versión On-line ISSN 0717-6538
Gayana (Concepc.) v.74 n.1 Concepción 2010
Gayana74(l): 74-77,2010. Comunicación breve ISSN 0717-652X
Usefulness of heterologous microsatellites obtained from Genypterus blacodes (Schneider 1801) in species Genypterus off the Southeast Pacifc
Utilidad de microsatélites heterólogos de Genypterus blacodes (Schneider 1801) en especies de Genypterus del Pacífco suroriental
Cristian B. Canales-Aguirre1'2*, Sandra Ferrada1'2, Cristian E. Hernández2 & Ricardo Galleguillos1
1Laboratorio de Genética y Acuicultura, Departamento de Oceanografía, Universidad de Concepción, Casilla 160-C Concepción, Chile.
2 Laboratorio de Diversidad Molecular y Filoinformática, Departamento de Zoología, Universidad de Concepción, Casilla 160- C Concepción, Chile. *E-mail:firstname.lastname@example.org
Analizamos la utilidad de cinco loci de microsatélites aislados en Genypterus blacodes de Australia en especies de Genypterus presentes en Chile (G. chilensis, G. maculatus y G. blacodes). Los resultados muestran que los loci microsatélites amplifcan positivamente en cada especie y podrían ser utilizados en futuros estudios poblacionales.
Microsatellites are currently one of the most popular and most used genetic markers in molecular ecology (Barbará et al. 2007). These markers are especially useful for evaluating patterns of populations genetic variability, and the microevolutionary mechanisms behind these patterns (e.g. Mundy et al. 1997). One strategy used to obtain microsatellite loci is the construction of a genomic library. Although cloning microsatellite loci for any taxon is relatively simple (Zane et al. 2002), the development of microsatellites still requires deep knowledge experts on molecular biology and a considerable amount of time and resources. An alternative strategy is to test primers described for microsatellite loci in closely related species (that is, cross-priming strategy or heterologous microsatellites). This cross-species amplifcation is only effective if the fanking regions of the primers are conserved among species. For example, highly conserved fanking regions have been reported in cetaceans (Schlótterer et al. 1991), turtles (FitzSimmons et al. 1995), and fsh (Angers & Bernatchez 1996, Rico et al. 1996). A potentially useful application of cross-species amplifcation of microsatellites could be its use with species of congeneric commercial importance fishes.
Within the order Ophidiiformes, Genypterus is the genus with the largest number of species of economic importance (Nielsen et al. 1999). In the southeast Pacifc Ocean three species of the genus canbe found: G. chilensis, G. maculatus, and G. blacodes; and despite their importance, there are few genetic studies on Genypterus species.
There are no existing genetic studies regarding G. chilensis, and only one in relation to G. maculatus through allozymes (Alay et al. 1993). There are a few studies involving G. blacodes (although not from the southeast Pacifc), two of these studies used allozymes (Smith 1979, Smith & Francis 1982), and two other studies use microsatellite loci (Ward & Reilly 2001, Ward et al. 2001). The lack of genetic studies regarding these species could result in inadequate measures of fshery management policies.
The availability of microsatellite loci isolated from G. blacodes from Australia could contribute to population studies regarding species of the genus off the southeast Pacifc. Therefore, the objective of the present study is to determine the usefulness of these markers in G. chilensis, G. maculatus and G. blacodes from the southeast Pacifc.
We tested five pairs of microsatellite primers previously isolated from G. blacodes in Australia (Ward & Reilly 2001: cmrGb4.2A, cmrGb4.2B, cmrGb4.11B, cmrGb5.2B, and cmrGb5.9) on six G. blacodes (from the southeast Pacific), five G. chilensis and seven G. maculatus individuáis. DNA was extracted from muscle tissue samples according to the protocol described by Grijalva-Chon et al. (1994). The quantity and quality DNA was measured by an Eppendorf® BioPhotometer and the DNA was diluted to 20 ng for amplifications. The PCR conditions for each locus were applied as described by Ward et al. (2001). The PCR producís were separated by means of electrophoresis in 6% polyacrylamide gel and were visualized with silver staining (Acrylamide:Bis-Acrylamide Kit; Winkler®). A 10 pb marker (Invitrogen, Life Technologies, USA) was used as a standard weight marker to evalúate the size of the amplified producís.
The genotypes were identified using the LabWork™ v4.6.00.0 software (UVP®, Inc), which estimates the size of alíeles based on electrophoretic mobility in relation to the mobility of the weight marker. Deviations from Hardy-Weinberg equilibrium between markers were tested using Arlequin v3.0 (Excoffier et al. 2005). The Genetix v4.05 program (Belkhir et al. 2000) was used to conduct a Factorial Correspondence Analysis (FCA). With this approach, we evaluated the usefulness of these markers in species differentiation. Additionally, a dendrogram was generated based on the Nei's standard genetic distance (Ds), using the UPGMA clustering method inthe TFPGAvl.3 program (Miller 1997).
The results showed positive amplifcation of microsatellite loci in the three species. The percentage of amplifcation, mean number of alíeles and size range of each locus varied between species (Table 1). Three loci showed signifcant deviation from Hardy-Weinberg equilibrium (Table 1). The percentage of variation in the FCA (Fig. 1) was explained by the frst two axes (Axisl: 51.45; Axis2: 48.55). With these results, G. blacodes can be differentiated from other species. The dendrogram constructed by means of UPGMA corroborates these results (Fig. 2). The most closely related species are G. chilensis and G. maculatus, and G. blacodes appears as sister group of both species.
Our results confrm that fanking regions of microsatellite loci have been conserved in closely related species of the genus Genypterus, as observed in other studies with congeneric species or other taxonomic groups (Schlótterer et al. 1991,FitzSimmonseía/. 1995, Angers &Bernatchez 1996, Rico et al. 1996). The percentage of amplifcation (Table 1) does not indicate a great loss of diversity among species (83.3% for G. blacodes, 88% for G. chilensis, and 97.1% for G. maculatus positive amplifcation), considering that a percentage of 57.2% and 57.3% of polymorphism and amplifcation, respectively, has been described for fsh species (Barbará et al. 2007). However, given the low number of analyzed samples, these results should be taken with precaution. Our observations differ from other studies, in which a decrease in genetic diversity between species has been observed when using heterologous microsatellites (Primmer & Merila 2002). The presence of nuil alíeles could reduce genetic variability with increasing phylogenetic distance from a focal species (e.g. in the oyster Crassostrea, Li et al. 2003). Particularly among fsh, the decrease of genetic variability is greater between genera of the same family, than between species of the same genus (Barbará et al. 2007), in conformity with the results obtained here.
tablE 1: Loci microsatellite used to evaluate interspecifc amplifcation. N: number of individuals; Na: number of alleles. Values in bold show signifcant deviation from Hardy-Weinberg equilibrium (P ≤ 0.05).
tabla 1: Marcadores microsatélites usados para evaluar amplifcación interespecífca. N: número de individuos; Na: número de alelos. Los valores en negrita muestran desviación signifcativa de equilibrio de Hardy-Weinberg (P ≤ 0,05).
The positive amplifcation of heterologous microsatellites in species of Genypterus from the southeast Pacifc indicates that these could be used to test polymorphism in other species of Genypterus spp., such as G. brasiliensis (southeast Atlantic), G. capensis (south of África) and G. tigerinus (Australia). Finally, fve of the microsatellite loci isolated from G. blacodes from Australia (Ward & Reilly 2001) proved to be useful for genetic studies of congeneric species.
Figure 1: Factorial Correspondence Analysis (FCA) between species of the genus Genypterus of the southeast Pacifc.
Figure 2: Dendrogram based on Nei's standard genetic distance using UPGMA. Valúes above branches depict the bootstrap support valúes.
Figura 2: Dendrograma basado en la distancia genética estándar de Nei usando UPGMA. Los valores sobre las ramas representan los valores de soporte de bootstrap.
Both C.B. Canales-Aguirre and S. Ferrada belong the doctoral program in systematic and biodiversity and both were supported by UdeC posgraduate Fellowships. We thank rwo anonymous referees for useful suggestions.
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