Exploring the chemotypic variability of Silybum marianum and Silybum eburneum by biochemical and genetic characterization

Laura Bassolino^1*Marianna Pasquariello¹Tommaso Martinelli²Roberta Paris¹Anna Moschella¹Roberto Colombo³Alice Di Bello¹Jessica Frigerio⁴Abdenour Kheloufi⁵Mohammad Amin Mirzaabolghasemi⁶Damiano Puglisi⁷Salvatore Esposito⁷Stefano Scalercio⁸Nino Virzì⁹Pasquale De Vita⁷Nicola Pecchioni⁷

• ¹Research Centre for Industrial Crops, Council for Agricultural and Economics Research (CREA), Bologna, Italy
• ²Research Centre for Plant Protection and Certification (CREA-DC), Loc. Cascine del Riccio, Via di Lanciola 12/A 50125 Firenze, Italy, Firenze, Italy
• ³Research Centre (CREA-AA), via di Corticella 133, 40128 Bologna, Italy, Bologna, Italy
• ⁴Department of Biotechnology and Biosciences, School of Sciences, University of Milano-Bicocca, Milano, Lombardy, Italy
• ⁵Department of Ecology and Environment, University Batna 2, 53, Route de Constantine. Fesdis, Batna 05078, Algeria., Batna, Algeria
• ⁶Section of Plant Breeding and Biotechnology, Department of Horticulture, Faculty of Agriculture, Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran., Rafsanjan, Iran
• ⁷Research Centre for Cereal and Industrial Crops, Council for Agricultural and Economics Research (CREA), Foggia, Apulia, Italy
• ⁸Research Centre for Forestry and Wood (CREA-FL), Via Settimio Severo 83, I87036 Rende, Italy, Rende, Italy
• ⁹, Research Centre for Cereal and Industrial Crops (CREA-CI), C.so Savoia 190, 95024, Acireale, CT, Italy, Acireale, Italy

The Silybum genus belonging to the Asteraceae family, is composed of two species, marianum and eburneum, although, in the past, their classification was not always appropriate. While Silybum marianum is very well known since ancient times for the medicinal properties of a blend of different flavonolignans contained in the achenes and named silymarin, very little information is available about Silybum eburneum chemodiversity. Here, we describe the biochemical characterization of a wide ex situ germplasm collection including 83 wild Silybum accessions collected during ad hoc sampling campaigns in Italy, Spain, Iran and Algeria as well as accessions acquired by seed GenBanks and studied at both population and single plant level. Interestingly, our results confirm the presence of only three chemotypes in S. marianum, namely A, B and C. Conversely, S. eburneum accessions, exhibit a distinct and stable chemotype (D) where isosilychristin is the predominant silymarin component.Additionally, DNA barcoding based on the ribosomal DNA region ITS2 combined with morphological phenotyping and chemotyping, successfully resolves frequently found mistakes in the identification of the two species. These findings significantly expand our knowledge of the global biodiversity of the Silybum genus and provide valuable insights for future breeding programs and potential applications in nutrition and human health sciences.