Development and characterization of novel tetranucleotide microsatellite markers in the noble crayfish (Astacus astacus) suitable for highly multiplexing and for detecting hybrids between the noble crayfish and narrow-clawed crayfish (A. leptodactylus)

Abstract 48 novel tetranucleotide microsatellite loci were developed for the noble crayfish ( Astacus astacus ) using Illumina MiSeq next generation sequencing technology. It was demonstrated that 25 loci were polymorphic and 19 loci could be co-amplified in a single multiplex polymerase chain reaction (PCR) assay and genotyped as a single panel on Applied Biosystems 3500 Genetic Analyser. The 19-plex assay was tested on 232 individuals of A. astacus originating from seven wild populations in Czech Republic and in Estonia. The multiplex assay designed in this study can be successfully applied in studies requiring high genetic resolution, such as population structuring, relatedness analysis, and stock identification. 21 loci were also successfully cross-amplified in the narrow-clawed crayfish ( Astacus leptodactylus ) from which 14 were polymorphic. In addition, 13 loci (both monomorphic and polymorphic) possessed species-specific allele size range in A. astacus and A. leptodactylus and can be applied for detecting possible hybrids between these sister species. Statement of relevance The novel 19-plex microsatellite assay can be applied for genetic management of captive stocks of the noble crayfish (selection of strains, planning of matings, avoiding of inbreeding) and in studies requiring high genetic resolution, such as parentage assessment, relatedness analysis or strain identification.