Development and characterization of fifteen polymorphic microsatellite loci in Bryconamericus aff . iheringii ( Teleostei : Characidae ) and cross-amplification in related Characidae species

Data on 15 novel microsatellite loci from the Neotropical fish Bryconamericus aff. iheringii are presented here. Analyses of 32 individuals from four different streams revealed 192 different alleles, ranging from four to 32 alleles per locus (mean of 12.8 per locus). Observed and expected heterozygosities ranged from 0.094 to 0.813 and 0.205 to 0.952, respectively. These loci showed high polymorphic information content and will be a resource for genetic studies of B. aff. iheringii. Furthermore, several loci also amplified other small Neotropical Characidae (Piabarchus stramineus and Piabina argentea) and should be useful for these species.


Introduction
Bryconamericus Eigenmann, 1907 is one of the most diverse genera of the Characidae family, occurring in many river systems in of the Neotropical region, from Costa Rica to Argentina (Vari, Siebert, 1990;Lima et al., 2003).They are small omnivorous fish (not exceeding 10 cm in length) and inhabit a range of environments, including streams, rivers and lakes (Britski et al., 1988).Species of this genus are important food sources for piscivorous fish (Britski et al., 1988) throughout the range of environments, and in the Paraná River basin (one of the main drainage systems in South America) the genus is a prey item of several piscivorous fish (Hahn et al., 1997;Lowe-McConnell, 1999).

Original article
Bryconamericus aff.iheringii microsatellite loci Neotropical Ichthyology, 16(1): e170135, 2018 2 e170135 [2] 1999; Winemiller et al., 2008).The high prevalence of small fish dominating the ichthyofauna raises questions about the evolutionary and population aspects of these species.According to Castro (1999), the small size of these species usually contributes to a low movement capacity along the drainages.This reduced movement capacity could be one of the underlying explanations for the many endemic species and small-scale population structure found in Neotropical areas (Castro et al., 2003;Ferreira et al., 2016).However, genetic data for these species are largely lacking (Sofia et al., 2006(Sofia et al., , 2008;;Ferreira et al., 2016).Biological data including genetic analyses of small fish, such as Bryconamericus spp., are essential for understanding biological and evolutionary properties, as well as for the management and conservation of the ichthyofauna of Neotropical streams (Agostinho et al., 2005;Dudgeon et al., 2006).Microsatellites are highly variable genetic markers suitable for cost-effective population genetic studies.However, particularly in the case of Neotropical fish species, the lack of suitable genetic markers has been one of the main obstacles to the development of more complete genetic studies involving this diverse group of fish.Thus, the present study aimed to identify and develop polymorphic microsatellite loci for Bryconamericus aff.iheringii and assess cross-species transferability to four other small Characidae from the Neotropical region.

Material and Methods
Microsatellite loci were obtained from an enriched genomic library using the methods described by Billotte et al. (1999) with some minor modifications (cf.Ferreira et al., 2013).The required genomic DNA was extracted from a sample of Bryconamericus aff.iheringii using the method of Almeida et al. (2001).Briefly, 5 μg was digested using 50 U of RsaI endonuclease and specific adapters RsaI-21 (5′CTCTTGCTTACGCGTGGACTA3′) and RsaI-25 (5′TAGTCCACGCGTAAGCAAGAGCACA3′) were linked to digested DNA using 5U of T4 DNA ligase (Invitrogen; www.invitrogen.com) in reaction buffer, including 10μM of each adapter in a final volume of 200μl and incubated at 20°C for 2 h.Fragments with putative microsatellite sequences were obtained using three probes bound to biotin: (AGA) 5 , (CT) 8 and (GT) 8 .These fragments were amplified by Polymerase Chain Reaction and cloned into pGem-T Easy (Promega; www.promega.com)vectors using 5μl of amplification product, 50 ng of vector and 1 U of T4 DNA ligase in reaction buffer at 4°C (overnight).Cloning products were used to transform Escherichia coli (DH5-α lineage) cells.
A total of 96 positive clones were amplified using M13 primers (F and R) and subsequently sequenced using a Big Dye Terminator 3.1 sequencing kit (Applied Biosystems; www.appliedbiosystems.com) on an automated sequencer (ABI3500 xL, Applied Biosystems).BioEdit v.7.0 software (Hall, 1999) was used to visualize sequences and the Primer3 program (Rozen, Skaletsky, 2000) to generate microsatellite locus specific primers.Tests for potential hairpin structures and primer-dimers were conducted in AutoDimer software (Vallone, Butler, 2004).

Results
Analyses of chromatograms for the 96 clones confirmed the presence of microsatellite repeats in a total of 46 sequenced clones.Based on sequence quality, microsatellite repeat structure, and primer suitability 15 loci (almost all dinucleotide repeats) were selected for further testing.These 15 loci all amplified successfully, showing polymorphisms that could be genotyped unambiguously.Information for these loci was submitted to the GenBank (accession numbers: MF693815-MF693829, Tab. 1).
Tab. 1. Description of 15 polymorphic microsatellite loci isolated from Neotropical fish Bryconamericus aff.iheringii.Ta , optimal annealing temperatures; k, number of alleles; allele size range (bp); H o , observed heterozygosity; H e , expected heterozygosity estimated from 32 individuals; PIC, polymorphic information content; Q, paternity exclusion probability; I, probability of genetic identity; F IS , endogamy coefficient; GenBank accession numbers.*Significant value of endogamy coefficient (F IS ).

Discussion
As expected, the mixture of individuals from different streams (possibly composing different populations) resulted in deviations from the Hardy-Weinberg equilibrium (HWE) and significant F IS values for all loci.This is likely an indication of a Wahlund effect caused by including elements of multiple genetic units in a single panel causing excess homozygosity and significant F IS estimates (Freeland, 2005).From the inflated homozygosity estimates, MicroChecker suggested null alleles were present at all loci.However, it is unlikely that the excess of homozygosity was caused by null alleles as it would require all analysed loci to be affected by null alleles.Instead we favour the hypothesis that the samples were derived from genetically distinct units and the pooling of these units caused a Wahlund effect.After correction for multiple tests (sequential Bonferroni correction, k= 105), thirteen loci combinations showed linkage disequilibrium, the locus Bih73 was linked to loci Bih30, Bih44, Bih45, Bih47and Bih77, the locus Bih45 linked to loci Bih31, Bih40 and Bih42, the locus Bih76 linked to loci Bih16 and Bih22, the locus Bih77 showed linkage with loci Bih30 and Bih44, and the locus Bih44 with Bih30.
The novel microsatellite loci developed in the present study constitute a highly variable marker set suitable for genetic studies of Bryconamericus aff.iheringii.Moreover, successful cross-species amplifications of some loci indicate that these microsatellites can be used for genetic studies of other small Characidae; Piabina argentea (12 polymorphic loci) and Piabarchus stramineus (nine polymorphic loci) (Tab.2).