Authors: MONIKA SZCZECINSKA, GRZEGORZ LAZARSKI, KATARZYNA BILSKA, JAKUB SAWICKI
Abstract: This study presents the first molecularly proven data on natural hybridization in the genus Pulsatilla, including the complete plastome sequence. A plant with morphological characteristics intermediate between P. patens and P. pratensis was found near the village of Bocheniec (southern Poland). In this study, several classes of markers based on genotyping (SSR and ISJ) and sequencing (Sanger and NGS) were used to confirm hybridization and its direction, and to determine the prospective parent species. Analysis of ISJ and ISSR genotyping data revealed that all amplified loci were polymorphic. The hybrid species was characterized by only 3 specific bands, and it shared the highest number of bands with P. pratensis. A total of 3,204,448 pair-end reads (2 × 250 bp) from Pulsatilla × hackelii were obtained in a single run of the MiSeq sequencer. The structure of the hybrid plastid genome did not differ from the previously identified genomes of the Pulsatilla species. A phylogenetic analysis of plastomes in the Pulsatilla species revealed three well-supported clades. The plastome of Pulsatilla × hackelii was included in the P. pratensis clade. P. hackelii and P. pratensis formed a well-supported clade, and their plastomes differed by 78 substitutions and 74 indels. Most of these differences were found in intergenic noncoding regions (52 substitutions and 67 indels) and introns (11 substitutions and 5 indels). The sequenced plastid genome of the hybrid species was most similar to Pulsatilla pratensis, which suggests that the analyzed P. pratensis specimen was pollinated by P. patens.
Keywords: Natural hybrid, nucleolus organizer region, simple sequence repeat, intron-exon splice junction, plastid genome, next-generation sequencing,
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