Cross-amplification and multiplexing of SSR markers for Alnus glutinosa and A-incana

We investigated 39 previously developed Betula, Alnus, and Corylus simple sequence repeat (SSR) markers for their utility in the cross-generic amplification of two European alder species, i.e., Alnus glutinosa and A. incana. Of these markers, ten loci had successful amplification within Alnus species. Finally, we designed two multiplexes composed of eight and nine loci for A. glutinosa and A. incana, respectively. Multiplexes were tested on 100 samples from five different populations of each species across Europe. The majority of loci had a relatively high genetic diversity, were in Hardy-Weinberg equilibrium, and showed low error rates and low occurrence of null alleles. By comparing sequences of source species and both Alnus species, we concluded that repeat motifs of five of these ten loci differed from those described for the source species. These differences represent mainly the modifications of the original motifs and affected compound or interrupted repeats as well as pure ones. The repeat motifs of three loci of the two alder species also differed. These mutations could lead to erroneous estimates of allele homology, because alleles with identical lengths will not have the same number of repeat units. Hence, before using microsatellite markers in studies comparing two or more species, they should be carefully examined and sequenced to ensure that allele homology is really stable and not affected by various inserts that change the sequence.