RAPD analysis of genetic diversities of three species of abalone.

RAPD (random amplified polymorphic DNA) technique was applied to detect the genetic diversity in 3 species of abalone (Haliotis discus hannai Ino, Haliotis discus discus Reeve and Haliotis diverscolar Reeve). Twenty random primers, screened out of 100, gave 213 clear and informative fragments, ranging from 250-2600 bp, under predetermined optimal reaction conditions. There existed genetic diversity not only between species but also among individuals. The percentage of polymorphic loci, genetic heterozygosity, genetic identity, and genetic distance were calculated to reveal the diversities among species by using the Popgene32 software and a phylogenetic tree was constructed with the method of UPGMA. The results indicated that genetic variation in the population of H. discus discus is relatively high. H. discus hannai and H. discus discus showed a very close relationship with each other but a relatively distant relationship with H. diverscolar.

KEY WORDS: abalone, RAPD, genetic diversity

INTRODUCTION

RAPD marker can detect the insertion, loss of the binding sites distributed throughout the target genome with the differences of the amplified fragments. Compared with other molecular markers, such as RFLP, RAPD marker is simpler, lower time- and money-consuming and requires less DNA template, and doesn't need prior knowledge of DNA sequence. It has been widely used in genetic variability analysis, identification of relationship and genetic breeding and so on (Liu & Xiang 1996, Liu et al. 1996, Li & Zou 1999, Zhang et al. 2000) since it was established in 1990 (Williams et al. 1990, Welsh & Mccland 1990). In recent years, there were some successful reports in China and elsewhere, such as American system project of HHS (high health shrimp), oyster (Liu & Dai 1998), and shrimp (Song et al. 1998, 1999).

Abalone belongs to Mollusca, Gastropoda, Prosobranchia, Haliotidae, Haliotis. It is a precious marine shellfish with great value both as a health food and medicine, living on rocks and reefs at about 10 m depth from the low water mark. It is reported that there are seven species of abalone in China. Two of them have high economic value, namely Haliotis discus hannai Ino and Haliotis diverscolar Reeve. The former lives in the northern sea of China, Japan and Korea (Hou 1998), with that from Japan being regarded as a geographical subspecies of Haliotis discus hannai, named Haliotis discus discus. The latter is a warm sea species distributed in Fujian, Taiwan, Guangdong of China, and the south part of Japan. Previous studies mostly focused on ultrastructure (Liu et al. 2000, Ke et al. 2003, Cui et al. 2004) and diseases diagnosis (Li et al. 1998, Chert et al. 2000), but there are few systematic studies on the genetic diversity of abalone in China. In the present study, the RAPD technique was applied to assess the genetic diversity and the relative relationship of the three species of abalone to provide some potential theoretical reference for ecologic protection and reasonable exploration of the marine resources.

MATERIALS AND METHODS

Samples

Twelve Haliotis discus hannai Ino were collected from Zhangzi Island of Dalian, Liaoning Province (named population Z), and nine cultured Haliotis discus discus provided by the Key Laboratory of Marine Bioengineering in Ningbo University, Zhejiang Province (population B), and 20 cultured Haliotis diverscolar Reeve from Xiamen, Fujian province sea area (population F).

Random Primers

Ten-Mer random primers were bought from Shenggong Biotechnology Ltd. Shanghai Co., 20 primers were screened from 100, using 1 animal from each group (Table 1).

Instruments & Reagents

Amplifications were carried out on the Gene Amp PCR system made by PERKIN ELMER Analyze Co. in US. Cary 100 Cone U-visible Spectrophotometer was used to test the purity and concentration of DNA. FR-980 biologic electrophoresis test system was used to take photographs of gels.

Proteinase-k, Taq DNA polymerase, and dNTP were the products of TaKaRa Co. The other A.R grade reagents were bought from local Chemical Reagents Co.

DNA Extraction

One hundred milligrams of abdominal foot muscle fixed with alcohol was incubated at 55[degrees]C for about 4 h in microcentrifuge tubes containing TEN (10 mM Tris-HCl, [Na.sub.2]-EDTA, 0.15 M NaCl, pH 8.3), 2% SDS and 200 [micro]g Proteinase-K, then extracted with phenol/Chloroform (1:1), and then Chloroform: isopentanol (24:1). The supernatant fractions were mixed with two volumes of cold 100% alcohol and 1/10 volume of 3 M sodium acetate. The DNA quality was tested by running the samples in 0.7% agarose gel containing ethidium bromide (Fig. 1). Its purity and concentration were tested with Cary 100 Cone system (Li et al. 2003).

[FIGURE 1 OMITTED]

PCR Amplification

PCR amplification was performed in a 25 [micro]L reaction volume containing 40 ng genomic DNA, 0.2 mM dNTP at 200 [micro]M final concentration. 2.5 [micro]L 10 x PCR buffer, 40 ng primer. 2 mM Mg[Cl.sub.2], 2.0 U of Taq DNA polymerase and dd[H.sub.2]O. Amplification was carried out in the Gene Amp PCR System at predenaturation at 94[degrees]C for 5 min, then total 40 cycles were run as follows: denaturation at 94[degrees]C for 1 min, annealing at 37[degrees] for 1 min, extension at 72[degrees] for 1 min. After the final cycle, reaction mixtures were incubated for a further extension at 72[degrees] 10 min. The amplification products were run in 1.3% agarose gel containing ethidium bromide (0.3 [micro]g/mL) in TBE buffer (89 mmoL/LTris-Hcl, 89 mM boric acid, 10 mM EDTA, pH 8.3). The gels were electrophoresed at 40 V for -2 h and photographed.

Data Analysis

PCR amplified DNA bands were recorded as 1 for presence and 0 for absence. Using the "0, 1" matricies, the percentage of polymorphic loci (P), average genetic heterozygosity (H) and genetic distance (D) of three species of abalone were calculated using Popgen32 software:

P = polymorphic fragments/total amplified fragments

D = [-In.sup.[Jxy/(Jx-Jy]1/2] (Nei & Li 1979), [J.sub.xy] is the average genetic identity between population x and y. [J.sub.x] and [J.sub.y] are the average genetic identity of population x and y respectively.

H = [n.summation over i=1] (1 - [SIGMA][Xi.sub.2])/n

(Nei 1978), [x.sub.i] is the frequency of allele Xi, n is the number of tested loci.

Cluster analysis is based on D with the UPGMA (unweighted pair group method using arithmetic mean).

RESULTS

RAPD Analysis

The screened 20 primers yielded 213 bands ranging from 250-2600 bp and most of them were 500-2000 bp. Each primer gave 0-11 bands for each individual. Every individual had some different bands from the others. Population F had 113 polymorphic bands, Z 93, and B 107. The genetic diversity between Z and B was relatively small whereas there was obvious diversity among F, Z, and B. The band of 600 bp for S01, the band of 278 bp for S02, and the band of 1,400 bp for S14 were specific fragments of population F. S12-900 bp and S12-1400 bp bands were hardly found in Z and B populations, but both were present in F. (Fig. 2 and Fig. 3). It is clear that S1-600 and S14-1400 are polymorphic bands of population F and S6-800 bp is the specific band of Z and B population. S13-750 bp of B is much clearer than that of Z and S5-1500 bp only emerges in B population (Fig. 4).

[FIGURES 2-4 OMITTED]

The percentage of polymorphic loci and the average genetic heterozygosity of every population were calculated according to the statistics of the amplified bands (Table 2).

Genetic Distance & Dendrogram 

According to the Nei's expressions, we obtained the genetic distances and the genetic identities among the 3 populations (Table 3). The genetic distances between F and the other two species (0.2998 and 0.02880) are greater than that between Z and B (0.0411). Simultaneously, the genetic identities between Z and B was higher than that between F and Z or B. Based on the genetic distances, a phylogenetic tree was constructed with the UPGMA method (Fig. 5). It showed that Z had much closer relationship with B than with F.

[FIGURE 5 OMITTED]

DISCUSSION

The average genetic heterozygosities of populations F, Z, and B were 0.1683, 0.1557, 0.1860 respectively, and the percentages of polymorphic loci were 53.05%, 43.66%, and 48.98%. The average genetic heterozygosity was the average number of all amplified loci, and Nei and Li (1979) suggested that genetic heterozygosity was more appropriate for genetic variation. Among the three populations, Z had a relatively lower genetic diversity than B and F. One explanation for this might be that the samples of Zhangzi Island were cultured and therefore had a relatively high homozygosity. Initially the analyses of genetic diversities on marine mollusca were mostly done by means of enzyme technique (Li et al. 2002, Yang et al. 2000). Later, RAPD analysis became a dominant method due to the fact that the RAPD marker is more sensitive than enzyme analysis. Song et al. (1999) analyzed the genetic diversities of Penaeus japonicus and found that the percentage of its polymorphic loci was 54.14% and 0.2157 for the average genetic heterozygosity. Wang et al. (2001) concluded that the percentage of polymorphic loci of Pseudosciaena crocea was 18.90% and the average genetic heterozygosity was 0.096. The average genetic heterozygosity of Penaeus chinensis is 0.2176 (Shi et al. 1999). Compared with the results with other marine creatures, abalone has an accessible genetic variation. Genetic diversity lays the foundation for creatures to survive and adapt to changing environments. Dropping of genetic variation results in lost of the ability of creatures to adjust to the changing living conditions. Protecting biologic diversity is mainly protecting genetic diversity.

The dendrogram constructed on the basis of the genetic distance indicates the systematic relationships among the 3 populations of abalone. Z and B have a very close relationship and their distance is only 0.0411. The distances between F and Z or B are 0.2898 and 0.2880 respectively. According to the traditional classification, F and Z are 2 species of abalone, and they have large differences in many aspects such as morphology, growth environment and growth rate. Z and B are regarded as the same species, but different geographical subspecies, and they have similar morphology and living habits. Hence, the result of present RAPD analysis is in agreement with the traditional taxonomy, and it is also identical with Thorp's (1982) viewpoint: two populations whose genetic distance is bigger than 0.15 are different species, and the genetic identity between the same species ranges from 0.2 to 0.8.

The RAPD technique has been gradually used in detection of genetic variability, identification of systematic relationship, etc., due to its advantages: prior knowledge of the molecular biology (genetic background) of the investigated organisms is not required; the required primers are easy to obtain. Our study again suggests that RAPD is an efficient and sensitive genetic marker. However, RAPD also has its defects: poor repetition and artificial identification of the amplified bands. To get reliable results, it is very important to pay attention to the strictness and consistency of experiment conditions, including DNA extraction, concentration test, PCR reaction system establishment etc. If possible the manipulations for all samples to be compared should be done at the same time.

TABLE 1.

Random primer sequence.

Primer        Sequence        Primer        Sequence

 S01      5'-GTTTCGCTCC-3'     S18      5'-CCACAGCAGT-3'
 S02      TGATCCCTGG           S20      GGACCCTTAC
 S04      GGACTGGAGT           S21      CAGGCCCTTC
 S05      TGCGCCCTTC           S22      TGCCGAGCTG
 S06      TGCTCTACCC           S41      ACCGCGAAGG
 S12      CCTTGACGCA           S46      ACCTGAACGG
 S11      TTCCCCCGCT           S55      CATCCGTGCT
 S14      TCCGCTCTGG           S60      ACCCGGTCAC
 S15      GGAGGGTGTT           S73      AAGCCTCGTC
 S17      AGGGAACGAG           S81      CTACGGAGGA

TABLE 2.

Percentage of polymorphic loci of three populations and
mean heterozygosity.

                          No. of                 Percentage of
Population    Sample    Polymorphic               Polymorphic
   Name        Size        Loci        Mean H      Loci (%)

    F           20          113        0.1653        53.05
    Z           12           93        0.1557        43.66
    B            9          107        0.1560        50.33

TABLE 3.

The genetic distances and the genetic identities among populations.

Population
   Name         F         Z         B

    F          ****     0.7484    0.7498
    Z         0.2898     ****     0.9597
    B         0.2880    0.0411     ****

Genetic identity (above diagonal, Genetic distance (below diagonal)

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TAIWU LI, (1) WENXIN YANG, (2) XIURONG SU, (1) ZHIBIAO YANG, (1) AND HAO GUO (3)

(1) Faculty of Life Science and Biotechnology, Ningbo University, Ningbo, 315211, China; (2) Life Science College, Liaoning Normal University, Dalian, 116029, China; (3) National Marine Environment Monitoring Center, Dalian, 116023, China

* Corresponding author. E-mail: [e-mail]