Development of SSR markers in polyploid species using ddRAD sequence data: a case study in the genus Mimosa

Abstract

Mimosa L. is a megadiverse genus where lower rank classification is challenging due to widespread morphological similarities. M. subser. Obstrigosae includes 12 species with different ploidy levels. Morphological boundaries between them are defined by a few traits influenced by the environment. The extreme case is displayed by M. tandilensis and M. ramulosa which are solely defined by differences in plant habit, a trait poorly documented in herbaria. To aid species delimitation and taxonomic resolution in M. subser. Obstrigosae, we intended to develop new microsatellite markers. A ddRAD library was constructed from one individual of each M. tandilensis and M. ramulosa species. NGS derived reads were bioinformatically analysed for SSR mining. In order to avoid other sources of variation rather than the intended allelic variants, manual curation was performed. The applied selection criteria retained 72 loci from 495 candidates. Over 22 primer pairs tested, 15 polymorphic SSR were successfully amplified in a 22 individual panel test. Transferability to other species with varying phylogenetic relatedness was successful. A total of 239 variable characters (alleles), scored as presence-absence matrix, allowed to recognize species membership among 30 individuals belonging to four species of M. subser. Obstrigosae; M. adpressa and M. obstrigosa constitute two clear groups, contrarily to M. tandilensis and M. ramulosa that grouped together in several clusters. This pattern supports the hypothesis that the two latter perform a taxonomic complex, coinciding with conflicts to distinguish them by means of morphology. These results suggest that the 15 loci selected are suitable for clarifying species delimitation within M. subser. Obstrigosae. In sum, SSR development based on NGS sequencing and the selection criteria contributed herein provide a valuable tool for the molecular characterization of Mimosa beyond subseries Obstrigosae and may aid SSR development in other polyploid species.