Development and characterization of novel microsatellite loci for Lusitanian toadfish,Halobatrachus didactylus

Abstract

The Lusitanian toadfish Halobatrachus didactylus is an eastern Atlantic polygynous species showing male paternal care. In this paper we describe 5 novel microsatellite loci obtained by 454 GS-FLX Titanium pyrosequencing of a microsatellite-enriched library. The number of alleles per polymorphic locus varied between 2 and 4, and the observed heterozygosity ranged from 0.082 to 0.600. No significant deviation from Hardy–Weinberg equilibrium was found and there was no evidence for linkage disequilibrium. These markers will be of great value for paternity studies and population genetics of this species.

Development and characterization of novel microsatellite markers by Next Generation Sequencing for the blue and red shrimpAristeus antennatus

“…Sequences of the contigs including these polymorphic markers were submitted to GenBank (Accession Numbers: KU195267 – KU195299 ) ( Table 1 and Table S1 ). The proportion of success (36%) in detecting polymorphic loci for putative microsatellite markers was consistent to that achieved in decapods (15%–68%) ( Meehan et al , 2003 ; Dao, Todd & Jerry , 2013 ; Dambach et al , 2013 ; Miller et al , 2013 ), in other marine species (e.g., 29% in a mollusk ( Pardo et al , 2011 ) and also in fishes (∼40%; Sousa-Santos, Fonseca & Amorim , 2015 ; Williams et al , 2015 )).…”

“…Nevertheless, microsatellites have proved their utility in identifying genetic stocks in marine penaeoid shrimps around the world ( Benzie , 2000 ; Brooker et al , 2000 ; Supungul, Sootanan & Klinbunga , 2000 ; Ball & Chapman , 2003 ; Maggioni, Rogers & Maclean , 2003 ; Waqairatu et al , 2012 ; Abdul-Aziz et al , 2015 ) and it is possible that a greater number of these might provide higher resolution in other Mediterranean and Mozambique populations. Next-generation sequencing (NGS) based on 454 pyrosequencing has enabled a high-throughput approach and has demonstrated its usefulness in the development of SSRs in non-model organisms ( Schoebel et al , 2013 ; Sousa-Santos, Fonseca & Amorim , 2015 ). Long reads generated by 454 sequencing are expected to contain SSRs and their flanking regions that are needed to develop methods for SSR screening among populations.…”

Feeding ecology and life-history strategy of nesting males in a fish with long parental care, Lusitanian toadfish (Halobatrachus didactylus, Batrachoididae)

“…Total genomic DNA from fin clips of nesting males and respective embryos was isolated using NucleoSpin tissue 96 kit (Macherey-Nagel) following the manufacturer's protocol. Five polymorphic markers (Table 1 in Sousa-Santos et al ., 2015) were amplified in 25 µL reactions containing 20 ng of template DNA, 1× reaction buffer, 37.5 pmol MgCl 2 , 6 pmol dNTP, 10 pmol of each primer, and 1 U Taq polymerase (FastStart – Roche Diagnostics). PCR conditions were the following: 95°C for 10 min [95°C for 30 s, 55°C for 30 s, 72°C for 1′] × 40 cycles and 72°C for 10 min.…”

“…Details on the laboratorial procedures involved are described in Sousa-Santos et al . (2015). Alleles were scored using GeneMapper v5.0 software (Applied Biosystems).…”

“…To estimate the proportion of offspring fathered by the nest holder (NHM) the ‘two-sex paternity’ model was used, which assumes multiple genetic mothers and fathers. This software requires two infiles, one with the allele frequencies of the population, which were calculated with GENEPOP () based on a sample of 85 sequenced individuals from the Tagus estuary (Sousa-Santos et al ., 2015), and a second data file, which was built for each putative father and included the male's genotype for the five polymorphic loci and the genotypes of all sampled eggs in the considered nest. As outputs, the software provides the number of eggs sired by the male, the most likely paternity estimate, the expected paternity estimate and its respective lower and upper bounds of the 95% confidence interval.…”