Microsatellites of Mytilus chilensis: a genomic print of its taxonomic status within Mytilus sp.
Article Type: Report
Subject: Mytilus edulis (Genetic aspects)
Mytilus edulis (Identification and classification)
Microsatellites (Genetics) (Identification and classification)
Zoology (Identification and classification)
Zoology (Research)
Authors: Ouagajjou, Yassine
Presa, Pablo
Astorga, Marcela
Perez, Montse
Pub Date: 08/01/2011
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2011 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: August, 2011 Source Volume: 30 Source Issue: 2
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: Spain Geographic Code: 4EUSP Spain
Accession Number: 268309686
Full Text: ABSTRACT The taxonomic status of the Chilean blue mussel Mytilus chilensis has been controversial for decades because of its phenotypic and genetic proximity to other species of the genus Mytilus from both hemispheres. This study reports the development of nine polymorphic microsatellite markers from the M. chilensis genome. The number of alleles per locus ranged between four and nine, and the observed heterozygosity ranged from 0.182-0.750 in a wild sample of 24 individuals from Caicaen (Chiloe Region, Chile). Lack of efficient amplification of many of these microsatellite loci in other Mytilus species suggests that M. chilensis is a valid, distinct species within the genus. These new markers would be useful in fine-scale population analyses of M. chilensis as well in the aquaculture management of this marine resource.

KEY WORDS: Mytilus chilensis, microsatellites, blue mussels, cross-priming


The Chilean blue mussel or "chorito," Mytilus chilensis (Hupe, 1854) is thought to be distributed between the Chilean localities of Arica (18[degrees] S) and Cape Horn (56[degrees] S) (Lancellotti & Vasquez 2000), with abundant natural banks south from Arauco. Chilean "mitiliculture" has been mainly developed on mass culture of Mytilus chilensis from natural banks of Los Lagos Region, Reloncavi Bay, Reloncavi Fiords and around Chiloe Island (39[degrees]15' S-44[degrees]04'S). During the past decade, the production of this resource has dramatically increased from 23,000 t in 2000 to 175,728 t in 2009 (Sernapesca 2009). Therefore, this blue mussel is one of the most promising aquaculture resources for coming decades in Chile.

The industrial and scientific sectors in Chile have recently expressed the necessity for a proper genetic management of this species using microsatellites as one of the most informative genetic markers at the intraspecific level (Estoup et al. 1993). The large polymorphism of microsatellite loci is commonly used to deal with intraspecific gene diversity in vertebrates (mapping, marker-assisted selection, parent--offspring assignment, gene diversity losses, and so forth) (Bouza et al. 2007) However, the use of microsatellites in invertebrates can be problematic because of the poor conservation of microsatellite-flanking sequences within species (Reece et al. 2004) and the low abundance of microsatellites in bivalve genomes (Cruz et al. 2005).

The first microsatellites within the genus Mytilus were developed by Presa et al. (2002). Although those microsatellites work properly in Mytilus galloprovincialis (see, for example, Diz and Presa (2008, 2009)), they do not amplify as consistently in its congeneric species as initially shown. This was probably a result of the sensitivity of the genotyping system used. For example, the AlfExpressII genotyping system (GE Healthcare) used to calibrate microsatellites by Presa et al. (2002) implemented a zoom-in function that allows visualization of all PCR amplicons even if they are extremely weak, as occurred in Mytilus edulis and Mytilus trossulus (see, for instance, Mech9 in M. edulis, Fig. 1B). To our knowledge, this is the explanation for the lack of results in cross-priming assays using instruments lacking the zoom-in property. Up-to-date microsatellite markers have been reported for various Mytilus species: 15 for M. galloprovincialis (7 by Presa et al. (2002) and 8 by Yu and Li (2007)), for M. trossulus with 4 of them cross-amplifying in M. edulis (Gardestrom et al. 2008), 10 for M. edulis (Lallias et al. 2009), and 9 for Mytilus californianus with some of them cross-amplifying in M. galloprovincialis, M. edulis, and M. trossulus (Vidal et al. 2009).

In this study we aimed to develop microsatellite markers for M. chilensis to be applied to the genetic management of its populations and aquaculture stocks. Because classic taxonomic studies have highlighted the difficulty of precisely distinguishing the species of genus Mytilus among each other (McDonald et al. 1991), we also tested the cross-amplification of microsatellites from M. chilensis in M. edulis, M. galloprovincialis, and M. trossulus. This cross-priming conservation could help to clarifying the taxonomic status of M. chilensis, if its randomly cloned microsatellites are either closer to M. edulis or to M. galloprovincialis, or if M. chilensis has its own taxonomic status as full species within the genus Mytilus.

Genomic DNA was extracted from the mantle tissue of 24 wild individuals of M. chilensis from the Chiloe region (Caicaen, Puerto Montt), following the protocol FENOSALT for fishes and molluscs (Perez & Presa 2011). A modification of the enrichment technique FIASCO (Zane et al. 2002) was used to isolate microsatellites. Genomic DNA (250 ng) was digested with MseI (New England Biolabs) at 37[degrees]C for 3 h. Digested DNA was ligated to 1 [micro]M of MseI AFLP adaptor (5'-TACTCAGGACTCAT-3'/5'-GACGATGAGTCCTGAG- 3') using Ready-to-go T4 DNA ligase (GE Healthcare). The ligation mixture was amplified in 40 [micro]L consisting of 20 [micro]L diluted DNA (1:10), 1x [NH.sub.4] Reaction Buffer (Bioline), 200 [micro]M of each dNTP, 4 U Taq DNA Polymerase (BIOTAQ), and 240 ng AFLP adaptor-specific primer (MseI-N). The PCR consisted of 26 cycles of 30 sec at 94[degrees]C, 1 min at 53[degrees]C, and 1 min at 72[degrees]C. About 750 ng (15 [micro]L) of the amplified product was hybridized to 80 pmol of the 5'-biotinylated probes [(CA).sub.13], [(GATA).sub.7], and [(GACA).sub.7] because they are known to be the most frequent motifs in molluscs (Cruz et al. 2005, Perez et al. 2005), in a volume of 100 [micro]L containing 4.2x SSC and 0.07% SDS. After a denaturation step of 3 min at 95[degrees]C, the annealing was performed at room temperature for 15 min. DNA molecules hybridized to biotinylated oligonucleotides were selectively captured by streptavidin-coated beads (Roche Diagnostics), the remaining protocol being identical to that described by Zane et al. (2002).


A cytochrome b amplicon of Merluccius merluccius was used to minimize the nonspecific binding of genomic DNA. The PCR products of 2 elution steps were cloned into pGEM-T Easy Vector System II (Promega). Inserts from 2,187 recombinant clones (1,629 [(CA).sub.n], 315 [(GATA).sub.n], and 243 [(GACA).sub.n]) were amplified by PCR in 20 [micro]L using 1 [micro]L of 1x [NH.sub.4] Reaction Buffer (Bioline), 1.5 mM Mg[Cl.sub.2], 20 pmol of each primer (T7 and SP6), and 1 U Taq DNA Polymerase (BIOTAQ). Inserts, ranging from 350-1,100 bp were used for Southern blot transfer and filter hybridization with the synthetic probes [(AC).sub.13], [(GATA).sub.7], and [(GACA).sub.7]. One hundred ninety-seven positive clones (9%) were lysed, their plasmid purified using the GFX Micro Plasmid Prep Kit (GE Healthcare), and then sequenced on both strands in an ABI Prism 3100 Sequencer (Applied Biosystems) using the BigDye Terminator method. One hundred twenty-one clones (61.4%) contained repeated motifs (86 tetranucleotides: 21 [(GACA).sub.7] and 65 [(GATA).sub.n], and 35 dinucleotides). Forty-three sequences (35.5% of all microsatellite containing sequences; 21.8% of the observed positives in Southern blot, and 1.96% of the initial plated cultures) were suitable for primer design using Oligo 4.05 (Rychlik & Rhoads 1989), and 30 microsatellite markers rendered an optimal PCR amplification.

Allelic variation was assessed in 24 individuals of M. chilensis from Caicaen (Chile). An intragenus cross-priming test was carried out on 12 individuals of each species M. edulis (Arhus, Ronde, Denmark), M. trossulus (Eckelo, Finland), and M. galloprovincialis (Vigo, Spain). About 100 ng per individual's DNA was used in a PCR of 15 [micro]L consisting of 1 U Taq DNA Polymerase (BIOTAQ) in 1x [NH.sub.4] Reaction Buffer (Bioline), 20 pmol of each primer, 200 [micro]M of each dNTP, and Mg[Cl.sub.2], ranging from 1.3-1.5 mM (Table 1). The amplification profile consisted of 1 cycle at 95[degrees]C for 5 min, followed by 35 cycles at the annealing temperature (Table 1) for 40 sec, 72[degrees]C for 1 min, and 94[degrees]C for 1 min, ending with a extension step at 72[degrees]C for 10 min. PCR products were visualized in 2% agarose gels and electrophoresed in acrylamide gels using an AlfExpressII sequencer (GE Healthcare). Alleles were characterized using internal size standards (80-114-180-230-402 bp), as well as external size controls from initial allele scorings of each marker.

Nine of 30 microsatellites were polymorphic in M. chilensis (Table 1). GENEPOP 3.4 (Raymond & Rousset 1995) was used to calculate observed and expected heterozygosities and to perform Hardy-Weinberg tests. Any of the tests performed between locus pairs for genotypic disequilibrium were significant (Fisher's exact test, GENEPOP 3.4), suggesting the independence of the 9 polymorphic markers reported herein. The putative presence of null alleles and other possible scoring errors were tested with MICRO-CHECKER (Van Oosterhout et al. 2004). The number of alleles per locus ranged from 4-9, and the observed heterozygosity ranged from 0.182-0.750. Three loci showed a significant deviation from Hardy-Weinberg expectations (P [less than or equal to] 0.05). The frequencies of putative null alleles in those 3 loci as estimated with the method of Brookfield1 (Brookfield 1996) were as follows: Mch2, 0.289; Mch4, 0.322; and Mch7, 0.189. Although the influence of a small sample size or other biological causes could underlie such Hardy-Weinberg departures, the presence of null alleles seems to be a widespread phenomenon in many types of markers so far applied in Mytilus (see, for example, Skibinski et al. (1983)). For instance, null alleles were the probable cause of the large [F.sub.IS] figures reported for the 6 microsatellites of M. trossulus (Gardestrom et al. (2008), for 8 of 10 microsatellites of M. edulis (Lallias et al. 2009), and for 6 microsatellites widely applied in phylogeographic studies of M. galloprovincialis (see, for example, Diz and Presa (2009)). Microsatellite identifications from data mining on EST libraries of Mytilus sp. have been performed in an attempt to characterize more stable flanking regions that could help to reduce priming failures and ensure higher transferability among species. However, EST-derived polymorphic microsatellites so far described for both M. galloprovincialis (8 markers (Yu & Li 2007)) and M. californianus (9 markers (Vidal et al. 2009)) showed significant departures from Hardy-Weinberg equilibrium at 6 of 8 and 5 of 9 loci, respectively. Noteworthy, the use of null allele-containing loci in interspecific cross-priming assays is only expected to worsen the per se high intraspecific rate of amplification failure. Consequently, the development of microsatellites in blue mussels (and bivalves in general) is at best useful within the cloning species, but it is generally useless for interspecific introgression studies characterizing hybrids or studying hybridization itself, as has been previously claimed (Lallias et al. 2009).

The same PCR conditions used to amplify the 9 micro-satellites in M. chilensis (Table 1) were applied in cross-amplification tests on 3 other species of Mytilus (Table 2 (Weir & Cockerham 1984)). Of the 9 loci, 7 amplified in M. edulis, 6 in M. galloprovincialis, and 5 in M. trossulus (Table 1, Fig. 1). The classic taxonomic literature describing Chilean marine bivalves considered the "chorito quilmahue" as the single representative of the genus Mytilus in Chile (Osorio et al. 1979). South American blue mussels were reported to contain isoenzymatic alleles of the 3 Mytilus species, but were morphologically more similar to M. edulis from the northern hemisphere (McDonald et al. 1991). Later, the Chilean blue mussel was proposed as an M. edulis subspecies (M. edulis chilensis) based on divergence found with M. edulis and M. trossulus in morphometric traits and PCR amplicons of the marker Glu-5 (Toro 1998).

Using factorial correspondence analyses on allozymic multi-locus genotypes, Carcamo et al. (2005) clearly identified 3 different gene pools corresponding to M. edulis, M. galloprovincialis from the northern hemisphere, and the Chilean blue mussel from Chiloe, which was proposed for taxonomic revision as M. galloprovincialis chilensis because of its genetic proximity to M. galloprovincialis. During the past decade, the species M. galloprovineialis was described in central Chile (Biobio region) (Daguin & Borsa 2000, Tarifeno et al. 2005), as well as in southern Chile (Toro et al. 2005). Recently, Galleguillos et al. (2009) found that the distribution of morphometric traits to classify the Chilean blue mussel was highly skewed by the size of specimens used, an additional bias to the previously described latitudinal shell shape variation in this species (Krapivka et al. 2007). The application of several genetic markers on samples from the Biobio region (Galleguillos et al. 2009) gave evidence of the cohabitation of two blue mussel species in Chile--M. chilensis and M. galloprovincialis--as has also been recently observed with mitochondrial DNA markers (M. Astorga, unpubl, data). Inspection of previous literature on South American blue mussels suggests that different authors and studies have probably sampled on different species or their hybrids along the Chilean coast, thus reporting a different taxonomic status for M. chilensis. Because interspecific alternate patchiness is not an uncommon feature in blue mussels (Skibinski 1985) and the existence of at least M. galloprovincialis and M. chilensis is confirmed in Chile, an interesting upcoming task is the definition of their distribution patterns along the Chilean coast.

The microsatellites developed herein could be a useful tool for assisting management and laboratory crosses in M. chilensis aquaculture. Also, they would enforce further population genetic analyses of M. chilensis aiming to describe finer genetic substructuring and dispersive patterns, as well as to interpret this novel information in the light of previous molecular scenarios (Toro et al. 2004). Currently, no evidence of discrete stocks seems to exist along the Chilean coast, except the divergence reported for the Punta Arenas population (southern Chile) using allozymes (Toro et al. 2006). Although, this phylogeographic homogeneity could be a natural scenario, the homogenizing consequence of farming transplantation is not negligible with regard to anthropogenically mediated gene flow through seed transplantation into cultivation areas (Winter et al. 1984).


This research was financed by the Chilean government through a grant by CORFO (INNOVA 07CN13PPD240) and Xunta de Galicia (grant IN845B-2010/l16). Y.O. is a PhD candidate of the Spanish Ministry of Foreign Affairs and Cooperation (Scholarship II-A from MAEC- AECID no. 0000502361), and M.P. is a contractual scientist from Xunta de Galicia (Program Isidro Parga Pondal).


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(l) University of Vigo, Faculty of Marine Sciences, Department of Biochemistry, Genetics and Immunology, 36310 Vigo, Spain; (2) Instituto de Acuicultura & Cien-Austral, Universidad Austral de Chile, Sede Puerto Montt, Chile

* Corresponding author. E-mail: montse.perez@vi.ieo.es

DOI: 10.2983/035.030.0218
Amplification characteristics for the 9 microsatellites
characterized in M. chilensis.

Locus         Motif             Primer Sequences (5'-3')

Mch1     [(CA).sub.6]      F: GAT GGC CGC ATC TGT AAT TC ([Xi])
                           R: TGT CGC ATG CTC ATT TCT TC
Mch2     [(GT).sub.6]      F: TAA TAA TTT AGA CAA GTG G ([Xi])
                           R: GGA TAA GGT AAA GAG TGC
Mch3     [(CAGA).sub.8]    F: AGA GGA GTT GCG ATG ATT ([Xi])
                           R: CGA AGT TGT GGA GGG TAT
Mch4     [(CAGA).sub.6]    F: ACC TGA AGC GGG AAG AA ([Xi])
                           R: AAC CTG GAC TTT TTT TCT CA
Mch5     [(CTGT).sub.6]    F: CTG TTG CTC AAT CCT TGC AG ([Xi])
                           R: GCG AAA AAT AGG AAA AGA TAA GCA
Mch6     [(GACA).sub.10]   F: CAA CGA AAC AAA CGG ACT GA ([Xi])
                           R: TCA GTA AAC ATT GAA GTG GAG CA
Mch7     [(CGGA).sub.7]    F: TTT CAA CTT GGC TAT CAT ([Xi])
                           R: GGG GGT ATA CTG TTT TAC
Mch8     [(CA).sub.6]      F: AAA CCT AAG TGC TGT TCA T ([Xi])
                           R: CAT TTA TTC GTC TGT CAC A
Mch9     [(GAAC).sub.13]   F: GGA CTG ACA AAC GAA T ([Xi])
                           R: TGT TTT CTG GTC TGT GC

                          Mg[Cl.sub.2]      GenBank
Locus    T ([degrees]C)       (MM)       Accession No.

Mch1           52             1.5          JF894123

Mch2           53             1.4          JF894124

Mch3           52             1.5          JF894125

Mch4           52             1.3          JF894126

Mch5           52             1.4          JF894127

Mch6           52             1.3          JF894128

Mch7           54             1.4          JF894129

Mch8           55             1.3          JF894130

Mch9           50             1.2          JF894131

F, forward primer; Mg[Cl.sub.2], PCR magnesium chloride; R, reverse
primer;  ([Xi]), oligonucleotide labeled with Cy5; T, PCR annealing

Amplification results of 9 microsatellites from M. chilensis
(Caicaen, Chile) in M. edulis, M. trossulus, and M. galloprovincialis.

                            M chilensis (n = 24)

Locus   SR (bp)   Na   Nis   [H.sub.E]   [H.sub.0]   [F.sub.IS]

Mch1    153-165    6   24      0.833       0.750      0.100
Mch2    121-145    7   24      0.814       0.250      0.693 *
Mch3    101-129    5   24      0.644       0.500      0.223
Mch4    298-318    5   22      0.809       0.182      0.775 *
Mch5    213-221    4   24      0.349       0.261      0.252
Mch6    125-153    8   24      0.654       0.541      0.172
Mch7    210-240    8   23      0.844       0.478      0.433 *
Mch8    193-207    9   24      0.860       0.750      0.128
Mch9    135-167    5   24      0.634       0.500      0.211

                            M. edulis (n = 12)

Locus   SR (bp)   Na   Nis   [H.sub.E]   [H.sub.0]   [F.sub.IS]

Mch1    147-157    6   12      0.701       0.416      0.405 *
Mch2    121-131    3   12      0.681       0.666      0.022
Mch3    101-125    5    5      0.806       0.750      0.070
Mch4      NA      --    0       --          --         --
Mch5    221-229    3   12      0.636       0.250      0.607 *
Mch6    145-151    4   12      0.598       0.250      0.582 *
Mch7      NA      --    0       --          --         --
Mch8    193-211    5   12      0.447       0.250      0.440 *
Mch9    121-165   11   12      0.928       0.583      0.371 *

                            M. trossulus (n = 12)

Locus   SR (bp)   Na   Nis   [H.sub.E]   [H.sub.0]   [F.sub.IS]

Mch1    143-153    6   12      0.750       0.583      0.222
Mch2    131-137    3   11      0.518       0.091      0.824 *
Mch3    101-125    6    5      0.859       0.272      0.682 *
Mch4      NA      --    0       --          --         --
Mch5      NA      --    0       --          --         --
Mch6    137-153    5   11      0.541       0.272      0.496 *
Mch7      NA      --    0        -           -         --
Mch8    184202     3   12      0.379       0.083      0.780 *
Mch9      NA      --    0       --          --         --

                       M. galloprovincialis (n = 12)

Locus   SR (bp)   Na   Nis   [H.sub.E]   [H.sub.0]   [F.sub.IS]

Mch1    153        1   12       --          --         --
Mch2      NA      --    0       --          --         --
Mch3    101-125    6   12      0.788       0.500      0.365 *
Mch4    286-310    8   12      0.871       0.333      0.617 *
Mch5    205-227    8   11      0.891       0.545      0.388 *
Mch6    147        1   12       --          --         --
Mch7      NA      --    0       --          --         --
Mch8    191-203    6   12      0.844       0.500      0.408 *
Mch9      NA      --    0       --          --         --

The number of individuals analyzed is given between parentheses
after the name of each species. [F.sub.IS], fixation index
(Weir & Cockerham 1984); [H.sub.E], expected heterozygosity;
[H.sub.0], observed heterozygosity; Na, number of alleles
observed; NA, no PCR amplification; Nis, number of individuals
successfully amplified; SR, allelic size range in base pairs.

* Significance at P [less than or equal to] 0.05. Indicative
adjusted nominal level was P [less than or equal to] 0.0025 for
M. chilensis and P [less than or equal to] 0.0031 for each of
the remaining species.
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