Genotyping by Amplicon Sequencing (GBAS) With Newly Developed SSR and EPIC Markers Reveals Structure in Populations of the Green Toad () Across Rural and Urban Environments.

Microsatellites (SSRs) are reliable markers for population genetic analyses but often suffer from null alleles caused by mutations in primer binding sites. Exon-primed intron-crossing (EPIC) markers address these limitations and serve as a complementary tool. In this study, we developed SSR and EPIC markers for the green toad (). We demonstrated their effectiveness using genotyping by high-throughput amplicon sequencing (GBAS) across urban and rural populations. Marker development followed an established in-house protocol. We then tested their functionality in singleplex-PCRs before employing them in multiplex reactions and preparing them for sequencing. We analyzed the resulting data using several variability measures as well as individual-based clustering methods. From the initial set of 48 markers of each type, 35 SSR and 46 EPIC markers consistently amplified across samples both in singleplex and multiplex assays. Data analysis did not corroborate the expectation of a continuous reduction of diversity in urban populations compared to rural ones. Clustering methods using EPIC markers revealed ecologically coherent results, showing weaker genetic structure in rural environments, whereas the SSRs' signal reflected drift-induced patterns. Our findings suggest that the green toad exhibits a degree of resilience to urban environments. Furthermore, EPIC markers not only complement SSRs by having fewer null alleles but also provide greater robustness to random drift events. We recommend their combined use, especially in fragmented environments prone to genetic drift, such as urban areas.