A polymorphic microsatellite from the Squalius alburnoides complex (Osteichthyes, Cyprinidae) cloned by serendipity can be useful in genetic analysis of polyploids

A new microsatellite locus (SAS1) for Squalius alburnoides was obtained through cloning by serendipity. The possible usefulness of this new species-specific microsatellite in genetic studies of this hybrid-species complex, was explored. The polymorphism exhibited by SAS1 microsatellite is an important addition to the set of microsatellites previously used in genetic studies in S. alburnoides complex, that mostly relied in markers described for other species. Moreover, the SAS1 microsatellite could be used to identify the parental genomes of the complex, complementing other methods recently described for the same purpose..

The taxon Squalius alburnoides is a small endemic cyprinid inhabiting the rivers of the Iberian Peninsula and is among the most complex polyploid systems known in vertebrates.
Based on molecular markers information, S. alburnoides is recognized as a hybrid taxon resulting from an ancient and unidirectional hybridization between S. pyrenaicus females (P genome) and males (A genome) of an extinct species sister to Anaecypris hispanica (reviewed in Alves et al. 2001, Robalo et al. 2006. For this reason, all the S.alburnoides fishes carry S.pyrenaicus mitochondrial DNA. Actually, the S. alburnoides complex combines the diploid (2n = 50) and polyploid (3n = 75 and 4n = 100) biotypes of both sexes and different nuclear genomes, that, by intercrossing, combine sexual and asexual reproductive modes.
The asexual modes range from clonal inheritance to hybridogenesis or meiotic hybridogenesis (in which one genome is excluded from gamete formation), whereby sympatric bisexual Squalius species act as sperm donors and contribute with new genetic material, i.e.., S. pyrenaicus, mainly in the southern basins of the Iberian Peninsula (P genome), and S. carolitertii in the northern (C genome)) (reviewed in Alves et al., 2001;Pala et al., 2009).
The predominant S. alburnoides specimens in nature are triploids with the sex ratio biased towards females, with the CAA biotype across the distribution range of S. carolitertii, and the PAA biotype across the S. pyrenaicus range.
As in other asexual complexes (Ambystoma: Bogart, 1989; Rana: Hotz et al.,1992;Phoxinus: Goddard and Schultz, 1993), the S. alburnoides complex have regenerated and maintained the extinct parental species genotype (AA, all males) through the fertilization of A ovocytes from PAA females by reduced A sperm produced by AA males of hybrid origin (Alves et al., 2002). This AA genotype is apparently absent from the northern populations Although in recent times, single nucleotide polymorphisms (SNPs) have disputed with microsatellites the role of a prominent tool in genetic studies (Coates et al. 2009), mainly through single nucleotide changes being universally comparable, polymorphic DNA microsatellites remain as a very useful (and less expensive) class of genetic markers in population genetics. Moreover, in general, microsatellites are more neutral than SNPs, since the latter frequently give evidence of selection Microsatellites, by addressing topics, such as genetic identification, population structure, parentage, kinship and population variability assessment (Jarne and Lagoda, 1996;Ellegren, 2004;Hamilton and Tyler, 2008), are traditionally considered as the markers of choice for genotyping, due to their abundance, polymorphism in repeat numbers and reliability (Jones et al. 1997).
Microsatellites display a very high content of genetic information, as they are codominant, with multiple alleles, and showing high expected heterozygosity values.
In addition to the abundance of microsatellites in vertebrate genomes (Weber, 1990;Jarne and Lagoda, 1996;Goldstein and Schlötterer, 1999), it is notable that, in fish, microsatellite-loci are longer, have a larger range in allele size, are more degenerated (i.e., contain more base substitutions or deletions), and are very abundant, when compared to mammals (e.g., Brooker et al.,1994;Colbourne et al., 1996;O'Reilly et al., 1996;Neff and Gross, 2001). The possibility of using microsatellites described for another closely related species (transferability of microsatellite loci) is a consequence of homology of flanking regions in simple sequence repeats (Turner et al., 2004). Studies in freshwater fish have already demonstrated the high rate of transferability of microsatellite loci among taxonomically related species (e.g. Huang et al., 2003;Salgueiro et al., 2003;Turner et al., 2004;Holmen et al., 2005).
Efforts to determine the copy number of microsatellite alleles in polyploid species have, in many cases, been unsuccessful (Falque et al., 1998) and in some cases no attempts have even been made to assign precise allelic configurations (Becher et al., 2000;Bockelmann et al., 2003). There are many applications where considerably more information would be gained from a proper quantification of the alleles in the loci analysed, such as population genetics and paternity analysis.
In the present report, the cloning by serendipity of a polymorphic microsatellite from a diploid S. alburnoides specimen from Estena River (Guadiana basin, Spain) is described, with a discussion of its possible application in studies of the characterization of genetic variability and parental assignment in this species complex.
The microsatellite was discovered during a series of trials for cloning short opsin fragments from S. alburnoides (Boto unpublished). Briefly, amplification of a short fragment of the exon five in the putative SWS1 opsin gene was attempted, by using a degenerate universal vertebrate forward primer OPF 5GCGAATTCGCNTCNACNCARA ARGCNGA 3 (Carleton et al., 2000) and a primer designed against a short Cyprinus carpio SWS1 sequence OPC1R 5CCTTGTTTGTATCCTCAGCA 3. DNA was extracted from fins preserved in ethanol, using standard methods (Sambrook et al., 1989).
An aliquot of 15 mL of a pooled mix of amplified fragments was precipitated with isopropanol and ligated to a PGEM-T vector. TOPO-competent bacteria were transformed with the ligation mix and plated onto LB/agar/ampicilin.
From the 18 transformants bearing an insert of compatible length with the expected fragment sequenced (ABI 3730), 15 bore an AG microsatellite sequence.
Three of these sequences presented 12 repeats of the AG motif, five 13, two 24 , two 25 and three 26. Seeing that DNA polymerase is capable of copying the same allele with different repeat numbers (Hauge and Litt, 1993;Clarke et al., 2001;Ellegren, 2004), the cloning of two different alleles (12-13 repeats and 24-26 repeats) from this microsatellite can be inferred.
A representative sequence, denominated SASI, is deposited in GenBank under accession number FJ652104.
In order to explore both the polymorphic character of this microsatellite, and its usefulness in further studies of the S. alburnoides complex, DNA from individuals of different geographic origin, genome composition and ploidy level, as well as several S.pyrenaicus and S. carolitertii samples, was amplified (Table 1), using OPF and OPC1R primers (the latter marked with FAM), at a hybridization temperature of 43.5°C. Fragments were analyzed with an ABI 3730 using GeneMapper v3.7.
After prior identification of the biotype, according to procedures by Cunha et al. (2008Cunha et al. ( , 2009, ploidy levels were determined through flow cytometry (FCM) of blood cells, as previously described (Collares-Pereira and Moreira de Costa, 1999).
As shown (Table 1), this microsatellite facilitates the discrimination between alleles coming from the genome of the extinct ancestor close to Anaecypris hispanica (Robalo et al., 2006) -genome A (allele length below 100 bp), and those from the P or C genomes corresponding to the S. pyrenaicus and S. carolitertii sperm donors (allele lengths above 100 bp). As such, this microsatellite could be used to differentiate individuals with hybrid genomes from individuals with a single genome.
This microsatellite does not allow distinguishing the alleles coming from the sperm donors, with genomes P or C, whose alleles overlap in length. However, this is a minor problem, since populations carrying P or C genomes are allopatric Due to manifest polymorphism, the SAS1 microsatellite became an important addition to those previously used in genetic studies of the S. alburnoides hybrid complex [n7k4, n7j4, e2f8 and e1g6 (Mesquita et al., 2003;Pala and Coelho, 2005) lco1, lco3, lco4 and lco5 (Turner et al. 2004) loci], since only two (e2f8 and e1g6) were really S.alburnoides-complex specific.
Furthermore, the identification of heterozygotes with the SAS1 microsatellite could be of use for detecting the genome copy number of intergeneric hybrids, despite the existence of new methods for quickly defining the genomic composition of Squalius alburnoides, based on determining the relative genome dosage by the semiquantitative polymerase chain reaction (PCR) method (Sousa-Santos et al., 2005;Inacio et al., 2010).
As shown above, the microsatellite loci previously used in genetic studies of S. alburnoides (Pala and Coelho, 2005;Crespo-López et. al., 2006, 2007Cunha et al., 2008) were mostly heterologous ones. This frequently leads to the appearance of null alleles (alleles which are not amplified) and a loss in polymorphism information in the species in which the marker was being tested. The increase in the number of microsatellite loci in genetic studies has been shown to be a beneficial strategy, through minimizing problems derived from characteristics of the microsatellites themselves (high mutation rate, presence of null alleles, size homoplasy, etc.). Hence, the addition of a new one constitutes a powerful tool for increasing knowledge