Внутригеномный полиморфизм внутренних транскрибируемых спейсеров 35S рДНК (ITS) у трёх видов и трёх межвидовых гибридов Pulsatilla (Ranunculaceae)

Morphological criteria do not always allow to determine the hybrid origin of samples. The study of intragenomic polymorphism of either protein-coding or rRNA-coding genes provides a more accurate assessment. ITS regions of 35S rDNA are often used as one of such markers. In the presented work, we performed a comparative analysis of ITS1 in three hybrids (nothospecies) of the genus Pulsatilla: P. × intermedia, P. × spuria and P. × hackelii and their parental species P. patens, P. vernalis, P. pratensis from Leningrad Region (NW Russia). The ITS1-5.8S rDNA-ITS2 region was sequenced by Sanger’s sequencing method. On the chromatograms of the ITS sequences of the parental species there were on average, 1.7 (P. pratensis) to 3.3 (P. vernalis) polymorphic sites (PSs) – positions where two different nucleotides were detected. All three studied nothospecies exhibited more PSs – from 4.7 (P. × intermedia) to 9.5 (P. × hackelii). Study of intragenomic polymorphism of the transcribed spacer ITS1 by locus-specific NGS confirmed an obviously assumption that polymorphic sites detected by Sanger sequencing actually reflect the presence in the genomes of different rDNA variants (ribotypes) that were received by the plant from its ancestors, if the proportion of the unusual ribotype is about 20 % or more. Rarer ribotype variants can be detected only by NGS. Among the major ribotypes (ZOTU) of the studied nothospecies there were found any specific ZOTU, which were not detected in the parental species. The intragenomic polymorphism of the ITS1 region revealed by either Sanger sequencing or NGS in parental species may indicate earlier events of homoploid hybridization accompanying speciation within the genus.