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Background  The species relationships within the genus  Linum  have already been studied several times by means of different molecular and phylogenetic approaches. Nevertheless, a number of ambiguities in phylogeny of  Linum  still remain unresolved. In particular, the species relationships within the sections  Stellerolinum  and  Dasylinum  need further clarification. Also, the question of independence of the species of the section  Adenolinum  still remains unanswered. Moreover, the relationships of  L. narbonense  and other species of the section  Linum  require further clarification. Additionally, the origin of tetraploid species of the section  Linum  (2n = 30) including the cultivated species  L. usitatissimum  has not been explored. The present study examines the phylogeny of blue-flowered species of  Linum  by comparisons of 5S rRNA gene sequences as well as ITS1 and ITS2 sequences of 35S rRNA genes.    Results  High-throughput sequencing has been used for analysis of multicopy rRNA gene families. In addition to the molecular phylogenetic analysis, the number and chromosomal localization of 5S and 35S rDNA sites has been determined by FISH.  Our findings confirm that  L. stelleroides  forms a basal branch from the clade of blue-flowered flaxes which is independent of the branch formed by species of the sect.  Dasylinum . The current molecular phylogenetic approaches, the cytogenetic analysis as well as different genomic DNA fingerprinting methods applied previously did not discriminate certain species within the sect.  Adenolinum.  The allotetraploid cultivated species  L. usitatissimum  and its wild ancestor  L. angustifolium  (2n = 30) could originate either as the result of hybridization of two diploid species (2n = 16) related to the modern  L. gandiflorum  and  L. decumbens , or hybridization of a diploid species (2n = 16) and a diploid ancestor of modern  L. narbonense  (2n = 14).    Conclusions  High-throughput sequencing of multicopy rRNA gene families allowed us to make several adjustments to the phylogeny of blue-flowered flax species and also reveal intra- and interspecific divergence of the rRNA gene sequences.    Electronic supplementary material  The online version of this article (doi: 10.1186/s12862-017-1105-x) contains supplementary material, which is available to authorized users.

Evolution of blue-flowered species of genus Linum based on high-throughput sequencing of ribosomal RNA genes