Development of microsatellite markers for use in breeding catfish, <i>Rhamdia</i> sp

Abstract

Microsatellites markers for catfish, Rhamdia sp. were developed using Next Generation. A shotgun paired-end library was prepared according to standard protocol of Illumina Nextera DNA Library Kit with dual indexing, paired-end reads of 100 base pairs and grouped with other species. From a single race, five million readings obtained were analyzed with the program PAL_FINDER_v0.02.03. perl script was used to extract readings containing microsatellites with di-, tri-, tetra-, penta- and hexanucleotides from five million readings obtained in sequencing. Readings were grouped and used in Primer3 version 2.0.0 to design primers when GC content was greater than 30%, melting temperatures was within 58 to 65°C, with 2°C maximum difference between primers, the last 2 nucleotides in 3’extremity are G or C, and maximum poli-N of 4 nucleotides. When all criteria were met, a single pair of primers was selected according to the highest score in Primer3 and with greater amplification region of the repeated sequence. We identified 6,331 microsatellite loci potentially amplifiable (microsatellites) of which 4,755 were dinucleotide, 728 trinucleotide, 729 tetranucleotide, 117 pentanucleotídeo and 2 were hexanucleotídeo. A group of 12 loci microsatellite (including di- and tetranucleotides) has been sequenced, using Sanger’s method, to obtain complete sequences for fragments between 140 and 200 pb and are currently being used to study the genetic diversity of catfish populations. The populations showed genetic variation with average number of alleles per locus of 6.14. Microsatellites acquired with Next-Generation Sequencing (NGS) are an efficient tool for obtaining highly polymorphic markers for non-model species.

Key words: Next-generation sequencing, simple sequence repeats, Rhamdia sp.