Rapid Development of Molecular Resources for a Freshwater Mussel, Villosa lienosa (Bivalvia: Unionidae) Using a RNA-seq-based Approach

Freshwater mussels (Unionidae) are among the most endangered groups of organisms in the world. North America, and, particularly, the southeastern United States, are hotspots of freshwater mussel diversity and areas of special concern due to habitat degradation by changes in land uses (stream alteration and impoundment), impact of exotic species, and loss of host fish necessary for completion of mussel life cycles. Efforts aimed at captive propagation and reintroduction are hindered, however, by significant gaps in our understanding of mussel assemblages, spatial arrangements of populations, naturally occurring sex ratios and male spawning contributions, and genetic variability within wild populations. To begin to fill these gaps, here we utilized an RNA-seq-based approach to develop molecular resources in Villosa lienosa, the little spectaclecase. Sequencing of barcoded, pooled tissue samples within a single lane of Illumina HiSeq followed by assembly using Trans-ABySS generated 778,234 contigs with average length of 707.5 bp from 162 million filtered reads. Data analysis allowed the identification of 23,742 unigene hits against the NCBI nr database, and 36,582 microsatellites with sufficient flanking sequence for primer design. Microsatellite validation indicated a 36% polymorphic rate (16/44 tested markers) in the tested population (26 individuals) with an average of five alleles per marker. Analysis of differentially expressed genes between heat-stressed and untreated controls allowed identification of 604 genes involved in the stress response pathways. Real-time RT-PCR validation of gene expression results utilizing individual samples generally confirmed RNA-seq patterns (correlation coefficient 0.847, p<0.001).