zhongbaoli@hhotmail.co

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