The 5S intergenic spacer
The 5S rRNA genes form gene arrays analogous to the 35S rDNA, localized on one or a few chromosomes, usually afar from the nucleolus organizer regions (NORs) of the 35S rDNA (http://www.plantrdnadatabase.com/; see Ribeiro et al. 2011 for selected oak and beech species). In higher plants, the number of repeats per genome ranges from hundreds to thousands (Cloix et al. 2000), and the 5S rRNA coding regions are separated by a generally variable, non-transcribed intergenic spacer (5S-IGS) with high phylogenetic resolution (e.g., Forest et al. 2005; Blattner et al. 2009; Grimm and Denk 2010; Garcia and Kovařik 2013; Mlinarec et al. 2016). Its inter-individual and intra-genomic variability is probably the main reason why it is little used. It cannot be directly sequenced but requires cloning and methodological frameworks that can make use of the often higher intra-genomic than inter-individual diversity (Göker and Grimm 2008; Potts et al. 2014; for an application using 5S-IGS data see e.g. Simeone et al. 2018). Therefore, large numbers of 5S-IGS variants should ideally be cloned and sequenced to identify intra- and inter-array polymorphism, and phylogenetic signals useful for inferring evolutionary patterns (Eidesen et al. 2007). In previous studies, Denk and Grimm (2010) and Simeone et al. (2018) produced a data set with more than 1000 Sanger-sequenced 5S-IGS clones (covering 30 species, nearly 300 individuals) and demonstrated the potential of this marker for the circumscription of most of the investigated oak species, to recognize hybridization, and to infer reticulation/introgression events.