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Genetic analysis of an artificially produced hybrid abalone (haliotis rufescens x haliotis discus hannai) in Chile.

ABSTRACT The Chilean abalone aquaculture industry is only supported with two species introduced during the late 1970s: red abalone Haliotis rufescens and Japanese abalone H. discus hannai. At the moment, red abalone accounts for 97% of total production due to its adaptability for full-cycle culture and faster growing than the Japanese species. However, Japanese abalone has a better acceptance and higher prices in Asian markets. These dualities have been merged by the successful hybridization between red and Japanese abalone. The goal of this study was to carry out a genetic analysis on red and Japanese abalone populations and their interspecific hybrids. Microsatellite markers were applied in three hatchery populations (HRed, HJap, and HHyb) to assess the genetic diversity and to certificate hybrid status. Allelic diversity was similar between HRed and HHyb populations (12 alleles), whereas for HJap it was significantly lower (7.8 alleles). Mean observed and expected heterozygosity (Ho, He) were 0.533 (0.045) and 0.786 (0.031), with no significant differences among populations (P [less than or equal to] 0.05). In most cases, Ho values were lower than He, indicating significant deviations from Hardy-Weinberg equilibrium. Comparison among populations showed that the hybrids are comparatively more similar to H. rufescens than H. discus hannai. Furthermore, hybrid status was confirmed by the presence of species-specific bands for each parental species of microsatellite locus Hco97. This work is the first approach to characterize genetically hybrids of H. rufescens x H. discus hannai produced in Chile.

KEY WORDS: abalone, Haliotis rufescens, Haliotis discus hannai, hybridization, microsatellites

INTRODUCTION

Currently, an alternative to conventional selective breeding methods of economically important aquaculture species is the production of intra- and interspecific hybrids, which have desirable phenotypic qualities to improve commercial traits (Chevassus 1983, Gaffney & Allen 1993, Elliott 2000). Hybridization has been successfully obtained in many species such as carp, catfish, salmonids, sparids, sunfish, oyster, and crayfish, among others, as a means of improving production traits like growth, survival, thermal tolerance, and disease resistance, as well as of manipulating sex ratios and producing sterile individual (Bartley et al. 2001, Hulata 2001). However there is not a general trend in hybridization results. Some hybrids can exhibit positive heterosis (hybrid vigor) in one or several quantitative traits, whereas others can present negative heterosis (Hulata 2001, Guo 2009). Nonetheless, even in the absence of hybrid vigor, hybridization can be used to express several trait combinations from either species or strains and improves characteristics such as texture, color, and taste (Elliott 2000). In worldwide abalone aquaculture, more than 50 crosses between simpatric and/ or allopatric species have been reported to be successfully produced; and most of the hybrid abalone evaluated have shown faster growth, high survival rate, particular adaptations to cultured conditions, and desirable market qualities (Lafarga-de la Cruz & Gallardo-Escfirate 2010). Despite the potential of hybrid abalone, currently there are only a few that are commercially produced and sold by China and Australia. These hybrid abalone are, the Dalian-I hybrid abalone, an intraspecific cross between the Chinese and Japanese stocks of Haliotis discus hannai (Guo 2009); the black abalone, hybrid of H. discus hannai x H. discus discus (Zhang et al. 2004); and the tiger abalone, hybrid of H. rubra x H. laevigata (Hamilton et al. 2009a). However, at least 10 additional hybrid crosses have been tested for its commercial production in countries like Thailand, Philippines, and Australia, being these hybrid a potential way to diversify and improve the abalone aquaculture (SEAFDEC/AQD 2008, Encena 2009).

From a genetic point of view, genetic characterization of those hybrids has rarely been done. Herein, molecular tools such as microsatellite marker, Randomly Amplified Polymorphic DNA (RAPD), and Restriction Fragment Length Polymorphism (RFLP) analysis has been applied to understand the genetic basis of heterosis, and to evaluate genetic diversity and distances between the parental species and their interspecific hybrids (Kim et al. 2000, Wan et al. 2001, Wan et al. 2003, Wan et al. 2004, Liu et al. 2007, Luo et al. 2009a). In regard to cytogenetic studies in hybrid abalone, there are only a few published data, revealing no differences in karyological characteristics when the parental species have the same chromosome number and their cellular DNA content is similar (Arai et al. 1982, An et al. 2007). However, karyological studies at the larva level of hybrids between H. diversicolor (2n = 32) and H. discus hannai (2n = 36) showed chromosome aneuploidy (Cai et al. 2009). On the other hand, genetic certification of hybrid status has been carried out using immunological analysis (Meyer 1967), allozymes (Fujino et al. 1980, Sasaki et al. 1980, Brown 1995, Hoshikawa et al. 1998, Ahmed et al. 2008), microsatellite analysis (Evans et al. 2000, Cruz et al. 2005, Ibarra et al. 2005, Luo et al. 2009b), and other molecular markers such as RAPD (Marin et al. 2007), sequences of DNA satellites (Muchmore et al. 1998, Hernandez-Ibarra et al. 2008), Restriction Fragment Length Polymorphism (RFLP) of 16s and Cytochrome Oxidase I gene (COI)(Ahmed et al. 2008), and molecular cytogenetic techniques as Genomic In Situ Hybridization (GISH) (Hernandez-Ibarra et al. 2005).

Compared with other molecular markers, microsatellites provide a high level of polymorphism and allow the differentiation between populations, species, and individuals by subtle observed differences. Microsatellite DNA markers have been successfully applied for molecular identification of hybrid abalone (Evans et al. 2000, Cruz et al. 2005, Ibarra et al. 2005, Luo et al. 2009b) and to assess genetic diversity of cultured abalone (Norris et al. 1999, Was & Wenne 2002, Hara & Sekino 2005, Sekino et al. 2005, Hara & Sekino 2007,; Marchant et al. 2009; Slabbert et al. 2009). Because of their characteristics such as relatively high genome abundance, high mutation rate, polymorphism, codominant Mendelian inheritance, and high reproducibility, microsatellite markers have been applied to a broad range of studies (Liu & Cordes 2004). In contrast to the microsatellite isolation process to develop DNA markers for a single species, the conservation level of sequences that flank the microsatellite regions allows the use primer pairs among closely related species (heterologous primers) (Evans et al. 2001). Heterologous microsatellite markers involve PCR primers designed for target species that can be used to analyze polymorphism in other related species, which can reduce time and cost of analyses significantly (Zane et al. 2002).

The Chilean abalone aquaculture industry is supported by only 2 species introduced during the late 1970s: red abalone H. rufescens Swainson 1822 and Japanese abalone H. discus hannai Ino 1953 (Flores-Aguilar et al. 2007). Red abalone account for 97% (418.3 MT) of the total production because of its adaptability for full-cycle culture and faster growing than the Japanese species. To the contrary, Japanese abalone have not adapted well because of its minor resistance to the Chilean culture conditions (Enriquez & Villagran 2008). However, Japanese abalone have a better acceptance and command higher prices in Asian markets (Gordon & Cook 2004). These dualities have been merged by the successful hybridization between red and Japanese abalone, which is an exceptional opportunity to diversify the Chilean abalone aquaculture. The goal of this study was to carry out a genetic analysis on red and Japanese abalone populations and their interspecific hybrid H. rufescens x H. discus hannai using 10 microsatellite DNA markers to estimate their genetic structure and diversity. In addition, the capability of these microsatellite loci to certify the hybrid status was tested.

MATERIALS AND METHODS

Samples and DNA Extraction

Three cultured abalone populations were sampled for DNA: red abalone (HRed; n = 66), Japanese abalone (HJap; n = 51), and their hybrids (HHyb; n = 32) produced by in vitro fertilization of red abalone female gametes with Japanese abalone male gametes. From 1-y abalone hybrids, epipodial tentacles were collected and immediately stored in 100% ethanol until DNA extraction (Slabbert & Roodt-Wilding 2006). About 30 mg preserved tissue was used for DNA extraction with the ENZA Tissue DNA kit (Omega Bio-Tek, Norcross, GA) according to the manufacturer's instructions. DNA quantity and purity was measured with a spectrophotometer (model ND1000; NanoDrop Technologies), and quality was checked running agarose 1% gel electrophoresis.

Genotyping

Ten heterologous primers, previously described and developed for H. corrugata (Diaz-Viloria et al. 2008), H. kamtschatkana (Miller et al. 2001), and H. discus hannai (Sekino et al. 2005) that showed successful cross-amplification in red and Japanese abalone were used. From those, microsatellite loci Hka80 and Awb062 were applied as red and Japanese positive controls. PCRs and conditions were performed as described by Perone-Millar et al. (2008). PCR products were sequence with a 400HD-standard in ABI3730xl sequencer (Applied Biosystem) by Macrogen Inc. (Korea), and the allelic score was assigned using GeneMarker (version 1.75; Softgenetics). Possible genotyping errors associated with microsatellite analysis (stutter bands, null alleles, and large allele dropout) were tested with MICRO-CHECKER (version 2.3.3) (Oosterhout et al. 2004), and the frequency of the null allele was calculated for each locus using ML-NullFreq (Kalinowski & Taper 2006).

Genetic Diversity and Differentiation

Genetic variability standard statistics, number of alleles (N), allelic frequencies, observed and expected heterozygosity ([H.sub.o], [H.sub.e]) were calculated with ARLEQUIN (version 3.11) (Excoffier et al. 2005). Allelic richness ([R.sub.S]) and F statistics ([F.sub.ST] and [F.sub.IS]) were estimated with FSTAT (version 2.9.3) (Goudet 1995). For the latter, [R.sub.ST] values ([F.sub.ST] analogous) were calculated using RSTCALC (version 2.2) (Goodman 1997). The observed genotype frequencies in each cultured population at each locus were tested for conformity to Hardy-Weinberg equilibrium by the Markov chain exact test (the Markov chain parameters used were 100,000 steps and a dememorization of 1,000) in ARLEQUIN (version 3.11). Corrections of the significance level for multiple tests were performed using the sequential Bonferroni procedure (Rice 1989). Kruskal-Wallis nonparametric analysis (Statistica version 6.1; StatSoft Inc.) was performed to test differences in N, [H.sub.o], [H.sub.e], and [F.sub.IS] among hatchery populations.

Genetic Identification of Hybrid Abalone

The PCR products of all microsatellite loci were visualized through horizontal electrophoresis at 60 V for 80 min in 1.8% agarose gel (1X Tris base Boric acid EDTA buffer), staining with ethidium bromide and compared with a molecular weight marker of 50 bp (New England Biolabs). Microsatellite locus Hco97 showed clear cross-amplification and a distinctive allelic range between red and Japanese abalone. Thus, it was evaluated as a potential molecular marker for genetic certification in hybrid abalone from several batches. As controls, 2 species-specific microsatellite markers (one for each parental species) were tested to confirm the genetic contributions of both parents. In addition, allelic distributions were depicted and analyzed to evaluate the presence of species-specific allelic bands as potential molecular DNA markers.

RESULTS

Genetic Diversity and Differentiation

Genetic variability parameters such as number of alleles (N), allelic richness ([R.sub.s]), heterozygosity observed ([H.sub.o]) and expected ([H.sub.e]), null allele frequency (Null), and the P values for Hardy-Weinberg equilibrium at 10 microsatellite loci analyzed for each cultured abalone population are shown in Table 1. Allelic diversity values, such as mean N and [R.sub.s], were similar between HRed and HHyb abalone populations: a mean of 12.6 (range, 3-19) and 11.1 (range, 5-17), respectively. In contrast, the HJap population showed a significantly lower number of alleles, with only 7.8 (range, 5-12 alleles). Population mean [H.sub.o] for samples HRed, HJap, and HHyb were 0.579 (range, 0.288-0.848), 0.534 (range, 0.294-0.784), and 0.488 (range, 0.125-1.00) respectively; whereas mean [H.sub.e] values were 0.766 (range, 0.4160.889), 0.771 (range, 0.630-0.882), and 0.823 (range, 0.660-0.936). Nonsignificant differences (P [less than or equal to] 0.05) were observed among the 3 populations in either [H.sub.o] or [H.sub.e]. However, significant differences between [H.sub.o] and He for most of the population loci cases evaluated were observed (22 of 28, 78.5%; Table 1), resulting in deviations from the Hardy-Weinberg equilibrium with an evident heterozygote deficit. Heterozygosity deficiency can be caused not only by biological processes (inbreeding, nonrandom mating, natural selection, and Wahlund-type effects), but also by technical problems in microsatellite amplification, such as the presence of null alleles. To discard this, null allele frequency was estimated, and allelic frequencies were corrected (Kalinowski & Taper 2006). Most of the population loci showed a low, inferred null allelic frequency (Dakin & Avise 2004). Only 1 microsatellite locus (Hka56) showed a high null allelic frequency in all populations analyzed (0.26, 0.27, 0.23) as detected also in wild red abalone populations of California (Gruenthal et al. 2007). However, no significant differences were observed between original and corrected data (not shown).

Based on 8 of the common microsatellite loci amplified in all the abalone hatchery populations, the resulting median inbreeding coefficient ([F.sub.IS]) values were 0.026, 0.269, and 0.451 for HRed, HJap, and HHyb, respectively. [F.sub.IS] values were not significantly different among populations (P [less than or equal to] 0.05). On the other hand, fixation indexes ([F.sub.ST], [R.sub.ST]) among the 3 abalone populations showed significant differences (P [less than or equal to] 0.05; Table 2). Comparisons among purebred parental species, HRed, and HJap showed the greatest genetic differences (P [less than or equal to] 0.01), with values of 0.183 and 0.253 for [F.sub.ST] and [R.sub.ST], respectively. Differences between HHyb and their parents showed that the hybrids are comparatively more similar to the HRed ([F.sub.ST] = 0.099, [R.sub.ST] = 0.113) than the HJap ([F.sub.ST] = 0.132, [R.sub.ST] = 0.170) broodstock population.

Microsatellite Markers for Genetic Identification of Hybrid Abalone

Microsatellite locus Hco97 showed species-specific bands for parental species, ranging from 76-90 bp for Japanese abalone and 200-258 bp for red abalone (Table 1 and Fig. 1). From the original hybrid sample (n = 32) for this study, 75% (n = 24) of the individuals analyzed showed clearly to be heterozygous for this locus, with an allelic range of 78-250 bp (Fig. 1), and were used to calculate the genetic parameters presented previously. Of the rest of the individuals (n = 8) shown to be homozygous for this locus, 6 presented only the characteristic band for Japanese abalone (76-90 bp) and 2 showed only the red abalone band (200-258 bp). Hence, the 2 positive parental control microsatellite markers (Hka80, Awb036) were used to identify them. In this regard, 4 individuals were identified as hybrids (positive for both controls), 2 were identified as Japanese abalone (positive for Japanese, negative for red), and 2 were identified as red abalone (positive for red, negative for Japanese). Homozygosity observed in locus Hco97 for these 4 later identified hybrids could be related to the high presence of null alleles for this locus in red abalone (0.32) and/or short allelic dominance (Wattier et al. 1998). Moreover, we had tested microsatellite locus Hco97 in other hybrid batches from our laboratory and found that usually more than 85% were heterozygotes (visualized in 1.8% agarose gels for a quick diagnosis). However, unidentified individuals need to be tested with microsatellite loci Hka80 and A wb036 to confirm genetically both parental species' genetic material contribution. Another approach undertaken to confirm hybrid status of the 8 unidentified individuals was to analyze their allelic composition. First, we compared allelic distributions among the 3 populations to recognize the number of species-specific alleles and common alleles for red-hybrid, Japanese-hybrid, and red-Japanese-hybrid for each microsatellite loci (Table 3). Results showed that 41% of the alleles found in the hybrids are common with red abalone, whereas 15% is common with Japanese abalone, and 29% with both species (not discriminating alleles). The remaining 15% were found only in hybrids (rare alleles). Second, we assigned to each allele in all loci a color tag-red, green, or black--based on whether they share with red, Japanese, or both species, respectively. This analysis corroborated the results described earlier: 4 hybrids, 2 red abalone, and 2 Japanese abalone.

[FIGURE 1 OMITTED]

DISCUSSION

Because the domestication of abalone species is a long-term process, the possibilities to increase production through appropriate hybridization will benefit the world abalone industry, as has been done in countries like Australia and China, where 50% and 90% of abalone cultures are tiger hybrids and intraspecific H. discus hannai hybrids, respectively (Guo 2009). However, as an example, in Australia, where blacklip (H. rubra Leach), greenlip (H. laevigata Donovan), and their interspecific hybrids are the principal species farmed, there is only a limited understanding of their genetic architecture (Hamilton et al. 2009a). In addition, hybrid abalone present highly variable phenotypes, because new genetic combinations occur in the hybrids, and their phenotypes can exceed the range of phenotypes in their parental species (Leighton 2000). In this scenario, the maintenance of pure lines and the establishment of a hybrid abalone breeding strategy becomes a challenge (Guo 2009, Hamilton et al. 2009b). In this study, an artificial hybrid between H. rufescens and H. discus hannai was produced in Chile, and genetic diversity and relationship with parental species were evaluated by using 10 heterologous microsatellite markers. Four culture red abalone populations in Chile have been genetically characterized before using this set of microsatellite loci to evaluate the genetic variability of intra- and interpopulations (Lafarga-de la Cruz and Gallardo-Escfirate 2010). Genetic diversity parameters were similar among these culture populations, but they have showed lower variability compared with wild red abalone populations of California. The inbreeding coefficient ([F.sub.IS]) value showed a tendency to panmixia (0.040-0.084) and a slight tendency to endogamy (0.270-0.380), perhaps as a result of differences in breeding management practices among hatchery facilities. With regard to the fixation index value ([F.sub.ST], [R.sub.ST]), results showed small genetic differences among red abalone culture populations: no evidence of geographical patterns was obtained. However, HRed genetic diversity results found in this study are within the values reported earlier. HJap results were, to some extent, different from those found by Marchant et al. (2009) for a hatchery population of H. discus hannai that is maintained under a family-based breeding program. We found reduced mean [H.sub.o] (0.536 vs. 0.716), but similar mean [H.sub.e] (0.771 vs. 0.700) values, resulting in a mean [F.sub.IS] index value of 0.259 in our study compared with -0.023 in the study by Marchant et al. (2009). This shows that the applied breeding program has been successful in controlling and maintaining heterozygosity. Differences in genetic variability and the inbreeding coefficient ([F.sub.IS]) between the red and Japanese abalone populations found here are probably the result of the comparative lower broodstock effective number and/or the matting of closely related individuals used to produce the large number of seeds for grow-out used in abalone farms. Only 2 of 25 operating abalone farms maintained Japanese abalone stocks in reduced numbers for more than 30 y (Sergio Ubillo, pers. comm.). However, microsatellite analysis showed that hybrid progeny had intermediate genetic diversity, lower heterozygosity, and a higher inbreeding coefficient when compared with parental populations of red and Japanese abalone. Contrary to these results, RAPD and AFLP analysis of hybrid abalone between H. discus hannai and H. discus discus show an increased genetic diversity, lower similarity indexes, and higher heterozygosity values in both reciprocal crosses compared with parental species (Wan et al. 2001, Wan et al. 2004). In reference to our results, 29% of the alleles were common between both parental species and hybrids, accounting for a mean allelic frequency among loci of 89 [+ or -] 10% in the hybrid population. So, inheritance of common and high-frequency alleles can be causing the high levels of homozygosity and the inbreeding coefficient (0.451) observed in the HHyb population in the current study. Rare alleles found in hybrids account just for 15% and less than 20% of the total allelic frequency. Moreover, AFLP marker segregation distortions of 30-50% have been reported for intra-and interspecific hybrids, perhaps maybe associated with the incompatibility of genes between the populations of abalone crossed (Liu et al. 2007; Luo et al. 2009b).

The fixation index ([F.sub.ST] and [R.sub.ST]) among all abalone populations showed significant differences; they were higher than those observed between red and Japanese abalone (>0.25), whereas differences between HHyb and its parents showed that hybrids are comparatively more similar to purebred H. rufescens, which is supported by the percentage of alleles shared between the hybrids and its maternal (red abalone) and paternal (Japanese abalone) parents (41% and 15%, respectively). Maternal effects were also reflected on phenotypic traits, such as hybrid growth and survival rate, shell weight-to-shell length ratio, and behavioral characteristics in cultures similar to purebred H. rufescens (Lafarga-De la Cruz and Gallardo-Escarate 2010). However, other adaptability responses to environmental conditions observed in hybrids, such as broader tolerance to higher temperatures, resemble its paternal parent, H. discus hannai (Nunez-Acuna 2009). According to Wan et al. (2001, 2004), with both RAPD and AFLP analysis, genetic distances among reciprocal hybrid abalone between H. discus discus and H. discus hannai and both parents were not equal, but were more similar to H. discus discus, regardless of the maternal and paternal material taken into account. In contrast, Kim et al. (2000) reported different results when study the phylogenetic relationship of 6 Haliotis sp. distributed in Korea with RAPD analysis, demonstrating that the hybrid H. discus discus X H. discus hannai is clustered with its parental species, and the distance coefficient is less with the paternal species H. discus hannai. However, a positive higher heterosis rate for fertilization, hatching, growth, and survival were obtained using H. discus hannai as maternal material.

The identification of interspecific hybrids among Haliotidae species has been performed by several approximations, from allozymes to microsatellite markers (Hoshikawa et al. 1998, Evans et al. 2000, Cruz et al. 2005, Ibarra et al. 2005, Ahmed et al. 2008). For example, Hoshikawa et al. (1998) identified wild hybrids between H. discus hannai and H. kamtschatkana by allozyme analysis, finding that hybrid abalone have heterozygous genotypes for allozymes AAT, GPI-2, IDH-1, MDH-1, MDH-2, and ME. Likewise, presumed wild hybrids between H.fulgens and H. rufescens were recognized with the presence of heterozygous genotypes for GPI, MDH-1, MDH-2, and SOD, and using a single microsatellite locus (Ibarra et al. 2005). Furthermore, Ibarra et al. 2005 reported that the microsatellite loci Hka28 and Hfu260 were not useful for hybrid certification because both parental abalones species share microsatellite alleles, overlapping their genotypes. Beside, the results showed overlapping in locus Hka56 with the allele 125. However, different frequencies between the parental species were estimated (0.18 for H. fulgens and 0.05 for H. rufescens). Herein, abalone hybrids were confirmed according their allele frequency. The assignment hybrids in most of the cases were identical with allozyme and microsatellite, with the exception of 2 individuals that had microsatellite alleles not found in the parental species (rare alleles). In the current study, microsatellite locus Hco97 as well as the species-specific loci for each parental species (Hka80, Awb036) allow genetic identification of hybrids in a quick diagnosis. For certain diagnoses (in particular, with regard to questionable individuals), the rest of the markers tested here can be used based on an analysis of allelic frequency and the presence or absence of alleles in the proposed color tag matrix. In addition, another molecular approach based on PCR RFLP analysis of partial sequences of the vitelline envelope receptor for lysin gene can identify reciprocal hybrids between H. rufescens and H. discus hannai (in prep.). As reported by Matin et al. (2007), the specific DNA fragments obtained by RAPD analysis can be used to differentiate both species and could be applied to molecular identification of individuals with ambiguous morphology and/or presumably abalone hybrids. In addition, phenotypic characterization could be helpful to some extent in clarifying hybrid status in juvenile and adult abalone, because red and Japanese abalone have a very characteristic shell and epipodial color and structure (Hanh 1989, Leighton 2000).

The genetic analyses performed here allowed us to characterize interspecific hybrids between H. rufescens and H. discus hannai, and also to assess relationships with their parental species. Future studies will be carried out to characterize the interspecific hybrids from other genetic points of view, such as molecular cytogenetic expression analysis and characterization of specific genes with dominance in offspring hybrids.

ACKNOWLEDGMENTS

This work has been supported by grant FONDEF-D06i1027, CONACYT-Mexico scholarship number 117673/217652, and CONICTY-Chile funding.

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FABIOLA LAFARGA DE LA CRUZ, (1,2) GABRIEL AMAR-BASULTO, (1,2) MIGUEL ANGEL DEL RIO-PORTILLA (3) AND CRISTIAN GALLARDO-ESCARATE (1) *

(1) Laboratorio de Biotecnologia Acuicola, Departamento de Oceanografia, Facultad de Ciencias Naturales y Oceanograficas, Centro de Biotecnologia, Universidad de Concepcion, Casilla 160-C, Concepcion, Chile; (2) Facultad de Ciencias Agronomicas, Universidad de Chile. Santa Rosa 11315, La Pintana, Santiago, Chile; (3) Departamento de Acuicultura, Division de Oceanologia, CICESE, Carretera Tij-Eda Km 107, BC, Ensenada, Mexico

* Corresponding author. E-mail: crisgallardo@udec.cl
TABLE 1.
Number of alleles (N), allelic richness ([R.sub.s]), heterozygosity
observed ([H.sub.o]), heterozygosity expected ([H.sub.e]), null allele
frequency (Null) and P value for Hardy-Weinberg equilibrium at 10
microsatellite loci analyzed in cultured populations of Red abalone,
Japanese abalone and their hybrids.

         Genetic                    Hatchery Populations
         Variability
Locus    Indices            HRed            HJap             HHyb

Hka3     N (size, bp)   17 (257-317)     5 (243-331)     13 (263-322)
         [R.sub.s]         17.00            4.84            13.00
         [H.sub.o]          0.848           0.529            0.500
         [H.sub.e]          0.846           0.630            0.876
         Null               0.010           0.086            0.191
         HWE                0.144           0.000 *          0.000 *
         [F.sub.IS]        -0.003           0.160            0.434
Hka28    N (size, bp)   16 (176-232)     7 (156-214)     10 (176-222)
         [R.sub.s]         16.00            7.00            10.00
         [H.sub.o]          0.636           0.608            0.667
         [H.sub.e]          0.889           0.821            0.829
         Null               0.138           0.065            0.079
         HWE                0.000 *         0.000 *          0.007 *
         [F.sub.IS]         0.286           0.078            0.199
Hka40    N (size, bp)   14 (127-167)     7 (133-177)     16 (125-179)
         [R.sub.s]         14.00            6.98            16.00
         [H.sub.o]          0.803           0.353            0.667
         [H.sub.e]          0.830           0.708            0.936
         Null               0.029           0.186            0.126
         HWE                0.012 *         0.026 *          0.000 *
         [F.sub.IS]         0.032           0.459            0.290
Hka56    N (size, bp)   15 (97-139)      12 (93-143)      13 (97-139)
         [R.sub.s]         15.00           11.86            13.00
         [H.sub.o]          0.379           0.333            0.417
         [H.sub.e]          0.875           0.882            0.857
         Null               0.259           0.266            0.226
         HWE                0.000 *         0.000 *          0.000 *
         [F.sub.IS]         0.569           0.575            0.519
Hka80    N (size, bp)   14 (104-148)         ND          11 (110-162)
         [R.sub.s]         14.00             ND             11.00
         [H.sub.o]          0.500            ND              0.542
         [H.sub.e]          0.888            ND              0.754
         Null               0.026            ND              0.139
         HWE                0.000 *          ND              0.0l5 *
         [F.sub.IS]         0.439            ND              0.286
Awh026   N (size, bp)    7(153-193)      7(143-215)       8(153-193)
         [R.sub.s]          7.00            6.98             8.00
         [H.sub.o]          0.530           0.294            0.417
         [H.sub.e]          0.541           0.681            0.791
         Null               0.015           0.208            0.196
         HWE                0.392           0.000 *          0.000 *
         [F.sub.IS]         0.019           0.554            0.467
Awb033   N (size, bp)   8 (146-173)     10 (151-185)      8 (155-182)
         [R.sub.s]          8.00            9.74             8.00
         [H.sub.o]          0.756           0.490            0.208
         [H.sub.e]          0.733           0.830            0.798
         Null               0.000           0.158            0.353
         HWE                0.742           0.203            0.000 *
         [F.sub.IS]        -0.034           0.378            0.797
Awb041   N (size, bp)   3 (201-210)      7 (195-219)      5 (201-219)
         [R.sub.s]          3.00            6.98             5.00
         [H.sub.o]          0.470           0.706            0.125
         [H.sub.e]          0.416           0.799            0.660
         Null               0.000           0.068            0.317
         HWE                0.744           0.001 *          0.000 *
         [F.sub.IS]        -0.13            0.114            0.814
Awb062   N (size, bp)        ND          8 (244-271)     10 (244-289)
         [R.sub.s]           ND             8.00            10.00
         [H.sub.o]           ND             0.784            0.333
         [H.sub.e]           ND             0.820            0.827
         Null                ND             0.000            0.261
         HWE                 ND             0.002 *          0.000 *
         [F.sub.IS]          ND            -0.066            0.602
Hco97    N (size, bp)   19 (200-258)      7 (76-90)       17 (78-250)
         [R.sub.s]         19.00            6.82            17.00
         [H.sub.o]          0.288           0.706            1.000
         [H.sub.e]          0.873           0.769            0.902
         Null               0.322           0.053            0.000
         HWE                0.000 *         0.000 *          0.279
         [F.sub.IS]         0.672           0.083           -0.112
Mean     N                 12.56            7.78            11.10
         [R.sub.s]         12.56            7.69            11.10
         [H.sub.o]          0.579           0.534            0.488
         [H.sub.e]          0.766           0.771            0.823
         Null               0.089           0.121            0.189

* Significant statistical deviation of Hardy-Weinberg equilibrium
(P < 0.05). ND, not data.

TABLE 2.
Genetic differentiation analysis between pairs of 3 kinds of
hatchery abalone populations based on [F.sub.st] (below diagonal)
and [R.sub.st] (above diagonal) estimations.

           HRed         HJap         HHyb

HRed        --        0.253 *      0.113 *
HJap     0.l83 *         --        0.170 *
HHyb     0.099 *      0.132 *         --

* Significant at P < 0.05.

Wide significant levels were applied using the sequential Bonferroni
correction.

HRed, red abalone H. rufescens; HJap, Japanese abalone H. discus
hannai; HHyb, hatchery hybrids abalone H. rufescens x H. discus
hannai.

TABLE 3.
Analysis of common alleles found between hatchery population of
hybrid abalone H. rufescens x H. discus hannai (HHyb) and each
parental species (HRed, red abalone H. rufescens; HJap, Japanese
abalone H. discus hannai), and among the 3 kinds of abalone in
Chile.

                        No. of Alleles (N)

Locus      HRed         HJap        HHyb        [SIGMA]

Hka3       17            7           13           24
Hka28       7            7           10           21
Hka40       8           10           16           22
Hka56       3            7           13           22
Awb026     17            5            8           13
Awb033     16            7            8           12
Awb041     14            7            5            8
Hco97      15           12           17           29

Mean       12.1          7.8         11.3         19
SD          5.4          2.2          4.2          7.1
Minimum     3            5            5            8
Maximum    17           12           17           29

                       Common Alleles (N)

                                    HHyb
           HHyb         HHyb        HRed
Locus      HRed         HJap        HJap        [SIGMA]

Hka3        7           0            2             9
Hka28       4           0            4             8
Hka40       9           3            1            13
Hka56       3           0            7            10
Awb026      4           1            2             7
Awb033      2           2            4             8
Awb041      1           2            2             5
Hco97      10           4            0            14

Mean        5.0         1.5          2.8           9.3
SD          3.3         1.5          2.2           3.0
Minimum     1           0            0             5
Maximum    10           4            7            14

                          Common Alleles (%)

                                        HHyb
              HHyb         HHyb         HRed
Locus         HRed         HJap         HJap       [SIGMA]

Hka3           54            0           15           69
Hka28          40            0           40           80
Hka40          56           19            6           81
Hka56          23            0           54           77
Awb026         50           13           25           88
Awb033         25           25           50          100
Awb041         20           40           40          100
Hco97          59           24            0           82

Mean           41           15           29           85
SD             16           15           20           11
Minimum        20            0            0           69
Maximum        59           40           54          100
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Author:De La Cruz, Fabiola Lafarga; Amar-Basulto, Gabriel; Del Rio-Portilla, Miguel Angel; Gallardo-Escarat
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:3CHIL
Date:Nov 1, 2010
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