Genetic variations and divergence of two Haliotis species as revealed by AFLP analysis.

ABSTRACT Amplified fragment length polymorphism (AFLP) technology was used to reveal genetic variations and divergence of Haliotis discus and H. diversicolor, the two economically important abalone species in China. Three sets of selective primers were used for amplification, and 350 bands ranging in size from 80 bp to about 500 bp were scored. The mean percentages of polymorphic bands in the four taxa varied from 51.7% to 62.7%. Similarity indices and genetic distances between individuals of the same species were greater than 0.8 and smaller than 0.2, respectively, whereas the values among individuals of different species were smaller than 0.3 and greater than 1.2, respectively. AFLP profiles of the two abalone species were distinct and could be used for species identification. Specific AFLP bands were also identified for discriminating the two subspecies of H. discus. Yet no diagnostic bands were found to differentiate the two subspecies of H. diversicolor. The lack of genetic divergence between these two subspecies supports results from a parallel study based on nuclear and mitochondrial DNA sequence analyses. We suggest that the subspecies status of H. diversicolor diversicolor and H. diversicolor supertexta is questionable. Results of this study are applicable to resource management and future genetic improvement of the two abalone species in China.

KEY WORDS: AFLP, abalone, genetic variation, genetic divergence, Haliotis discus, Haliotis diversicolor, subspecies

INTRODUCTION

Haliotis discus Reeve, 1846 and Haliotis diversicolor Reeve, 1846 (Mollusca; Gastropoda; Orthogastropoda; Vetigastropoda; Haliotoidea; Haliotidae; Haliotis) are two economically important abalone species that are extensively cultured in China and Japan (Chen 1984). Based on morphologic characters, two subspecies have been identified from each species (Chen 1984, Nie 1989, Okutani 2001). They are H. discus discus Reeve, 1846 and H. discus hannai Ino, 1952, and H. diversicolor diversicolor Reeve, 1846 and H. diversicolor supertexta Lischke, 1871 (also named H. diversicolor aquatilis Reeve, 1846). However, analyses based on sequences of mitochondrial and nuclear DNA markers show that the subspecies of each species are not genetically distinct, although the difference between the two species is evident (Wang et al. 2004b, this issue). Because the taxonomic status of the subspecies of H. discus and H. diversicolor is controversial, we aim to further elucidate any genetic differences between these taxa using molecular markers with a higher resolving power.

AFLP is a powerful technique applicable for genomic DNA fingerprinting (Vos et al. 1995). It has a higher resolving power and sensitivity than RAPD analysis in revealing allelic polymorphism (Barker et al. 1999) and thus has been suggested to be one of the appropriate techniques for revealing the genetic variation within species. This technique has been widely applied in studies of genotyping, population differentiation, and genetic diversity for a wide variety of organisms (Liu et al. 1998, Lerceteau & Szmidt 1999, Mueller & Wolfenbarger 1999, Seki et al. 1999, Keiper & McConchie 2000, Wang et al. 2000). In the present study, the genetic variation and divergence within and between the two abalone species H. discus and H. diversicolor were investigated using AFLP technique. For breeding purpose and genetic resource management, it is essential to clarify the genetic variation and relationship of these two commercially important taxa.

MATERIALS AND METHODS

Sample Collection and DNA Extraction

Specimens of H. discus discus, H. discus hannai, H. diversicolor supertexta, were collected from abalone farms in Putian, Fujian Province (introduced from Japan), Qingdao, Shandong Province, and Dongshan, Fujian Province (introduced from Taiwan), China, respectively. Specimens of H. diversicolor diversicolor were collected from the wild stock in Sanya, Hainan Province. The specimens were either preserved in 95% ethanol or stored in -80[degrees]C prior to analysis. Total DNA was extracted from foot muscle (20-25 mg) using proteinase-K digestion and DNA binding columns (QIAGEN QIAamp DNA Mini Kit) according to the manufacturer's instructions. The quality of extracted DNA was assessed using 1.0% agarose gel electrophoresis, and the DNA concentration was measured with an UV spectrophotometer (Hitachi U-2001).

AFLP Reactions

Procedures and reagents for AFLP analysis were applied as described in Vos et al. (1995). AFLP banding patterns were visualized on 5% denaturing polyacrylamide gel using silver staining technique. DNA templates for AFLP reactions were generated by restriction digestion and ligation. About 100 ng of total DNA was digested with 5U of EcoRI and MseI (New England BIOLABS) in 1X NE buffer2 at 37[degrees]C for 2 h. The digested DNA fragments were ligated with 2.5 pmol of EcoRI and 25 pmol MseI adapters in a reaction mixture containing 0.25 mg BSA, 5 pmol ATP, 0.04U T4Dnase, and 10X NE buffer2 at 37[degrees]C for 6 h. The ligated DNA fragments were preamplified using a MJ Thermocycler (PTC-100) with a pair of primers containing a single selective nucleotide. The preamplification PCR reaction was conducted at an annealing temperature of 53[degrees]C for 30 sec. The 20-[micro]L PCR product mixture was diluted 10-fold with distilled water and used as templates for the subsequent selective PCR amplification. The selective amplification was performed using 3 pairs of primers (E-AAG/M-CGA, E-AGG/M-CTG and E-AGC/M-CTT).

Gel Electrophoresis and Silver Staining

After selective amplification, the PCR products were mixed with 10 [micro]L AFLP loading buffer (99% formamide, 10 mM EDTA, 0.05% bromophenol and 0.05% xylene cyanol). The product mixtures were denatured and concentrated at 90[degrees]C for 25 min, and cooled down in an ice bath immediately after denaturation. A 5% denaturing polyacrylamide gel (4.75% acrylamide, 0.25% bis acrylamide, 7 mol x [1.sup.-1] urea and 0.5X TBE) was prerun at 145 W for 30 min. Each well was loaded with 2.5 [micro]L of sample. The gel was electrophoresed in a BioRad Sequi-Gen GT DNA sequencing cell (38 x 50 cm) at 110 W and 50[degrees]C for 2.5 h.

Bands on the gel were visualized using silver staining method derived from Merril et al. (1979). After electrophoresis, the gel was fixed in 1% ethanoic acid for 30 min. The fixed gel was rinsed in distilled water and stained with a mixture of 0.2% silver nitrate and 0.007% benzene sulphonic acid for 30 min. The stained gel was rinsed again and immersed in a developing solution (2.5% sodium carbonate, 0.037% formaldehyde, 0.002% sodium thiosulphate). When the bands became clear, the development was stopped with 1% ethanoic acid. The bands were scored using the Gel Imaging Analyzing System (Kodak Digital Science, EDAS120) and the band sizes were estimated with a standard AFLP DNA ladder (Invitrogen, Life Technologies).

Data Analysis

AFLP bands were scored for presence (1) or absence (0), and transformed into 0/1 binary character matrix. Based on the binary matrix, genotypes, percentage of polymorphic bands, similarity indices, and genetic distance were obtained. Similarity indices were calculated using the formula S = 2[N.sub.ab]/([N.sub.a] + [N.sub.b]) (Nei & Li 1979), where [N.sub.a] and [N.sub.b] are the number of bands in individuals a and b, respectively and [N.sub.ab] is the number of shared bands. Genetic distances were computed using the formula D = -ln S (Nei & Li 1979). For the calculation of the above values, we have developed a Microsoft Excel-based BASIC program (AFLP Data Analyser v1.3). Cluster analysis based on UPGMA was conducted using the software MEGA 2 (Kumar et al. 2001) based on the distance matrix.

RESULTS

AFLP was performed using three pairs of selective primers on a total of 38 individuals belonged to four abalone taxa. A total of 350 different bands ranged from 80 to 500 bp in size were scored. The number of bands generated from a single individual varied from 116 to 163. Because no two individuals shared exactly the same banding pattern, the number of genotypes identified was the same to the number of individuals used in the study (Table 1). The number of bands generated from each taxon varied from 195 to 231 and the percentage of polymorphism ranged from 51.7% (in H. discus hannai) to 62.7% (in H. discus discus).

A representative AFLP fingerprint from the amplification using one of the primer sets (Fig. 1) shows that the fingerprint was quite similar among individuals of the same species but distinct between different species. Among the 350 bands scored, only 10 bands (2.9%) were shared among all individuals of the four taxa. There were 79 bands common to all individuals of H. discus, constituting 32% of the total number of bands scored in this species. On the other hand, 77 bands were common to all H. diversicolor, accounting for 36% of the total number of bands scored in this species. There were 41 bands specific to all individuals of H. discus but not found in H. diversicolor, and 34 bands were specific to H. diversicolor. Some of these bands are indicated in Figure 1. These species-specific bands could be used for the development of molecular markers applicable in the identification of these two species of abalone. Six diagnostic bands were also identified between H. discus discus and H. discus hannai, with three specific to each of the taxa. Yet for H. diversicolor, no bands specific to each of the subspecies could be identified.

[FIGURE 1 OMITTED]

The similarity indices and genetic distances within or between the four taxa of abalone based on AFLP banding patterns are shown in Table 2. The similarity indices between individuals of the two abalone species were all lower than 0.3, with genetic distances greater than 1.3. The mean similarity index between individuals of H. discus discus and H. discus hannai (0.78) was considerably lower than those among individuals of the same subspecies. It is evident that the two subspecies of H. discus are genetically distinct. However, the similarity index and genetic distance between individuals of H. diversicolor diversicolor and H. diversicolor supertexta were very similar to values among individuals of the same subspecies, suggesting a lack of genetic divergence between these two taxa.

A phylogenetic tree of the 38 individuals of the four abalone taxa was constructed using UPGMA method (Fig. 2). Individuals of H. discus discus and H. discus hannai were separated into two distinct clusters but individuals of H. diversicolor diversicolor or H. diversicolor supertexta did not cluster together.

[FIGURE 2 OMITTED]

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 & Szmidt 1999, Mueller & Wolfenbarger 1999, Seki et al. 1999, Keiper & McConchie 2000, Wang et al. 2000, Nicholas et al. 2001, Ogden & Thorpe 2002). In the present study, we identified 195 to 231 different bands from a single abalone taxon by using three primer sets in the four taxa. Each primer set generates 65 to 77 bands from a single taxon, with polymorphism higher than 50%. Further, AFLP patterns of the individuals differed from each other, and the pattern from a single primer set could be used to distinguish all the individuals from the same taxon. Previous studies, based on analyses of allozymes and RFLP of mtDNA, suggest low genetic diversity in abalone populations, particularly the cultured stocks (Fujino 1978, Smith & Conroy 1992). No genetic variations were found in H. discus hannai, H. discus discus, and H. diversicolor supertexta in a recent study in our laboratory using 12 allozyme loci and 15 individuals of each taxon (Ke et al. 2003). Yet, more than half of the AFLP bands scored in the present study were found to be polymorphic (the same specimens of H. discus discus and H. diversicolor supertexta were used in both studies), indicating AFLP as a more effective approach than allozyme analysis in detecting genetic diversity in abalone. Moreover, the results suggest that there are no differences in genetic diversity between the wild stock of H. diversicolor diversicolor and the cultured stocks of the other three taxa. It is difficult to distinguish whether the wild stock of H. diversicolor diversicolor exhibits low genetic diversity due to over-fishing, or the cultured stocks of the other taxa do not suffer from a reduction of genetic diversity. Further studies on genetic differentiation among abalone populations are needed to understand the extent of genetic polymorphism in wild stocks, as well as effect of aquaculture on genetic diversity of abalone. Nevertheless, the present study establishes AFLP as the appropriate method in elucidating and monitoring genetic polymorphism in abalone.

AFLP can be applicable in discriminating different species or subspecies but also elucidating their evolutionary relationships (Tsoi et al. 2003, Wang et al. 2004a). The present study shows that the difference in AFLP banding pattern between H. discus and H. diversicolor is apparent, giving a genetic distance of higher than 1.3 between the two species. Our phylogenetic analysis also shows that the individuals of these two taxa are clearly separated into two clusters. The results are consistent with the analyses based on morphologic traits or mitochondrial DNA (Wang et al., 2004b, this issue), thus indicating AFLP technique as a potentially useful method for species identification and phylogenetic analysis among species. We have isolated some of the AFLP bands specific to a particular abalone taxon for sequencing analysis and the development of sequence tagged sites (STS) markers. These markers will be most useful for the genetic identification and breeding studies of the abalone taxa under study.

Haliotis discus discus and H. discus hannai, and H. diversicolor diversicolor and H. diversicolor supertexta have been designated as subspecies of their respective species based on morphologic traits (Chert 1984, Nie 1989, Okutani 2001). Results from the present study show that AFLP banding patterns of H. diversicolor diversicolor and H. diversicolor supertexta are similar. No bands specific to each taxon could be identified. The genetic distance between these two taxa is similar to that within each taxon. Phylogenetic analysis shows that individuals within each subspecies do not cluster together, suggesting H. diversicolor diversicolor and H. diversicolor supertexta are genetically similar. Recent studies in our laboratory on these two taxa using eight additional primer sets also support results from the present study using three primer sets. A parallel study on the sequence analysis of nuclear and mitochondrial (mt) genes (18S rRNA, ITS-1, mt 16S rRNA, and cytochrome oxidase I) also shows a lack of divergence between H. diversicolor diversicolor and H. diversicolor supertexta (Wang et al. 2004b, this issue). Investigations based on morphologic traits and allozymes also cannot establish differences between these two taxa (Lu 1978, Ke et al. 2003 and unpublished data).

In contrast to our AFLP data on H. diverscolor, AFLP analysis on the two subspecies of H. discus show that they are genetically distinct. Diagnostic bands could be identified from both Haliotis discus discus and H. discus hannai, and genetic distance among subspecies is higher than values within subspecies. Phylogenetic analysis also demonstrates that individuals of these two subspecies are separated into two different clusters. Yet it should be noted that nuclear and mtDNA sequence analyses failed to distinguish these two subspecies (Wang et al. 2004b, this issue), showing that AFLP is a more sensitive assay for discriminating genetic divergence in closely related taxa. Taken together, results from the present study suggest that because the subspecies status of Haliotis discus discus and H. discus hannai may be warranted, the distinction between H. diversicolor diversicolor and H. diversicolor supertexta is put into question. The latter conclusion supports Geiger's (2000) assertion that the two subspecies of H. diversicolor are synonymous. Yet Hara and Fijio (1992) also argued that divergence of the two subspecies of H. discus only represents population differences. It should be pointed out that only a single population from each of the four abalone taxa was included in the present study. A more extensive study based on population aggregation analysis (Davis & Nixon 1992) over the geographical range of these abalone taxa is necessary to establish the presence or absence of genetic divergence of the subspecies in question.

ACKNOWLEDGMENTS

The authors thank Dr. Fuhua Li (Institute of Oceanology, Chinese Academy of Sciences) for collecting the specimens of Haliotis discus hannai used in this study. The work described in this article was fully supported by grants from Hi-Tech Research and Development (863) Program of China (Project nos. 2001AA620108 & 2003AA603240, and an AoE Fund from The Chinese University of Hong Kong.

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