Rapid development of molecular resources for a freshwater mussel, Villosa lienosa (Bivalvia:Unionidae), using an RNA-seq-based approach

Freshwater mussels (Unionidae) are among the most endangered groups of organisms in the world, and their conservation and recovery are priorities throughout North America, especially the southeastern USA. We used a ribonucleic acid sequencing (RNA-seq)-based approach to develop molecular resources for Villosa lienosa, the little spectaclecase. We sequenced barcoded samples (Illumina HiSeq 2000 platform) and assembled (Trans-ABySS) 778,234 contigs (average length = 707.5 base pairs [bp]) from 162 million filtered reads. We identified 23,742 unigene hits against the National Center for Biotechnology Information nonredundant 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; mean = 5 alleles/marker). Analysis of differentially expressed genes between heat-stressed and untreated controls enabled us to identify 604 genes involved in stress-response pathways. Real-time polymerase chain reaction validation of gene-expression results using individual samples confirmed RNA-seq patterns (r = 0.847, p < 0.001). RNA-seq is a powerful tool for rapid development of molecular resources in nonmodel species, and our study is the first large-scale transcriptome project in freshwater mussels. The validated microsatellite set and stress-associated genes are being used in parentage analysis and health-assessment surveys to support mussel conservation.