Epigenetic variability among saffron crocus (Crocus sativus L.) accessions characterized by different phenotypes.

Saffron (Crocus sativus L.) is a sterile triploid (2n=3x=24), initially assumed to be of autotriploid origin, although a growing number of evidences support alloploidy as the most probable mechanism to have occurred. The crop vegetatively multiplies year by year by means of corms. Corm multiplication does not generate genome variations with the exception of some spontaneous mutations that in a triploid saffron population are not easily detectable. At the present time, the real level of genetic variability inside saffron is still debated and in literature it is possible to recover contradictory articles providing contrasting results about if the species is monomorphic or not. In a preliminary characterisation of 50 saffron accessions of the WSCC (World Saffron and Crocus Collection, located in the Bank of Plant Germplasm of Cuenca), characters related to phenology (date of sprouting and flowering, duration of flowering), floral morphology (length and width of tepals, and length of stamen filaments and anthers) and saffron production (percentage of flowering corms, number of flowers per corm, saffron spice weight per flower) were measured and a big variation detected. This raises the question about the origin of such variability, and, considering that gene expression can be influenced both by genetic and epigenetic changes, epigenetic variation could be a possible origin of the alternative phenotypes. In order to have a deeper insight in the epigenetic of saffron, the present study was devoted to the analysis of the cytosine methylation among saffron accessions with different geographic origin and cultivated for at least three consecutive years in the same conditions inside the saffron “CrocusBank” collection. The analysis of the methylation was carried out by using the High C+G Patch (HCGP) Filtration method coupled with high throughput sequencing. The accessions have been selected based on geographic origin, different phenotypes, and different agronomic characters and were characterized by high or low saffron production, early and late flowering time. The presence of high epigenetic variability in DNA regions associated with gene expression was detected. Finally, in order to gain information on the stability along the years of the epigenetic in a vegetatively propagated plant, saffron epigenetics of 17 different accessions stored in the “CrocusBank” collection was analysed in 4 consecutive years from 2013 to 2016. Each accession, despite the cultivation in proximity in the same field and despite the presence of intra- and inter-accession variability, tended to maintain a proper epigenotype clearly different from the other accessions.