First insight into the genomes of the Pulmonaria officinalis group (Boraginaceae) provided by repeatome analysis and comparative karyotyping

Abstract

Background: The genus Pulmonaria (Boraginaceae) represents a taxonomically complex group of species in which morphological similarity contrasts with striking karyological variation. The presence of different numbers of chromosomes in the diploid state suggests multiple hybridization/polyploidization events followed by chromosome rearrangements (dysploidy). Unfortunately, the phylogenetic relationships and evolution of the genome, have not yet been elucidated. Our study focused on the P. officinalis group, the most widespread species complex, which includes two morphologically similar species that differ in chromosome number, i.e. P. obscura (2n = 14) and P. officinalis (2n = 16). Ornamental cultivars, morphologically similar to P. officinalis (garden escapes), whose origin is unclear, were also studied. Here, we present a pilot study on genome size and repeatome dynamics of these closely related species in order to gain new information on their genome and chromosome structure.

Results: Flow cytometry confirmed a significant difference in genome size between P. obscura and P. officinalis, corresponding to the number of chromosomes. Genome-wide repeatome analysis performed on genome skimming data showed that retrotransposons were the most abundant repeat type, with a higher proportion of Ty3/Gypsy elements, mainly represented by the Tekay lineage. Comparative analysis revealed no species-specific retrotransposons or striking differences in their copy number between the species. A new set of chromosome-specific cytogenetic markers, represented by satellite DNAs, showed that the chromosome structure in P. officinalis was more variable compared to that of P. obscura. Comparative karyotyping supported the hybrid origin of putative hybrids with 2n = 15 collected from a mixed population of both species and outlined the origin of ornamental garden escapes, presumably derived from the P. officinalis complex.

Conclusions: Large-scale genome size analysis and repeatome characterization of the two morphologically similar species of the P. officinalis group improved our knowledge of the genome dynamics and differences in the karyotype structure. A new set of chromosome-specific cytogenetic landmarks was identified and used to reveal the origin of putative hybrids and ornamental cultivars morphologically similar to P. officinalis.

Keywords: Pulmonaria; Comparative karyotyping; Genome size; Repeatome; Satellite DNA.

Fig. 1

Distribution map of the Pulmonaria officinalis group. (A) Distribution map of the P. officinalis group sampled and analyzed in this study (large dots; see Supplementary Table 1), including published chromosome reports (758 records in total, small dots; see Supplementary Table 2): P. obscura in yellow (2n = 14) and P. officinalis s.str. (2n = 16) in red. Illustrative images of (B) P. obscura (B473.1), (C) P. officinalis s.str. (B470.1) and (D) P. saccharata-like accession (B15.1)

Fig. 2

Genome size and GC content variation in Pulmonaria obscura and P. officinalis. (A) Variation in absolute genome size (2C-value); and (B) genomic GC content detected in the P. officinalis group: P. obscura (POBS), P. officinalis (POFF). Rectangles define the 25th and 75th percentiles, horizontal lines show median values, whiskers are 10–90 percentiles

Fig. 3

Repeatome composition in analyzed Pulmonaria accessions. Genome proportion of individual repeat type was obtained as the ratio of reads specific to the individual repeat type to all reads used for the clustering analysis by the RepeatExplorer2 pipeline. P. obscura (POBS; B473.1); P. officinalis (POFF; B470.3); an interspecific natural hybrid P. obscura × P. officinalis (HYBR; B481.1); P. saccharata-like accessions (PSAC1–PSAC3; B465.1; B472.1; B15.1)

Fig. 4

Graph structure of 5S rDNA sequence reads from the comparative analysis of RepeatExplorer2. (A) Graph structure obtained from all sequence reads homologous to the 5S rDNA. (B) The position of the 5S genic region on the graph topology is highlighted in yellow. (C–H) Cluster graph with annotated read origin: P. obscura in green (C); P. officinalis in orange (D); reads specific for P. obscura × P. officinalis (B481.1) in blue (E); reads specific for P. saccharata-like accession B15.1 are highlighted in purple (F), B465.1 in pink (G), and B472.1 in red (C)

Fig. 5

Idiograms of analyzed Pulmonaria accessions

Fig. 6

Chromosomal localization of newly identified satDNAs and rDNA sequences in P. obscura (2n = 14). (A, B, C, D) P. obscura (B473.1) with probes for: (A) 45S rDNA (yellow), PulTR01_29 (red) and 5S rDNA (green); (B) 45S rDNA (yellow), PulTR02_305 (red) and 5S rDNA (green); (C) PulTR01_29 (red), PulTR03_308 (orange), and 5S rDNA (green); (D) PulTR02_305 (yellow), 5S rDNA (red) and PulTR05_70 (green): yellow arrows indicate signals of PulTR02_305 and green arrows indicate signals of PulTR05_70. (E, F, G, H) P. obscura (B467.2) with probes for: (E) 45S rDNA (yellow), PulTR01_29 (red) and 5S rDNA (green): red arrow indicate subtelomeric signals of PulTR01_29; (F) 45S rDNA (yellow), PulTR02_305 (red) and 5S rDNA (green): red arrows indicate signals of PulTR02_305; (G) PulTR01_29 (red), PulTR03_308 (orange), and 5S rDNA (green); and (G) PulTR02_305 (yellow), 5S rDNA (red) and PulTR05_70 (green): yellow arrows indicate signals of PulTR02_305 and green arrows indicate signals of PulTR05_70. White arrows indicate 5S rDNA in all figures. Chromosomes were counterstained with DAPI (blue). Bars = 5 μm

Fig. 7

Chromosomal localization of newly identified satDNAs and rDNA sequences in P. officinalis (2n = 16). (A, B, C, D, E) P. officinalis (B470.3) with probes for: (A) 45S rDNA (yellow), PulTR01_29 (red) and 5S rDNA (green); (B) 45S rDNA (yellow), PulTR01_29 (red) and PulTR04_420 (green): green arrows indicate PulTR04_420 and; (C, D) the same plate with the signals for (C) 45S rDNA (red) and PulTR03_308 (green), and (D) co-localization of PulTR03_308 (green) and PulTR04_420 (red): green arrows indicate PulTR03_308, and red arrows indicate PulTR04_420; (E) 45S rDNA (yellow), PulTR05_70 (red), 5S rDNA (green): red arrows indicate signals of PulTR05_70. (F, G, H, I) P. officinalis (B100.2) with probes for: (F) 45S rDNA (yellow) and PulTR01_29 (red); (G) 45S rDNA (yellow), PulTR03_308 (red) and 5S rDNA (green): red arrows indicate PulTR03_308; and (H) 45S rDNA (yellow), PulTR04_420 (red), and 5S rDNA (green): red arrows indicate PulTR04_420; (I) 45S rDNA (yellow), PulTR05_70 (red) and PulTR03_308 (green): red arrows indicate signals of PulTR05_70 and green arrows point to PulTR03_308. White arrows indicate signals of 5S rDNA and yellow arrows point at interstitial 45S rDNA clusters. Chromosomes were counterstained with DAPI (blue). Bars = 5 μm

Fig. 8

Chromosomal localization of new satDNAs and rDNA sequences in natural hybrid P. obscura × P. officinalis (B481.1; 2n = 15). (A) Probes for 45S rDNA (yellow), PulTR02_305 (red) and 5S rDNA (green): red arrow points at the locus of PulTR02_305; (B) PulTR01_29 (red), PulTR03_308 (orange) and 5S rDNA (green): orange arrows indicate PulTR03_308; (C) 45S rDNA (yellow), PulTR01_29 (red) and PulTR04_420 (green): green arrows indicate PulTR04_420; red arrow points to PulTR01_29 and yellow arrow points at 45S rDNA locus indicating co-localization of these two probes; (D) 45S rDNA (yellow), PulTR05_70 (red) and 5S rDNA (green): red arrows indicate signals of PulTR05_70. White arrows indicate 5S rDNA signals. Chromosomes were counterstained with DAPI (blue). Bars = 5 μm

Fig. 9

Chromosomal localization of new satDNAs and rDNA sequences in P. saccharata-like accessions. (A, B, C, D, E) P. saccharata-like accession B465.1 (2n = 16) with probes for: (A) 45S rDNA (yellow), PulTR01_29 (red) and PulTR04_420 (green): green arrows indicate PulTR04_420; (B) PulTR01_29 (red) and 5S rDNA (green); (C) 45S rDNA (red) and PulTR03_308 (green): green arrows point at PulTR03_308; and (D) PulTR03_308 (green) and PulTR04_420 (red): green arrows point at PulTR03_308 and red arrows indicate PulTR04_420; (E) 45S rDNA (yellow), PulTR05_70 (red) and 5S rDNA (green): red arrows point to signals of PulTR05_70. (F, G, H, I) P. saccharata-like accession B472.1 (2n = 16) with probes for: (F) 45S rDNA (yellow), PulTR01_29 (green) and PulTR04_420 (red): red arrows point at PulTR04_420; (G) 45S rDNA (yellow), PulTR03_308 (green) and PulTR04_420 (red): green arrows point at PulTR03_308, and red arrows indicate PulTR04_420; (H) 45S rDNA (yellow), PulTR03_308 (red) and 5S rDNA (green); (I) 45S rDNA (yellow), PulTR05_70 (red) and PulTR03_308 (green): red arrows indicate signals of PulTR05_70 and green arrows point to PulTR03_308. (J, K, L, M) P. saccharata-like accession B15.1 (2n = 15) with probes for: (J) 45S rDNA (yellow), PulTR02_305 (red) and 5S rDNA (green): red arrow points at a signal of PulTR02_305; (K) 45S rDNA (yellow), PulTR01_29 (red) and PulTR04_420 (green): green arrows point at PulTR04_420; (L) PulTR03_308 (red) and 5S rDNA (green); (M) 45S rDNA (yellow), PulTR05_70 (red) and 5S rDNA (green): red arrows point to signals of PulTR05_70. White arrows indicate 5S rDNA signals and yellow arrows point at interstitial 45S rDNA clusters. Chromosomes were counterstained with DAPI (blue). Bars = 5 μm