S05 - Session O6 - Putting rose microsatellites into orbit: development and assessment of an SSR sequencing method

Authors: Clovis Pawula *, Olivier Lepais, Erwan Guichoux, Annie Chastellier, Emilie Chancerel, Benjamin Tyssandier, Valéry Malécot, Agnès Grapin, Jérémy Clotault, Alix Pernet

Microsatellite s , also named s imple 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 ( SSR ce ) have been previously developed for roses. However, this technic suffer s 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 evaluat e a firs t 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. Ninety-five rose individuals (with varying ploidy levels) were genotyped by sequencing 60 selected SSRseq markers. The genotyping was independently repeated to identify reliable markers. Alleles were called considering all sequence information (SNP, InDel and SSR motif, id est 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 a nd 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 will present the comparisons of diversity and structure analyses 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 Rosa gallica L. accessions to investigate the origins of French populations.