Microsatellite markers developed in the stingless bee Melipona fasciculata by next-generation sequencing and an exploratory analysis of geographic genetic variation

Background: Native meliponines are currently threatened by increased human impacts. The assessment of their genetic variation by microsatellite DNA markers can assist in the conservation of populations and help in the planning and establishment of efficient management strategies. Next generation sequencing has proven to be useful for identifying microsatellite loci from the large amounts of sequence data generated. Methods: . The purpose of this study was to develop the first set of microsatellite markers for Melipona fasciculata , selected from partial genome assembly of Illumina paired-end reads. Contigs were created from the resulting paired-end sequence data and these were analyzed with specialized software to extract those reads that contained microsatellite loci. Primer pairs were designed for each detected locus at their flanking regions. Bee samples were genotyped from two different locations for markers characterization and validation. Results: . A total of 17 microsatellite loci displayed polymorphism from two different populations of Northeastern Brazil. Mean H E and H O heterozygosities were 0.453 and 0.536, respectively. PIC across all loci ranged from 0.108 to 0.714. A genetic diversity analysis revealed high values for population differentiation estimates (F ST = 0.194, R ST = 0.230, and D est = 0.162). PCoA and Bayesian clustering showed a separation of the species into two distinct clusters. Discussion: The Illumina paired-end sequencing system provided a large number of microsatellite loci from the M. fasciculata genome. From the genotyped data this study was able to reveal high F ST and R ST estimates and suggest the existence of genetic structure. These microsatellite markers have demonstrated strong potential for population-level genetic studies and can be used effectively as a molecular tool. Moreover, the exploratory analysis of the genetic diversity in M. fasciculata provides provisional evidence of significant population differentiation between the two studied populations.