Putting rose microsatellites into orbit: development and assessment of an SSR sequencing method

Microsatellites, also named simple sequence repeat (SSR), markers have been used for accession identification, parentage analysis, genetic diversity estimation and genetic map construction, on wild and cultivated roses. Capillary electrophoresis genotyping of SSR markers (SSRce) have been previously developed for roses. However, this technic suffers from low to medium throughput, homoplasy and moderate portability across laboratories. To overcome these drawbacks, recent studies applied amplicon sequencing of loci harbouring an SSR motif, a technic called SSR sequencing (SSRseq). Here we design and evaluate a first SSRseq panel for roses. We aim this set of markers to be informative and usable both at the Rosa genus level and at Rosa gallica L. (4x) species level, an important wild relative of cultivars. Available rose genome sequences were used to design primer pairs and to check in silico their transferability between Rosa species. Seventy-eight rose individuals (with varying ploidy levels) were genotyped by sequencing 60 selected SSRseq markers. The reproducibility was assessed on technical replicates. Alleles were called considering all sequence information (SNP, InDel and SSR motif, i.e., SSRseq approach) or the amplicon lengths only (mimicking SSRce genotyping). To estimate information gain of the SSRseq approach, genetic analyses were performed using one or the other allele calling method and previously obtained SSRce data. Forty SSRseq markers were validated. Transferability rate varied among markers. The SSRseq approach revealed much more alleles than SSRce mimicking approach, highlighting a high amount of size-homoplasy. We present the comparisons of pairwise distances calculations performed with either SSRseq or SSRce data. For the first time we have developed an SSRseq method for Rosa species and we compared SSRseq to SSRce data in polyploids. We will use this set on a large panel of wild R. gallica accessions to investigate the origins of French populations.