The goal of this project was to compare the genetic diversity of a wild source population of Three-ridge mussels (Amblema plicata) to the cohort of juveniles propagated from three gravid females from the wild population. The juveniles were subsampled directly after transformation from glochidia (“young juveniles”) and subsampled again after one year of being raised in the hatchery (“old juveniles”).  After correcting for different sample sizes, the young juveniles exhibited the greatest allelic richness, followed by the source mussels and then the old juveniles. All three groups exhibited private alleles. Private alleles in the juveniles indicated the dams (mothers) were likely fertilized by males living upstream of the source population, outside of the sampling effort of this study. High levels of multiple paternity were observed in the young and old juveniles. In total, 89 juveniles were estimated to have been sired by 58 males, increasing the amount of genetic variability in the produced population. The old juvenile samples were taken from a random subsample and were found to have been produced nearly entirely from a single dam, indicating differential mortality in the hatchery which reduced the amount of genetic variability in the released population. The old juvenile sample was significantly differentiated from the source population (p<2.04e-26), suggesting the released juvenile population did not fully represent the source population.

We collected non-destructive samples from 50 Three-ridge mussels in the Upper Cedar River near Lansing, Minnesota using buccal swabs, which were then stored in ethanol (the ‘source’ sample set). Three gravid females from the source population were used for propagation at the Center for Aquatic Mollusk Programs in Lake City, MN. Hatchery staff infested individual walleye (Sander vitreus) host fish with the glochidia from a single dam. After transformation and dropping from their hosts, 20 juveniles from each dam were collected and preserved in ethanol for a total of 60 samples (‘young juveniles’). Staff combined the remaining juveniles and reared them at the hatchery for one year. On July 22, 2020, staff collected 50 more juveniles and preserved them in ethanol (‘old juveniles’).

Genomic DNA was isolated from the buccal swabs of the adult mussels using QIAGEN’s Purgene Buccal Cell Core Kit A following the ‘DNA Purification from a Buccal Brush’ protocol (Qiagen). Genomic DNA was isolated from the young juvenile samples using Chelex 100 Resin (BioRad) following a modified version of the protocol outlined in Singh, Kumari, and Iyengar (2018). Tissue samples were taken from the old juveniles, and genomic DNA was isolated using the QIAamp DNA mini kit according to the ‘Tissue’ protocol (Qiagen). All extracted DNA was quantified using a Nanodrop ND1000 spectrophotometer and stored at 4° C. We used 16 microsatellite markers developed for the Fat Threeridge (Amblema neislerii) (Díaz-Ferguson, Williams, and Moyer 2011) to genotype the mussel samples collected (Table 1).

We conducted polymerase chain reaction (PCR) amplification using the BIOLASE PCR kit (Bioline, Boston, MA). Each 10 microliter (μl) reaction contained 6.6 μl of sterile deionized water, 1 μl of Biolase NH4 reaction buffer (10x), 0.6 μl of MgCl2 (50 mM), 0.8 μl of dNTP’s (2.5 mM each), 0.1 μl of M13 labeled forward primer (20 mM), 0.1 μl of reverse primer (20 mM), 0.05 μl of M13 labeled oligo (20 mM), 0.05 μl of Biolase DNA Taq polymerase (5 U/ μl), and 1 μl of template DNA (approximately 2 nanograms (ng)/ μl). Reactions were completed in Eppendorf Master Cycler Thermocyclers under the following conditions: 95⁰C/ 5:00 min; (94⁰C/ 00:30 seconds, touchdown beginning at 56⁰C and dropping by 0.6⁰C per cycle/ 1:00 minute, 72⁰C/ 00:30 seconds) x11; (94⁰C/ 00:30 seconds, 55⁰C / 1:00 minute, 72⁰C/ 00:30 seconds) x25; 72⁰C / 20:00 minutes. A negative control was performed with each reaction. PCR products were visualized on 1.5% agarose gels against a 100 bp DNA ladder to confirm the success of the reactions and to ensure the negative control showed no contamination. Allele sizes were determined with capillary electrophoresis on an Applied Biosystems 3500 Genetic Analyzer by the Iowa State University DNA Facility. We scored raw data with the software Gene Marker (Version 3.0.1). We checked all loci for null alleles with MICRO-CHECKER (Van Oosterhout et al. 2004). Loci with possible null alleles were excluded from the data set.