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Latin american journal of aquatic research

versión On-line ISSN 0718-560X

Lat. Am. J. Aquat. Res. vol.40 no.4 Valparaíso nov. 2012

http://dx.doi.org/10.3856/vol40-issue4-fulltext-23 

Lat. Am. J. Aquat. Res., 40(4): 1077-1084, 2012

Short Communication

Genetic composition of Mytilus species in mussel populations from southern Chile

Composición genética de especies de Mytilus en poblaciones de mejillón del sur de Chile

 

María Angélica Larraín1, Nelson F. Díaz2, Carmen Lamas2 Carlos Vargas2 & Cristián Araneda2

1Departamento de Ciencia de los Alimentos y Tecnología Química Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile Vicuna Mackenna 20, Santiago, Chile.
2Departamento de Producción Animal, Facultad de Ciencias Agronómicas P.O. Box 882 08 08, Santiago, Chile
.


ABSTRACT. Mussels are one of the most cultivated and commercialized bivalves worldwide and in southern Chile its culture represent an important economic activity. The species identification within the Mytilus genera, by morphological features, is unreliable, so we used a polymorphism RFLP in the gene encoding the polyphenolic adhesive protein as a species-specific genetic marker to describe Mytilus species diversity in southern Chile, and evaluate possible applications in traceability, food quality and safety. Using Me 15-16 marker most mussels were M. chilensis, finding no other pure individuals; however, putative hybrids of M. chilensis x M. trossulus and M. chilensis x M. galloprovincialis were detected. There was no evidence of M. edulis. The presence of the M. trossulus allele, faraway from its distribution area, demands further analysis with different genetic markers to allow a better understanding of its origin. In addition, the correspondence between markers that distinguishes northern from southern hemisphere M. galloprovincialis, with those who discriminates between M. chilensis and M. galloprovincialis would contribute to the taxonomic status of Chilean blue mussels. In Chile, the genetic composition of Mytilus indicates that geographical origin of mussels and its traceability cannot be established merely from the identification of the species. The use of other markers would be required.

Keywords: Mytilus, mussels, genetic identification, Me 15-16, PCR-RFLP, southern Chile.


RESUMEN. Los mejillones son una de las especies de bivalvos más cultivadas y comercializadas, en el sur de Chile donde su cultivo representa una actividad económica importante. La identificación de la especie dentro del género Mytilus, basada en las características morfológicas no es confiable por lo que se utilizó un polimorfismo RFLP en el gen que codifica la proteína adhesiva polifenólica como marcador genético específico de la especie para describir la diversidad de especies Mytilus en el sur de Chile, y evaluar posibles aplicaciones en trazabilidad, calidad e inocuidad de los alimentos. Usando el marcador Me 15-16, la mayoría de los mejillones fueron M. chilensis, no se encontraron individuos puros de otras especies. Sin embargo, se detectaron híbridos putativos M. chilensis x M. trossulus y M. chilensis x M. galloprovincialis. No se encontró evidencia de M. edulis. La presencia del alelo de M. trossulus, lejos de su área de distribución, requiere de análisis posteriores con diferentes marcadores genéticos para entender su origen. Además, conocer la correspondencia entre marcadores que distinguen M. galloprovincialis del hemisferio norte y sur con aquellos que discriminan entre M. chilensis y M. galloprovincialis contribuirá al estatus taxonómico del mítilido chileno. La composición genética de Mytilus en Chile no permite establecer su origen geográfico ni la trazabilidad, basándose solo en la identificación de la especie y se requiere el uso de otros marcadores.

Palabras clave: Mytilus, mejillones, identificación de especies, Me 15-16, PCR-RFLP, sur de Chile.


 

Marine mussels are one of the most cultivated and marketed bivalves, being the genus Mytilus widely used in prepared food products. There are various Mytilus species at higher latitudes in all the oceans and major seas of the world, in temperate zones of both hemispheres, but not reported in equatorial regions (Hilbish et al., 2000). In addition, many genetic studies describe their ability to generate hybrid individuals when two species are in contact (Dias et al., 2009; Kijewski et al., 2009; Gardner & Westfall, 2012). Mussels develop for weeks, even months, in planktonic larval stages and thereby potentially dispersed over large geographical areas by marine currents or human-mediated activities (Hilbish et al., 2002; Toro et al., 2004). Biochemical and molecular characterization divided the morphologically classified Mytilus edulis Linnaeus, 1758 into a complex of three sibling species termed the Mytilus edulis species complex: M. edulis, M. trossulus Gould, 1850 and M. galloprovincialis Lamarck, 1819 (Westfall & Gardner, 2010). The genetic composition of these three taxa, now globally well defined, in the northern hemisphere (Wonham, 2004; Gérard et al., 2008); in contrast, in southern hemisphere the composition is still under analysis.

The taxonomic status of Chilean blue mussel M. chilensis has been controversial for decades because of its phenotypic and genetic proximity to other species of the genus Mytilus from both hemispheres. Toro (1998), suggested the taxonomical status of M. edulis chilensis; then, Cárcamo et al. (2005) proposed M. galloprovincialis chilensis based on 30 allozyme loci. Current reports suggest that M. chilensis is a valid, distinct species within the genus Mytilus. Ouagajjou et al. (2011 based on nine microsatellite developed for M. chilensis, suggest that this is a distinct valid species within the genus, but recently other authors (Borsa et al., 2012) proposed that native Chilean smooth-shelled Mytilus, should be named M. edulisplatensis D'Orbigny, 1846.

In southern Chile, mussel culture is an important economic activity that yielded a 12.1% of the world's mitylid production in 2009 (FAO, 2011). Irrespective of the taxonomic status of Chilean mussels, regulatory and commercial interest to differentiate them from northern hemisphere M. galloprovincialis is a major concern to prevent improperly labeling [Regulations (CE)104/2000 and 2065/2001] ENREF 20, to protect consumers rights, to achieve food traceability, and to fulfill other quality objectives, such as designation of origin. To differentiate both species, Santaclara et al. (2006) developed a PCR-RFLP assay in polyphenolic adhesive protein cutting only M. galloprovincialis PCR product into two, allowing such discrimination.

The interest in this differentiation has led to the recent developing of alternative PCR-RFLP methods for mussel Mytilus species identification (Fernández-Tajes et al., 2011).

The objective of this study was to identify the species of Mytilus individuals using polyphenolic adhesive protein locus and RFLP-Aci I to describe Mytilus species diversity in southern Chile and assess potential applications in traceability and food quality and safety.

Samples of mussels (n = 50) were collected in southern Chile covering 11 sites from 41°31'S, 72°20'W to 50°50'S, 74°00'W (Fig. 1): one wild population, five seed collection centers and five on-growing centers, these last ones were supplied with seed from three collection centers. The samples were collected sub tidally. Shell size of 15-25 mm was considered seed and higher sizes as adult (Table 1). Control samples of M. chilensis and M. gallopro-vincialis were obtained from the hatchery of Universidad de Concepción in Dichato and commercial samples of M. galloprovincialis from Galicia (Spain). Up to 24 h after collection, mussels were dissected and a small piece of mantle edge tissue (approximately 200 mg) was removed, placed in a 1.5 mL Eppendorf tube, fixed with 95% ethanol and stored at -20°C. DNA extraction was based on the simplified protocol of Taggart et al. (1992), approximately 50-100 mg tissue was coarsely chopped and digested overnight in 385 μL of lysis buffer (50 mM Tris HCl pH 8.0; 100 mM EDTA (ethylendiaminetetraacetic acid) pH 8.0; 100 mM NaCl; 1% SDS (sodium dodecyl sulphate) with 10 μL proteinase K (US Biologicals®) at 37°C. The solution was digested for 1 h with 200 μg RNAse (US Biologicals®) at 37°C. A precipitation step with 130 μL of saturated NaCl solution (6.1 M) followed by centrifugation (13,400 g x 10 min) was performed to remove mucopolysaccharides. The solution transferred to a clean tube, and 400 μL phenol was added and vigorously stirred for 10 s and then gently stirred during 20 min afterwards, 400 μL of chloroform-isoamyl alcohol (24:1) was added following the same agitation procedure. After centrifugation (9,300 g x 5 min), 380 μL of upper phase was taken to a clean tube and was ethanol precipitated. DNA extracted was resuspended in 100 μL of TE buffer (10 mM Tris, 1 mM EDTA; pH 8.0) until complete dissolution. DNA integrity verified by 0.7% agarose gel electrophoresis, showed no degradation. Concentration and purity controls were done in spectrophotometer (NanoDrop, ND-1000), using samples with absorbance ratio 260 280-1 nm higher than 1.5. Concentration was adjusted to 20 ng μυ1 with 0.1 TE buffer. All DNA samples were stored at -18°C.


Figure 1. Location of sampling sites in southern Chile. Allele composition of Mytilus samples are shown as frequencies of alleles for the nuclear DNA marker Me 15-16 - RFLP Aci I.

Figura 1. Ubicación de los sitios de muestreo en el sur de Chile. Composición de alelos de las muestras de Mytilus se muestran como frecuencia de alelos para el marcador de ADN nuclear Me 15-16 - RFLP Aci I.

To prevent in the samples the unintended presence of individuals from other genera inhabiting the same geographical region -Cholga (Aulacomya ater) and Choro zapato (Choromytilus chorus)- a genus assignment was made. PCR-RFLP technique was accomplished according to Santaclara et al. (2006), using MusRFLP F and R primers to amplify a fragment of the gene encoding the small subunit rDNA (18S rDNA) and later digestion with endonuclease Bsa HI (New England Biolabs®).


Species identification was performed by PCR using the primers Me 15-16 of Inoue et al. (1995). RFLP-Aci I (Fermentas®) was used to differentiate between M. galloprovincialis and M. chilensis (Santaclara et al., 2006). Each PCR was undertaken in 25 μL. A negative control without DNA template added and a positive control with DNA from control samples of M. chilensis and M. galloprovincialis were included in all PCR assays.

Currently DNA amplifications were performed in a thermocycler Techne TC-412 (Bibby Scientific Ltd®, UK), with high quality recombinant Taq DNA polymerase (RBC Bioscience®), and PCR-grade water. PCR products were visualized in agarose gels (1.8%) in TBE buffer with 10 mg mL-1 of ethidium bromide for band detection under ultraviolet light and in Polyacrylamide gels (8%), with silver staining. For every gel, the size of amplified fragments was estimated from 10 bp DNA ladder (Invitrogen®) or Hyperladder V (BioLine®).

Amplicons of 168 bp and 126 bp from CB-Canal Coldita-Piedra Blanca, were isolated from the gel, reamplified, purified with FavorPrep® Gel purification kit (Favorgen®) and sent to be sequenced in both directions with primers Me 15-16. Consensus sequences were obtained using Multalin (http://multalin.toulouse.inra.fr/multalin/multalin.html), and in order to verify their identity, alignments with sequences of polyphenolic adhesive protein of M. trossulus (DQ640589.1) and Mytilus sp. (AF489933.1) were performed with BLAST (bl2seq) (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi).

Scoring with the Me 15-16 primers was possible for all 550 Mytilus genera individuals sampled from the 11 localities. After the RFLP-Aci I digestion of all individuals showing a single 126 bp PCR product, M. chilensis alleles were the most (98-100%) in all collection places, as expected from this native species in commercial production in Chile. In six sites (IP-Isla Peel, PI-Pichicolo, CN-Canutillar, CP-Canal Coldita-Patagonia, EC-Estero Chauquiar y EH-Estero Chope) only M. chilensis alleles (one fragment 126 bp and no restriction site for Aci I) were found. Only homozygous individuals were identified for M. chilensis alleles, no other Mytilus homozygous were found in this study (Table 2).


Table 2. Number of individuals of each genotype -Mytilus chilensis (Mch), M. chilensis x M. galloprovincialis apparent hybrids (Mch x Mg), M. chilensis x M. trossulus apparent hybrids (Mch x Mt)- found in samples (n = 50) from the 11 sites studied.

Tabla 2. Número de individuos de cada genotipo -Mytilus chilensis (Mch), hibridos aparentes M. chilensis x M. galloprovincialis (Mch x Mg), híbridos aparentes M. chilensis x M. trossulus (Mch x Mt)- encontrados en las muestras (n = 50) de los 11 lugares estudiados.


M. galloprovincialis allele (126 bp and restriction site for Aci I) was identified only in PA-Piedra Azul c o l lection center, in a single individual (Figs. 2 and 3) that after the digestion showed three fragments of 126, 75 and 51 bp and was considered a putative hybrid M. chilensis x M. galloprovincialis. The same restriction pattern size (75 and 51 bp) was found in control samples of M. galloprovincialis from Dichato-Chile, and commercial samples from 0alicia-Spain (Fig. 3). The difference in the restriction fragment size described by Santaclara et al. (2006) and the one observed in our study was probably due to the different methods employed to estimate size (sequencing vs 8% polyacrylamide gel electro-phoresis). Pure M. galloprovincialis individuals were not found in the studied area, as was the case in Scottish shellfish farms and in the Oostershelde estuary in the Netherlands (Dias et al., 2009; Kijewski et al., 2009). The low frequency of the M. gallopro-vincialis allele found in our survey and the limited sample size obtained in each location (n = 50), could cause underreport of M. galloprovincialis in southern Chile. There might be a wider distribution of this species within the sampled locations.



Figure 2. Polyacrylamide gel (8%) with Me 15-16 PCR products: 1. Molecular size standard (25 bp) Lanes 2-6. Control individuals, 2. M. chilensis from Dichato-Chile 3-4. M. galloprovincialis from Dichato-Chile 5-6. M. galloprovincialis from Galicia-Spain 7. Heterozygous mussel with 126 bp and 180 bp alleles from Galicia-Spain 8-11 and 13-16. Heterozygous mussel with 126 bp and 168 bp allele from southern Chile 12. Heterozygous mussel with 126 bp allele and restriction site for Aci I from southern Chile 17. Molecular size standard (10 bp).

Figura 2. Gel de poliacrilamida (8%) con los productos de PCR Me 15-16: 1. Estándar de tamańo molecular (25 pb) Lineas 2-6. Individuos control 2. M. chilensis de Dichato-Chile 3-4. M. galloprovincialis de Dichato-Chile 5-6. M. galloprovincialis de Galicia-Espańa 7. Mejillón heterocigoto de Galicia-Espańa con alelos de 126 pb y 180 pb 8-11 and 13-16. Mejillones heterocigotos del sur de Chile con alelos de 126 pb y 168 pb 12. Mejillón heterocigoto del sur de Chile con alelo de 126 pb y sitio de restricción para Aci I 17. Estándar de tamańo molecular (10 pb).



Figure 3. Polyacrylamide gel (8%) of PCR Me 15-16 -RFLP Aci I analysis: Lanes 1-4. Control individuals 1. M.chilensis from Dichato-Chile 2. M. galloprovincialis from Dichato-Chile 3. M. galloprovincialis from Galicia-Spain 4. Heterozygous mussel with 126 bp and 180 bp alleles from Galicia-Spain 5-7. Mussels with 126 bp allele from southern Chile 8. Molecular size standard (10 bp). 9-16. After digestion with Aci I. Lanes 9-12. Control individuals 9. M. chilensis from Dichato-Chile 10. M. galloprovincialis from Dichato-Chile 11. M. galloprovincialis from Galicia-Spain 12. Heterozygous mussel with 126 bp and 180 bp alleles from Galicia-Spain 13 and 15. Mussels with 126 bp allele from southern Chile 14. Heterozygous mussel with 126 bp allele and restriction site for Ac iI from southern Chile 16. Molecular size standard (25 bp).

Figure 3. Gel de polyacrylamida (8%) del análisis PCR Me 15-16 -RFLP Aci I: Lineas 1-4. Individuos control 1. Mytilus chilensis de Dichato-Chile 2. M. galloprovincialis de Dichato-Chile 3. M. galloprovincialis de Galicia-Espańa 4. Mejillón heterocigoto de Galicia-Espańa con alelos 126 pb y 180 pb 5-7. Mejillones del sur de Chile con alelos 126 pb 8. Estándar de tamańo molecular (10 pb). 9-16. Después de digestión con Aci I. Lineas 9-12. Individuos control 9. M. chilensis de Dichato-Chile 10. M. galloprovincialis de Dichato-Chile 11. M. galloprovincialis de Galicia-Espańa 12. Mejillón heterocigoto con alelos de 126 pb y 180 pb de Galicia-Espańa 13 y 15. Mejillones del sur de Chile con alelo de 126 pb 14. Mejillón heterocigoto del sur de Chile con alelo 126 pb y sitio de restricción para Aci I 16. Estándar de tamańo molecular (25 pb).


We did not find M. edulis alleles (180 bp). A maximum of two alleles from different species were found per sampling location. In this study, no site presented the three alleles altogether (Table 2).

M. trossulus alleles (168 bp) were detected together with M. chilensis alleles in 4% of individuals from collection centers (LA-Caleta La Arena and CB-Canal Coldita-Piedra Blanca) and on-growing centers (CC-Canal Caicaén and CH-Canal Chidguapi); these apparent hybrids exhibited amplicons of 126 bp and 168 bp. There was no clear relationship between on-growing centers and seed collection centers regarding the presence of the 168 bp allele. The aligned sequences of amplicons of 168 bp and 126 bp obtained from CB-Canal Coldita-Piedra Blanca, supposedly M. trossulus and M. chilensis alleles respectively (Table 3), showed high similitude with the polyphenolic protein gene sequences from GenBank, permitting to verify their identity.


M. trossulus is circumpolar in the northern hemisphere, but ambiguously been identified in the southern hemisphere (Wonham, 2004). Previous information (McDonald et al., 1991), described that South American blue mussels contain iso-enzymatic alleles of the three Mytilus species and recently, Fernández-Tajes et al. (2011), found two individuals with M. chilensis and M. trossulus alleles in canned samples of M. chilensis, and confirmed their identity by sequencing the M. trossulus amplicon. Faraway from M. trossulus distribution area, the presence of the 168 bp allele is quite surprising. Further analysis with different genetic markers on larger number of specimens and sequencing of the Me 15-16 fragment would allow a better understanding of the origin of the 168 bp allele in the studied region.

Testing correspondence between the 16s rRNA RFLP marker, that distinguishes the northern from southern hemisphere M. galloprovincialis, and markers that allowed the discrimination between M. chilensis and M. galloprovincialis, Me 15-16 RFLP Aci I and the COIXbarF/R RFLP Xba I recently developed by Fernández-Tajes et al. (2011), in a wider range of southern hemisphere Mytilus samples would contribute to the taxonomic status of Chilean blue mussel. Our results, and the previously discussed studies, about the presence of M. galloprovincialis in Chile indicated that the geographical origin of mussels and its traceability, cannot be established merely from the identification of the species but would require the use of other markers such as microsatellites (Presa & Diz, 2002; Gardestrom et al., 2007).

ACKNOWLEDGEMENTS

The authors thank Dr. Eduardo Tarifeńo, Professor Alicia Rodríguez, Dr. Santiago Aubourg, Dr. Carlos Varela, Eugenio Yokota, Marcela Cárcamo, Armando Salinas and José Villarroel for help in samples obtaining. Dr. Elie Poulin -Universidad de Chile, Facultad de Ciencias- for access to spectrophotometer NanoDrop. To Juan Vidal, from Instituto Geográfico Militar, for his assistance in the preparation of maps. Special thanks to Professor Claudio Telha for checking and improving the English of this manuscript. This research was supported by Universidad de Chile - Vice Rectoría de Investigación - Proyecto Domeyko - Alimentos and CONICYT -Programa de formación de capital humano avanzado Grant AT 24110174. M.A. Larraín recived PhD scholarship from Universidad de Santiago de Chile, VIRD - DIGEGRA.

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Received: 12 December 2011; Accepted: 28 September 2012.

Corresponding author: María Angélica Larraín (mlarrain@uchile.cl)

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