Genetic structure of cultured Haliotis diversicolor supertexta (reeve) populations.ABSTRACT Genetic structure of four cultured Haliotis diversicolor supertexta populations were investigated using the assay of vertical slab polyacrylamide gel electrophoresis polyacrylamide gel electrophoresis n. A technique for determining the molecular weight of proteins, in which proteins that have been coated in an anionic detergent undergo electrophoresis in a polyacrylamide gel. . 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 aquaculture, the raising and harvesting of fresh- and saltwater plants and animals. The most economically important form of aquaculture is fish farming, an industry that accounts for an ever increasing share of world fisheries production. 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 Population genetics The study of both experimental and theoretical consequences of mendelian heredity on the population level, in contradistinction to classical genetics which deals with the offspring of specified parents on the familial level. (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 abalone (ăbəlō`nē), popular name in the United States for a univalve gastropod mollusk of the genus Haliotis, members of which are also called ear shells, or sea ears, as their shape resembles the human ear. 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 ho·mog·e·nize v. ho·mog·e·nized, ho·mog·e·niz·ing, ho·mog·e·niz·es v.tr. 1. To make homogeneous. 2. a. To reduce to particles and disperse throughout a fluid. b. with Tris-HC1 extract solution (pH = 7.0). The homogenates were centrifuged at 12000 rpm for 15 min at 4[degrees]C. The supernatant supernatant /su·per·na·tant/ (-na´tant) the liquid lying above a layer of precipitated insoluble material. supernatant the liquid lying above a layer of precipitated insoluble material. 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 lactate dehydrogenase n. Abbr. LDH Any of a class of enzymes found in the liver, kidneys, striated muscle, and heart muscle that catalyze the reversible conversion of pyruvate and lactate. (LDH LDH -lactate dehydrogenase. LDH abbr. lactate dehydrogenase LDH lactic acid dehydrogenase; see lactate dehydrogenase. , E.C.1.1.1.27), superoxide dismutase superoxide dismutase n. An enzyme that catalyzes the decomposition of a superoxide into hydrogen peroxide and oxygen. superoxide dismutase (SOD, E.C.1.15.1.1), esterase esterase /es·ter·ase/ (es´ter-as) any enzyme which catalyzes the hydrolysis of an ester into its alcohol and acid. es·ter·ase n. Any of various enzymes that catalyze the hydrolysis of an ester. (EST EST electroshock therapy. EST abbr. electroshock therapy , E.C.3.1.1-), aspartate aminotransferase aspartate aminotransferase n. Abbr. AST See SGOT. aspartate aminotransferase an enzyme that catalyzes the reversible transfer of an amino group: $$\eqalign $$ (AAT Alpha-1-antitrypsin (AAT) A blood component that breaks down infection-fighting enzymes such as elastase. Mentioned in: Chronic Obstructive Lung Disease , E.C.2.6.1.1), alcohol dehydrogenase alcohol dehydrogenase /al·co·hol de·hy·dro·gen·ase/ (ADH) (de-hi´dro-jen-as) an enzyme that catalyzes the reversible oxidation of primary or secondary alcohols to aldehydes; the reaction is the first step in the metabolism of alcohols by (ADH ADH: see antidiuretic hormone. , E.C.1.1.1.), malic enzyme malic enzyme an adaptive enzyme involved in lipogenesis. Catalyses the irreversible conversion of malate to pyruvate + CO2 in the cytosol. Called also the NADP-linked malate dehydrogenase. (M E, E.C.1.1.1.40), malate dehydrogenase malate dehydrogenase n. An enzyme that catalyzes, by means of NAD or NADP, the dehydrogenation of malate to oxaloacetate or the decarboxylation of maleate to pyruvate. (MDH MDH Minnesota Department of Health MDH Mälardalens Högskola (Swedish) MDH Malate Dehydrogenase MDH Manila Doctors' Hospital MDH Carbondale, IL, USA - Southern Illinois Airport (Airport Code) , E.C.1.1.1.37), sorbital dehydrogenase dehydrogenase /de·hy·dro·gen·ase/ (de-hi´dro-jen-as?) an enzyme that catalyzes the transfer of hydrogen or electrons from a donor, oxidizing it, to an acceptor, reducing it. de·hy·dro·gen·ase n. (SDH (Synchronous Digital Hierarchy) The European counterpart to SONET. See SONET. SDH - Synchronous Digital Hierarchy , E.C.1.1.1.14), isocitrate dehydrogenase i·so·cit·rate dehydrogenase n. Either of two enzymes that catalyze the oxidative decarboxylation of isocitrate during the Krebs cycle. isocitrate dehydrogenase see isocitrate dehydrogenase. (IDH IDH Isocitric Dehydrogenase IDH intradialytic hypotension IDH Instituto do Homem (Brasil) IDH Indirect Hire IDH Interface Design Handbook IDH Id Help , E.C. 1.1.1.42), and amylase amylase (ăm`əlās'), enzyme having physiological, commercial, and historical significance, also called diastase. It is found in both plants and animals. Amylase was purified (1835) from malt by Anselme Payen and Jean Persoz. (AMY A`my´ n. 1. A friend. ,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 allele (əlēl`): see genetics. allele Any one of two or more alternative forms of a gene that may occur alternatively at a given site on a chromosome. 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 A dendrogram is a tree diagram frequently used to illustrate the arrangement of the clusters produced by a clustering algorithm (see cluster analysis). Dendrograms are often used in computational biology to illustrate the clustering of genes. from the matrix of genetic distances was constructed using unweighted pair group method with arithmetic averaging (UPGMA UPGMA Unweighted Pair Group Method, Arithmetic Mean , 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 heterozygosity /het·ero·zy·gos·i·ty/ (het?er-o-zi-gos´i-te) the state of possessing different alleles at a given locus in regard to a given character.heterozy´gous het·er·o·zy·gos·i·ty n. 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 isozyme /iso·zyme/ (i´so-zim) one of the multiple forms in which an enzyme may exist in an organism or in different species, the various forms differing chemically, physically, or immunologically, but catalyzing the same reaction. 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 subspecies, also called race, a genetically distinct geographical subunit of a species. See also classification. , 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. 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Nei, M. 1987. Molecular evolutionary genetics. New York: Columbia University Press Columbia University Press is an academic press based in New York City and affiliated with Columbia University. It is currently directed by James D. Jordan (2004-present) and publishes titles in the humanities and sciences, including the fields of literary and cultural studies, . 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 ho·mo·zy·gos·i·ty n. 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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 The University of Chicago Press is the largest university press in the United States. It is operated by the University of Chicago and publishes a wide variety of academic titles, including The Chicago Manual of Style, dozens of academic journals, including . 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 dang interj. Used to express dissatisfaction or annoyance. adv. & adj. Damn. tr.v. danged, dang·ing, dangs To damn. n. 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 Chen - Peter 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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