Development and characterization of eighty-four microsatellite markers for the red snapper (Lutjanus campechanus) using Illumina paired-end sequencing Adrienne E. Norrell a , Danielle Crawley a , Kenneth L. Jones b , Eric A. Saillant a, ⁎ a Department of Coastal Sciences, The University of Southern Mississippi, Gulf Coast Research Laboratory, Ocean Springs, MS 39564, United States b Department of Biochemistry and Molecular Genetics, University of Colorado, School of Medicine, Aurora, CO 80045, United States abstract article info Article history: Received 11 December 2013 Received in revised form 4 April 2014 Accepted 6 April 2014 Available online 13 April 2014 Keywords: Lutjanus campechanus Red snapper Microsatellites PAL_FINDER Illumina sequencing Eighty-four microsatellite loci were isolated from red snapper, Lutjanus campechanus, using Illumina paired-end sequencing and a direct ‘Seq-to SSR’ approach. The number of alleles per locus ranged from 7 to 30 and estimates of expected heterozygosity ranged from 0.466 to 0.967 (n = 30). Genotype frequencies at 7 loci deviated signif- icantly from Hardy-Weinberg expectations; the departure at 4 of these loci likely reflects the segregation of null alleles. The markers characterized in this work will be applied to linkage mapping and genetic studies involved in the domestication and stock enhancement of red snapper. Development of the 84 microsatellites required screening approximately 1.5 M paired-end reads, highlighting the potential of this method for rapid and cost- effective development of new homologous microsatellites in emerging aquaculture species. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Microsatellites are among the most widely used DNA markers in aquaculture genetics (Liu and Cordes, 2004). Recent advances in next- generation sequencing have rendered the discovery of novel microsat- ellite loci much more effective through screening of sequencing reads for potentially amplifiable microsatellites. This ‘Seq-to-SSR’ approach can be implemented directly on raw sequencing reads without the need for post-sequencing assembly (Castoe et al., 2012), thereby greatly simplifying the bioinformatics treatment involved in the discovery of new loci. In this note, we report a practical implementation of this method using Illumina paired-end sequencing to develop a panel of new microsatellites in an economically important marine fish, the red snapper Lutjanus campechanus. Briefly, the red snapper is a tropical reef fish intensively exploited by recreational and commercial fisheries in the Gulf of Mexico region. Be- cause of its economic importance and overfished status (SEDAR, 2013), the red snapper is identified as a primary candidate for marine aquaculture in the United States (Blaylock et al., 2000; Saillant et al., 2013). Genomic tools needed for the sustainable management of red snapper aquaculture operations are currently being developed and in- clude a high density linkage map that will be based, in part, on a large panel of single nucleotide polymorphism (SNP) loci. The addition of hypervariable markers such as microsatellites will improve the accuracy of the map, particularly during the combination of multiple SNP data sets (Ball et al., 2010). The markers used to create the map should ideal- ly provide genome-wide coverage, making it necessary to expand the existing panel of homologous microsatellites designed by Gold et al. (2001). In this work, 84 new loci were therefore developed and charac- terized and will be available for genome mapping in red snapper. This study illustrates the outcome and effectiveness of the ‘Seq-to SSR’ meth- od for the development of new microsatellites in a non-model species. 2. Materials and methods Genomic DNA was extracted from the spleen tissue of one red snap- per individual using a standard phenol chloroform protocol (Sambrook et al., 1989). An Illumina paired-end library was prepared as described by Castoe et al. (2012) and sequenced on one lane of the Illumina HiSeq platform, producing a total of 166,174,049 million reads. Raw reads were screened for di-, tri-, tetra-, penta-, and hexanucleotide mi- crosatellite arrays in the program PAL_FINDER_v.0.02.03 (Castoe et al., 2012). Loci containing between 12 and 25 uninterrupted repeats were preferentially selected and PCR primers were designed in the regions flanking each microsatellite using the software Primer3 (Untergrasser et al., 2012). A total of 150 primer pairs could be designed through screening ap- proximately 1.5 M raw reads in PAL_FINDER. The primer pairs were test- ed for Polymerase Chain Reaction (PCR) amplification success and Aquaculture 430 (2014) 128–132 ⁎ Corresponding author. Tel.: +1 228 818 8007; fax: +1 228 872 4204. E-mail address: eric.saillant@usm.edu (E.A. Saillant). http://dx.doi.org/10.1016/j.aquaculture.2014.04.005 0044-8486/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online