Villosa lienosa Transcriptome or Gene expression

Freshwater mussels (Unionidae) are among the most endangered groups of organisms in the world. North America, and, particularly, the southeastern United States, are both 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. The conservation and recovery of freshwater mussel species is now a priority throughout North America, with particular focus in the southeast U.S. 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 23,742unigene 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). The present work demonstrates the power of RNA-seq for rapid development of molecular resources in non-model species and represents the first large-scale transcriptome project in freshwater mussels. The validated microsatellite set and stress-associated genes are currently being utilized in ongoing parentage analysis and health assessment surveys, respectively, in support of ongoing mussel conservation efforts.