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

First insight into the genomes of the Pulmonaria officinalis group (Boraginaceae) provided by repeatome analysis and comparative karyotyping | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article First insight into the genomes of the Pulmonaria officinalis group (Boraginaceae) provided by repeatome analysis and comparative karyotyping Lucie Kobrlová, Jana Čížková, Veronika Zoulová, Kateřina Vejvodová, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4148849/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Sep, 2024 Read the published version in BMC Plant Biology → Version 1 posted 12 You are reading this latest preprint version Abstract Background The genus Pulmonaria (Boraginaceae) represents a taxonomically complex group of species in which morphological similarity contrasts with striking karyological variability. 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 (2 n = 14) and P. officinalis (2 n = 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 partial Illumina sequencing 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 landmarks, represented by satellite DNAs, showed that the chromosome structure in P. officinalis was more variable compared to that of P. obscura . Comparative karyotyping strongly supported the hybrid origin of putative hybrids with 2 n = 15 collected from a mixed population of both species and outlined the origin of ornamental garden escapes, confirming their derivation 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 . Comparative karyotyping Genome size Pulmonaria Repeatome Satellite DNA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background The genus Pulmonaria L. (Boraginaceae, sensu [ 1 ]) is a taxonomically complex group of species in which the rather similar morphology contrasts with striking karyological variability. In total, 16 different somatic chromosome counts ranging from 2 n = 14 to 2 n = 38 are currently reported in the genus [Kobrlová unpubl. ], with x = 7 proposed as the basic chromosome number [e.g. 2, 3, 4, 5, 6]. According to the traditional morphology- and karyology-based taxonomy [cf. 3, 6], about 30 taxa are recognized (16 species and 14 subspecies), all found in Europe, with the exception of the P. mollis group, which extends as far as north-eastern and eastern Asia [ 5 , 7 , 8 , 9 ]. Although almost nothing is known about the mechanisms of the chromosomal evolution of the genus, such a karyological diversity is indicative of extensive chromosomal rearrangements, i.e. the hypothesis of ancient episodes of polyploidization/hybridization and subsequent dysploidal chromosome reorganizations [cf. 6, 10]. Considerable variability in chromosome number and genome size has been reported within the Boraginaceae family [summarized in 11, 12], but its role in species diversification and evolution has mostly only been hinted at or discussed [but see e.g. 10, 13, 14, 15]. To date, only a few attempts have been made to explore the evolutionary history of the genus Pulmonaria , focusing on a limited or specific group of species [ 10 , 16 , 17 , 18 , 19 ]. These studies have highlighted the significant role of hybridization (e.g. the recent hybrid origin of P. helvetica Bolliger [ 19 ] or the P. hirta complex [ 10 ]), and provided evidences for the involvement of introgression and dysploidy in the speciation process [ 10 , 18 ]. In horticulture, Pulmonaria species are known to be crossable (no geographical isolation, flowering synchrony, similar pollinator preferences), giving rise to a wide variety of cultivars [see 20]. Even in the contact zones of some taxa, intermediate chromosome numbers have rarely been documented [ 2 , 3 , 21 , 22 ]. However, most of the evolutionary relationships are still unknown, and the question remains as to what lies behind the observed variability in their morphology and chromosome number. Along with chromosome number, genome size is another relevant biodiversity trait that can indicate significant genomic events and evolutionary changes [ 23 , 24 ]. Diversity in genome size is driven by multiple evolutionary processes such as polyploidy, the proliferation of repetitive DNA sequences and/or drastic reduction of non-genic DNA [ 23 , 25 , 26 , 27 ]. Typically, post-polyploid diploidization is thought to be associated with extensive loss of DNA or 'genome downsizing' [ 28 , 29 , 30 , 31 , 32 , 33 , 34 ]. However, the mechanisms, rates and selection pressures driving these changes in DNA content remain unknown [cf. 33]. Genome size can also be a useful tool for the practical botany, i.e. distinguishing between cryptic taxa, identifying hybrids and resolving taxonomies in critical groups [e.g. 35, 36, 37, 38]. According to the available literature, the genome size values of Pulmonaria species have only been reported sporadically [summarized in 39], without any systematic study. As already mentioned, karyological studies in Pulmonaria have mainly focused on determining the number of chromosomes and their significance for taxonomy [e.g. 3, 21]. A more detailed analysis of chromosome structure at the cytogenetic level has not yet been carried out. The most important technique in plant cytogenetic research is fluorescence in situ hybridization (FISH). The major limiting factor of the FISH techniques in non-model plant species is the lack of powerful DNA probes [reviewed in 40, 41]. Recent development of whole genome sequencing technologies and bioinformatics facilitates fast progress of cytogenomics. In non-model plants, the first step is identification of suitable cytogenetic landmarks, which provide specific pattern on chromosomes and can be used to identify individual chromosomes, to compare karyotypes and chromosomal rearrangements in closely related species and inter-specific hybrids, and to provide important information on the plant evolution through chromosome changes [e.g. 42, 43, 44, 45, 46]. The best performing cytogenetic probes include rRNA genes and satellite DNAs (satDNA). Both of them are present in high copy numbers of tandem organized repetitive units in the genomes, and very often provide species-specific patterns on chromosomes [e.g. 27, 47, 48, 49, 50]. To identify such cytogenetic probes, whole genome sequencing data can be utilized and further analyzed by the RepeatExplorer pipeline [ 51 ]. RepeatExplorer pipeline is based on a graph-based clustering algorithm and enables identification and characterization of repetitive DNA from small quantities of short read sequencing data [ 52 , 53 ]. The pipeline has been used for repeat identification in many model and non-model plant species [ 54 , 55 ]. The new version of RepeatExplorer2 provides de novo repeat identification and annotation of transposable elements, comparative repeat analysis in large sets of species, and identification of satellite DNA with the aim to develop cytogenetic probes. The analyses are complemented by visualization of the results in the SeqGrapheR visualization tool [ 55 ]. Our study is focused on the P. officinalis group, the most widespread European species complex [ 3 , 56 , 57 ]. The P. officinalis group contains two morphologically relatively distinct species differing in chromosome numbers, namely P. obscura Dumort. (2 n = 14) and P. officinalis L. (2 n = 16 [e.g. 3, 22, 58, 59, 60]). Within the latter, two subspecies are sometimes recognized [ 61 , 62 , 63 ]. As the P. officinalis complex has been cultivated for medicinal and ornamental purposes for a very long time [ 64 , 65 ], it has probably become even more widespread. In some regions it may also be non-native as a result of frequent cultivation and possible garden escapes [ 60 , 65 ]. “Pure” Pulmonaria species have often been crossed in cultivation to produce more attractive varieties [cf. 20], which are rarely found in nature. Many of these plants are listed under the name P. saccharata in the horticultural trade. Their origin is not clear, but it can be assumed that some of them are derived from P. officinalis (esp. plants with distinctly white-spotted leaves, cordate at the base). However, they are not “true” P. saccharata sensu Miller, whose taxonomic status has long been debated, and are most likely related to the P. hirta complex [cf. 10, 66, 67]. In this work, we performed the analysis of genome size and comparative analysis of the repeatomes in closely related species of the P. officinalis group in order to shed light on the karyotype structure and variability. Specifically, we have analyzed repeatomes and karyotypes of P. obscura and P. officinalis s.str. individuals from pure separate populations, their putative hybrid accessions with 2 n = 15, which have been collected in a mixed population where P. obscura and P. officinalis grew together, and three populations of ornamental cultivars, morphologically similar to P. officinalis , that have escaped into the wild where they were collected (here listed as P. saccharata -like). Genome size has been studied and compared on a wider geographical scale. This pilot study can serve as a springboard for future cytogenetic and genomic studies, to understand the role of chromosomal rearrangements in the evolution of this genus. Results Genome size and chromosome number variation in the P. officinalis group A total number of 196 accessions from 65 populations of the Pulmonaria officinalis group were collected throughout Europe and analyzed by flow cytometry (Fig. 1 A). The analyses resulted in good quality histograms with distinct peaks and low coefficients of variation (below 5%) for both PI and DAPI staining (Supplementary Table 1). The flow cytometry data were confirmed by chromosome counts, some of which were obtained de novo (see below), but previously published reports were also revised. A total of 754 published chromosome records were found and revised for the P. officinalis complex (Supplementary Table 2). Of these, 287 chromosome reports with 2 n = 14 were related to P. obscura and 450 with 2 n = 16 to P. officinalis s.str., respectively (Fig. 1 A). Five records were excluded as they probably belong to another species of the genus. In one case 2 n = 17 have been reported for P. officinalis . In addition to this, there are even karyological records (16 in total) that refer to hybrids of P. obscura and P. officinalis . In all cases, 15 chromosomes were counted in these plants (Supplementary Table 2). P. obscura (2 n = 14; mean 2C = 2.92 ± 0.10 pg) and P. officinalis s.str. (2 n = 16; mean 2C = 3.21 ± 0.13 pg) differed significantly in holoploid genome size (2C value, Fig. 2 A) as well as in the genomic GC content (Fig. 2 B). Their DNA amount ranges are listed in Table 1 . The genome size value of the presumed hybrid plants (B481) with 2 n = 15 (2C = 3.08 ± 0.02 pg) was positioned between their values (within the theoretically expected intermediate genome size based on the genome sizes of P. obscura and P. officinalis ). The GC content was also intermediate (Table 1 ). Among P. saccharata -like populations, B465.1 with 2 n = 16 had the genome size value (2C = 3.13 pg) in the range of P. officinalis . The remaining two populations (B15: mean 2C = 3.74 ± 0.03 pg; B472.1: 2C = 3.63 pg) had larger genome sizes than P. obscura and P. officinalis . Interestingly, these populations differ in the number of chromosomes (B15: 2 n = 15 vs. B472.1: 2 n = 16). The similar pattern was detected in the genomic GC content, only in B472.1 the GC content was lower than in B15 (Table 1 ). Table 1 Summary of flow cytometric analyses of the Pulmonaria officinalis group. Taxon 2 n N pop N ind Mean 2C [pg] Min Max 1C [Mbp] C/ n [pg] GC [%] P. obscura 14 25 83 2.92 ± 0.10 2.73 3.08 1 428 0.21 35.89 P. officinalis s.str. 16 36 103 3.21 ± 0.13 2.95 3.48 1 570 0.20 35.09 P. obscura × P. officinalis (B481) 15 1 3 3.08 ± 0.02 3.06 3.09 1 511 0.21 35.42 P. saccharata -like (B15) 15 1 5 3.74 ± 0.03 3.71 3.78 1 829 0.25 35.64 P. saccharata -like (B465.1) 16 1 1 3.13 - - 1 531 0.20 35.16 P. saccharata -like (B472.1) 16 1 1 3.63 - - 1 775 0.23 35.19 Genome-wide repeatome analysis To identify the major types of repetitive sequences and to compare their genome representation in P. officinalis group, the comparative mode of RepeatExplorer2 pipeline was used. The analysis was performed on partial Illumina sequencing data of P. obscura (2 n = 14; B473.1, Fig. 1 B), P. officinalis (2 n = 16; B470.3, Fig. 1 C), their putative interspecific hybrid (2 n = 15; B481.1) and three P. saccharata -like accessions (B15.1; 2 n = 15, Fig. 1 D and B465.1, B472.1; both 2 n = 16). The identified repetitive sequences accounted for 58.48% of the P. obscura genome and 47.86% of the P. officinalis genome. A similar and overall highest proportion of repetitive sequences was found in repeatomes of the putative interspecific hybrid B481.1 (64.10%) and P. saccharata -like accession B15.1 (64.53%), while the repetitive DNA content of the other two P. saccharata -like accessions (B465.1 and B472.1) was around 50% (Table 2 , Fig. 3 ). In all studied taxa, LTR retroelements were the most abundant repeats, accounting for 46.68% of P. obscura genome, 34.52% of P. officinalis genome and 33.80-49.96% of the other genomes. The genome proportion of Ty3/Gypsy elements, which were mainly represented by Tekay lineage, ranged from 23.90% in P. saccharata -like (B472.1) to 35.06% in putative interspecific hybrid (B481.1). Ty1/Copia elements were about twice less abundant compared to Ty3/Gypsy superfamily, and were mostly represented by elements of SIRE and Angela clades (Table 2 , Fig. 3 ). DNA transposons and long interspersed nuclear elements (LINE elements) were found in low copy numbers in all analyzed groups, with genome proportions ranging from 0.14–0.23% and from 0.16–0.22%, respectively (Table 2 ). rDNA sequences accounted for about 3.00–5.00%, and other tandem organized repeats represented 1.25% to more than 4.00% of the Pulmonaria genomes (Table 2 ). Table 2 Proportion of repetitive DNA sequences identified de novo in Pulmonaria taxa Repeat Lineage/class Proportion of repeats in genomes [%] POBS POFF HYBR PSAC PSAC PSAC B473.1 B470.3 B481.1 B465.1 B472.1 B15.1 LTR retroelements Ty1/Copia SIRE 12.05 6.47 10.38 6.48 6.18 9.83 Angela 2.63 2.18 3.01 2.17 2.21 3.07 Tork 1.00 0.93 1.00 0.99 0.96 1.03 TAR 0.03 0.03 0.03 0.02 0.03 0.03 Ale 0.07 0.07 0.08 0.08 0.06 0.07 Total Ty1/Copia 15.78 9.68 14.50 9.74 9.48 14.03 Ty3/Gypsy Ogre 0.82 0.82 0.92 0.80 0.74 0.91 Athila 0.04 0.09 0.08 0.10 0.08 0.07 Tekay 29.69 23.41 33.97 24.84 23.00 32.67 Chromovirideae 0.07 0.08 0.09 0.08 0.08 0.08 Total Ty3/Gypsy 30.62 24.40 35.06 25.82 23.90 33.73 Unclassified LTR elements 0.28 0.44 0.40 0.43 0.42 0.47 Total LTR 46.68 34.52 49.96 35.99 33.8 48.23 Other LINE 0.23 0.17 0.23 0.16 0.14 0.21 DNA transposons 0.16 0.20 0.18 0.22 0.18 0.22 Tandem repeats 3.78 1.25 4.33 3.33 2.12 3.60 rRNA genes 3.78 4.36 2.78 4.52 4.73 5.09 Unclassified repeats 3.85 7.36 6.62 7.84 7.61 7.18 Total repetitive DNA content 58.48 47.86 64.1 52.06 48.58 64.53 POBS: P. obscura ; POFF: P. officinalis ; HYBR: P. obscura × P. officinalis , putative interspecific hybrid; PSAC: P. saccharata -like Variability in the Satellite DNAs and rDNA loci Considering satellite (tandem organized) repeats as preferable cytogenetic landmarks, which can be species- or even subspecies-specific, we inspected the individual P. obscura and P. officinalis datasets by TAREAN program. Five putative satellites (tandem organized repeats) were observed, three of them (PulTR01_29, PulTR03_308, PulTR05_70) were shared by both species. One satDNA (PulTR02_305) was only found in P. obscura , and another one (PulTR04_420) was only detected in silico in the P. officinalis genome. Highly abundant tandem organized repeat PulTR05_70 provided strong cluster signals distributed along whole chromosomes with enrichments in pericentromeric regions, and cannot be used as a chromosome-specific cytogenetic landmark (Suppl. Figure 1). The other tandem repeats produced chromosome-specific signals and were used together with rDNA sequences to create karyotypes of the Pulmonaria accessions studied. Tandem organized character is also typical for rRNA genes, which are present in the genomes in high copy numbers and localized in specific chromosomal regions. The 45S rRNA gene unit is usually about 10 kb long and is often represented by several cluster graphs after the RepeatExplorer analysis, as it was observed in the case of all the Pulmonaria accessions analyzed. To reconstruct the whole 45S unit, we followed the procedure described in Kapustová et al. [ 68 ]. The unit length of the Pulmonaria 45S rDNA unit varied from 9.1 kb to 9.6 kb and was found to be highly conserved at the sequence level within all accessions studied (Suppl. Figure 2). The intergenic spacer (IGS) region contained two different tandemly organized repeats (with repetitive units of 117 and 150-nt long) which were identical in all the accessions studied, with the exception of a P. saccharata -like plant B472.1, which contained tandem regions with shorter repetitive units (79 and 150-nt long). A higher variability was found in the IGS, which contained two relatively large INDEL regions that differed between P. obscura and P. officinalis (Suppl. Figure 3A). To support this observation, we performed read mapping of Pulmonaria accessions to the assembled P. obscura 45S rDNA reference (Suppl. Figure 3B). The differences in read coverage along the 45S rDNA unit are evident in the IGS, indicating the variability at the sequence level. It should be pointed out that the assembly of the 45S rDNA unit was obtained from short Illumina sequences, so the resulting consensus sequence may represent the most abundant sequence type. The 5S rRNA gene unit is typically up to 1kb in length in plant species [ 69 ], so the graph-based clustering algorithm of the RepeatExplorer2 pipeline allowed its complete reconstruction, even in the comparative analysis (Fig. 4 ). The graph region representing the 5S rRNA gene was shared by all Pulmonaria accessions studied (Fig. 4 B). Three variable graph loops emanating from this conserved graph region correspond to three types of 5S rDNA unit, differing in the length of their IGS (Fig. 4 A, B). Mapping of sample-specific sequencing reads onto the graph topology revealed the presence of different 5S rDNA units between P. obscura and P. officinalis (Fig. 4 C, D). The cluster layout of B481.1, the putative hybrid between P. obscura and P. officinalis , was represented by all three loops specific to its presumed progenitors (Fig. 4 E). A similar situation was observed for the P. saccharata -like accession B15.1 (Fig. 4 F). The other P. saccharata -like individuals (B465.1 and B472.1) shared the same graph layout as P. officinalis (i.e. B470.3; Fig. 4 G, H). The analysis of 5S rDNA specific graph layouts observed by clustering analysis of individual accessions confirmed the observation of the comparative analysis. All Pulmonaria individuals studied were composed of at least two different types of 5S rDNA genes, which differed in the IGS (Suppl. Figure 4). Comparative karyotyping in the P. officinalis group Molecular karyotyping was performed using newly identified satellites (PulTR01_29, PulTR02_305, PulTR03_308 and PulTR04_420) and 5S and 45S rDNA sequences. In general, in situ hybridization confirmed the results obtained by repeatome analysis. FISH analysis with the probes for rDNAs and four satDNAs resulted in well visible cluster signals on specific chromosomes in the genome of the analyzed plants of the P. officinalis complex. P. obscura , P. officinalis and their putative natural interspecific hybrid FISH analysis of P. obscura plants (all 2 n = 14) collected from three different populations (B467, B469, B473; Suppl. Table S1 ) provided highly consistent results. The only exception was observed for plant B473.1 in the number of the most abundant satDNA (PulTR01_29). The 45S rDNA was located into terminal NOR regions on four chromosome pairs (Fig. 5 ; Fig. 6 A, B, D, E). 5S rDNA loci were detected on two chromosome pairs in pericentromeric regions. One chromosome pair contained signals of 5S, 45S rDNA and satellite PulTR02_305 (Fig. 5 ; Fig. 6 B, E). In B473.1, the probe for PulTR01_29 provided very strong signals in subtelomeric regions only on one chromosome pair. The same chromosome pair also contained a signal of PulTR03_308 in the pericentromeric region (Fig. 6 C). In B467.2 and B469.1, one additional subtelomeric signal of PulTR01_29 was found, located on a chromosome arm with 5S rDNA locus (Fig. 5 ; Fig. 6 A, D, F). As expected from the results of the RepeatExplorer2 analysis, signals of PulTR04_420 were not detected in any of the P. obscura plants analyzed. In general, the genome of P. officinalis contained one additional pair of chromosomes (i.e. 2 n = 16) compared to P. obscura . Molecular karyotype was studied in individuals from two different populations (B100, B470; Suppl. Table S1 ). A higher variability in the chromosomal distribution of the probes was detected within representatives of P. officinalis compared to P. obscura (Suppl. Figure 5). Terminal NORs (45S rDNA) were found only on three chromosome pairs in both analyzed individuals (B100.2, B470.1). These terminal 45S rDNA loci were often fragile and broken off from the chromosomes (Fig. 7 A, F). Interstitial 45S rDNA clusters were detected on one chromosome pair, on the same arm which also contained signals of satDNA PulTR04_420 and PulTR01_29 (Fig. 5 ; Fig. 7 B, F). The genome of B100.2 contained an additional weak signal of interstitial 45S rDNA (Fig. 5 ; Fig. 7 D, E, F). Variability in the number of signals specific to 5S rDNA loci was detected. While 5S rDNA loci were identified in pericentromeric regions on seven chromosomes of B470.3, only six chromosomes were bearing these loci in B100.2. The signal of PulTR03_308 was detected on one NOR bearing chromosome pair in both individuals with various combinations of co-localization with PulTR04_420 and 5S rDNA (Fig. 5 ; Fig. 7 C, E, G). Signals of PulTR02_305 were not detected on chromosomes of P. officinalis , supporting the results of comparative repeatome analysis by RepeatExplorer2. FISH analysis of the other representative of the same population B100 provided slightly different molecular karyotype (Suppl. Figure 5). Karyotype analysis of the putative natural hybrid between P. obscura and P. officinalis (B481.1; Suppl. Table S1 ), collected in a mixed population of both species, confirmed the expected chromosome number 2 n = 15. In this case, eight 45S rDNA clusters were found, seven in terminal chromosomal regions and one in an interstitial position. 5S rDNA loci were detected on five chromosomes in pericentromeric regions. One chromosome pair contained signals of 45S rDNA and 5S rDNA, another individual chromosome contained 45S and 5S rDNA and PulTR02_305 (found only as one single locus in the genome). Additional two chromosomes contained one or two signals of 5S rDNA, respectively (Fig. 5 ; Fig. 8 A, B). Five signals of the most abundant satellite PulTR01_29 were detected on five chromosomes, one of which co-localized with the 45S rDNA locus, one signal was detected on the chromosome bearing weak interstitial signal of 45S and one with signal of PulTR03_308 and PulTR04_420, respectively. One chromosome pair with the remaining two signals of PulTR01_29 also contained a signal of 5S rDNA. SatDNAs PulTR03_308 and PulTR04_420 co-localized on one chromosome pair, containing also PulTR01_29, or joint signal of 45S rDNA and PulTR01_29 (Fig. 8 C). Ornamental garden escapes, morphologically similar to P. officinalis Karyotype analysis of P. saccharata -like plants from three populations, which were collected in the wild but apparently escaped from cultivation (B15, B465, B472; Suppl. Table S1 ), revealed variability in chromosome number. While B465.1 and B472.1 were characterized by 2 n = 16, 2 n = 15 was detected in B15.1. The karyotypes of B465.1 and B472.1 were very similar, containing terminal NORs on three chromosome pairs and one interstitial 45S rDNA cluster on one additional chromosome pair (Fig. 8 G, I, K, L, M). Two chromosome pairs were bearing PulTR01_29 in subtelomeric regions and other two chromosome pairs contained 5S rDNA clusters in pericentromeric regions (Figure G, H, K). The signal of PulTR04_420 was found on two chromosome pairs (Fig. 8 G, J, K, L). One of these chromosome pairs also contained signals of PulTR01_29 and an interstitial signal of 45S rDNA, while the other chromosome pair bearing terminal 45S rDNA and the PulTR04_420 co-localized with PulTR03_308 (Fig. 5 ; Fig. 7 ; Fig. 8 G, J, K, L). However, these two accessions differed in the presence of an additional 5S rDNA and PulTR01_29 interstitial loci (Fig. 5 ; Fig. 7 ). The karyotype of B15.1 was similar to that of the putative interspecific hybrid (B481.1) with the same number of chromosomes, indicating a similar hybrid character. 45S rDNA loci were located as strong signals in terminal regions of three chromosome pairs, one additional chromosome contained a weak terminal signal and another contained an interstitial signal (Fig. 8 D, E). Odd number of signal localizations was detected for 5S rDNA (5 chromosomes with interstitial signals), PulTR01_29 (three chromosomes with the signals in terminal regions), PulTR04_420 (three chromosomes with interstitial signals), and for PulTR02_305 (found only on one chromosome) (Fig. 8 D, E, F). Finally, PulTR03_308 provided signals on one chromosome pair co-localizing with PulTR04_420 (Fig. 5 ; Fig. 7 ). Discussion The genus Pulmonaria is karyologically highly variable [e.g. 3, 6], with 16 different somatic chromosome numbers reported (Kobrlová unpubl. ) and about 30 taxa recognized, growing in Europe and northeastern and eastern Asia [ 5 , 7 , 8 , 9 ]. However, the mechanism, origin and evolutionary consequences of genome size and karyotype variability remain unexplored [but see 10]. Apparently, chromosomal rearrangements have played an important role in the evolution of this genus [cf. 10, 19, 39], but how and to what extent has never been clearly demonstrated. In this work, we studied the P. officinalis complex (Fig. 1 ), which includes two morphologically similar, closely related species that differ in chromosome number [ 3 , 10 , 18 , 59 , 60 ]. The present study confirmed previously reported chromosomal data, which were consistent across the whole of Europe. The predominant 2 n = 16 reports correspond to P. officinalis s.l., whereas only 2 n = 14 were documented in P. obscura populations (Fig. 1 ). Only in one case, 2 n = 17 was documented from the “pure” population of P. officinalis [ 22 ], but no explanation was given. Despite the morphological and karyological differences observed, the origin of this species group remains unknown. To uncover the differences in the karyotype evolution, we used complex methodological approaches involving partial Illumina sequencing followed by bioinformatic analysis and characterization of repeatomes in the P. officinalis group. We identified a new set of chromosome-specific cytogenetic landmarks and performed comparative karyotyping within and between the two species, their putative natural hybrid from a population where both species occur, and ornamental cultivars morphologically similar to P. officinalis , which are also rarely found in nature. Impact of DNA repeats dynamics on genome size Genome size can reflect some aspects of the evolutionary history of taxa by allowing us to understand the influence of DNA gain/loss between related species [e.g. 70, 71]. Despite the apparent karyological variability of several Boraginaceae genera [see e.g. 14, 72, 73], there have been almost no complex analyses of genome size variation and the evolutionary pathways behind the observed diversity [but see 74, 75, 76]. Given the sparse DNA content records in the Boraginaceae family, the only comprehensive study has been published, providing the first genome size reports for most taxa [ 12 ]. Our study represents the first large-scale investigation of interspecific genome size variation in Pulmonaria . As already shown in a pilot study by Kobrlová & Hroneš [ 12 ], genome size is effective in delimiting morphologically similar taxa of the Boraginaceae, which is also true for the P. officinalis group. We found a significant difference in genome size between P. obscura and P. officinalis , corresponding to the number of chromosomes (Fig. 2 ), confirming previous results [cf. 12], but on a larger geographical scale. The suitability of using flow cytometry to revise the distribution of the P. officinalis group (i.e. relative genome size) has already been documented in the Bohemian Forest and adjacent foothills [ 60 ]. So far, the genome size has only been estimated for eight Pulmonaria taxa, including P. officinalis and P. obscura , ranging from 2.27 to 4.27 pg (i.e. very small/small genomes according to the categories of Leitch et al. [ 77 ], Supplementary Table 3). Only minor differences were observed when comparing previously analyzed genome sizes of P. obscura and P. officinalis with our data, most likely due to different methodologies used (i.e. nuclei isolation buffer, reference standard, plant organ [cf. 12, 39]). The only exception is the study by Šmarda et al. [ 78 ], where almost the same 2C values are presented for P. obscura and P. officinalis , probably as a consequence of taxa misidentification. DNA transposons have been shown to be the major contributor to the enormous variation in genome size in plants [e.g. 27, 79, 80, 81, 82, 83]. To shed light on genome size dynamics and relationships between P. officinalis and P. obscura species, we performed a genome-wide comparison of their repeatomes. We found that most of the repetitive elements in the genomes of the Pulmonaria taxa studied were dispersed repeats represented by LTR retrotransposons [cf. 27, 81], with higher proportion of Ty3/Gypsy elements, which were twice more abundant than Ty1/Copia. Ty3/Gypsy retroelements were almost exclusively represented by Tekay retrotransposons (Chromoviridea clade), whereas SIRE elements were the most abundant types of the Ty1/Copia superfamily (Table 2 ). Ty3/Gypsy elements represent one of the major classes of LTR retrotransposons and are dominant in many plant groups, such as the family Poaceae [e.g. 82, 84, 85, 86] or the tribe Fabeae [ 27 , 87 ]. Unfortunately, a genome-wide analysis of DNA repeats and their impact on genome size has not been performed in any other species of the Boraginaceae family. However, the higher proportion of Ty3/Gypsy retroelements have also been found in genera of the closely related Solanaceae family, such as Solanum , Nicotiana and Capsicum [88, 89, 90, 91, 92, 93). In contrast, recent studies in the genus Salvia , a member of the closely related Lamiaceae family, have shown that the nuclear genomes of different species contain different proportions of Ty3/Gypsy and Ty1/Copia retroelements [ 94 , 95 , 96 ], indicating a proliferation of different types of DNA repeats during the evolution of individual species. In comparison, the Pulmonaria species analyzed contained a similar proportion of the repeat lineages and individual clusters were represented by reads from all specimens analyzed. This indicates a high degree of genome homology within the P. officinalis complex, suggesting that the evolution of this species group was not accompanied by a dramatic diversification of DNA transposons, as previously shown in other plant species [e.g. 82]. To better understand the proliferation of DNA repeats during genome evolution and its impact on genome size variation and speciation, analysis of a larger data set of Pulmonaria species from different phylogenetic groups is required. Satellite DNAs and their use in comparative karyotyping Repeatome analysis using the RepeatExplorer pipeline also allows the identification of putative satellite DNAs, which together with tandem organized ribosomal genes are the best cytogenetic landmarks. SatDNAs are usually species- or subspecies-specific, provide chromosome-specific labeling patterns and can therefore be used not only to generate and compare molecular karyotypes, but also to identify putative chromosomal structural changes [ 42 , 45 , 97 ]. The use of the FISH technique revealed different patterns of chromosomal localization of the tandem repeats and rDNA loci examined, both between and within P. obscura and P. officinalis (Fig. 5 ). The almost identical cytogenetic pattern of satDNAs and rDNA sequences in P. obscura (2 n = 14), collected from three different populations, suggests karyotype stability in this diploid species (Fig. 5 ). In comparison, the chromosome structure in P. officinalis appears to be more dynamic, as individuals from two different populations differ slightly in the cytogenetic pattern of the satDNAs and rDNA sequences. Odd number of signals of some satDNAs as well as of rDNA sequences, and interstitial 45S rDNA loci were found in both diploid accessions (2 n = 16), indicating chromosomal structural changes involved in the origin and evolution of P. officinalis (Fig. 5 ). It is generally accepted that n = 7 is the basic chromosome number in Pulmonaria [e.g. 2, 3, 4, 5, 6], which raises the question of how the species represented by different chromosome numbers arose. In the case of P. officinalis , there are two possible scenarios. There could have been chromosome fission leading to 2 n = 16, or a more complex karyotype evolution with polyploidization and further diploidization by chromosome rearrangements. Large differences between the molecular karyotypes of P. obscura and P. officinalis suggest that diploid P. obscura most probably could not give a rise to P. officinalis by chromosome fissions (Fig. 5 ). Thus, we can only speculate, that the evolution and speciation in the P. officinalis group could be influenced by polyploidy and/or hybridization followed by post-polyploid diploidization process, which can result in numerous chromosomal rearrangements [see 98, 99, 100] and the origin of Pulmonaria species differing in their basic chromosome number. A similar evolutionary scenario involving chromosome multiplication, hybridization and, in particular, structural rearrangements of chromosomes (dysploidy) has been outlined by Liu et al. [ 10 ] for the origin of the P. hirta complex on the Italian peninsula and the Swiss endemic P. helvetica [ 19 ], respectively. Evidence of hybridization within the P. officinalis complex The evolution of the genus Pulmonaria remains unresolved, due to a lack of rigorous phylogenomic studies. However, several molecular studies have been published highlighting the important role of hybridization and introgression in the evolution of the genus [ 10 , 18 , 19 ]. The high level of chromosomal variation may also support the hypothesis of ancient hybridization events and subsequent chromosomal rearrangements [cf. 6, 7, 10, 18]. In addition, some species groups exhibit weak ecological and geographic isolation, near-synchronous phenology and pollinator sharing, all of which may facilitate the hybridization [cf. 18]. This is particularly true for the P. officinalis complex, which is widespread in Europe and therefore often in secondary contact with other Pulmonaria species (cf. 56, 101]. As the ranges of P. obscura and P. officinalis partly overlap (Fig. 1 A), the co-occurrence of both species in the same habitat can be expected. Some authors have occasionally reported mixed populations, with morphological intermediates rarely observed [ 21 , 22 ]. The extent of hybridization between these two species is still controversial. Nevertheless, several karyological data referring to as P. obscura × P. officinalis with an intermediate number of chromosomes (i.e. 2 n = 15), may provide convincing evidence of an ongoing hybridization between these two species [ 18 , 21 , 22 ]. In our study, we analyzed presumed hybrids from a mixed population (B481) of P. obscura and P. officinalis . Chromosome counting in all three analyzed individuals confirmed 2 n = 15, and their hybrid origin was also supported by their genome sizes, halfway between those of the parents (Table 1 ). The cytogenetic mapping of the set of satDNAs and rDNA sequences also strongly supports the hybrid origin, by the presence of P. obscura and P. officinalis species-specific satDNAs (PulTR_305 and PulTR_420) in haploid state, and also their pattern on chromosomes (Fig. 7 ). The hybrid status of these individuals was further confirmed by detailed analysis of the 5S rDNA sequences. As recently shown, graph-based clustering of the RepeatExplorer pipeline enables reconstruction of complete 5S rDNA sequences from partial Illumina sequencing data and provides clues to the evolutionary history of interspecific hybrids and allopolyploids [ 69 , 102 ]. The detailed analysis of the 5S rDNA cluster shapes of all Pulmonaria accessions examined revealed the presence of both P. obscura - and P. officinalis -specific 5S rDNA sequences in the genome of a putative hybrid clone B481.1 (Fig. 4 ). Given the overlapping ranges of some Pulmonaria species groups [see 56, 101], the possibility of interspecific hybridization might be expected to be relatively common. So far, however, natural hybrids have only occasionally been identified by chromosome counting [ 3 , 21 ] or distinguished on the basis of intermediate morphology [e.g. 103]. Questions also remain about the hybrid's fertility and their longevity in populations. Although the hybridization is apparently rare, hybrids can potentially persist and spread clonally at the locality [cf. 104], as Pulmonaria species partly reproduce by vegetative propagation (i.e. creeping rhizomes). Origin of ornamental cultivars morphologically similar to P. officinalis As a valuable medicinal and ornamental plant, P. officinalis is represented in horticulture by several cultivars and has also been used to generate new artificial hybrids [cf. 20]. This seems to be the case for plants with distinctly white-spotted leaves, cordate at the base, which are sometimes offered commercially as P. saccharata . However, they are not “true” P. saccharata sensu Miller [see 10, 66, 67]. These plants often escape into the wild and are sometimes confused with P. officinalis . The origin of these cultivars is unknown, they only resemble P. officinalis complex in their morphology (i.e. cordate bases of leaves, Fig. 1 D). In total, we analyzed three populations of these garden escapees (B15, B465 and B472). Our cytogenetic analysis and detailed examination of the reconstructed 5S rDNA sequence indicate that two analyzed P. saccharata -like accessions (B465.1 and B472.1) are derived from the P. officinalis . Both plants had 2 n = 16 as P. officinalis . They also shared the general cytogenetic pattern of satDNAs and rDNA sequences typical of P. officinalis , and contain only P. officinalis -specific, not P. obscura -specific, 5S rDNA sequence types (Fig. 4 , 5 ). In addition, the genome size value of the B465.1 plant was also in the range of P. officinalis , only in the B472.1 plant was slightly larger (Table 1 ). This may confirm the assumption that these plants are derived from P. officinalis . On the other hand, an interesting cytogenetic pattern was observed in the third P. saccharata -like plant analyzed (B15.1). The karyotype of this plant was similar to that of the interspecific hybrid B481.1, with the same chromosome number 2 n = 15 and P. obscura - and P. officinalis -specific 5S rDNA sequences in its genome (Fig. 4 ). Furthermore, even the cytogenetic pattern of the probes and the presence of P. obscura - and P. officinalis -specific satDNAs (PulTR_305 and PulTR_420) in the haploid state also suggest a hybrid origin (Fig. 5 ). In contrast, the genome size of B15.1 (the whole population, respectively) was considerably larger, the largest found in the whole data set presented (Table 1 ). However, unlike the population B481, population B15 was collected in the area where only P. obscura occurs naturally and where no population of P. officinalis has been confirmed (Kobrlová, pers. obs. ). The morphology of the plants was also typical for cultivated P. saccharata -like plants. Their origin therefore requires further investigation, although the cytogenetic data presented suggest a hybrid origin between P. obscura and P. officinalis (e.g. phylogenetic revision and analysis of a larger data set of Pulmonaria species from different phylogenetic groups). As this population is a garden escape, its geographical origin is unclear and it cannot be ruled out that it was originally collected from a mixed population of both species. Our data undoubtedly demonstrate that molecular karyotyping is a powerful method for identifying the mode of karyotype evolution and the hybrid origin of Pulmonaria taxa. Conclusions Our study provides comprehensive information on genome size variability and repeatome dynamics of the two morphologically similar species of the P. officinalis group. Large-scale genome size analysis using flow cytometry confirmed a significant difference in DNA content between P. obscura and P. officinalis , corresponding to the number of chromosomes. Partial sequencing of six accessions, including putative natural hybrid of P. obscura and P. officinalis , and ornamental garden escapes resembling P. officinalis , showed that a large proportion of their genomes is represented by various types of DNA transposons, with Ty3/Gypsy elements being the most abundant. Comparative analysis of the repeatomes revealed no species-specific retrotransposons or striking differences in their copy number between the species, suggesting a common evolutionary history. Cytogenetic analysis using probes for rDNA sequences and newly identified satellite DNAs allows the origin of the Pulmonaria accessions to be determined, or at least outlined. Thus, in combination with a robust phylogenetic framework, it can contribute to the elucidation of the evolutionary history of Pulmonaria relatives. In our case, comparative karyotyping strongly supported the hybrid origin of putative hybrids with 2 n = 15, collected from a mixed population of P. obscura and P. officinalis , and also outlined the origin of ornamental garden escapes, confirming their derivation from the P. officinalis complex. Finally, databases of repeats were created, which can be used for repeat identification (or masking) in future sequencing projects. Materials and methods Plant material A total of 196 plants from 65 populations of the Pulmonaria officinalis group (Fig. 1 A), representing typical populations of P. obscura and P. officinalis s. str. (Fig. 1 B, 1 C), including their potential hybrids and several garden escapes of cultivars morphologically similar to P. officinalis (in horticulture often referred to as P. saccharata , here listed as P. saccharata -like, Fig. 1 D), were included in this study (see Supplementary Table 1). These samples were collected between 2014 and 2023 from natural populations across Europe, some of which were cultivated in the experimental garden of Palacký University in Olomouc, Czech Republic, or deposited in the Herbarium of Palacký University in Olomouc (OL). Flow cytometry: genome size and GC content Estimation of the nuclear DNA content, i.e. absolute genome size (AGS [ 105 ]), and DNA base composition (GC content [ 106 ]) were estimated using Partec PAS and Partec ML instruments, with PI (propidium iodide) and DAPI (4,6-diamidino-2-phenylindole) staining. The same methodology as in Kobrlová and Hroneš [ 12 ] was followed, using fresh, rarely silica dried, leaves for sample preparation. Pisum sativum L. ‘Ctirad’ (2C = 9.09 pg [ 107 ]; GC content = 38.5% [ 108 ]) was selected as a primary internal standard, since it has non-overlapping genome size with neither G1 and nor G2 phase of all studied samples. The conversion from picograms (pg) to base pairs (bp) followed Doležel et al. [ 109 ], using 1 pg DNA = 978 Mbp. DNA base content was estimated using the protocol and GC content calculation tool of Šmarda et al. [ 106 ]. The data analyses were performed using the NCSS 9 statistical software [ 110 ]. The non-parametric Kruskal-Wallis test was used to test for differences between population means of genome size/GC content of P. obscura and P. officinalis . DNA extraction and sequencing Genomic DNA was isolated using alkyltrimethylammonium bromide (MATAB) lysis: after sorbitol washes, the ground plant material was incubated in 2% (w/v) MATAB for 20 min at 65°C, immediately after the incubation, the same volume of chloroform:isoamyl alcohol (24:1) was added, gently but thoroughly mixed and centrifuged at 10,000 g for 3 min at 4°C. After centrifugation, aqueous upper phase was collected to a new tube and this step was repeat until the upper phase was clear. Genomic DNA was precipitated by adding 0.7 volume of isopropanol, centrifuged at 10,000 g for 3 min at 4°C. Finally, the pellet was washed by cold 70% and 96% ethanol, air dried and diluted in TE buffer, pH 8. Genomic DNA was sheared by Bioruptor Plus (Diagenode, Liege, Belgium) to achieve an insert size of about 500 bp. Libraries for sequencing were prepared from 2 µg of fragmented DNA using TruSeq® DNA PCR-free kit (Illumina) and sequenced on a NovaSeq 6000 (Illumina), producing 2 × 100-bp or 2 × 150-bp paired-end reads to achieve at least 3Gb of nucleotide sequence per each genotype. Raw data were trimmed for low-quality bases and adapter sequences and to the same length using fastp v.0.20.1 [ 111 ]. Analysis and characterization of DNA repeats Random datasets corresponding to 0.1× coverage of the individual accessions were used for reconstruction and characterization of DNA repeats using RepeatExplorer2 [ 55 ], that includes TAREAN analysis tool for identification of tandemly organized repeats [ 53 ]. RepeatExplorer2 and TAREAN analyses were also used to perform comparative analysis of Pulmonaria repeatomes on a merged dataset containing all studied individuals (1 mil. reads per accession), marked by specific prefixes. In both cases, the resulting clusters of repeats were characterized by various tools, including BLASTN and BLASTX, and phylogenetic analysis of the repetitive elements’ coding domains [ 52 , 112 ]. The presence of tandemly organized repeats within the clusters identified by TAREAN was confirmed with Dotter [ 113 ]. The results of the clustering were then used to create repetitive databases. Databases of Illumina reads were deposited in the Sequence Read Archive (project number: PRJNA1076467). Assembled contigs from different types of repetitive DNA elements are publicly available online ( https://olomouc.ueb.cas.cz/en/content/dna-repeats ). The sequences of newly identified tandem organized repeats and 5S rDNA which were used as cytogenetic markers were deposited in GenBank (accessions: PP457292–PP457296). Cluster graphs of 5S rDNA sequences were visualized using SeqGrapheR visualization tool [ 55 ]. Preparation of chromosome spreads Mitotic metaphase chromosome spreads were prepared from root meristems by a dropping method according to Šimoníková et al. [ 44 ]. Briefly, actively growing root tips of Pulmonaria were collected and pre-treated in 0.05% (w/v) colchicine for three hours at room temperature, fixed in 3:1 ethanol:acetic acid fixative overnight at 4°C and stored in 70% ethanol at − 20°C. Chromosome preparations were prepared using the drop technique according to Kato et al. [ 114 , 115 ], with minor modifications: After washing in 75 mM KCl and 7.5 mM EDTA (pH 4), root tip segments were digested in a mixture of 2% (w/v) cellulase and 2% (w/v) pectinase in 75 mM KCl and 7.5 mM EDTA (pH 4) for 45 min at 37°C. The cell suspension was dropped onto glass slides in a box lined with wet paper towels and let dried. Probe design and fluorescence in situ hybridization Consensus sequences of TAREAN analysis which contained tandemly organized repeats were used for specific primer design using the Primer3 program [ 116 ]. Probes for newly identified tandem repeats were labeled by PCR either directly with Cy5 fluorochrome (Thermo Fisher Scientific), DEAC (Jena Biosciences, Jena, Germany), or indirectly with biotin-dUTP or digoxigenin-dUTP (Sigma Aldrich/Roche Applied Science, Mannheim, Germany) using primers listed in Supplementary Table S4 and P. obscura DNA as template. The 25µl of PCR mix contained 30 ng of genomic DNA, 200 µM dNTPs including directly- or indirectly-labeled dUTP, 1 µM primers and 0.5 U of Q5 High-Fidelity DNA polymerase and appropriate reaction buffer (New England Biolabs, Massachusetts, USA). Plasmid pTa71 (45S rDNA) containing 9-kb fragment from Triticum aestivum with 18S-5.8S-26S rDNA and intergenic spacers [ 117 ] was labeled by nick translation (Sigma Aldrich) using Cy5 fluorochrome (Thermo Fisher Scientific). Hybridization mixture containing 50% (v/v) formamide, 10% (w/v) dextran sulfate in 2 × SSC and 10 ng/µl of labeled probes was added onto slide and denatured for 30s at 80°C, followed by overnight hybridization performed in a humid chamber at 37°C. If the chromosome structure was damaged after the denaturation step, the slides with chromosome spreads were post-fixed in 4% (v/v) formaldehyde in 2 × saline-sodium citrate (SSC) for 10 min at room temperature, washed in 2 × SSC for 2 × 5 min, and dehydrated using ethanol series. The sites of digoxigenin- and biotin-labeled probes were detected using anti-digoxigenin-FITC (Sigma Aldrich/Roche Applied Science) and streptavidin-Cy3 (Thermo Fisher Scientific/Invitrogen, Carlsbad, CA, USA), respectively. Chromosomes were counterstained with DAPI and mounted in Vectashield Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA). Microscopic and image analysis Slides were examined using Axio Imager Z.2 Zeiss microscope (Zeiss, Oberkochen, Germany) equipped with a Cool Cube 1 camera (Metasystems, Altlussheim, Germany) and appropriate optical filters, and a PC running ISIS software 5.4.7 (Metasystems). The final image adjustment was performed in Adobe Photoshop CS5, and idiograms and final pictures were created in Adobe Photoshop CS5 and GIMP (GNU Image Manipulation Program) v2.10.34. A minimum of ten preparations with mitotic metaphase chromosome spreads and different probe combinations were used for the final karyotype reconstruction of each genotype. Declarations Availability of data and materials The datasets supporting the results of this article are included within the article and its supplementary information files. Databases of Illumina reads are deposited in the Sequence Read Archive (project number: PRJNA1076467). Assembled contigs from different types of repetitive DNA elements are publicly available online (https://olomouc.ueb.cas.cz/en/content/dna-repeats). The sequences of newly identified tandem organized repeats were deposited in GenBank (accessions: PP457292–PP457296). Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This study was financially supported by an internal grant from Palacký University, Olomouc (IGA PrF-2024-001). Authors’ contributions E.H. and L.K. conceived the project. K.V. provided plant material from the B481 population. L.K. performed flow cytometry and analyzed the results. J.Č., L.K. and E.H. provided and analyzed sequencing data. J.Č. and V.Z. performed the cytogenetic part of the work. L.K., J.Č. and E.H., wrote the original draft, and V.Z. and K.V. revised and edited the manuscript. All authors have read and approved the final manuscript. Acknowledgements We thank Eva Jahnová, Radomíra Tušková, and Petr Navrátil for excellent technical assistance. The computing was supported by the e-INFRA CZ project (ID:90254), supported by the Ministry of Education, Youth and Sports of the Czech Republic, and the ELIXIR-CZ project (ID:90255), part of the international ELIXIR infrastructure. 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Primer3--new capabilities and interfaces. Nucleic Acids Res. 2012;40(15):e115. doi:10.1093/nar/gks596. Gerlach WL, Bedbrook JR. Cloning and characterization of ribosomal-RNA genes from wheat and barley. Nucleic Acids Res. 1979;7(7):1869-1885. doi:10.1093/nar/7.7.1869 Additional Declarations No competing interests reported. Supplementary Files SuppFigure1.tiff Supplementary Figure 1. Chromosomal distribution of the newly identified satDNA PulTR05_70 in Pulmonaria accessions analyzed. (A) P. officinalis , B470.3; (B) putative natural interspecific hybrid P. obscura × P. officinalis , B481.1; and (C) ornamental P. saccharata -like plant, B15.1. Probes for PulTR05_70 are visualized in red. Chromosomes were counterstained with DAPI (blue). Bars = 5 µm. SupplFigure2.tiff Supplementary Figure 2. Dotter analysis of 45S rDNA units from all Pulmonaria accessions analyzed, reconstructed from Illumina reads using RepeatExplorer2. SupplFigure3.tiff Supplementary Figure 3. Dotter analysis of 3.5 kb long part of IGS. (A) P. obscura , (B) P. officinalis , and (C) their comparison. The first part of inverted regions in the IGS, and two larger indels between P. obscura and P. officinalis are marked by pink arrows. SupplFigure4.tiff Supplementary Figure 4. Graph structure of 5S rDNA sequence reads obtained by analysis of individual datasets with RepeatExplorer2.Graph layout of the 5S rDNA sequence reads of (A) P. obscura , B473.1; (B) P. officinalis , B470.3; (C) P. obscura × P. officinalis natural hybrid, B481.1; (D) ornamental P. saccharata -like plants, B15.1, (E) B465.1, and (F) B472.1. Single reads are represented by vertices (nodes) and the sequence overlaps by edges. The 5S coding gene on the graph layout is highlighted in yellow, and intergenic spacers are highlighted as gray vertices. SupplFigure5.tiff Supplementary Figure 5. Chromosomal localization of newly identified satDNAs and rDNA sequences in Pulmonaria officinalis . (A) Idiogram of P. officinalis (B100.3); FISH with probes for: (B) 45S rDNA (yellow), PulTR03_308 (red), and 5S rDNA (green): red arrows point at signals of PulTR03_308; (C) 45S rDNA (yellow), PulTR04_420 (red), and PulTR01_29 (green): red arrows indicate signals of PulTR04_420; and (D) 45S rDNA (yellow), PulTR01_29 (red), and 5S rDNA (green). The white arrows indicate the 5S rDNA loci. Chromosomes were counterstained with DAPI (blue). Bars = 5 µm. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4148849","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":283227189,"identity":"04bce09b-a6ee-46b5-85e0-d94c2d3779e5","order_by":0,"name":"Lucie Kobrlová","email":"","orcid":"","institution":"Palacký University","correspondingAuthor":false,"prefix":"","firstName":"Lucie","middleName":"","lastName":"Kobrlová","suffix":""},{"id":283227191,"identity":"e99b6d81-2b9b-4bdc-9ac3-a968b91e02e7","order_by":1,"name":"Jana Čížková","email":"","orcid":"","institution":"Institute of Experimental Botany","correspondingAuthor":false,"prefix":"","firstName":"Jana","middleName":"","lastName":"Čížková","suffix":""},{"id":283227192,"identity":"4b17a254-4aa1-465f-a423-a6907586d4f4","order_by":2,"name":"Veronika Zoulová","email":"","orcid":"","institution":"Institute of Experimental Botany","correspondingAuthor":false,"prefix":"","firstName":"Veronika","middleName":"","lastName":"Zoulová","suffix":""},{"id":283227194,"identity":"da9cae7c-9e6c-4e7b-acdf-3739b3352098","order_by":3,"name":"Kateřina Vejvodová","email":"","orcid":"","institution":"University of South Bohemia","correspondingAuthor":false,"prefix":"","firstName":"Kateřina","middleName":"","lastName":"Vejvodová","suffix":""},{"id":283227197,"identity":"00ee9fd6-64b1-432f-a72c-bc156670bf82","order_by":4,"name":"Eva Hřibová","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYBACAwbGNhAtx8DAQ6IWY1K0MLCB6MQGorWYSx9ue/CxzS59w/HeAww/auoY+PsP4Ndi2ZfYbjizLTl3w5lzCYw9xw4zSBwgoMXgDGObNM8Z5twNN3IMmBnYDjAYMDYQpaU+3eD+G6CWf3UMQJIYLRWHEwxu8BgwM7YxMxiwEdbSbjij4rjhzDN5CQd7+w7zSJwhqIX92YMPBtXyfMfPHnzw41udHMEQQwEgtcTGzigYBaNgFIwCfAAAo3NAz/EZn0sAAAAASUVORK5CYII=","orcid":"","institution":"Institute of Experimental Botany","correspondingAuthor":true,"prefix":"","firstName":"Eva","middleName":"","lastName":"Hřibová","suffix":""}],"badges":[],"createdAt":"2024-03-22 10:05:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4148849/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4148849/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-024-05497-4","type":"published","date":"2024-09-13T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53365311,"identity":"73d0e3fb-63cd-48cc-9bee-b765f796de43","added_by":"auto","created_at":"2024-03-25 06:04:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2797519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution map of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePulmonaria officinalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e group. \u003c/strong\u003e(A) Distribution map of the \u003cem\u003eP. officinalis \u003c/em\u003egroup sampled and analyzed in this study (large dots; see Supplementary Table 1), including published chromosome reports (744 records in total, small dots; see Supplementary Table 2): \u003cem\u003eP. obscura\u003c/em\u003ein green and \u003cem\u003eP. officinalis\u003c/em\u003e s.str. in green. Illustrative images of (B) \u003cem\u003eP. obscura\u003c/em\u003e (B473.1), (C) \u003cem\u003eP. officinalis \u003c/em\u003e(B470.1) and (D) \u003cem\u003eP. saccharata\u003c/em\u003e-like accession (B15.1).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/25dd2dfe21c6e47d5d08287b.png"},{"id":53365306,"identity":"cb52c71a-47de-4f2e-b8e5-3e83b084874c","added_by":"auto","created_at":"2024-03-25 06:04:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":95804,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenome size and GC content variation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePulmonaria obscura\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. officinalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(A) Variation in absolute genome size (2C-value); and (B) genomic GC content detected in the \u003cem\u003ePulmonaria officinalis\u003c/em\u003e group: \u003cem\u003eP. obscura\u003c/em\u003e(POBS), \u003cem\u003eP. officinalis\u003c/em\u003e (POFF). Rectangles define the 25th and 75th percentiles, horizontal lines show median values, whiskers are 10–90 percentiles.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/26bbc75f0e85fc443dee91e3.png"},{"id":53365307,"identity":"64370872-768f-4a65-a198-3aa2155b8e35","added_by":"auto","created_at":"2024-03-25 06:04:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":38342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepeatome composition in analyzed \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePulmonaria \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eaccessions.\u003c/strong\u003e 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. \u003cem\u003eP. obscura\u003c/em\u003e (POBS; B473.1); \u003cem\u003eP. officinalis\u003c/em\u003e (POFF; B470.3); an interspecific natural hybrid \u003cem\u003eP. obscura\u003c/em\u003e × \u003cem\u003eP. officinalis\u003c/em\u003e (HYBR; B481.1); \u003cem\u003eP. saccharata\u003c/em\u003e-like accessions (PSAC1–PSAC3; B465.1; B472.1; B15.1).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/7e8cb8d2223ca148765cdefc.png"},{"id":53365309,"identity":"638d4a22-16b3-41a3-9892-4e1081fcb5d9","added_by":"auto","created_at":"2024-03-25 06:04:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53452,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraph structure of 5S rDNA sequence reads from the comparative analysis of RepeatExplorer2.\u003c/strong\u003e (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: \u003cem\u003eP. obscura \u003c/em\u003ein green (C); \u003cem\u003eP. officinalis\u003c/em\u003e in orange (D); reads specific for \u003cem\u003eP. obscura\u003c/em\u003e × \u003cem\u003eP. officinalis\u003c/em\u003e (B481.1)\u003cem\u003e \u003c/em\u003ein blue (E); reads specific for \u003cem\u003eP. saccharata\u003c/em\u003e-like accession B15.1 are highlighted in purple (F), B465.1 in pink (G), and B472.1 in red (H).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/5ed18282b359f0fc39c007d1.png"},{"id":53365312,"identity":"102591cf-434d-44a5-bfc6-80625a6c3663","added_by":"auto","created_at":"2024-03-25 06:04:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":56075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdiograms of analyzed \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePulmonaria \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eaccessions.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/d84340adb2a51b14bb0b0d4d.png"},{"id":53365315,"identity":"4f8cd648-c791-44df-b485-d82167b18ca5","added_by":"auto","created_at":"2024-03-25 06:04:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2928526,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromosomal localization of newly identified satDNAs and rDNA sequences in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. obscura \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(2\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 14). \u003c/strong\u003e(A, B, C) \u003cem\u003eP. obscura\u003c/em\u003e (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, E, F) \u003cem\u003eP. obscura\u003c/em\u003e (B467.2) with probes for: (D) 45S rDNA (yellow), PulTR01_29 (red) and 5S rDNA (green); (E) 45S rDNA (yellow), PulTR02_305 (red) and 5S rDNA (green); and (F) PulTR01_29 (red), PulTR03_308 (orange), and 5S rDNA (green). The white arrows indicate 5S rDNA loci and red arrows in (E) indicate loci of PulTR02_305. Chromosomes were counterstained with DAPI (blue). Bars = 5 µm.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/4da95f5255e2f076f5950df3.png"},{"id":53365318,"identity":"d495cf26-263c-4ce6-8bf3-9acb88905311","added_by":"auto","created_at":"2024-03-25 06:04:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3708863,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromosomal localization of newly identified satDNAs and rDNA sequences in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. officinalis \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(2\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 16). \u003c/strong\u003e(A, B, C, G) \u003cem\u003eP. officinalis\u003c/em\u003e (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 the loci of PulTR04_420 and; (C, G) the same plate with the signals for (C) 45S rDNA (red) and PulTR03_308 (green), and (G) co-localization of PulTR03_308 (green) and PulTR04_420 (red): green arrows indicate PulTR03_308, and red arrows indicate PulTR04_420. (D, E, F) \u003cem\u003eP. officinalis\u003c/em\u003e (B100.2) with probes for: (D) 45S rDNA (yellow) and PulTR01_29 (red); (E) 45S rDNA (yellow), PulTR03_308 (red) and 5S rDNA (green): red arrows indicate PulTR03_308; and (F) 45S rDNA (yellow), PulTR04_420 (red), and 5S rDNA (green): red arrows indicate PulTR04_420. The white arrows indicate the 5S rDNA loci and yellow arrows point at minor interstitial loci of 45S rDNA. Chromosomes were counterstained with DAPI (blue). Bars = 5 µm.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/6c64ba9f1fe7bee8695f35b4.png"},{"id":53365431,"identity":"e5c6abd7-5f09-43a7-829d-4c9092c16e44","added_by":"auto","created_at":"2024-03-25 06:12:07","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5859451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromosomal localization of new satDNAs and rDNA sequences in natural hybrid and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. saccharata\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-like accessions. \u003c/strong\u003e(A, B, C) Putative natural hybrid between \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP.\u003c/em\u003e \u003cem\u003eofficinalis \u003c/em\u003e(B481.1; 2\u003cem\u003en \u003c/em\u003e= 15) with probes for: (A) 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 loci of 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, E, F,) \u003cem\u003eP. saccharata\u003c/em\u003e-like accession B15.1 (2\u003cem\u003en\u003c/em\u003e = 15) with probes for: (D) 45S rDNA (yellow), PulTR02_305 (red) and 5S rDNA (green): red arrow points at a signal of PulTR02_305; (E) 45S rDNA (yellow), PulTR01_29 (red) and PulTR04_420 (green): green arrows point at PulTR04_420; (F) PulTR03_308 (red) and 5S rDNA (green). (G, H, I, J) \u003cem\u003eP. saccharata\u003c/em\u003e-like accession B465.1 (2\u003cem\u003en\u003c/em\u003e = 16) with probes for: (G) 45S rDNA (yellow), PulTR01_29 (red) and PulTR04_420 (green): green arrows indicate PulTR04_420; (H) PulTR01_29 (red) and 5S rDNA (green); (I) 45S rDNA (red) and PulTR03_308 (green): green arrows point at PulTR03_308; and (J) PulTR03_308 (green) and PulTR04_420 (red): green arrows point at PulTR03_308 and red arrows indicate PulTR04_420. (K, L, M) \u003cem\u003eP. saccharata\u003c/em\u003e-like accession B472.1 (2\u003cem\u003en\u003c/em\u003e= 16) with probes for: (K) 45S rDNA (yellow), PulTR01_29 (green) and PulTR04_420 (red): red arrows point at PulTR04_420; (L) 45S rDNA (yellow), PulTR03_308 (green) and PulTR04_420 (red): green arrows point at PulTR03_308, and red arrows indicate PulTR04_420; (M) 45S rDNA (yellow), PulTR03_308 (red) and 5S rDNA (green). The white arrows indicate the 5S rDNA loci and yellow arrows point at minor interstitial loci of 45S rDNA. Chromosomes were counterstained with DAPI (blue). Bars = 5 µm.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/6da1e5f724dc872539260675.png"},{"id":64619003,"identity":"8bb8653b-2ac0-4345-8c9f-5910d4bbe984","added_by":"auto","created_at":"2024-09-16 16:10:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23840654,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/90b1ee7e-38f7-4668-8774-a599c4083474.pdf"},{"id":53365310,"identity":"8fecd0ed-e1d9-432e-ad6e-fbd8d4063e1f","added_by":"auto","created_at":"2024-03-25 06:04:06","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1416046,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. Chromosomal distribution of the newly identified satDNA PulTR05_70 in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePulmonaria\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e accessions analyzed. \u003c/strong\u003e(A) \u003cem\u003eP. officinalis\u003c/em\u003e, B470.3; (B) putative natural interspecific hybrid \u003cem\u003eP. obscura\u003c/em\u003e × \u003cem\u003eP.\u003c/em\u003e \u003cem\u003eofficinalis\u003c/em\u003e, B481.1; and (C) ornamental \u003cem\u003eP. saccharata\u003c/em\u003e-like plant, B15.1. Probes for PulTR05_70 are visualized in red. Chromosomes were counterstained with DAPI (blue). Bars = 5 µm.\u003c/p\u003e","description":"","filename":"SuppFigure1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/73ebb4af33b129e55f393821.tiff"},{"id":53365308,"identity":"9e1e0240-4492-4f2d-a72b-2c7b862d58a9","added_by":"auto","created_at":"2024-03-25 06:04:06","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":144158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Dotter analysis of 45S rDNA units from all \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePulmonaria \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eaccessions analyzed, reconstructed from Illumina reads using RepeatExplorer2.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplFigure2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/dae4f726cdce5d8455beda48.tiff"},{"id":53365313,"identity":"27afbec5-051a-45b3-a057-93dfceb766d9","added_by":"auto","created_at":"2024-03-25 06:04:07","extension":"tiff","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":883574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. Dotter analysis of 3.5 kb long part of IGS. \u003c/strong\u003e(A) \u003cem\u003eP. obscura\u003c/em\u003e, (B) \u003cem\u003eP. officinalis\u003c/em\u003e, and (C) their comparison. The first part of inverted regions in the IGS, and two larger indels between \u003cem\u003eP. obscura\u003c/em\u003e and P. \u003cem\u003eofficinalis \u003c/em\u003eare marked by pink arrows.\u003c/p\u003e","description":"","filename":"SupplFigure3.tiff","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/72157aa2000036b4943a36a1.tiff"},{"id":53365316,"identity":"dd8a20da-6e20-41c0-afad-c9c62402c68b","added_by":"auto","created_at":"2024-03-25 06:04:07","extension":"tiff","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":184670,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4. Graph structure of 5S rDNA sequence reads obtained by analysis of individual datasets with RepeatExplorer2.\u003c/strong\u003eGraph layout of the 5S rDNA sequence reads of (A) \u003cem\u003eP. obscura\u003c/em\u003e, B473.1; (B) \u003cem\u003eP. officinalis\u003c/em\u003e, B470.3; (C) \u003cem\u003eP. obscura \u003c/em\u003e× \u003cem\u003eP. officinalis\u003c/em\u003e natural hybrid, B481.1; (D) ornamental \u003cem\u003eP. saccharata\u003c/em\u003e-like plants, B15.1, (E) B465.1, and (F) B472.1. Single reads are represented by vertices (nodes) and the sequence overlaps by edges. The 5S coding gene on the graph layout is highlighted in yellow, and intergenic spacers are highlighted as gray vertices.\u003c/p\u003e","description":"","filename":"SupplFigure4.tiff","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/70c15f93f7fcc737bcee8444.tiff"},{"id":53365314,"identity":"1fed7b33-fa88-456f-b403-326c6637de9b","added_by":"auto","created_at":"2024-03-25 06:04:07","extension":"tiff","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3098852,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 5. Chromosomal localization of newly identified satDNAs and rDNA sequences in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePulmonaria officinalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) Idiogram of \u003cem\u003eP. officinalis\u003c/em\u003e (B100.3); FISH with probes for: (B) 45S rDNA (yellow), PulTR03_308 (red), and 5S rDNA (green): red arrows point at signals of PulTR03_308; (C) 45S rDNA (yellow), PulTR04_420 (red), and PulTR01_29 (green): red arrows indicate signals of PulTR04_420; and (D) 45S rDNA (yellow), PulTR01_29 (red), and 5S rDNA (green). The white arrows indicate the 5S rDNA loci. Chromosomes were counterstained with DAPI (blue). Bars = 5 µm.\u003c/p\u003e","description":"","filename":"SupplFigure5.tiff","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/b6dab87b11dc54dad270e942.tiff"},{"id":53365317,"identity":"2e11fc6a-e61d-4cad-bb79-7486ecf221c8","added_by":"auto","created_at":"2024-03-25 06:04:07","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":177885,"visible":true,"origin":"","legend":"","description":"","filename":"SupplTableS1S4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4148849/v1/df670f79ef249c8049ed1e17.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"First insight into the genomes of the Pulmonaria officinalis group (Boraginaceae) provided by repeatome analysis and comparative karyotyping","fulltext":[{"header":"Background","content":"\u003cp\u003eThe genus \u003cem\u003ePulmonaria\u003c/em\u003e L. (Boraginaceae, \u003cem\u003esensu\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]) is a taxonomically complex group of species in which the rather similar morphology contrasts with striking karyological variability. In total, 16 different somatic chromosome counts ranging from 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14 to 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;38 are currently reported in the genus [Kobrlov\u0026aacute; \u003cem\u003eunpubl.\u003c/em\u003e], with \u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7 proposed as the basic chromosome number [e.g. 2, 3, 4, 5, 6]. According to the traditional morphology- and karyology-based taxonomy [cf. 3, 6], about 30 taxa are recognized (16 species and 14 subspecies), all found in Europe, with the exception of the \u003cem\u003eP. mollis\u003c/em\u003e group, which extends as far as north-eastern and eastern Asia [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Although almost nothing is known about the mechanisms of the chromosomal evolution of the genus, such a karyological diversity is indicative of extensive chromosomal rearrangements, i.e. the hypothesis of ancient episodes of polyploidization/hybridization and subsequent dysploidal chromosome reorganizations [cf. 6, 10]. Considerable variability in chromosome number and genome size has been reported within the Boraginaceae family [summarized in 11, 12], but its role in species diversification and evolution has mostly only been hinted at or discussed [but see e.g. 10, 13, 14, 15].\u003c/p\u003e \u003cp\u003eTo date, only a few attempts have been made to explore the evolutionary history of the genus \u003cem\u003ePulmonaria\u003c/em\u003e, focusing on a limited or specific group of species [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These studies have highlighted the significant role of hybridization (e.g. the recent hybrid origin of \u003cem\u003eP. helvetica\u003c/em\u003e Bolliger [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] or the \u003cem\u003eP. hirta\u003c/em\u003e complex [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]), and provided evidences for the involvement of introgression and dysploidy in the speciation process [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In horticulture, \u003cem\u003ePulmonaria\u003c/em\u003e species are known to be crossable (no geographical isolation, flowering synchrony, similar pollinator preferences), giving rise to a wide variety of cultivars [see 20]. Even in the contact zones of some taxa, intermediate chromosome numbers have rarely been documented [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, most of the evolutionary relationships are still unknown, and the question remains as to what lies behind the observed variability in their morphology and chromosome number.\u003c/p\u003e \u003cp\u003eAlong with chromosome number, genome size is another relevant biodiversity trait that can indicate significant genomic events and evolutionary changes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Diversity in genome size is driven by multiple evolutionary processes such as polyploidy, the proliferation of repetitive DNA sequences and/or drastic reduction of non-genic DNA [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Typically, post-polyploid diploidization is thought to be associated with extensive loss of DNA or 'genome downsizing' [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, the mechanisms, rates and selection pressures driving these changes in DNA content remain unknown [cf. 33]. Genome size can also be a useful tool for the practical botany, i.e. distinguishing between cryptic taxa, identifying hybrids and resolving taxonomies in critical groups [e.g. 35, 36, 37, 38]. According to the available literature, the genome size values of \u003cem\u003ePulmonaria\u003c/em\u003e species have only been reported sporadically [summarized in 39], without any systematic study.\u003c/p\u003e \u003cp\u003eAs already mentioned, karyological studies in \u003cem\u003ePulmonaria\u003c/em\u003e have mainly focused on determining the number of chromosomes and their significance for taxonomy [e.g. 3, 21]. A more detailed analysis of chromosome structure at the cytogenetic level has not yet been carried out. The most important technique in plant cytogenetic research is fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (FISH). The major limiting factor of the FISH techniques in non-model plant species is the lack of powerful DNA probes [reviewed in 40, 41]. Recent development of whole genome sequencing technologies and bioinformatics facilitates fast progress of cytogenomics. In non-model plants, the first step is identification of suitable cytogenetic landmarks, which provide specific pattern on chromosomes and can be used to identify individual chromosomes, to compare karyotypes and chromosomal rearrangements in closely related species and inter-specific hybrids, and to provide important information on the plant evolution through chromosome changes [e.g. 42, 43, 44, 45, 46]. The best performing cytogenetic probes include rRNA genes and satellite DNAs (satDNA). Both of them are present in high copy numbers of tandem organized repetitive units in the genomes, and very often provide species-specific patterns on chromosomes [e.g. 27, 47, 48, 49, 50].\u003c/p\u003e \u003cp\u003eTo identify such cytogenetic probes, whole genome sequencing data can be utilized and further analyzed by the RepeatExplorer pipeline [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. RepeatExplorer pipeline is based on a graph-based clustering algorithm and enables identification and characterization of repetitive DNA from small quantities of short read sequencing data [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The pipeline has been used for repeat identification in many model and non-model plant species [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The new version of RepeatExplorer2 provides \u003cem\u003ede novo\u003c/em\u003e repeat identification and annotation of transposable elements, comparative repeat analysis in large sets of species, and identification of satellite DNA with the aim to develop cytogenetic probes. The analyses are complemented by visualization of the results in the SeqGrapheR visualization tool [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study is focused on the \u003cem\u003eP. officinalis\u003c/em\u003e group, the most widespread European species complex [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The \u003cem\u003eP. officinalis\u003c/em\u003e group contains two morphologically relatively distinct species differing in chromosome numbers, namely \u003cem\u003eP. obscura\u003c/em\u003e Dumort. (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14) and \u003cem\u003eP. officinalis\u003c/em\u003e L. (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16 [e.g. 3, 22, 58, 59, 60]). Within the latter, two subspecies are sometimes recognized [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. As the \u003cem\u003eP. officinalis\u003c/em\u003e complex has been cultivated for medicinal and ornamental purposes for a very long time [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], it has probably become even more widespread. In some regions it may also be non-native as a result of frequent cultivation and possible garden escapes [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. \u0026ldquo;Pure\u0026rdquo; \u003cem\u003ePulmonaria\u003c/em\u003e species have often been crossed in cultivation to produce more attractive varieties [cf. 20], which are rarely found in nature. Many of these plants are listed under the name \u003cem\u003eP. saccharata\u003c/em\u003e in the horticultural trade. Their origin is not clear, but it can be assumed that some of them are derived from \u003cem\u003eP. officinalis\u003c/em\u003e (esp. plants with distinctly white-spotted leaves, cordate at the base). However, they are not \u0026ldquo;true\u0026rdquo; \u003cem\u003eP. saccharata sensu\u003c/em\u003e Miller, whose taxonomic status has long been debated, and are most likely related to the \u003cem\u003eP. hirta\u003c/em\u003e complex [cf. 10, 66, 67].\u003c/p\u003e \u003cp\u003eIn this work, we performed the analysis of genome size and comparative analysis of the repeatomes in closely related species of the \u003cem\u003eP. officinalis\u003c/em\u003e group in order to shed light on the karyotype structure and variability. Specifically, we have analyzed repeatomes and karyotypes of \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e s.str. individuals from pure separate populations, their putative hybrid accessions with 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15, which have been collected in a mixed population where \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e grew together, and three populations of ornamental cultivars, morphologically similar to \u003cem\u003eP. officinalis\u003c/em\u003e, that have escaped into the wild where they were collected (here listed as \u003cem\u003eP. saccharata\u003c/em\u003e-like). Genome size has been studied and compared on a wider geographical scale. This pilot study can serve as a springboard for future cytogenetic and genomic studies, to understand the role of chromosomal rearrangements in the evolution of this genus.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eGenome size and chromosome number variation in the P. officinalis group\u003c/h2\u003e\n\u003cp\u003eA total number of 196 accessions from 65 populations of the \u003cem\u003ePulmonaria officinalis\u003c/em\u003e group were collected throughout Europe and analyzed by flow cytometry (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). The analyses resulted in good quality histograms with distinct peaks and low coefficients of variation (below 5%) for both PI and DAPI staining (Supplementary Table\u0026nbsp;1). The flow cytometry data were confirmed by chromosome counts, some of which were obtained \u003cem\u003ede novo\u003c/em\u003e (see below), but previously published reports were also revised. A total of 754 published chromosome records were found and revised for the \u003cem\u003eP. officinalis\u003c/em\u003e complex (Supplementary Table\u0026nbsp;2). Of these, 287 chromosome reports with 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14 were related to \u003cem\u003eP. obscura\u003c/em\u003e and 450 with 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16 to \u003cem\u003eP. officinalis\u003c/em\u003e s.str., respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Five records were excluded as they probably belong to another species of the genus. In one case 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17 have been reported for \u003cem\u003eP. officinalis\u003c/em\u003e. In addition to this, there are even karyological records (16 in total) that refer to hybrids of \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e. In all cases, 15 chromosomes were counted in these plants (Supplementary Table\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. obscura\u003c/em\u003e (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14; mean 2C\u0026thinsp;=\u0026thinsp;2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 pg) and \u003cem\u003eP. officinalis\u003c/em\u003e s.str. (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16; mean 2C\u0026thinsp;=\u0026thinsp;3.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 pg) differed significantly in holoploid genome size (2C value, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) as well as in the genomic GC content (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Their DNA amount ranges are listed in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The genome size value of the presumed hybrid plants (B481) with 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15 (2C\u0026thinsp;=\u0026thinsp;3.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 pg) was positioned between their values (within the theoretically expected intermediate genome size based on the genome sizes of \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e). The GC content was also intermediate (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Among \u003cem\u003eP. saccharata\u003c/em\u003e-like populations, B465.1 with 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16 had the genome size value (2C\u0026thinsp;=\u0026thinsp;3.13 pg) in the range of \u003cem\u003eP. officinalis\u003c/em\u003e. The remaining two populations (B15: mean 2C\u0026thinsp;=\u0026thinsp;3.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 pg; B472.1: 2C\u0026thinsp;=\u0026thinsp;3.63 pg) had larger genome sizes than \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e. Interestingly, these populations differ in the number of chromosomes (B15: 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15 vs. B472.1: 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16). The similar pattern was detected in the genomic GC content, only in B472.1 the GC content was lower than in B15 (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eSummary of flow cytometric analyses of the \u003cem\u003ePulmonaria officinalis\u003c/em\u003e group.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTaxon\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e2\u003cem\u003en\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eN pop\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eN ind\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMean 2C [pg]\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMin\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMax\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e1C [Mbp]\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eC/\u003cem\u003en\u003c/em\u003e [pg]\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGC [%]\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eP. obscura\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e83\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.73\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 428\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35.89\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eP. officinalis\u003c/strong\u003e \u003cstrong\u003es.str.\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e36\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e103\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.95\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 570\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35.09\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eP. obscura\u003c/strong\u003e \u003cstrong\u003e\u0026times;\u003c/strong\u003e \u003cstrong\u003eP. officinalis\u003c/strong\u003e \u003cstrong\u003e(B481)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 511\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35.42\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eP. saccharata\u003c/strong\u003e\u003cstrong\u003e-like (B15)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.71\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.78\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 829\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35.64\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eP. saccharata\u003c/strong\u003e\u003cstrong\u003e-like (B465.1)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 531\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35.16\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eP. saccharata\u003c/strong\u003e\u003cstrong\u003e-like (B472.1)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 775\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35.19\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eGenome-wide repeatome analysis\u003c/h2\u003e\n\u003cp\u003eTo identify the major types of repetitive sequences and to compare their genome representation in \u003cem\u003eP. officinalis\u003c/em\u003e group, the comparative mode of RepeatExplorer2 pipeline was used. The analysis was performed on partial Illumina sequencing data of \u003cem\u003eP. obscura\u003c/em\u003e (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14; B473.1, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), \u003cem\u003eP. officinalis\u003c/em\u003e (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16; B470.3, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC), their putative interspecific hybrid (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15; B481.1) and three \u003cem\u003eP. saccharata\u003c/em\u003e-like accessions (B15.1; 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD and B465.1, B472.1; both 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16). The identified repetitive sequences accounted for 58.48% of the \u003cem\u003eP. obscura\u003c/em\u003e genome and 47.86% of the \u003cem\u003eP. officinalis\u003c/em\u003e genome. A similar and overall highest proportion of repetitive sequences was found in repeatomes of the putative interspecific hybrid B481.1 (64.10%) and \u003cem\u003eP. saccharata\u003c/em\u003e-like accession B15.1 (64.53%), while the repetitive DNA content of the other two \u003cem\u003eP. saccharata\u003c/em\u003e-like accessions (B465.1 and B472.1) was around 50% (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn all studied taxa, LTR retroelements were the most abundant repeats, accounting for 46.68% of \u003cem\u003eP. obscura\u003c/em\u003e genome, 34.52% of \u003cem\u003eP. officinalis\u003c/em\u003e genome and 33.80-49.96% of the other genomes. The genome proportion of Ty3/Gypsy elements, which were mainly represented by Tekay lineage, ranged from 23.90% in \u003cem\u003eP. saccharata\u003c/em\u003e-like (B472.1) to 35.06% in putative interspecific hybrid (B481.1). Ty1/Copia elements were about twice less abundant compared to Ty3/Gypsy superfamily, and were mostly represented by elements of SIRE and Angela clades (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). DNA transposons and long interspersed nuclear elements (LINE elements) were found in low copy numbers in all analyzed groups, with genome proportions ranging from 0.14\u0026ndash;0.23% and from 0.16\u0026ndash;0.22%, respectively (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). rDNA sequences accounted for about 3.00\u0026ndash;5.00%, and other tandem organized repeats represented 1.25% to more than 4.00% of the \u003cem\u003ePulmonaria\u003c/em\u003e genomes (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eProportion of repetitive DNA sequences identified \u003cem\u003ede novo\u003c/em\u003e in \u003cem\u003ePulmonaria\u003c/em\u003e taxa\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eRepeat\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLineage/class\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"6\" align=\"left\"\u003e\n\u003cp\u003eProportion of repeats in genomes [%]\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePOBS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePOFF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHYBR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePSAC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePSAC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePSAC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eB473.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eB470.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eB481.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eB465.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eB472.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eB15.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eLTR retroelements\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTy1/Copia\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSIRE\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.83\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAngela\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.07\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTork\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.93\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.99\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.96\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.03\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTAR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAle\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e \u003cstrong\u003eTy1/Copia\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e15.78\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e9.68\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e14.50\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e9.74\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e9.48\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e14.03\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTy3/Gypsy\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOgre\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.82\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.82\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.92\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.80\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.74\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.91\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAthila\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTekay\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e29.69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e23.41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33.97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24.84\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e23.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e32.67\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChromovirideae\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.08\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e \u003cstrong\u003eTy3/Gypsy\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e30.62\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e24.40\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e35.06\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e25.82\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e23.90\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e33.73\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eUnclassified LTR elements\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.28\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.44\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.40\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.43\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.42\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.47\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTotal LTR\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e46.68\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e34.52\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e49.96\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e35.99\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e33.8\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e48.23\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eOther\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eLINE\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.23\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.17\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.23\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.16\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.14\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.21\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDNA transposons\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.16\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.20\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.18\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.22\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.18\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.22\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTandem repeats\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e3.78\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e1.25\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e4.33\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e3.33\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.12\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e3.60\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003erRNA genes\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e3.78\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e4.36\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.78\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e4.52\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e4.73\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e5.09\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eUnclassified repeats\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e3.85\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e7.36\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e6.62\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e7.84\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e7.61\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e7.18\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTotal repetitive DNA content\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e58.48\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e47.86\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e64.1\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e52.06\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e48.58\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e64.53\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"9\"\u003ePOBS: \u003cem\u003eP. obscura\u003c/em\u003e; POFF: \u003cem\u003eP. officinalis\u003c/em\u003e; HYBR: \u003cem\u003eP. obscura\u003c/em\u003e \u0026times; \u003cem\u003eP. officinalis\u003c/em\u003e, putative interspecific hybrid; PSAC: \u003cem\u003eP. saccharata\u003c/em\u003e-like\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eVariability in the Satellite DNAs and rDNA loci\u003c/h2\u003e\n\u003cp\u003eConsidering satellite (tandem organized) repeats as preferable cytogenetic landmarks, which can be species- or even subspecies-specific, we inspected the individual \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e datasets by TAREAN program. Five putative satellites (tandem organized repeats) were observed, three of them (PulTR01_29, PulTR03_308, PulTR05_70) were shared by both species. One satDNA (PulTR02_305) was only found in \u003cem\u003eP. obscura\u003c/em\u003e, and another one (PulTR04_420) was only detected \u003cem\u003ein silico\u003c/em\u003e in the \u003cem\u003eP. officinalis\u003c/em\u003e genome. Highly abundant tandem organized repeat PulTR05_70 provided strong cluster signals distributed along whole chromosomes with enrichments in pericentromeric regions, and cannot be used as a chromosome-specific cytogenetic landmark (Suppl. Figure\u0026nbsp;1). The other tandem repeats produced chromosome-specific signals and were used together with rDNA sequences to create karyotypes of the \u003cem\u003ePulmonaria\u003c/em\u003e accessions studied.\u003c/p\u003e\n\u003cp\u003eTandem organized character is also typical for rRNA genes, which are present in the genomes in high copy numbers and localized in specific chromosomal regions. The 45S rRNA gene unit is usually about 10 kb long and is often represented by several cluster graphs after the RepeatExplorer analysis, as it was observed in the case of all the \u003cem\u003ePulmonaria\u003c/em\u003e accessions analyzed. To reconstruct the whole 45S unit, we followed the procedure described in Kapustov\u0026aacute; \u003cem\u003eet al.\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e]. The unit length of the \u003cem\u003ePulmonaria\u003c/em\u003e 45S rDNA unit varied from 9.1 kb to 9.6 kb and was found to be highly conserved at the sequence level within all accessions studied (Suppl. Figure\u0026nbsp;2). The intergenic spacer (IGS) region contained two different tandemly organized repeats (with repetitive units of 117 and 150-nt long) which were identical in all the accessions studied, with the exception of a \u003cem\u003eP. saccharata\u003c/em\u003e-like plant B472.1, which contained tandem regions with shorter repetitive units (79 and 150-nt long). A higher variability was found in the IGS, which contained two relatively large INDEL regions that differed between \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e (Suppl. Figure\u0026nbsp;3A). To support this observation, we performed read mapping of \u003cem\u003ePulmonaria\u003c/em\u003e accessions to the assembled \u003cem\u003eP. obscura\u003c/em\u003e 45S rDNA reference (Suppl. Figure\u0026nbsp;3B). The differences in read coverage along the 45S rDNA unit are evident in the IGS, indicating the variability at the sequence level. It should be pointed out that the assembly of the 45S rDNA unit was obtained from short Illumina sequences, so the resulting consensus sequence may represent the most abundant sequence type.\u003c/p\u003e\n\u003cp\u003eThe 5S rRNA gene unit is typically up to 1kb in length in plant species [\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e], so the graph-based clustering algorithm of the RepeatExplorer2 pipeline allowed its complete reconstruction, even in the comparative analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The graph region representing the 5S rRNA gene was shared by all \u003cem\u003ePulmonaria\u003c/em\u003e accessions studied (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Three variable graph loops emanating from this conserved graph region correspond to three types of 5S rDNA unit, differing in the length of their IGS (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Mapping of sample-specific sequencing reads onto the graph topology revealed the presence of different 5S rDNA units between \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). The cluster layout of B481.1, the putative hybrid between \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e, was represented by all three loops specific to its presumed progenitors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). A similar situation was observed for the \u003cem\u003eP. saccharata\u003c/em\u003e-like accession B15.1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF). The other \u003cem\u003eP. saccharata\u003c/em\u003e-like individuals (B465.1 and B472.1) shared the same graph layout as \u003cem\u003eP. officinalis\u003c/em\u003e (i.e. B470.3; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG, H).\u003c/p\u003e\n\u003cp\u003eThe analysis of 5S rDNA specific graph layouts observed by clustering analysis of individual accessions confirmed the observation of the comparative analysis. All \u003cem\u003ePulmonaria\u003c/em\u003e individuals studied were composed of at least two different types of 5S rDNA genes, which differed in the IGS (Suppl. Figure\u0026nbsp;4).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eComparative karyotyping in the P. officinalis group\u003c/h2\u003e\n\u003cp\u003eMolecular karyotyping was performed using newly identified satellites (PulTR01_29, PulTR02_305, PulTR03_308 and PulTR04_420) and 5S and 45S rDNA sequences. In general, \u003cem\u003ein situ\u003c/em\u003e hybridization confirmed the results obtained by repeatome analysis. FISH analysis with the probes for rDNAs and four satDNAs resulted in well visible cluster signals on specific chromosomes in the genome of the analyzed plants of the \u003cem\u003eP. officinalis\u003c/em\u003e complex.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. obscura\u003c/em\u003e, \u003cem\u003eP. officinalis\u003c/em\u003e and their putative natural interspecific hybrid\u003c/p\u003e\n\u003cp\u003eFISH analysis of \u003cem\u003eP. obscura\u003c/em\u003e plants (all 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14) collected from three different populations (B467, B469, B473; Suppl. Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e) provided highly consistent results. The only exception was observed for plant B473.1 in the number of the most abundant satDNA (PulTR01_29). The 45S rDNA was located into terminal NOR regions on four chromosome pairs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, B, D, E). 5S rDNA loci were detected on two chromosome pairs in pericentromeric regions. One chromosome pair contained signals of 5S, 45S rDNA and satellite PulTR02_305 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB, E). In B473.1, the probe for PulTR01_29 provided very strong signals in subtelomeric regions only on one chromosome pair. The same chromosome pair also contained a signal of PulTR03_308 in the pericentromeric region (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). In B467.2 and B469.1, one additional subtelomeric signal of PulTR01_29 was found, located on a chromosome arm with 5S rDNA locus (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, D, F). As expected from the results of the RepeatExplorer2 analysis, signals of PulTR04_420 were not detected in any of the \u003cem\u003eP. obscura\u003c/em\u003e plants analyzed.\u003c/p\u003e\n\u003cp\u003eIn general, the genome of \u003cem\u003eP. officinalis\u003c/em\u003e contained one additional pair of chromosomes (i.e. 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16) compared to \u003cem\u003eP. obscura\u003c/em\u003e. Molecular karyotype was studied in individuals from two different populations (B100, B470; Suppl. Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). A higher variability in the chromosomal distribution of the probes was detected within representatives of \u003cem\u003eP. officinalis\u003c/em\u003e compared to \u003cem\u003eP. obscura\u003c/em\u003e (Suppl. Figure\u0026nbsp;5). Terminal NORs (45S rDNA) were found only on three chromosome pairs in both analyzed individuals (B100.2, B470.1). These terminal 45S rDNA loci were often fragile and broken off from the chromosomes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA, F). Interstitial 45S rDNA clusters were detected on one chromosome pair, on the same arm which also contained signals of satDNA PulTR04_420 and PulTR01_29 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB, F). The genome of B100.2 contained an additional weak signal of interstitial 45S rDNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD, E, F). Variability in the number of signals specific to 5S rDNA loci was detected. While 5S rDNA loci were identified in pericentromeric regions on seven chromosomes of B470.3, only six chromosomes were bearing these loci in B100.2. The signal of PulTR03_308 was detected on one NOR bearing chromosome pair in both individuals with various combinations of co-localization with PulTR04_420 and 5S rDNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC, E, G). Signals of PulTR02_305 were not detected on chromosomes of \u003cem\u003eP. officinalis\u003c/em\u003e, supporting the results of comparative repeatome analysis by RepeatExplorer2. FISH analysis of the other representative of the same population B100 provided slightly different molecular karyotype (Suppl. Figure\u0026nbsp;5).\u003c/p\u003e\n\u003cp\u003eKaryotype analysis of the putative natural hybrid between \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e (B481.1; Suppl. Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e), collected in a mixed population of both species, confirmed the expected chromosome number 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15. In this case, eight 45S rDNA clusters were found, seven in terminal chromosomal regions and one in an interstitial position. 5S rDNA loci were detected on five chromosomes in pericentromeric regions. One chromosome pair contained signals of 45S rDNA and 5S rDNA, another individual chromosome contained 45S and 5S rDNA and PulTR02_305 (found only as one single locus in the genome). Additional two chromosomes contained one or two signals of 5S rDNA, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA, B). Five signals of the most abundant satellite PulTR01_29 were detected on five chromosomes, one of which co-localized with the 45S rDNA locus, one signal was detected on the chromosome bearing weak interstitial signal of 45S and one with signal of PulTR03_308 and PulTR04_420, respectively. One chromosome pair with the remaining two signals of PulTR01_29 also contained a signal of 5S rDNA. SatDNAs PulTR03_308 and PulTR04_420 co-localized on one chromosome pair, containing also PulTR01_29, or joint signal of 45S rDNA and PulTR01_29 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eOrnamental garden escapes, morphologically similar to P. officinalis\u003c/h2\u003e\n\u003cp\u003eKaryotype analysis of \u003cem\u003eP. saccharata\u003c/em\u003e-like plants from three populations, which were collected in the wild but apparently escaped from cultivation (B15, B465, B472; Suppl. Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e), revealed variability in chromosome number. While B465.1 and B472.1 were characterized by 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16, 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15 was detected in B15.1. The karyotypes of B465.1 and B472.1 were very similar, containing terminal NORs on three chromosome pairs and one interstitial 45S rDNA cluster on one additional chromosome pair (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eG, I, K, L, M). Two chromosome pairs were bearing PulTR01_29 in subtelomeric regions and other two chromosome pairs contained 5S rDNA clusters in pericentromeric regions (Figure G, H, K). The signal of PulTR04_420 was found on two chromosome pairs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eG, J, K, L). One of these chromosome pairs also contained signals of PulTR01_29 and an interstitial signal of 45S rDNA, while the other chromosome pair bearing terminal 45S rDNA and the PulTR04_420 co-localized with PulTR03_308 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eG, J, K, L). However, these two accessions differed in the presence of an additional 5S rDNA and PulTR01_29 interstitial loci (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe karyotype of B15.1 was similar to that of the putative interspecific hybrid (B481.1) with the same number of chromosomes, indicating a similar hybrid character. 45S rDNA loci were located as strong signals in terminal regions of three chromosome pairs, one additional chromosome contained a weak terminal signal and another contained an interstitial signal (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD, E). Odd number of signal localizations was detected for 5S rDNA (5 chromosomes with interstitial signals), PulTR01_29 (three chromosomes with the signals in terminal regions), PulTR04_420 (three chromosomes with interstitial signals), and for PulTR02_305 (found only on one chromosome) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD, E, F). Finally, PulTR03_308 provided signals on one chromosome pair co-localizing with PulTR04_420 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe genus \u003cem\u003ePulmonaria\u003c/em\u003e is karyologically highly variable [e.g. 3, 6], with 16 different somatic chromosome numbers reported (Kobrlov\u0026aacute; \u003cem\u003eunpubl.\u003c/em\u003e) and about 30 taxa recognized, growing in Europe and northeastern and eastern Asia [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the mechanism, origin and evolutionary consequences of genome size and karyotype variability remain unexplored [but see 10]. Apparently, chromosomal rearrangements have played an important role in the evolution of this genus [cf. 10, 19, 39], but how and to what extent has never been clearly demonstrated.\u003c/p\u003e \u003cp\u003eIn this work, we studied the \u003cem\u003eP. officinalis\u003c/em\u003e complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which includes two morphologically similar, closely related species that differ in chromosome number [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The present study confirmed previously reported chromosomal data, which were consistent across the whole of Europe. The predominant 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16 reports correspond to \u003cem\u003eP. officinalis\u003c/em\u003e s.l., whereas only 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14 were documented in \u003cem\u003eP. obscura\u003c/em\u003e populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Only in one case, 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17 was documented from the \u0026ldquo;pure\u0026rdquo; population of \u003cem\u003eP. officinalis\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], but no explanation was given. Despite the morphological and karyological differences observed, the origin of this species group remains unknown. To uncover the differences in the karyotype evolution, we used complex methodological approaches involving partial Illumina sequencing followed by bioinformatic analysis and characterization of repeatomes in the \u003cem\u003eP. officinalis\u003c/em\u003e group. We identified a new set of chromosome-specific cytogenetic landmarks and performed comparative karyotyping within and between the two species, their putative natural hybrid from a population where both species occur, and ornamental cultivars morphologically similar to \u003cem\u003eP. officinalis\u003c/em\u003e, which are also rarely found in nature.\u003c/p\u003e\n\u003ch3\u003eImpact of DNA repeats dynamics on genome size\u003c/h3\u003e\n\u003cp\u003eGenome size can reflect some aspects of the evolutionary history of taxa by allowing us to understand the influence of DNA gain/loss between related species [e.g. 70, 71]. Despite the apparent karyological variability of several Boraginaceae genera [see e.g. 14, 72, 73], there have been almost no complex analyses of genome size variation and the evolutionary pathways behind the observed diversity [but see 74, 75, 76]. Given the sparse DNA content records in the Boraginaceae family, the only comprehensive study has been published, providing the first genome size reports for most taxa [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study represents the first large-scale investigation of interspecific genome size variation in \u003cem\u003ePulmonaria\u003c/em\u003e. As already shown in a pilot study by Kobrlov\u0026aacute; \u0026amp; Hroneš [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], genome size is effective in delimiting morphologically similar taxa of the Boraginaceae, which is also true for the \u003cem\u003eP. officinalis\u003c/em\u003e group. We found a significant difference in genome size between \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e, corresponding to the number of chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), confirming previous results [cf. 12], but on a larger geographical scale. The suitability of using flow cytometry to revise the distribution of the \u003cem\u003eP. officinalis\u003c/em\u003e group (i.e. relative genome size) has already been documented in the Bohemian Forest and adjacent foothills [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSo far, the genome size has only been estimated for eight \u003cem\u003ePulmonaria\u003c/em\u003e taxa, including \u003cem\u003eP. officinalis\u003c/em\u003e and \u003cem\u003eP. obscura\u003c/em\u003e, ranging from 2.27 to 4.27 pg (i.e. very small/small genomes according to the categories of Leitch et al. [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e], Supplementary Table\u0026nbsp;3). Only minor differences were observed when comparing previously analyzed genome sizes of \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e with our data, most likely due to different methodologies used (i.e. nuclei isolation buffer, reference standard, plant organ [cf. 12, 39]). The only exception is the study by Šmarda \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e], where almost the same 2C values are presented for \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e, probably as a consequence of taxa misidentification.\u003c/p\u003e \u003cp\u003eDNA transposons have been shown to be the major contributor to the enormous variation in genome size in plants [e.g. 27, 79, 80, 81, 82, 83]. To shed light on genome size dynamics and relationships between \u003cem\u003eP. officinalis\u003c/em\u003e and \u003cem\u003eP. obscura\u003c/em\u003e species, we performed a genome-wide comparison of their repeatomes. We found that most of the repetitive elements in the genomes of the \u003cem\u003ePulmonaria\u003c/em\u003e taxa studied were dispersed repeats represented by LTR retrotransposons [cf. 27, 81], with higher proportion of Ty3/Gypsy elements, which were twice more abundant than Ty1/Copia. Ty3/Gypsy retroelements were almost exclusively represented by Tekay retrotransposons (Chromoviridea clade), whereas SIRE elements were the most abundant types of the Ty1/Copia superfamily (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Ty3/Gypsy elements represent one of the major classes of LTR retrotransposons and are dominant in many plant groups, such as the family Poaceae [e.g. 82, 84, 85, 86] or the tribe Fabeae [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Unfortunately, a genome-wide analysis of DNA repeats and their impact on genome size has not been performed in any other species of the Boraginaceae family. However, the higher proportion of Ty3/Gypsy retroelements have also been found in genera of the closely related Solanaceae family, such as \u003cem\u003eSolanum\u003c/em\u003e, \u003cem\u003eNicotiana\u003c/em\u003e and \u003cem\u003eCapsicum\u003c/em\u003e [88, 89, 90, 91, 92, 93). In contrast, recent studies in the genus \u003cem\u003eSalvia\u003c/em\u003e, a member of the closely related Lamiaceae family, have shown that the nuclear genomes of different species contain different proportions of Ty3/Gypsy and Ty1/Copia retroelements [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e, \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e], indicating a proliferation of different types of DNA repeats during the evolution of individual species. In comparison, the \u003cem\u003ePulmonaria\u003c/em\u003e species analyzed contained a similar proportion of the repeat lineages and individual clusters were represented by reads from all specimens analyzed. This indicates a high degree of genome homology within the \u003cem\u003eP. officinalis\u003c/em\u003e complex, suggesting that the evolution of this species group was not accompanied by a dramatic diversification of DNA transposons, as previously shown in other plant species [e.g. 82]. To better understand the proliferation of DNA repeats during genome evolution and its impact on genome size variation and speciation, analysis of a larger data set of \u003cem\u003ePulmonaria\u003c/em\u003e species from different phylogenetic groups is required.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSatellite DNAs and their use in comparative karyotyping\u003c/h2\u003e \u003cp\u003eRepeatome analysis using the RepeatExplorer pipeline also allows the identification of putative satellite DNAs, which together with tandem organized ribosomal genes are the best cytogenetic landmarks. SatDNAs are usually species- or subspecies-specific, provide chromosome-specific labeling patterns and can therefore be used not only to generate and compare molecular karyotypes, but also to identify putative chromosomal structural changes [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e]. The use of the FISH technique revealed different patterns of chromosomal localization of the tandem repeats and rDNA loci examined, both between and within \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The almost identical cytogenetic pattern of satDNAs and rDNA sequences in \u003cem\u003eP. obscura\u003c/em\u003e (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14), collected from three different populations, suggests karyotype stability in this diploid species (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In comparison, the chromosome structure in \u003cem\u003eP. officinalis\u003c/em\u003e appears to be more dynamic, as individuals from two different populations differ slightly in the cytogenetic pattern of the satDNAs and rDNA sequences. Odd number of signals of some satDNAs as well as of rDNA sequences, and interstitial 45S rDNA loci were found in both diploid accessions (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16), indicating chromosomal structural changes involved in the origin and evolution of \u003cem\u003eP. officinalis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). It is generally accepted that \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7 is the basic chromosome number in \u003cem\u003ePulmonaria\u003c/em\u003e [e.g. 2, 3, 4, 5, 6], which raises the question of how the species represented by different chromosome numbers arose. In the case of \u003cem\u003eP. officinalis\u003c/em\u003e, there are two possible scenarios. There could have been chromosome fission leading to 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16, or a more complex karyotype evolution with polyploidization and further diploidization by chromosome rearrangements. Large differences between the molecular karyotypes of \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e suggest that diploid \u003cem\u003eP. obscura\u003c/em\u003e most probably could not give a rise to \u003cem\u003eP. officinalis\u003c/em\u003e by chromosome fissions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Thus, we can only speculate, that the evolution and speciation in the \u003cem\u003eP. officinalis\u003c/em\u003e group could be influenced by polyploidy and/or hybridization followed by post-polyploid diploidization process, which can result in numerous chromosomal rearrangements [see 98, 99, 100] and the origin of \u003cem\u003ePulmonaria\u003c/em\u003e species differing in their basic chromosome number. A similar evolutionary scenario involving chromosome multiplication, hybridization and, in particular, structural rearrangements of chromosomes (dysploidy) has been outlined by Liu \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] for the origin of the \u003cem\u003eP. hirta\u003c/em\u003e complex on the Italian peninsula and the Swiss endemic \u003cem\u003eP. helvetica\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEvidence of hybridization within the P. officinalis complex\u003c/h2\u003e \u003cp\u003eThe evolution of the genus \u003cem\u003ePulmonaria\u003c/em\u003e remains unresolved, due to a lack of rigorous phylogenomic studies. However, several molecular studies have been published highlighting the important role of hybridization and introgression in the evolution of the genus [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The high level of chromosomal variation may also support the hypothesis of ancient hybridization events and subsequent chromosomal rearrangements [cf. 6, 7, 10, 18]. In addition, some species groups exhibit weak ecological and geographic isolation, near-synchronous phenology and pollinator sharing, all of which may facilitate the hybridization [cf. 18]. This is particularly true for the \u003cem\u003eP. officinalis\u003c/em\u003e complex, which is widespread in Europe and therefore often in secondary contact with other \u003cem\u003ePulmonaria\u003c/em\u003e species (cf. 56, 101]. As the ranges of \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e partly overlap (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), the co-occurrence of both species in the same habitat can be expected. Some authors have occasionally reported mixed populations, with morphological intermediates rarely observed [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The extent of hybridization between these two species is still controversial. Nevertheless, several karyological data referring to as \u003cem\u003eP. obscura\u003c/em\u003e \u0026times; \u003cem\u003eP. officinalis\u003c/em\u003e with an intermediate number of chromosomes (i.e. 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15), may provide convincing evidence of an ongoing hybridization between these two species [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, we analyzed presumed hybrids from a mixed population (B481) of \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e. Chromosome counting in all three analyzed individuals confirmed 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15, and their hybrid origin was also supported by their genome sizes, halfway between those of the parents (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The cytogenetic mapping of the set of satDNAs and rDNA sequences also strongly supports the hybrid origin, by the presence of \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e species-specific satDNAs (PulTR_305 and PulTR_420) in haploid state, and also their pattern on chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The hybrid status of these individuals was further confirmed by detailed analysis of the 5S rDNA sequences. As recently shown, graph-based clustering of the RepeatExplorer pipeline enables reconstruction of complete 5S rDNA sequences from partial Illumina sequencing data and provides clues to the evolutionary history of interspecific hybrids and allopolyploids [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e]. The detailed analysis of the 5S rDNA cluster shapes of all \u003cem\u003ePulmonaria\u003c/em\u003e accessions examined revealed the presence of both \u003cem\u003eP. obscura\u003c/em\u003e- and \u003cem\u003eP. officinalis\u003c/em\u003e-specific 5S rDNA sequences in the genome of a putative hybrid clone B481.1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the overlapping ranges of some \u003cem\u003ePulmonaria\u003c/em\u003e species groups [see 56, 101], the possibility of interspecific hybridization might be expected to be relatively common. So far, however, natural hybrids have only occasionally been identified by chromosome counting [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] or distinguished on the basis of intermediate morphology [e.g. 103]. Questions also remain about the hybrid's fertility and their longevity in populations. Although the hybridization is apparently rare, hybrids can potentially persist and spread clonally at the locality [cf. 104], as \u003cem\u003ePulmonaria\u003c/em\u003e species partly reproduce by vegetative propagation (i.e. creeping rhizomes).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOrigin of ornamental cultivars morphologically similar to P. officinalis\u003c/h2\u003e \u003cp\u003eAs a valuable medicinal and ornamental plant, \u003cem\u003eP. officinalis\u003c/em\u003e is represented in horticulture by several cultivars and has also been used to generate new artificial hybrids [cf. 20]. This seems to be the case for plants with distinctly white-spotted leaves, cordate at the base, which are sometimes offered commercially as \u003cem\u003eP. saccharata\u003c/em\u003e. However, they are not \u0026ldquo;true\u0026rdquo; \u003cem\u003eP. saccharata sensu\u003c/em\u003e Miller [see 10, 66, 67]. These plants often escape into the wild and are sometimes confused with \u003cem\u003eP. officinalis\u003c/em\u003e. The origin of these cultivars is unknown, they only resemble \u003cem\u003eP. officinalis\u003c/em\u003e complex in their morphology (i.e. cordate bases of leaves, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eIn total, we analyzed three populations of these garden escapees (B15, B465 and B472). Our cytogenetic analysis and detailed examination of the reconstructed 5S rDNA sequence indicate that two analyzed \u003cem\u003eP. saccharata\u003c/em\u003e-like accessions (B465.1 and B472.1) are derived from the \u003cem\u003eP. officinalis\u003c/em\u003e. Both plants had 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16 as \u003cem\u003eP. officinalis\u003c/em\u003e. They also shared the general cytogenetic pattern of satDNAs and rDNA sequences typical of \u003cem\u003eP. officinalis\u003c/em\u003e, and contain only \u003cem\u003eP. officinalis\u003c/em\u003e-specific, not \u003cem\u003eP. obscura\u003c/em\u003e-specific, 5S rDNA sequence types (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In addition, the genome size value of the B465.1 plant was also in the range of \u003cem\u003eP. officinalis\u003c/em\u003e, only in the B472.1 plant was slightly larger (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This may confirm the assumption that these plants are derived from \u003cem\u003eP. officinalis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eOn the other hand, an interesting cytogenetic pattern was observed in the third \u003cem\u003eP. saccharata\u003c/em\u003e-like plant analyzed (B15.1). The karyotype of this plant was similar to that of the interspecific hybrid B481.1, with the same chromosome number 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15 and \u003cem\u003eP. obscura\u003c/em\u003e- and \u003cem\u003eP. officinalis\u003c/em\u003e-specific 5S rDNA sequences in its genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, even the cytogenetic pattern of the probes and the presence of \u003cem\u003eP. obscura\u003c/em\u003e- and \u003cem\u003eP. officinalis\u003c/em\u003e-specific satDNAs (PulTR_305 and PulTR_420) in the haploid state also suggest a hybrid origin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In contrast, the genome size of B15.1 (the whole population, respectively) was considerably larger, the largest found in the whole data set presented (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, unlike the population B481, population B15 was collected in the area where only \u003cem\u003eP. obscura\u003c/em\u003e occurs naturally and where no population of \u003cem\u003eP. officinalis\u003c/em\u003e has been confirmed (Kobrlov\u0026aacute;, \u003cem\u003epers. obs.\u003c/em\u003e). The morphology of the plants was also typical for cultivated \u003cem\u003eP. saccharata\u003c/em\u003e-like plants. Their origin therefore requires further investigation, although the cytogenetic data presented suggest a hybrid origin between \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e (e.g. phylogenetic revision and analysis of a larger data set of \u003cem\u003ePulmonaria\u003c/em\u003e species from different phylogenetic groups). As this population is a garden escape, its geographical origin is unclear and it cannot be ruled out that it was originally collected from a mixed population of both species. Our data undoubtedly demonstrate that molecular karyotyping is a powerful method for identifying the mode of karyotype evolution and the hybrid origin of \u003cem\u003ePulmonaria\u003c/em\u003e taxa.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study provides comprehensive information on genome size variability and repeatome dynamics of the two morphologically similar species of the \u003cem\u003eP. officinalis\u003c/em\u003e group. Large-scale genome size analysis using flow cytometry confirmed a significant difference in DNA content between \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e, corresponding to the number of chromosomes. Partial sequencing of six accessions, including putative natural hybrid of \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e, and ornamental garden escapes resembling \u003cem\u003eP. officinalis\u003c/em\u003e, showed that a large proportion of their genomes is represented by various types of DNA transposons, with Ty3/Gypsy elements being the most abundant. Comparative analysis of the repeatomes revealed no species-specific retrotransposons or striking differences in their copy number between the species, suggesting a common evolutionary history. Cytogenetic analysis using probes for rDNA sequences and newly identified satellite DNAs allows the origin of the \u003cem\u003ePulmonaria\u003c/em\u003e accessions to be determined, or at least outlined. Thus, in combination with a robust phylogenetic framework, it can contribute to the elucidation of the evolutionary history of \u003cem\u003ePulmonaria\u003c/em\u003e relatives. In our case, comparative karyotyping strongly supported the hybrid origin of putative hybrids with 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15, collected from a mixed population of \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e, and also outlined the origin of ornamental garden escapes, confirming their derivation from the \u003cem\u003eP. officinalis\u003c/em\u003e complex. Finally, databases of repeats were created, which can be used for repeat identification (or masking) in future sequencing projects.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eA total of 196 plants from 65 populations of the \u003cem\u003ePulmonaria officinalis\u003c/em\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), representing typical populations of \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e s. str. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), including their potential hybrids and several garden escapes of cultivars morphologically similar to \u003cem\u003eP. officinalis\u003c/em\u003e (in horticulture often referred to as \u003cem\u003eP. saccharata\u003c/em\u003e, here listed as \u003cem\u003eP. saccharata\u003c/em\u003e-like, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), were included in this study (see Supplementary Table\u0026nbsp;1). These samples were collected between 2014 and 2023 from natural populations across Europe, some of which were cultivated in the experimental garden of Palack\u0026yacute; University in Olomouc, Czech Republic, or deposited in the Herbarium of Palack\u0026yacute; University in Olomouc (OL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry: genome size and GC content\u003c/h2\u003e \u003cp\u003eEstimation of the nuclear DNA content, i.e. absolute genome size (AGS [\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e]), and DNA base composition (GC content [\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e]) were estimated using Partec PAS and Partec ML instruments, with PI (propidium iodide) and DAPI (4,6-diamidino-2-phenylindole) staining. The same methodology as in Kobrlov\u0026aacute; and Hroneš [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] was followed, using fresh, rarely silica dried, leaves for sample preparation. \u003cem\u003ePisum sativum\u003c/em\u003e L. \u0026lsquo;Ctirad\u0026rsquo; (2C\u0026thinsp;=\u0026thinsp;9.09 pg [\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e]; GC content\u0026thinsp;=\u0026thinsp;38.5% [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e]) was selected as a primary internal standard, since it has non-overlapping genome size with neither G1 and nor G2 phase of all studied samples. The conversion from picograms (pg) to base pairs (bp) followed Doležel \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e], using 1 pg DNA\u0026thinsp;=\u0026thinsp;978 Mbp. DNA base content was estimated using the protocol and GC content calculation tool of Šmarda \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e]. The data analyses were performed using the NCSS 9 statistical software [\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e]. The non-parametric Kruskal-Wallis test was used to test for differences between population means of genome size/GC content of \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and sequencing\u003c/h2\u003e \u003cp\u003eGenomic DNA was isolated using alkyltrimethylammonium bromide (MATAB) lysis: after sorbitol washes, the ground plant material was incubated in 2% (w/v) MATAB for 20 min at 65\u0026deg;C, immediately after the incubation, the same volume of chloroform:isoamyl alcohol (24:1) was added, gently but thoroughly mixed and centrifuged at 10,000 \u003cem\u003eg\u003c/em\u003e for 3 min at 4\u0026deg;C. After centrifugation, aqueous upper phase was collected to a new tube and this step was repeat until the upper phase was clear. Genomic DNA was precipitated by adding 0.7 volume of isopropanol, centrifuged at 10,000 \u003cem\u003eg\u003c/em\u003e for 3 min at 4\u0026deg;C. Finally, the pellet was washed by cold 70% and 96% ethanol, air dried and diluted in TE buffer, pH 8.\u003c/p\u003e \u003cp\u003eGenomic DNA was sheared by Bioruptor Plus (Diagenode, Liege, Belgium) to achieve an insert size of about 500 bp. Libraries for sequencing were prepared from 2 \u0026micro;g of fragmented DNA using TruSeq\u0026reg; DNA PCR-free kit (Illumina) and sequenced on a NovaSeq 6000 (Illumina), producing 2 \u0026times; 100-bp or 2 \u0026times; 150-bp paired-end reads to achieve at least 3Gb of nucleotide sequence per each genotype. Raw data were trimmed for low-quality bases and adapter sequences and to the same length using fastp v.0.20.1 [\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis and characterization of DNA repeats\u003c/h2\u003e \u003cp\u003eRandom datasets corresponding to 0.1\u0026times; coverage of the individual accessions were used for reconstruction and characterization of DNA repeats using RepeatExplorer2 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], that includes TAREAN analysis tool for identification of tandemly organized repeats [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. RepeatExplorer2 and TAREAN analyses were also used to perform comparative analysis of \u003cem\u003ePulmonaria\u003c/em\u003e repeatomes on a merged dataset containing all studied individuals (1 mil. reads per accession), marked by specific prefixes. In both cases, the resulting clusters of repeats were characterized by various tools, including BLASTN and BLASTX, and phylogenetic analysis of the repetitive elements\u0026rsquo; coding domains [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e]. The presence of tandemly organized repeats within the clusters identified by TAREAN was confirmed with Dotter [\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results of the clustering were then used to create repetitive databases. Databases of Illumina reads were deposited in the Sequence Read Archive (project number: PRJNA1076467). Assembled contigs from different types of repetitive DNA elements are publicly available online (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://olomouc.ueb.cas.cz/en/content/dna-repeats\u003c/span\u003e\u003cspan address=\"https://olomouc.ueb.cas.cz/en/content/dna-repeats\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e The sequences of newly identified tandem organized repeats and 5S rDNA which were used as cytogenetic markers were deposited in GenBank (accessions: PP457292\u0026ndash;PP457296). Cluster graphs of 5S rDNA sequences were visualized using SeqGrapheR visualization tool [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of chromosome spreads\u003c/h2\u003e \u003cp\u003eMitotic metaphase chromosome spreads were prepared from root meristems by a dropping method according to Šimon\u0026iacute;kov\u0026aacute; \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Briefly, actively growing root tips of \u003cem\u003ePulmonaria\u003c/em\u003e were collected and pre-treated in 0.05% (w/v) colchicine for three hours at room temperature, fixed in 3:1 ethanol:acetic acid fixative overnight at 4\u0026deg;C and stored in 70% ethanol at \u0026minus;\u0026thinsp;20\u0026deg;C. Chromosome preparations were prepared using the drop technique according to Kato \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e, \u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e], with minor modifications: After washing in 75 mM KCl and 7.5 mM EDTA (pH 4), root tip segments were digested in a mixture of 2% (w/v) cellulase and 2% (w/v) pectinase in 75 mM KCl and 7.5 mM EDTA (pH 4) for 45 min at 37\u0026deg;C. The cell suspension was dropped onto glass slides in a box lined with wet paper towels and let dried.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eProbe design and fluorescence in situ hybridization\u003c/h2\u003e \u003cp\u003eConsensus sequences of TAREAN analysis which contained tandemly organized repeats were used for specific primer design using the Primer3 program [\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e]. Probes for newly identified tandem repeats were labeled by PCR either directly with Cy5 fluorochrome (Thermo Fisher Scientific), DEAC (Jena Biosciences, Jena, Germany), or indirectly with biotin-dUTP or digoxigenin-dUTP (Sigma Aldrich/Roche Applied Science, Mannheim, Germany) using primers listed in Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e and \u003cem\u003eP. obscura\u003c/em\u003e DNA as template. The 25\u0026micro;l of PCR mix contained 30 ng of genomic DNA, 200 \u0026micro;M dNTPs including directly- or indirectly-labeled dUTP, 1 \u0026micro;M primers and 0.5 U of Q5 High-Fidelity DNA polymerase and appropriate reaction buffer (New England Biolabs, Massachusetts, USA). Plasmid pTa71 (45S rDNA) containing 9-kb fragment from \u003cem\u003eTriticum aestivum\u003c/em\u003e with 18S-5.8S-26S rDNA and intergenic spacers [\u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e] was labeled by nick translation (Sigma Aldrich) using Cy5 fluorochrome (Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003eHybridization mixture containing 50% (v/v) formamide, 10% (w/v) dextran sulfate in 2 \u0026times; SSC and 10 ng/\u0026micro;l of labeled probes was added onto slide and denatured for 30s at 80\u0026deg;C, followed by overnight hybridization performed in a humid chamber at 37\u0026deg;C. If the chromosome structure was damaged after the denaturation step, the slides with chromosome spreads were post-fixed in 4% (v/v) formaldehyde in 2 \u0026times; saline-sodium citrate (SSC) for 10 min at room temperature, washed in 2 \u0026times; SSC for 2 \u0026times; 5 min, and dehydrated using ethanol series. The sites of digoxigenin- and biotin-labeled probes were detected using anti-digoxigenin-FITC (Sigma Aldrich/Roche Applied Science) and streptavidin-Cy3 (Thermo Fisher Scientific/Invitrogen, Carlsbad, CA, USA), respectively. Chromosomes were counterstained with DAPI and mounted in Vectashield Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopic and image analysis\u003c/h2\u003e \u003cp\u003eSlides were examined using Axio Imager Z.2 Zeiss microscope (Zeiss, Oberkochen, Germany) equipped with a Cool Cube 1 camera (Metasystems, Altlussheim, Germany) and appropriate optical filters, and a PC running ISIS software 5.4.7 (Metasystems). The final image adjustment was performed in Adobe Photoshop CS5, and idiograms and final pictures were created in Adobe Photoshop CS5 and GIMP (GNU Image Manipulation Program) v2.10.34. A minimum of ten preparations with mitotic metaphase chromosome spreads and different probe combinations were used for the final karyotype reconstruction of each genotype.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the results of this article are included within the article and its supplementary information files. Databases of Illumina reads are deposited in the Sequence Read Archive (project number: PRJNA1076467). Assembled contigs from different types of repetitive DNA elements are publicly available online (https://olomouc.ueb.cas.cz/en/content/dna-repeats). The sequences of newly identified tandem organized repeats were deposited in GenBank (accessions: PP457292\u0026ndash;PP457296).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by an internal grant from Palack\u0026yacute; University, Olomouc (IGA PrF-2024-001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.H. and L.K. conceived the project. K.V. provided plant material from the B481 population. L.K. performed flow cytometry and analyzed the results. J.Č., L.K. and E.H. provided and analyzed sequencing data. J.Č. and V.Z. performed the cytogenetic part of the work. L.K., J.Č. and E.H., wrote the original draft, and V.Z. and K.V. revised and edited the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Eva Jahnov\u0026aacute;, Radom\u0026iacute;ra Tu\u0026scaron;kov\u0026aacute;, and Petr Navr\u0026aacute;til for excellent technical assistance. The computing was supported by the e-INFRA CZ project (ID:90254), supported by the Ministry of Education, Youth and Sports of the Czech Republic, and the ELIXIR-CZ project (ID:90255), part of the international ELIXIR infrastructure.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChac\u0026oacute;n J, Luebert F, Hilger HH, Ovchinnikova S, Selvi F, Cecchi L, et al. The borage family (Boraginaceae s.s.): a revised infrafamilial classification based on new phylogenetic evidence, with emphasis on the placement of some enigmatic genera. Taxon 2016;65(3):523-546. doi:10.12705/653.6\u003c/li\u003e\n\u003cli\u003eMerxm\u0026uuml;ller H, Grau J. Dysploidie bei \u003cem\u003ePulmonaria\u003c/em\u003e. Rev Roum Biol-Botanique. 1969;14(1):57-63.\u003c/li\u003e\n\u003cli\u003eSauer W. Karyo-systematische Untersuchungen an der Gattung \u003cem\u003ePulmonaria \u003c/em\u003e(Boraginaceae). 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Nucleic Acids Res. 1979;7(7):1869-1885. doi:10.1093/nar/7.7.1869\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Comparative karyotyping, Genome size, Pulmonaria, Repeatome, Satellite DNA","lastPublishedDoi":"10.21203/rs.3.rs-4148849/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4148849/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe genus \u003cem\u003ePulmonaria\u003c/em\u003e (Boraginaceae) represents a taxonomically complex group of species in which morphological similarity contrasts with striking karyological variability. 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 \u003cem\u003eP. officinalis\u003c/em\u003e group, the most widespread species complex, which includes two morphologically similar species that differ in chromosome number, i.e. \u003cem\u003eP. obscura\u003c/em\u003e (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14) and \u003cem\u003eP. officinalis\u003c/em\u003e (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16). Ornamental cultivars, morphologically similar to \u003cem\u003eP. officinalis\u003c/em\u003e (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.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFlow cytometry confirmed a significant difference in genome size between \u003cem\u003eP. obscura\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e, corresponding to the number of chromosomes. Genome-wide repeatome analysis performed on partial Illumina sequencing 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 landmarks, represented by satellite DNAs, showed that the chromosome structure in \u003cem\u003eP. officinalis\u003c/em\u003e was more variable compared to that of \u003cem\u003eP. obscura\u003c/em\u003e. Comparative karyotyping strongly supported the hybrid origin of putative hybrids with 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15 collected from a mixed population of both species and outlined the origin of ornamental garden escapes, confirming their derivation from the \u003cem\u003eP. officinalis\u003c/em\u003e complex.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eLarge-scale genome size analysis and repeatome characterization of the two morphologically similar species of the \u003cem\u003eP. officinalis\u003c/em\u003e 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 \u003cem\u003eP. officinalis\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"First insight into the genomes of the Pulmonaria officinalis group (Boraginaceae) provided by repeatome analysis and comparative karyotyping","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-25 06:04:01","doi":"10.21203/rs.3.rs-4148849/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-03T09:50:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-28T22:13:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-26T20:32:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f3b38e3f-1d03-4b95-83a9-aebebf5866a7","date":"2024-04-15T09:15:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-11T15:09:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"385d27dc-ed6d-49d8-b1ba-2c1800f4b2ab","date":"2024-03-30T21:52:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4f48802c-61d0-470c-b40c-d6fe7cdde281","date":"2024-03-29T10:17:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-26T08:17:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-24T13:06:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-24T13:05:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-24T13:05:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-03-22T10:02:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"16b6c10c-9e58-473d-af4b-065b7f64ed95","owner":[],"postedDate":"March 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-16T16:00:23+00:00","versionOfRecord":{"articleIdentity":"rs-4148849","link":"https://doi.org/10.1186/s12870-024-05497-4","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2024-09-13 15:57:14","publishedOnDateReadable":"September 13th, 2024"},"versionCreatedAt":"2024-03-25 06:04:01","video":"","vorDoi":"10.1186/s12870-024-05497-4","vorDoiUrl":"https://doi.org/10.1186/s12870-024-05497-4","workflowStages":[]},"version":"v1","identity":"rs-4148849","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4148849","identity":"rs-4148849","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}