Red and brown seaweeds are species with high ecological and economic importance. Here we report the feasibility of cost-effective molecular marker development in 6 species from different clades. Microsatellites markers of two brown seaweed species Alaria esculenta, Pylaiella littoralis, and of four red seaweed species Calliblepharis jubata, Gracilaria gracilis, Gracilaria dura and Palmaria palmata were identified and characterized using genomic sequences of Double-Digest Restriction site Associated DNA (ddRAD). A total of 64,623,186 reads were generated from two runs of multiplexed Illumina Miseq sequencing for which 30,636 reads containing microsatellites and 15,443 microsatellite loci with primers pairs were found. Five hundred seventy-six primers pairs were selected for amplification trials and levels of polymorphism. From the 338 that gave a positive amplification, 142 primers pairs were polymorphic. For genetic analyses two or three populations per species from 13 different geographic locations were used. A total of 28 usable polymorphic markers for A. esculenta, 18 for P. littoralis, 11 for C. jubata, 14 for G. gracilis, 21 for G. dura and 13 for P. palmata were developed. The overall number of alleles per locus ranged from 2 to 22. These 105 new microsatellite markers will be useful for further studies of population genetics, breeding programs and conservation genetics of these species. Compared with traditional approaches, our study yielded thousands of microsatellite loci in a short time with affordable costs in six different species. This study based on ddRAD-sequencing for the development of microsatellite markers provides preliminary data using a few individuals from two distinct populations on the genetic structure and reproduction mode of a non-model species as shown with the detection of clonality for the two red algae, C. jubata and G. dura and the detection of highly genetically divergent populations corresponding probably to different cryptic species under the name of P. littoralis.

Two individuals of each of the 6 species of algae were used to construct 2 genomics libraries (Lib1 and Lib2) according to the methods described by Peterson et al., 2012. Sage Science Pippin-Prep (sagescience.com) method was used to make the size selection to obtain fragments of interest between 350 and 550 bp (between 480 and 680 bp with the full library construction fragments). Size selection and quality of both libraries were checked using Bioanalyzer and High Sensitivity DNA Chips (Agilent Technologies), and the final concentrations were estimated through quantitative PCR (qPCR) using KAPA Library Quantification Kit (Roche Molecular Systems, Inc) and LightCycler® 480 System (Roche Molecular Systems, Inc). Sequencing was performed in two runs on a MiSeq sequencer (Illumina, San Diego, CA, USA) using the Miseq Reagent Kit v3 600 cycles (2 x 300 paired-end reads) and libraries at 15pM mixed with 5% of Phix control v3 (Illumina, San Diego, CA, USA).

STACKS software version 2.52 (Catchen et al., 2013); https://catchenlab.life.illinois.edu/stacks/