Bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix) are invasive species and listed as US federally injurious species under the Lacy Act. They have established populations in much of the Mississippi River Basin (MRB; Mississippi, Illinois, and Missouri rivers) and are capable of producing fertile hybrids and complex introgression. Characterizing the composition of this admixture requires a large set of high-quality, evolutionarily conserved, diagnostic genetic markers to aid in the identification and management of these species in the midst of morphological ambiguity. Restriction site-associated DNA (RAD) sequencing of 45 barcoded bighead and silver carp from the United States and China produced reads that were aligned to the silver carp transcriptome yielded 261 candidate single nucleotide polymorphisms (SNPs) with fixed allelic differences between the two species. We selected the highest quality 112 SNP loci for validation using 194 putative pure-species and F1 hybrids from the MRB and putative bighead carp and silver carp pure species from China (Amur, Pearl and Yangtze rivers). Fifty SNPs were omitted due to design/amplification failure or lack of diagnostic utility. A total of 57 species-diagnostic SNPs conserved between carp species in US and Chinese rivers were identified; 32 were annotated to functional gene loci. Twenty-seven of the 181 (15%) putative pure species were identified as hybrid backcrosses after validation, including three backcrosses from the Amur River, where hybridization has not been documented previously. The 57 SNPs identified through RAD sequencing provide a diagnostic tool to detect population admixture and to identify hybrid and pure-species Asian carps in the United States and China.

SNP genotype file for validationData was generated on the MassARRAY 4 analyzer system (Sequenom, Inc., San Diego, CA) and tissue was collected in the field from the authors and other personnel listed in the acknowledgements. The SNP ID's and primer sequences are added in the supplementary information of the manuscript for reference. The top rows indicate species specific genotypes for Hypophthalmichthys nobilis (BH) and H. molitrix (SV). The remaining rows and columns give the location, field ID, genetic ID, and genotypes of individual fishes at 57 SNP loci.