Evaluation of ploidy level and endoreduplication in carnation (Dianthus spp.)

Plant Science 201–202 (2013) 1–11

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Evaluation of ploidy level and endoreduplication in carnation (Dianthus spp.) María Ángeles Agulló-Antón a , Enrique Olmos b , José Manuel Pérez-Pérez c , Manuel Acosta a,∗ a b c

Departamento de Biología Vegetal (Fisiología Vegetal), Universidad de Murcia, 30100 Murcia, Spain Departamento de Biología del Estrés y Patología Vegetal, CEBAS (CSIC), 30100 Murcia, Spain Instituto de Bioingeniería, Universidad Miguel Hernández, Elche 03202, Alicante, Spain

a r t i c l e

i n f o

Article history: Received 10 September 2012 Received in revised form 26 October 2012 Accepted 19 November 2012 Available online 28 November 2012 Keywords: Endoreduplication Mixoploidy Polyploidy C-value Flow-cytometry Organ size

a b s t r a c t Carnation (Dianthus caryophyllus L.) is one of the fifth most important ornamental species worldwide. Many desirable plant characteristics, such as big size, adaptation under stress, and intra or interspecific hybridization capability, are dependent on plant ploidy level. We optimized a quick flow cytometry method for DNA content determination in wild and cultivated carnation samples that allowed a systematic evaluation of ploidy levels in Dianthus species. The DNA content of different carnation cultivars and wild Dianthus species was determined using internal reference standards. The precise characterization of ploidy, endoreduplication and C-value of D. caryophyllus ‘Master’ makes it a suitable standard cultivar for ploidy level determination in other carnation cultivars. Mixoploidy was rigorously characterized in different regions of several organs from D. caryophyllus ‘Master’, which combined with a detailed morphological description suggested some distinctive developmental traits of this species. Both the number of endoreduplication cycles and the proportion of endopolyploid cells were highly variable in the petals among the cultivars studied, differently to the values found in leaves. Our results suggest a positive correlation between ploidy, cell size and petal size in cultivated carnation, which should be considered in breeding programs aimed to obtain new varieties with large flowers. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Cultivated carnation is one of the most important ornamental species worldwide [1]. High-quality commercial carnation cultivars are usually obtained by inter or intraspecific hybridizations involving Dianthus caryophyllus L. and other related species [2–4]. The success of such hybridizations depends on the ploidy levels of the parentals used [2,4]. The chromosome number in the genus Dianthus follows a simple ploidy series of basic chromosome set of x = 15, where different species may contain a different number of chromosome sets [5,6]. Many desirable plant characteristics, such as size and adaptation under stress, are also related to plant ploidy levels [7]. For many breeding companies, the ploidy of cultivars selected for crosses is unknown due to the technical equipment required and the lack of quick and standardized protocols for a systematic determination of ploidy levels in Dianthus species. Ploidy level determination is usually achieved by direct counting of chromosomes or by flow cytometry methods [8,9]. Karyotyping is a time consuming process that requires certain expertise in cytological techniques; and since a limited number of individuals can be studied at once, the systematic analysis of seedlings to detect the

∗ Corresponding author. Tel.: +34 868884940; fax: +34 868883963. E-mail address: [email protected] (M. Acosta). 0168-9452/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2012.11.006

presence of individuals with undesirable ploidy levels is a long and tedious method [10]. Besides, chromosome counting is difficult in some species, such as Dianthus spp., with abundant and small chromosomes [6,11]. On the contrary, flow cytometry methods, based on the detection of the fluorescence emitted by compounds that specifically bind to DNA, are cheaper, faster and very effective to estimate ploidy levels in plants [12,13]. Many plant organs contain mixoploid tissues in which cell populations with different ploidy levels coexist [14]. These cells are the result of somatic polyploidization processes, such as the endoreduplication cycle or endocycle in which cells increase their ploidy through repeated rounds of DNA replication that are not followed by cytokinesis [15]. Endoreduplication is an effective strategy to regulate plant organ growth [16–18]. Cell size in plants is positively correlated with their nuclear DNA content [19,20] and the transition from cell proliferation to cell differentiation is usually accompanied by a genetic switch from the mitotic cycle to the endocycle [17,21]. Additionally, recent investigations indicate an active role of endoreduplication in maintaining the differentiation status of specialized leaf cells [22]. Given that, the characterization of mixoploidy in different tissues is a simple approach to describe cellular patterns in the context of organ growth and development. To the best of our knowledge, studies that correlate desirable breeding characteristics with ploidy or endopolyploidy levels in cultivated carnation are lacking.

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Table 1 DNA content and ploidy estimation in wild and cultivated Dianthus spp. Species

Cultivar

D. caryophyllus

‘Arcos’

D. caryophyllus

‘Pilar’

D. caryophyllus

‘Master’

D. caryophyllus

‘Roble’

D. caryophyllus

‘Reina’

D. subbaeticus

–

D. hispanicus

–

D. anticarius subsp. saorinii

–

D. broteri

–

Co-ordinates ◦





◦





B&B 37 34 50 N, 1 46 35 W (395 m) B&B 37◦ 34 50 N, 1◦ 46 35 W (395 m) B&B 37◦ 34 50 N, 1◦ 46 35 W (395 m) B&B 37◦ 34 50 N, 1◦ 46 35 W (395 m) B&B 37◦ 34 50 N, 1◦ 46 35 W (395 m) Collado de la Magra, Moratalla, Murcia, Spain 38◦ 15 17 N, 2◦ 1 53 W (700 m) Sierra de la Pila, Abarán, Murcia, Spain 38◦ 18 1 N, 1◦ 17 19 W (650 m) Sierra de Almenara, Águilas, Murcia, Spain 37◦ 30 35 N, 1◦ 37 19 W (510 m) Sierra de la Pila, Abarán, Murcia, Spain 38◦ 18 1 N, 1◦ 17 19 W (650 m)

2C DNA content (pg)a

DNA ploidy levelb

Standard

1.37 ± 0.06

2x (670 ± 29)

S. lycopersicum ‘Stupické’

1.38 ± 0.02

2x (673 ± 11)

S. lycopersicum ‘Stupické’

1.40 ± 0.02

2x (685 ± 11)

S. lycopersicum ‘Stupické’

2.58 ± 0.02

4x (1259 ± 11)

S. lycopersicum ‘Stupické’

2.60 ± 0.01

4x (1269 ± 6)

S. lycopersicum ‘Stupické’

1.66 ± 0.09

2x (813 ± 46)

Glycine max ‘Polanka’ and Raphanus sativus ‘Saxa’

1.72 ± 0.14

2x (842 ± 67)

Glycine max ‘Polanka’ and Raphanus sativus ‘Saxa’

1.75 ± 0.11

2x (854 ± 54)

Glycine max ‘Polanka’ and Raphanus sativus ‘Saxa’

4.72 ± 0.00

6x (2305 ± 1)

S. lycopersicum ‘Stupické’

B&B: Barberet & Blanc (Puerto Lumbreras, Murcia, Spain). a Values are mean ± standard deviation (n = 4 samples). b Estimated genome size (1C) ± standard deviation, in Mbp, is shown between parenthesis.

Therefore, a procedure based on flow cytometry was optimized for ploidy analysis in carnation. First, the DNA contents of different carnation cultivars and wild species from the genus Dianthus were determined using internal reference standards and were compared with previously determined karyotypes [11] to establish a suitable carnation standard (D. caryophyllus ‘Master’) for high-throughput analysis of DNA content in this genus. Next, samples from 30 different regions taken from leaves, stems and flowers from the ‘Master’ standard carnation were analyzed by flow cytometry. The degree of endoreduplication found in the different tissues analyzed (i.e., mixoploidy), combined with the morphological description of their cellular architecture, allowed us the identification of substantial developmental attributes representative of this species. Finally, DNA ploidy levels were measured in leaves and flowers from seven representative carnation cultivars in order to find associations between these ploidy features and morphological traits of commercial interest. Our results suggest a positive correlation between DNA content, cell size and petal size in cultivated carnation, which should be considered in breeding programs aimed to obtain new varieties with large flowers.

analysis. In all cases, ploidy measurements were determined in the following two days after sample excision from the mother plants. Seeds from four wild Dianthus species (Dianthus anticarius subsp. saorinii Sánchez-Gómez et al.; Dianthus broteri Boiss. & Reut.; Dianthus subbaeticus Fern. Casas and Dianthus hispanicus Asso) (Table 1) were obtained from the germplasm collection of the “Dirección General de Patrimonio Natural y Biodiversidad” (Murcia, Spain). Solanum lycopersicum ‘Stupické’ (2C = 1.96 pg), Glycine max ‘Polanka’ (2C = 2.50 pg) and Raphanus sativus ‘Saxa’ (2C = 1.11 pg) were a generous gift of Dr. Dolezel (Czech Republic) and were used as DNA content calibration standards [12]. Surface-sterilized seeds from wild Dianthus and the DNA content calibration standard species were sowed on Murashige and Skoog agar medium (MS medium) supplemented with 2% sucrose and grown for two weeks at 25 ± 2 ◦ C, 50% RH and 14:10 h (light:dark) photoperiod with an average photosynthetic photon flux density (PPFD) of 100 ␮mol m−2 s−1 until the second pair of true leaves became visible (>5 mm).

2. Materials and methods

Representative samples from 30 different zones of cuttings and flowers from D. caryophyllus ‘Master’ were studied. Leaf samples were collected from the five youngest leaf pairs of the cuttings, representing juvenile (first-node) to mature (fifth-node) leaves (Fig. 2A–E). For flower analysis, sepals and representative petals located in three concentric regions of the corolla were collected. For each organ studied (leaf, sepal and petal), three regions were identified along its proximodistal axis: proximal, central and distal regions (Figs. 2 and 3). Leaf epidermis and parenchyma were collected separately in the central region of mature leaves. Epidermis, cortex and pith tissue were manually excised and collected separately from transverse sections of the basal stem of the cuttings. In addition, samples taken from the proximal region of mature leaves of cuttings and from the central region of petals at the outer corolla were studied for the seven cultivated varieties. Samples from juvenile leaves of wild Dianthus species and calibration standard species were also collected.

2.1. Plant material Cultivated varieties from D. caryophyllus L. belonged to the mother plant collection of Barberet & Blanc (Puerto Lumbreras, Murcia, Spain), and were grown in a glasshouse under environmental conditions at 37◦ 34 50 N, 1◦ 46 35 W and 395 m of height. Disease-free certified cuttings from five standard (large single flower per stem: ‘Hugo’, ‘Master’, ‘Pilar’, ‘Reina’ and ‘Roble’) and two spray (several flowers per stem; ‘Arcos’ and ‘Suprema’) varieties were harvested from their mother plants, wrapped in plastic bags to keep them moist and stored on a cold chamber at 5 ± 2 ◦ C, 60% relative humidity (RH) and complete darkness until they were used for the measurements. Fresh cut flowers from these seven cultivars were collected from mother plants, and kept watered at the same conditions described above until their

2.2. Sample collection

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Table 2 Nuclei ploidy levels in different tissues of D. caryophyllus ‘Master’. Organ

Region

Nuclei (%) 2C

4C

16C

32C

First-node leaves

Distal Proximal

63.57 ± 3.09 a 61.41 ± 0.93 A

32.22 ± 3.55 a 32.27 ± 1.21 A

3.56 ± 0.40 a 4.69 ± 0.26 A*

0.58 ± 0.16 a 1.30 ± 0.17 A*

0.07 ± 0.05 a 0.32 ± 0.03 A*

Fifth-node leaves

Distal Proximal

41.54 ± 5.01 b 32.40 ± 1.48 B*

48.41 ± 4.51 b 51.38 ± 4.29 B

8.44 ± 0.63 b 15.99 ± 3.82 B*

1.55 ± 0.39 b 0.22 ± 0.09 B*

0.06 ± 0.05 a 0.00 ± 0.01 B

Petals

Distal Proximal

27.34 ± 4.27 18.70 ± 1.63*

64.78 ± 2.46 34.51 ± 3.15*

7.34 ± 1.99 41.65 ± 2.81*

0.49 ± 0.15 4.84 ± 0.86*

0.04 ± 0.02 0.29 ± 0.07*

Sepals

Distal Proximal

29.02 ± 1.65 29.13 ± 3.72

42.71 ± 3.41 43.88 ± 4.97

24.18 ± 1.15 24.84 ± 5.80

3.93 ± 1.18 2.08 ± 0.16*

0.16 ± 0.15 0.07 ± 0.07

8C

Values are mean ± standard deviation of a minimum of 4 samples. Different letters indicate significant differences (p < 0.05) between leaf pairs for the specified leaf zone. Nuclear DNA content distribution was obtained as indicated in Section 2. * Significant differences (p < 0.05) between proximal and distal zone.

2.3. Nuclei isolation and flow-cytometry analysis Nuclei were prepared from freshly harvested tissues with a DNA staining kit (Partec CyStain UV Precise P, Partec GmbH, Germany) according to manufacturer’s instructions. Briefly, 25 mm2 of plant material (about 20 mg fresh weight) were chopped with a razor blade in 400 ␮l cold nuclear isolation buffer (solution A of the kit). After a short incubation (∼3 min), the cell suspension was filtered through a 50-␮m mesh CellTrics filter (Partec), and stained with 1600 ␮l of staining solution containing the 4 ,6 -diamidino2-phenylindole (DAPI) fluorescent dye (solution B of the kit). The nuclear DNA content distribution was then analyzed with a Partec PAS (Partec) flow cytometer equipped with a UV lamp for DAPI excitation (365 nm) and fluorescence detection at 450 nm. Data were processed using the WinMDI 2.9 software (Joseph Trotter). Ten thousand nuclei counts were performed per sample. Our fast ploidy determination procedure was then validated by measuring some tissues from D. caryophyllus ‘Master’ using a different nuclei staining method and flow cytometer (Table 2). For this purpose, samples from leaves, petals and sepals were excised and chopped with a razor blade in 500 ␮l of cold nuclear isolation buffer [23]. The cell suspension was then filtered over 30 ␮m nylon mesh, treated with RNase A (200 ␮g mL−1 ) and stained with propidium iodide (50 ␮g mL−1 ) during 10 min. The nuclear DNA content distribution was then analyzed with a FACS Canto II flow cytometer and the data obtained were processed using the FACSDiva 6.1.2 software (BD Biosciences, Franklin Lakes, NJ, USA). A minimum of three biological replicates including 10,000 nuclei counts each were performed per sample. 2.4. DNA content estimation DNA content (C-value) of five carnation cultivars and four wild Dianthus species (Table 1) was determined by flow cytometry using internal calibration standards as recommended by Bennett et al. [24]. In each case, similar weights of young leaf tissues from a Dianthus spp. problem sample and the appropriate standard were mixed and nuclei were isolated, stained and analyzed as described above. C-values were calculated according to Dolezel et al. [12], as follows: 2C DNA (pg) = (mean of the problem sample G1 peak × 2C DNA content of the standard (pg))/mean of the standard G1 peak. At least four biological replicates were measured per sample. 2.5. Study of leaf epidermis The adaxial (upper) and abaxial (lower) epidermises from different longitudinal regions (proximal, central and distal) of mature leaves were manually isolated using a scalpel and tweezers.

Isolated epidermal tissues were placed on a glass slide and covered after the addition of a few drops of 0.1 M sodium phosphate buffer, pH 7.2. Preparations were observed immediately using a bright-field Leica DMR microscope (Leica Microsystems, Wetzlar, Germany). The selected images were captured with a video camera and digitized using the computer software Leica IM1000 1.20 (Leica Microsystems AG, Heerbrugg, Switzerland). 2.6. Scanning electron microscopy Fresh petal and mature leaf samples were directly observed using a JEOL JSM-6360 LV (JEOL, Peabody; Massachusetts, USA, Inc.) scanning electron microscope operated at 20 kV and high vacuum. Five mm-long fragments from the central region of mature leaves were frozen in liquid N2 and freeze-dried before being mounted and coated with gold. Samples were studied using a JEOL JSM6100 scanning electron microscope operated at 15 kV as previously described [25]. 2.7. Morphometric analysis Photographs for the study of organ morphology were captured using an Olympus digital camera. The area of mature leaves of the cuttings and petals from the outer region of the corolla was measured from digital images captured with a scanner and analyzed using the software ImageJ 1.43u (Waine Rasband, National Institutes of Health, USA), in at least 20 samples per cultivar. Corolla diameter was determined using a digital caliper in a minimum of 5 flowers per cultivar. Scanning electron microscopy images from petals and leaf epidermises were used to quantify cell areas and interstomatal distances, respectively, as follows. Images from distal and proximal regions of each organ were used to obtain diagrams by drawing cell limits or lines between adjacent stomata on a Wacom Bamboo Pen tablet (Wacom Company Ltd., Tokyo, Japan) and using the software Adobe Photoshop CS3 Extended (Adobe Systems Incorporated, San José, CA, USA). A minimum of 40 cell areas and interstomatal distances were measured from the resulting diagrams using the software ImageJ 1.43u. 2.8. Light microscopy Two mm-long fragments from the central region of mature leaves and 5 mm-long segments of the basal stem were sectioned from ‘Master’ carnation cuttings. Samples were fixed at room temperature in 4% (v/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 2.5 h. The samples were washed in the same buffer three times for 15 min and transferred to 1% (v/v) osmium tetroxide in 0.1 M sodium phosphate buffer (pH 7.2) for 2 h. After postfixation, samples were

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washed three times for 15 min and were kept at 4 ◦ C in the same buffer overnight. Fixed samples were progressively dehydrated at room temperature using a graded ethanol series (35, 50, 75, 96 and 100% (v/v) ethanol; 30 min each). Dehydrated samples were transferred consecutively to propylene oxide (30 min), propylene oxide:Spurr resin (1:1) (1.5 h) and Spurr resin, where they remained overnight at 4 ◦ C. Samples were then transferred to rubber molds filled with Spurr resin and polymerized for 24 h in an oven at 68 ◦ C. Cross-semithin sections (0.5–0.7 ␮m) were prepared using a Leica EM UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany) and stained with 0.5% (w/v) toluidine blue for optical microscopy observation using a bright-field Leica DMR microscope (Leica Microsystems, Wetzlar, Germany). The selected images were captured with a video camera and digitized using the computer software Leica IM1000 1.20 (Leica Microsystems AG, Heerbrugg, Switzerland).

were also estimated. Resulting values ranged from 670 Mbp in D. caryophyllus ‘Arcos’ to 2305 Mbp in D. broteri (Table 1). A small intraspecific variation was observed for the haploid DNA content of the D. caryophyllus cultivars studied, and the haploid genome sizes of wild Dianthus spp. were always higher than in the cultivated carnation varieties (Table 1). For a quick determination of ploidy in different cultivars of a single species, Dolezel et al. [12] suggested the use of an external standard from the same species. Our results about the DNA content of several carnation varieties (Table 1) provide a battery of potential standards for routine measurements. From the studied cultivars, ‘Master’ was selected as the most suitable external standard cultivar for ploidy determination in cultivated carnation due to its commercial availability and wide use in breeding programs. 3.2. Morphology of cultivated carnation leaves

2.9. Statistical analysis Means and standard deviations (SD) were calculated. Statistical analysis of the data was performed using the Statistica 8.0 software (StatSoft, Inc., Tulsa, OK, USA). The differences between the data groups were analyzed by t test (p ≤ 0.05) when only two groups were compared. In the other cases, data were subjected to analysis of variance (ANOVA) and the mean values were compared by a Newman–Keuls test (p ≤ 0.05). Non-parametric tests were used when necessary. In that case median was represented instead of mean. The differences between the data groups were analyzed by Mann–Whitney U test (p ≤ 0.05) when only two groups were compared. In the other cases, data were subjected to Kruskal–Wallis test (p ≤ 0.05). Correlations were studied using Spearman’s rank correlation coefficient (R). 3. Results 3.1. DNA content and ploidy level in carnation cultivars and wild Dianthus species Using a quick and reliable flow cytometry protocol (see Section 2), DNA content was determined in five D. caryophyllus cultivars and four wild Dianthus species (Table 1). Suitable internal calibration standards were selected for each problem sample as to avoid the overlapping of G1 peaks for problem and standard nuclei (Table 1). The mean DNA content of cells at G1 phase in the distal region of first-node (youngest) leaves of D. caryophyllus cuttings ranged from 1.37 pg in ‘Arcos’ to 2.60 pg in ‘Reina’ (Table 1). Results from previous studies in the Dianthus genus, in which karyotypes and DNA content were independently assessed [11], were used to deduce the ploidy level of the studied cultivars. Whereas ‘Arcos’, ‘Master’ and ‘Pilar’ showed DNA content close to the diploid range described for D. broteri (1.70–1.99 pg) [11], ‘Reina’ and ‘Roble’ doubled the DNA content of the other studied cultivars (Table 1), indicating that these two cultivars are likely tetraploid. DNA content was also determined in the youngest leaves of four wild carnation species (Dianthus spp.) grown from in vitro-germinated seeds in a controlled environment (Table 1). The mean DNA content of cells at G1 phase for the wild species ranged from 1.66 in D. subbaeticus to 4.72 in D. broteri. Comparing these results with those of previous studies [11] we propose that, except for D. broteri, whose DNA content of cells at G1 phase seems to be close to those of the hexaploid populations described by [11] for the same species (5–5.61 pg), all the other wild Dianthus spp. populations studied were diploid (Table 1). Based on the C-values obtained (pg) and the known genome sizes (Mbp) of the internal standard species used [12], the haploid genome sizes of the Dianthus cultivars and the wild species studied

Leaves in cuttings of cultivated carnation ‘Master’ are arranged in pairs in an opposite and decussate manner along a developmental series, where younger (first-node) leaves are located apically on the stem and mature leaves are toward the stem base (Figs. 1A and 2A–E). Carnation leaves are linear with a triangular blade, distally ending on a sword-shape tip, and that lack a recognizable petiole (Fig. 1A–C), as the leaf blade is directly attached to the stem by a proximal sheath-like tissue (Fig. 1B). Carnation leaves display parallel venation since primary and secondary veins are mostly aligned to the proximodistal axis of the leaf, with only a few interconnections through higher order veins (Fig. 1D). We studied cell morphology at both leaf epidermises (adaxial and abaxial) by means of light (Fig. 1E and F) and scanning electron (Supplementary Fig. 1) microscopy. We found similar stomatal density, at the proximal region of mature (fifth-node) leaves, for both the adaxial (23 ± 5 stomata per square mm) and the abaxial (24 ± 2 stomata per square mm) epidermises. In the carnation epidermis, stomata are normally arranged in linear files in which they alternate with pavement cells, with a regular spacing of an average of two pavement cells between adjacent stomata (Fig. 1E and F and Supplementary Fig. 1). In this species, stomata (Fig. 1G) are of the diacytic type, characteristic of Caryophyllaceae, with guard cells oriented perpendicular to the large axis of the neighboring pavement cells. Each stomata consists of two symmetrical kidney-shape guard cells that delimit a pore, and two subsidiary cells of smaller sizes (Fig. 1H and I). Interestingly, pavement cells are rectangular-shaped and lack marginal lobes (Fig. 1E and F). Since adjacent stomata are regularly spaced on each cell column at the leaf epidermis, we determined the distance between adjacent stomata in different regions of mature leaves as an indirect estimation of cell size (see Section 2). We found that interstomatal distances were significantly higher in the proximal region of leaves (277.3 ± 131.4 ␮m) as compared with the interstomatal distances at their distal region (114.1 ± 34.3 ␮m). The smaller epidermal cell size found in the distal region of mature leaves suggest that cell maturation progress acropetally in carnation, from the base to the tip of the leaf. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.plantsci.2012.11.006. Consistent with the glabrous nature of carnation leaves, no trichome cells were found in their epidermises (Supplementary Fig. 1). Transverse sections of mature leaves from the fifth-node allowed us to study their internal anatomy (Fig. 1H–K). A thick cuticle and the two epidermises delimit several layers of photosynthetic palisade mesophyll cells. The inner mesophyll do not show apparent morphological differences between the adaxial and the abaxial leaf domains, with the exception of the large and

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Fig. 1. Morphological description of D. caryophyllus stem cuttings and leaves. (A) A stem cutting of D. caryophyllus ‘Master’. (B) Longitudinal section of the apical region of the stem cutting that highlights a decussate leaf phyllotaxis (asterisks indicate the insertion points for three successive leaves). (C) Different regions identified along the proximodistal axis in the first-node (young, top) and fifth-node (mature, bottom) leaves. (D) Detailed micrograph of the venation pattern in the proximal region of a mature leaf. The white arrowheads indicate the interconnections between parallel veins. (E) Light micrograph of the adaxial epidermis in the center of a mature leaf. (F) Light micrograph of the abaxial epidermis in the center of a mature leaf. Black arrowhead marks the stomata precursors. (G) Scanning electron micrograph of a stomata complex. (H and I) Detail of adaxial transverse leaf sections showing the structure of (H) an open stomata and (I) a closed stomata. (J) Light micrograph of a cross-section of a carnation leaf showing the margin. Black arrowheads indicate stomata in the adaxial (Ad) epidermis; the asterisk indicates the non-photosynthetic internal mesophyll. (K) Magnification of a leaf vascular bundle at the midvein. (L) Light micrograph of a transverse section at the base of the stem. Ab: abaxial; ad: adaxial; am: apical meristem; bsc: bundle sheath cells; ca: cambium; co: cortex; ep: epidermis; gc: guard cell; in: internode; p: pore; pc: pavement cell; pl: phloem; pm: palisade mesophyll; SC: substomatal cavity; sc: subsidiary cell; xl: xylem. Photographs and micrographs captured as described in Section 2.

non-photosynthetic mesophyll cells that are located in the central region of the leaf (asterisk in Fig. 1J). Carnation leaves have collateral vascular bundles, with xylem vessels located in the adaxial region of the vascular bundle and phloem in the abaxial region, separated by the cambium (Fig. 1K). A variable number of bundle-sheath cells enclose the vascular tissues within the leaf (Fig. 1J and K). Some of the features described here for cultivated carnation leaves (presence of a sheath-like region, epidermal patterning and morphology, stomatal density, unifacial mesophyll, etc.) are characteristic and distinctive traits of monocotyledon leaves [26]. 3.3. Mixoploidy in cultivated carnation leaves Next, we aimed to characterize the mixoploidy extent in different organs and tissues of our standard D. caryophyllus ‘Master’. Leaves from the first-node (apical) to the fifth-node (basal) of the cutting (Fig. 2), representing young to mature stages of leaf development, respectively, were collected and individual samples selected from proximal, central and distal regions of these leaves were processed for the determination of their DNA content by flow cytometry (see Section 2). With this experimental design up to 13 leaf developmental zones where distinguished, that included temporal (from young to mature) and spatial (from base, proximal, to tip, distal) information about endoreduplication levels (Fig. 2A–E and Table 2).

In the central region of the youngest leaves analyzed (first-node leaves), most of the nuclei were 2C and 4C (97.4% of total nuclei) and only a low proportion of endoreduplicated nuclei >4C was found (2.6% of total nuclei) (Fig. 2A). Since the 4C nuclei population includes both mitotic diploid cells in the G2 /M phase (4C) and tetraploid endoreduplicated cells (4n), we assumed that nuclei with higher ploidy levels (>4C) arise only by endoreduplication (i.e., successive DNA replication cycles without intervening mitoses) [17]. For a given leaf, irrespectively of its age, the proportion of 2C nuclei increased over the proximal-to-distal axis of the leaf at the expense of the proportion of endopolyploid nuclei (Fig. 2A–E). Additional analyses using a different flow cytometer and DNA staining method (see Section 2) confirmed the observed trend of an increase in the proportion of 2C nuclei from the leaf base toward the leaf apex followed by a concomitant decrease in the proportion of 8C, 16C and 32C endopolyploid nuclei (Table 2). Additionally, we found that in carnation leaves, as previously reported for other plant species [19], the proportion of endoreduplicated cells positively increased with the age of the leaves. For example, in their proximal region (Fig. 2A–E and Table 2), mature (fifth-node) leaves contain higher proportion of nuclei >4C (16.21% of total nuclei) than younger (first-node) leaves (6.31% of total nuclei), and the proportion of endoreduplicated 8C nuclei varied up to 4-fold in the proximal region of mature (15.99% of total nuclei) versus the proximal region of young leaves (4.69% of total nuclei). To assess whether different tissues within a given leaf

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Fig. 2. Mixoploidy characterization of D. caryophyllus cuttings. Proportion (%) of nuclei with different DNA content in several tissues of carnation cuttings ‘Master’. Nuclei populations are colored as follows: 2C (brown), 4C (orange), 8C (light green) and 16C (dark green). Ploidy measurements were taken for the five younger consecutive leaves, (A)–(E), at three regions along their proximodistal axis: proximal (pr), central (ce) and distal (di). (F) Ploidy levels at the adaxial leaf epidermis and parenchyma were measured separately at the central region of mature (fifth-node) leaves. (G) Ploidy levels of epidermis, cortex and pith tissues were analyzed separately at the basal stem of the cutting. Median values are shown. See Section 2 for the determination of ploidy levels by flow cytometry.

display heterogeneity in the DNA content of their cells, the leaf epidermises were detached from the underlying parenchyma and the nuclei ploidy levels were quantified in the adaxial epidermis and the parenchyma independently (see Section 2). The adaxial epidermis isolated from the central region of mature (fifth-node) leaves (Fig. 2F), contained a higher proportion of endoreduplicated nuclei (35.2% of total nuclei were >4C) compared with those found in the parenchyma (3.0% of total nuclei were >4C), and even a low, but significant proportion of 16C nuclei (1.2%) were detected in the adaxial epidermis. The epidermis tissue of the leaves was also characterized by a 10-fold increase in the 8C nuclei population than the parenchyma and a small population of dividing (2C + 4C) nuclei (Fig. 2F).

3.4. Morphology and mixoploidy in cultivated carnation basal stems A transverse section of the basal stem of the cutting allowed us to study the internal anatomy of the stem, which is characterized from the outside to the inside by a single layer of epidermis, a cortex composed of many (n > 16) layers of parenchymal cells that surround the vasculature (phloem, cambium and xylem) and the internal pith tissue composed of several layers of large parenchymal cells (Fig. 1L) [27]. Nuclei ploidy levels were independently determined in different tissues that were isolated from the basal stem, such as the epidermis, the cortex and the pith (see Section 2) (Fig. 2G). Contrary to that found in leaves, the internal tissues of the

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Fig. 3. Mixoploidy characterization of D. caryophyllus flowers. (A) Representative image of a mature flower from D. caryophyllus ‘Master’. (B) Ploidy level determination in three regions along the proximodistal axis of sepals. (C–E) Ploidy level determination in petals collected from three concentric whorls of the corolla, representing young (inner whorl) to mature (outer whorl) developmental stages of petals. As in (B), the proportions of nuclei populations were studied in up to three different regions along the proximodistal axis of these petals. Median values are shown. See Fig. 2 legend for details.

stem (cortex and pith) contained a larger proportion of nuclei populations with ploidy levels >4C (16.3% for cortex and 65.6% for pith) than those in the stem epidermis (6.8% of total nuclei were >4C). Interestingly, in the pith region the 2C nuclei population was not detected and nuclei populations of 4C, 8C and 16C (one to three endoreduplication cycles) were found (Fig. 2G). In addition, the overall 2C + 4C nuclei population decreased from the outer (epidermis) to the inner (pith) tissues of the stem, results that are compatible with the hypothesis that cell proliferation in carnation cuttings is confined to the outer layers of the stem that contain the cambium, while the pith contains large parenchyma cells with a main function on storage and support. 3.5. Morphology and mixoploidy in cultivated carnation flowers In the flowers of cultivated carnation, the reproductive structures (pistil and stamens) are covered by several whorls of spirally arranged colorful petals (Fig. 3). In ‘Master’, the calix consists of five fused sepals that protect the proximal region of the petals and

the reproductive structures (Fig. 3). For the analysis of DNA content and mixoploidy extent in flower organs, we collected sepals from the calix and representative petals of the corolla at three different developmental stages (young to mature), which were isolated from three concentric whorls of the corolla (inner to outer). For each organ studied in flowers (sepals or petals), nuclei ploidy distribution was characterized along its proximodistal axis as in leaves: the proximal, the central and the distal region were analyzed (see Section 2). In the sepals, the proportion of 2C and 4C nuclei did not differ significantly along their proximodistal axis (Fig. 3A and Table 2). Petals were collected at three developmental stages (Fig. 3): old petals were collected from the outer whorl of the corolla, mature petals were collected from the medial whorls of the corolla, and young petals were collected from the inner whorl of the corolla. For a given petal, the proportion of endoreduplicated cells >4C significantly increased toward the base, reaching up to 39.5% of total nuclei >4C in the proximal region of the petals from the outer whorl (Fig. 3B). In addition, 16C nuclei populations were only detected in

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Fig. 4. Endopolyploidy and organ size in cultivated carnation. (A) Size of petals from the outer corolla in several commercial carnation cultivars. Median values from 20 samples are represented. Different letters indicate significant differences (p ≤ 0.05) between cultivars. (B) Representative mature petals from four different cultivars. (C) Proportion of nuclei with different DNA content in the central region of the outer petals of the corolla in different carnation cultivars. DNA amounts are expressed as multiples of the monoploid C-value of the diploid ‘Master’ (CM ), as follows: 2CM (brown), 4CM (orange), 8CM (light green) and 16CM (dark green). (D and E) Scanning electron micrographs of proximal and distal regions from petals in the outer corolla from (D) ‘Master’ and (E) ‘Roble’. (F) Proportion of nuclei with different DNA content in the proximal region of the fifth pair of leaves in carnation cuttings from different cultivars. DNA amounts expressed as multiples of the monoploid C-value of the diploid ‘Master’ as indicated above. (G) Size of leaves from the fifth pair (older) in several commercial carnation cultivars. See Section 2 for the determination of ploidy levels by flow cytometry. Median values are shown. Photographs and micrographs were captured as described in Section 2.

the proximal region of the studied petals, irrespectively of their age (Fig. 3). As for the leaves, mature petals from the outer whorls of the corolla always displayed higher proportion of endoreduplicated nuclei >4C than younger petals located in the inner whorl (Fig. 3), which is in agreement with the outer petals as being the oldest petals in the flower. We also confirmed these results with a different flow cytometer and DNA staining method (Table 2). Interestingly, the proportion of 4C nuclei, which is an indirect estimate for cell proliferation, was much higher in the distal region of the old petals than in any other tissue or region studied within the flower (Fig. 3 and Table 2). The study of scanning electron micrographs from the proximal and distal epidermises of mature petals in ‘Master’ (Fig. 4D and E and Table 4) revealed significant differences in the size of their pavement cells: large and elongated cells (1191.9 ± 336.3 ␮m2 ) were found at the proximal region of the petals, where the higher endopolyploidy values had been observed (Fig. 3 and Table 2), while smaller (397.0 ± 115.1 ␮m2 ) and isodiametric pavement cells were

located in the distal region of the petals, with lower proportion and levels of endoreduplication (Fig. 3 and Table 2). 3.6. Organ size, cell size and DNA ploidy level in cultivated carnation To assess whether the incidence of polyploidy and endoreduplication levels in cultivated carnation correlate with the size and morphology of different organs in this species, we quantified the corolla diameter, the area of old petals from the outer whorl, and the area of mature (fifth-node) leaves in the seven cultivars studied (see Section 2) (Fig. 4A and Table 3). We found that the corolla diameters and the area of the old petals were significantly and positively correlated (Spearman correlation coefficient  = 0.886; p < 0.05). Corolla diameters ranged between 41 mm in the spray ‘Arcos’ and 76 mm in the standard ‘Reina’, and the area of their old petals varied between 4.4 cm2 and 13.3 cm2 , respectively (Fig. 4A

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Table 3 Petal and leaf dimensions for different cultivars. Cultivar

Petal area (cm2 )

‘Suprema’ ‘Arcos’ ‘Master’ ‘Hugo’ ‘Pilar’ ‘Roble’ ‘Reina’

5.3 4.4 7.5 8.6 10.2 9.0 13.3

± ± ± ± ± ± ±

1.3 (5.3) a 0.4 (4.4) a 1.3 (7.6) ab 0.5 (8.5) bc 0.5 (10.3) cd 1.0 (9.0) bc 1.6 (13.5) d

Corolla diameter (cm) 4.2 4.1 6.5 6.5 7.1 7.2 7.6

± ± ± ± ± ± ±

0.7 (4.0) ab 0.1 (4.0) a 0.5 (6.6) ac 0.1 (6.5) ac 0.5 (7.3) c 0.3 (7.2) bc 0.3 (7.5) c

Leaf area (cm2 ) 6.4 6.0 9.5 5.4 5.4 5.7 5.2

± ± ± ± ± ± ±

1.6 (6.4) a 0.8 (6.1) a 1.4 (9.5) b 1.3 (5.7) a 1.4 (5.2) a 1.5 (5.4) a 1.3 (5.2) a

Cutting length (cm) 19.1 14.9 18.9 14.8 18.1 18.2 15.9

± ± ± ± ± ± ±

1.8 (18.9) a 0.7 (15.0) b 1.3 (18.8) a 1.3 (15.0) b 1.7 (18.3) a 1.2 (18.0) a 1.3 (15.9) b

Basal stem diameter (mm) 3.6 3.2 3.2 2.7 3.0 3.1 3.4

± ± ± ± ± ± ±

0.9 (3.0) a 0.6 (3.0) ab 0.41 (3.0) ab 0.6 (3.0) b 0.7 (3.0) ab 0.6 (3.0) ab 0.8 (3.0) a

Values are mean ± standard deviation (minimum n = 20 samples for cutting parameters, minimum n = 15 samples for petal area, minimum n = 4 samples for corolla diameter). Median values indicated between parentheses. Different letters indicate significant differences between cultivars for the specified parameter.

Table 4 Petal cell size for different cultivars. Cultivar

Cell area (␮m2 ) Distal zone of petal

‘Master’ ‘Hugo’ ‘Roble’ ‘Reina’

397.0 611.6 763.4 982.2

± ± ± ±

115.1 (383.5) a 256. 3 (555.7) b 282.4 (731.1) bc 238.2 (969.7) c

Proximal zone of petal 1191.9 1102.1 1637.8 2333.7

± ± ± ±

336.3 (1163.8) a* 302.3 (1124.6) a* 662.8 (1724.0) b* 735.9 (2174.9) b*

Values are mean ± standard deviation (minimum n = 40 samples taken from, at least, 2 images). Median values indicated between parentheses. Different letters indicate significant differences (p < 0.05) between cultivars for the specified petal zone. * Significant differences (p < 0.05) between proximal and distal zone.

and B and Table 3). Non-significant differences were found for the fifth-node leaf areas of the varieties studied (Fig. 4G and data not shown), except for ‘Master’ that displayed larger leaves (9.5 cm2 ). Besides, the DNA content and the extent of mixoploidy for these cultivars were determined in the central region of old petals from the outer whorl (Fig. 4C) and in the proximal region of mature (fifth-node) leaves (Fig. 4F), using ‘Master’ as an external standard with known DNA ploidy level (2CM ; see Section 2). The first nuclei populations identified in mature leaves and old petals for ‘Roble’ and ‘Reina’ were 4CM (Fig. 4C and F), suggesting that both cultivars are tetraploid (2C ∼ = 2.6 pg). In both tissues, nuclei populations from two endoreduplication levels were found: 4CM and 8CM for the diploid cultivars and 8CM and 16CM for the tetraploid cultivars. No significant differences were observed in the proportion of the nuclei populations identified in the proximal region of mature leaves among the seven cultivars studied (Fig. 4F), in agreement with the non-significant correlations between fifth-node leaf area and ploidy or mixoploidy extent in these tissues (data not shown). In the central region of the old petals, the proportions of the nuclei populations found differed significantly among the studied cultivars (Fig. 4C). The diploid cultivars ‘Hugo’ and ‘Pilar’ showed a higher proportion of endoreduplicated nuclei (8CM and 16CM ) and a concomitant reduction in the levels of 4CM nuclei than the other diploid cultivars (‘Suprema’, ‘Arcos’ and ‘Master’). For the two tetraploid cultivars studied, ‘Reina’ showed a higher proportion of endoreduplicated nuclei (37.0% of total nuclei were >8CM ) than ‘Roble’ (7.6% of total nuclei were >8CM ), the later with an increased proportion of 4CM nuclei instead (Fig. 4C). Our results indicate some degree of intraspecific variation among carnation cultivars for the proportion of endoreduplicated cells at the central region of outer petals, irrespectively of the ploidy level of the cultivar. To estimate the endoreduplication index in the outer petals for the different cultivars studied, the ratio between the proportion of cells with DNA content >4C (8C + 16C) and the proportion of cells with DNA content ≤4C (2C + 4C) was calculated. The endoreduplication index, estimated in this way, increased with the petal area both in the diploid standard cultivars (0.40, 0.79 and 1.03 for ‘Master’, ‘Hugo’ and ‘Pilar’, respectively) and in the tetraploid cultivars (0.08 and 0.59 for ‘Roble’ and ‘Reina’, respectively). In the two spray

cultivars analyzed, where no significant differences between them were observed for petal and corolla areas, their endoreduplication indexes were similar (0.48 for ‘Suprema’ and 0.49 for ‘Arcos’). Interestingly, a positive and significant correlation was found in diploid cultivars between the proportions of 16C nuclei and the areas of the old petals (Spearman correlation coefficient  = 0.894, p < 0.05). Also, tetraploid cultivars with high endoreduplication (‘Reina’) display the largest petals (and flowers). Additionally, cell areas were measured in mature petals from two diploid (‘Master’ and ‘Hugo’) and two tetraploid cultivars (‘Roble’ and ‘Reina’) (Table 4). Within the tetraploid cultivars, cell size at their proximal region was significantly higher in ‘Reina’ than in ‘Roble’, which correlates with the higher endoreduplication levels found in ‘Reina’. Our data suggest that, in cultivated carnation cultivars, flower size depends on the size of their constituent petals, which is primarily influenced by the proportion of the cells that undergo endoreduplication, and secondarily by the C-value of a given cultivar. 4. Discussion Carnation is, after rose, the most important species on the worldwide market of cut flowers, with a yearly sales volume of almost 200 million plants [1]. Commercial carnation cultivars are vegetatively propagated from stem cuttings containing 6–8 pairs of leaves. The rooted cuttings are transferred to hardening chambers before transplanting them to production fields. Breeding strategies to improve the quality of cultivated carnation need to consider a large number of characteristics from the cultivars chosen for production, such as rooting ability, suitability for long-term storage, disease resistances, flower morphology and vase life, among many others. The genetic complexity of some of these (polygenic) traits, makes breeding for desirable characteristics quite problematic [28]. Wild species are a reservoir for genetic variation, which is often used to transfer desirable traits to commercial cultivars from the same or related species [29]. For an optimal design of breeding programs in carnation, one should consider the ploidy levels of the cultivars and wild species being used and their genetic compatibilities [4,30]. We found that the distal region of the youngest leaves in carnation stem cuttings is the most suitable tissue for the highthroughput determination of ploidy levels by flow cytometry in this species, and we provided extensive data for the mixoploidy extent found in different tissues of D. caryophyllus ‘Master’, which can be used as a reference standard in carnation ploidy determination. In addition, we determined the C-value for five commercial carnation cultivars and four Dianthus species taken from the wild. The C-value that we found for D. caryophyllus L. was 0.7 pg, representing a haploid genome size of about 670 Mbp, which is in agreement with previous results [30–32]. The DNA content for the D. broteri population analyzed (4.72 pg) was consistent with those previously described for the hexaploid neighboring populations (5.00–5.61 pg) [11], which confirms the diversification scenario of D. broteri along its geographical ranged as previously proposed [33].

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We found that some of the features defining carnation leaves, such as a distinctive sheath and blade regions along the proximodistal axis, their parallel venation, a linear pattern of cell arrangement at the epidermis or their unifacial mesophyll, are a distinctive feature of monocot leaves [26,34,35]. Similar to that found in Arabidopsis [19,21], carnation leaves contained a mixture of cells with 2C to 16 nuclei (zero to three endocycles) and the proportion of endopolyploid nuclei increased with the developmental age of the leaves. Also, the larger proportion of endopolyploidy was found in the epidermal layer. Given that the increase of endopolyploidy within a particular tissue is positively correlated with the aging of their constituent cells [23], our results on the mixoploidy extent and the interstomatal distances in different regions along the proximodistal axis suggest that, in carnation leaves, contrary to most angiosperm plants [36,37], cell maturation progresses acropetally, so that the youngest tissues, with smaller cells, are located at the distal region of the leaves. In plants, systemic polyploidization directly influences organ size. Many crops, such as cotton, wheat, and strawberry, have highploidy (>2C) varieties, which produce larger flowers, grains, or fruits than those of their diploid counterparts [18]. Endoreduplication, which consist in the doubling of the nuclear genome in the absence of cell division, is a highly developmentally regulated process in both plants and animals [38]. Cell size is usually correlated with the level of endoploidy [19,39] hence it has been proposed that the developmental regulation of endoreduplication might contribute to set organ size in plants [18]. Whether cellular processes, such as cell division, cell expansion and endoreduplication, drive organ growth or whether organ growth controls cellular processes is still an open question [20,40,41]. Our ploidy analyses of carnation petals indicate that cells at the proximal region display increased endoreduplication levels, up to 32C (four endocycles), than those in the more distal regions where most of the cells (>90%) are 2C and 4C. Consistent with the latter being arrested in the G1/S phases of the cell cycle, epidermal cells at the distal part of the mature petals were small and homogeneous while cells toward the base of the petals were large and extremely elongated. In addition, younger petals contain less endoreduplicated cells than mature petals, and these differences are larger in the proximal region, suggesting that endoreduplication in the carnation petals is developmentally regulated, both in space and time. Our results are comparable to those previously found for petal development of cabbage [42]. We found similar differences in the sizes of the epidermal cells between the proximal and the distal regions on the tetraploid cultivars studied, which also shown larger cell size than those in the diploid cultivars studied. These and previous results [19,43] confirmed the positive correlation found between the size of cells and their DNA content during the development of plant organs. According to the nucleo-cytoplasmic volume ratio theory [44], endoreduplication might trigger organ growth through the positive regulation of cell expansion. In this sense, the proportional increase of the genome size, as that produced by endoreduplication, has been proposed to increase metabolic activity, rRNA synthesis and transcriptional activity [42], and as such cell growth [45,46]. However, the alternative hypothesis where endoreduplication is under the control of a surveillance mechanism of organ growth [20,40,41] has been recently confirmed in leaves by using a large dataset from 200 Arabidopsis genotypes, including both genetically modified lines and recombinant lines [47]. Despite a limited number of carnation cultivars in the study, our results suggest a positive correlation between the size of mature petals and their endoreduplication index, measured as the proportion of cells >4C. Indeed, among the tetraploid cultivars, petals in ‘Reina’ contained higher proportion of endoreduplicated cells than those in ‘Roble’, which were of smaller size too. Interestingly, petal size in the diploid ‘Hugo’ and the tetraploid ‘Roble’ was similar,

which might be due to the higher proportion of endoreduplication found in ‘Hugo’. However, the petal size differences between standard and spray diploid cultivars are likely attributed to differences in their production method. On the other hand, leaf size in the carnation cultivars studied was not significantly correlated neither with the ploidy nor with the endoreduplication levels of their constituent cells, suggesting that leaf size in this species is not directly dependent on the DNA content of their cells, as has been shown in Arabidopsis [47]. Indeed, the organismal theory proposed for plant organ growth [40] has been shown to operate in some vegetative organs but not in others [48]. Still, more research is needed to demonstrate if a similar surveillance mechanism operates in the growth of other laminar plant organs, such as petals, as well as the signals and molecular pathways involved in such mechanism. Since a positive and significant correlation was found between the size of the flowers and the size of the mature petals in all carnation cultivars studied, one way to obtain new carnation varieties with large flowers is by increasing their proportion of endoreduplicated cells, either through defined crosses with cultivars with high endoreduplication indexes or by experimentally increasing their endoreduplication index. Acknowledgements This work was supported by the “Ministerio de Ciencia e Innovación” (MICINN/FEDER) of Spain [grant numbers AGL200802472 and AGL2012-33610] and the “Consejería de Universidades, Empresa e Investigación” (Murcia, Spain) [CARM/PEPLAN S4 and S13-E005-06], with fellowships to M.A.A.-A. We are especially indebted to Dr J. Dolezel (Olomouc, Czech Republic) for the supply of standard plants (ploidy calibration); Drs E. Cano and G. Garrido (Barberet & Blanc, Puerto Lumbreras, Murcia) for cultivated plant material; F.J. Sánchez-Saorín and Dr P. Sánchez-Gómez for wild plant material and valuable comments. We thank to E. GonzálezGómez and A. Gómez-Martinez for help with the cytometers set-up and to R. Granja Camarasa for scanning electron microscopy. Dr Nieves Fernández-García (CEBAS-CSIC) gave her special support for optical microscopy. References [1] V.L. Sheela, Carnation, in: Flowers for Trade, New India Publishing, New Delhi, 2008, pp. 95–112. [2] M.K. Gatt, K.R.W. Hammett, K.R. Markham, B.G. Murray, Yellow pinks: interspecific hybridization between Dianthus plumarius and related species with yellow flowers, Sci. Hortic. 77 (1998) 207–218. [3] I. Andersson-Kottö, Interspecific crosses in the genus Dianthus, Genetica 13 (1931) 77–122. [4] M. Nimura, J. Kato, M. Mii, K. Ohishi, Cross-compatibility and the polyploidy of progenies in reciprocal backcrosses between diploid carnation (Dianthus caryophyllus L.) and its amphidiploid with Dianthus japonicus Thunb, Sci. Hortic. 115 (2008) 183–189. [5] H. Weiss, C. Dobe, G.M. Schneeweiss, J. Greimler, Occurrence of tetraploid and hexaploid cytotypes between and within populations in Dianthus sect. Plumaria (Caryophyllaceae), New Phytol. 156 (2002) 85–94. [6] R.C. Carolin, Cytological and hybridization studies in the genus Dianthus, New Phytol. 56 (1957) 81. [7] D.A. Levin, The Role of Chromosomal Change in Plant Evolution, Oxford University Press, New York, USA, 2002. [8] D.W. Galbraith, K.R. Harkins, J.M. Maddox, N.M. Ayres, D.P. Sharma, E. Firoozabady, Rapid flow cytometric analysis of the cell cycle in intact plant tissues, Science 220 (1983) 1049–1051. [9] A.M.M. de Laat, W. Gohde, M.J.D.C. Vogelzakg, Determination of ploidy of single plants and plant populations by flow cytometry, Plant Breed. 99 (1987) 303–307. [10] A. Kato, J.M. Vega, F. Han, J.C. Lamb, J.A. Birchler, Advances in plant chromosome identification and cytogenetic techniques, Curr. Opin. Plant Biol. 8 (2005) 148–154. [11] F. Balao, R. Casimiro-Soriguer, M. Talavera, J. Herrera, S. Talavera, Distribution and diversity of cytotypes in Dianthus broteri as evidenced by genome size variations, Ann. Bot. 104 (2009) 965–973. [12] J. Dolezel, J. Greilhuber, J. Suda, Estimation of nuclear DNA content in plants using flow cytometry, Nat. Protoc. 2 (2007) 2233–2244.

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