Introduction
The genus Populus (poplar) is a globally significant model system for woody plant biology, valued for its rapid growth, ecological importance and fully sequenced genome (Tuskan et al. Reference Tuskan, Difazio, Jansson, Bohlmann, Grigoriev, Hellsten, Putnam, Ralph, Rombauts, Salamov and Schein2006). Its clonal propagation makes it a cornerstone for commercial forestry and genetic improvement programs (Kong et al. Reference Kong, Ma, Du and Zhang2024). The success of these programs fundamentally relies on identifying superior clones with traits like high yield and stress resilience, a process contingent upon a deep understanding of existing genetic diversity (Hedrick Reference Hedrick2004; Espahbodi et al. Reference Espahbodi, Mirzaie- Nodoushan, Tabari and Akbarinia2006).
While the strategic importance of genetic diversity is well-established, a critical bottleneck remains in its efficient translation into breeding outcomes. Traditional phenotypic selection is slow, and commonly used molecular markers (e.g., SSRs (Simple Sequence Repeats), SNPs (Single Nucleotide Polymorphisms)) may not directly capture fundamental aspects of genomic architecture crucial for long-term clonal stability, such as chromosomal integrity and ploidy consistency (Schubert and Lysak Reference Schubert and Lysak2011). This represents a significant research gap: a lack of direct, cytogenetically based markers that can predict genomic stability and breeding potential at an early stage. In general, chromosomal differences reflect disparities in genetic resources, whereas morphological, physiological and chemical variations indicate differences in the products of gene expression, which can be modified by environmental influences.
Chromosomal and cytogenetic investigations play a vital role in understanding genetic diversity (Knight Reference Knight2009). Cytogenetic and karyotypic analysis provides this foundational layer of genomic information, offering precise data on chromosome number, structure and behaviour that is essential for phylogenetic, taxonomy and understanding hybrid viability (Badr and El-Shazly, Reference Badr and El-Shazly2021; Hesamzadeh Hejazi and Ziaei-Nasab Reference Hesamzadeh Hejazi and Ziaei-Nasab2010, Reference Hesamzadeh Hejazi and Ziaei-Nasab2024). Previous cytogenetic studies in Populus have been invaluable but are often limited in scope-focusing on single species, specific hybrids or lacking integration of detailed karyometric data with ploidy analysis across a broad germplasm panel (e.g., Kim et al. Reference Kim, Kwon, Kang, Yoem, Kim, Kim and Kang2020; Liu et al. Reference Liu, Wang, Tao, Liu, Qu and Dou2021). Although the genus Populus is recognized as a genetic-woody model, a major knowledge gap exists in understanding the quantitative relationship between karyotypic structure, genomic stability and ploidy levels. Most previous studies have focused on morphological assessments or diversity at the molecular marker level, while the simultaneous and quantitative investigation of advanced cytogenetic indices alongside ploidy in a broad, multi-species germplasm collection has been less frequently conducted.
To address this gap, the primary novelty of this study lies in its comprehensive, comparative cytogenetic framework. We move beyond basic characterization to perform an integrated analysis of both numerical (ploidy) and structural (karyotype symmetry, chromosome size) chromosomal stability across a diverse collection of 40 clones from 10 species. This approach allows us to quantify core genomic stability traits and propose them as practical, microscopy-based proxies for clonal robustness.
The specific objectives of this research were to: (1) determine the chromosome base number, ploidy levels and assess karyotypic diversity among the studied clones; (2) establish detailed idiograms and quantify intra-chromosomal asymmetry; (3) analyse relationships between clones using multivariate statistical methods (principal component analysis [PCA] and cluster analysis) based on cytogenetic parameters; (4) identify clones exhibiting exceptional karyotypic stability, which may serve as valuable candidates for breeding programs and for understanding genomic adaptation within the genus; (5) quantitatively estimate and compare chromosomal volume and nuclear DNA content implications between diploid and triploid clones. By linking cytogenetic profiles to potential phenotypic performance, this work aims to bridge the gap between raw genetic diversity and actionable selection criteria for poplar improvement.
Materials and methods
Plant material and germplasm
The germplasm under investigation consisted of 40 clones from 10 different poplar species (Table 1). Following a study of their distribution, cuttings were obtained from the Karaj research station Poplar Collection and cultivated in the research greenhouse of the Biotechnology Department at the Research Institute of Forests and Rangelands (RIFR), Iran. Rooted cuttings were maintained in wind-blown sand under humid conditions at 24°C prior to cytogenetic analysis.
Specifications of 40 poplar clones from 10 species under investigation
Table 1 Long description
This table presents specifications of 40 poplar clones from 10 different species, including their clone names and genders. Notably, P. euramericana (canadensis) and P. nigra species have multiple clones, with a mix of male and female genders. P. euramericana (canadensis) clones are predominantly female, while P. nigra clones are mostly male. Other species like P. alba and P. × unknown show a balanced gender distribution. The table highlights the diversity in clone names and gender distribution across species, which may be relevant for studies on genetic variation and breeding programs. The data does not indicate any specific trends or patterns beyond the gender distribution.
Cytogenetic preparation and slide preparation
To observe metaphase plates, the following procedures were carried out: pretreatment (0.5% saturated α-bromonaphthalene at 4°C for 2 hours), fixation (10% formaldehyde and 1% chromic acid (1:1) for a minimum of 16 hours at 24°C, Levitsky’s solution), hydrolysis was carried out with 1N NaOH at 60°C for 8–10 minutes, stained with haematoxylin-iron for 4–5 hours and finally squashed in a droplet of 45% glacial acetic acid and lactic acid (10:1 ratio) (Hesamzadeh Hejazi and Ziaei-Nasab Reference Hesamzadeh Hejazi and Ziaei-Nasab2010).
Karyotyping and chromosome measurement
Photomicrography of clear metaphase plates was performed using an Olympus BX-40 light microscope with a magnification of 1980×. Karyotypes were prepared using Adobe Photoshop CS version 8.0 software, and chromosome measurements were conducted using MicroMeasure version 3.3 software. A minimum of five well-spread metaphase cells (replicates) were analysed for each clone.
Karyotypic parameters and indices
The following parameters were measured or calculated for each chromosome: long arm length (LA), short arm length (SA), long arm relative length percentage (%LA), short arm relative length percentage (%SA), total chromosome length (TL) [LA + SA], arm ratio (AR) [LA/SA], centromeric index (CI) [SA/(LA + SA)], total form percentage (%TF) [(ΣSA/ΣTL) × 100] (Huziwara Reference Huziwara1962); difference of relative length (DRL) [MaxRL% − MinRL%] (Levan et al. Reference Levan, Fredga and Andberg1964); intra-chromosomal asymmetry index (A1) [1 − Σ(SA/LA)/n] and inter-chromosomal asymmetry index (A2) [Sd/
$\bar{\text{X}}$] (Romero Zarco Reference Romero Zarco1986); value of relative chromatin (VRC) [ΣTL/n] (Levan et al. Reference Levan, Fredga and Andberg1964); dispersion index (DI) (Lavania and Srivastava Reference Lavania and Srivastava1992) and Karyotype formulas and Stebbins’ symmetry classes (SC) (Stebbins Reference Stebbins1971) were determined based on centromere position (Levan et al. Reference Levan, Fredga and Andberg1964).
Estimation of chromosomal and genomic volume
Chromosomal volume (V_chr) was estimated for representative diploid and triploid clones to quantitatively assess genomic size differences. The volume of an average metaphase chromosome was approximated using the cylinder volume formula V = πr 2h, where h is the mean chromosome length (µm) and r is the mean chromatid radius (0.4 µm) where metaphase chromosome diameter typically ranges 0.8–1.0 µm (Schubert and Lysak Reference Schubert and Lysak2011). Total metaphase chromosomal volume per cell was calculated as V_total = V_chr × n, where n is the somatic chromosome number. These volumetric estimates serve as a proxy for comparative nuclear DNA content and genome size between ploidy levels (Greilhuber Reference Greilhuber2005).
Statistical analysis
Analysis of variance (ANOVA) was performed on normalized data, and parameter means were compared using Duncan’s test. PCA was conducted to evaluate the contribution of each karyotypic parameter to the ordination of clones. Clustering was performed using the average method following the calculation of the cophenetic correlation coefficient (r) to examine karyotypic similarity among clones. All numerical analyses were performed using SAS version 6.12 (1996), JMP version 3.1.2 (1995) and StatistiXL version 1.7 (Reference Statisti2007) software.
Results
Ploidy levels and basic karyotypic features
Mitotic metaphase plates and corresponding karyotypes for all investigated clones are presented in Fig. 1, with detailed karyotypic characteristics provided in Table 2. All studied clones shared a base chromosome number of x = 19, with both diploid and triploid ploidy levels observed. With the exception of two triploid clones (2n = 3x = 57), P. euramericana (canadensis) var. triplo: (7) and P. euramericana (canadensis) var. 45/51: 10(17), all other clones exhibited a diploid ploidy level (2n = 2x = 38).
Metaphase images of the 40 examined clones along with their karyotypes. Scale 10 µm (
).
Figure 1 Long description
The image displays metaphase images of 40 examined clones, each accompanied by its karyotype. The clones are organized in a grid format, with each row showing a set of metaphase plates and corresponding karyotypes. The scale is indicated as 10 micrometers. Each clone is labeled with its name and identifier, such as P. canadensis var. vernirubensis 1 left parenthesis 16 right parenthesis, P. canadensis var. I-214 2 left parenthesis 36 right parenthesis and so on. The chromosome arrangements are clearly visible, with both diploid and triploid ploidy levels observed. The karyotypes are presented in a linear format below each metaphase image, showing the specific chromosome count and arrangement for each clone. The image provides a detailed visual representation of the genetic characteristics of the studied clones, highlighting variations in chromosome number and structure across different varieties.
Karyotype characteristics of 40 clones of different Populus species along with classification of karyotypic traits using Duncan’s method at the 0.05 probability level
Table 2 Long description
This table presents karyotype characteristics of 40 clones from various Populus species, focusing on somatic chromosome number, total chromosome length, and arm lengths. Key metrics include arm ratio, centromeric index, and asymmetry indices, which are used to classify karyotypic traits. Notable findings include variations in chromosome number, with P. eura,triplo having 57 chromosomes, and differences in symmetry classes, ranging from 1A to 3B. The clones exhibit diverse karyotype formulas, indicating a mix of metacentric, submetacentric, and subtelocentric chromosomes. Comparisons reveal trends in chromosome length and asymmetry, with implications for species classification and genetic diversity. Values sharing the same letters in columns belong to the same group, suggesting statistical significance in trait classification.
Notes: 2n: Somatic chromosome number; TL: total chromosome length; LA: long arm length; SA: short arm length; AR: arm ratio; CI: centromeric index; %LA: long arm relative length percentage; % SA: short arm relative length percentage; %TF: total form percentage; DRL: difference of relative length; VRC: value of relative chromatin; A1: intra-chromosome asymmetry index; A2: inter-chromosome asymmetry index; DI: dispersion index; SC: symmetry classes of Stebbins and K.F: karyotype formula (m: metacentric, sm: submetacentric, st: subtelocentric). P. eura: P. euramericana (Canadensis). In each column, values sharing the same letters belong to the same group.
Spectrum of karyotypic symmetry and chromosome size
Regarding intra-chromosomal symmetry, the clone P. euramericana (canadensis) var. vernirubensis: 1(16) possessed the most symmetric karyotype, characterized by the highest value of the %TF parameter (43.80) and the lowest value of the A1 parameter (0.21). The presence of the highest number of metacentric chromosomes in the karyotype formula of this clone (18m + 1sm) and its classification within Stebbins’ class 1A further corroborate this finding. In contrast, the clone P. nigra var. 63/135: 20(5) exhibited the most asymmetric karyotype, indicated by the lowest %TF value (23.81) and the highest A1 value (0.63). The complete absence of metacentric chromosomes in its chromosomal formula (13sm + 6st) confirms this observation. Concerning inter-chromosomal symmetry, the clone P. euramericana (canadensis) var. costanzo: 3(18), with the highest values for the DRL (4.93) and A2 (0.20) parameters, and the clone P. nigra var. betolifolia: 21(32), with the lowest values for DRL (2.27) and A2 (0.11), possessed the most asymmetric and most symmetric karyotypes, respectively. In terms of chromosome size, it is noteworthy that the clones P. euramericana (canadensis) var. vernirubensis: 1(16) (VRC = 1.55) and P. caspica: (35) (VRC = 1.04) possessed the largest and smallest chromosomes, respectively.
ANOVA and diversity of karyotypic parameters
Based on the results of the ANOVA, significant differences (p ≤ 0.01) among the studied clones for all measured karyotypic traits, with the exception of DRL, A2 and DI (Table 3). This indicates substantial karyotypic diversity among the investigated clones.
Mean squares from the analysis of variance (ANOVA) of karyotypic traits in the studied clones
Table 3 Long description
The table presents ANOVA mean squares for karyotypic traits in clones, highlighting significant differences in traits such as Leaf Area (LA), Stem Area (SA), Total Length (TL), Aspect Ratio (AR), and Chromosome Index (CI), all at the 1% significance level. The percentage of Total Form (TF) is also significantly different among clones. However, traits like Distal Recombination Length (DRL), A2, and Dispersion Index (DI) show non-significant differences. The error variance is relatively low for most traits, indicating reliable measurements. The coefficient of variation (%C.V) ranges from 9.7% to 23.74%, suggesting moderate variability in the data. These results suggest that while some karyotypic traits vary significantly among clones, others do not, which could impact genetic studies and breeding programs.
Note:
** and ⁿˢ indicate significance at the 1% level and non-significance, respectively.
Multivariate analysis of karyotypic variation
To determine the importance of each karyotypic trait in the diversity among clones, a PCA was performed (Table 4). Based on this, the first four principal components accounted for 54.66%, 20.20%, 13.33% and 7.05% of the total variance among clones, respectively. Collectively, these four components explained 95.25% of the total diversity observed among the studied clones. The values of the eigenvector coefficients in the first component indicated that the traits SA, AR, CI, %TF and intra-chromosomal asymmetry index (A1) had the highest contributions to the formation of this component. This component can be termed the intra-chromosomal symmetry component, as it groups clones based on their evolutionary status and intra-chromosomal symmetry. In the second component, the traits TL and LA contributed the most to the variance among clones. This component can thus be identified as the chromosome length component, as it differentiates clones based on the size of their chromosomes. The traits DRL, inter-chromosomal asymmetry index (A2) and DI were predominantly associated with the third component. Therefore, this component can be designated as the inter-chromosomal asymmetry component. The ploidy level trait loaded exclusively onto the fourth component, indicating that this component plays a significant role in distinguishing clones based on their ploidy level. A scatter plot of the clones based on the first two principal components and the third and fourth principal component is presented in Fig. 2. Based on this figure, two clones P. euramericana triplo (7) and P. euramericana 45(51) (10/17) are completely separated from the rest of the clones’ duo to triploidy. Based on the scatter plot of clones according to the first two principal components, it appears that parameters of intra-chromosomal symmetry caused the distinction of these clones from others, as they possessed the highest %TF and lowest A1 values, indicating greater intra-chromosomal symmetry compared to other clones. It is noteworthy that the observed intra-specific diversity among these clones is also attributed to chromosomal length parameters. The two clones P. euramericana (canadensis) var. triplo: (7) and P. euramericana (canadensis) var. 45/51: 10(17) were separated and placed in distinct classes due to their difference in ploidy level compared to other clones of P. euramericana (canadensis). All three clones belonging to the species P. deltoides were grouped into a single class due to their similar chromosome size and intra-chromosomal symmetry indices. The various clones of P. alba were placed in separate classes due to their high karyotypic diversity. Inter-chromosomal symmetry indices appear to have played a significant role in this differentiation.
Scatter plot of the clones based on the (first and second) and (third and fourth) principal components derived from principal component analysis (PCA).
Figure 2 Long description
The left scatter plot displays clones based on Pin1 and Pin2 axes, with values ranging from negative five to positive seven on the Pin1 axis and negative three to positive three on the Pin2 axis. Clones are marked with numbers, such as 1, 2, 3 and so on, with some clusters and isolated points visible. The right scatter plot shows clones based on Pin3 and Pin4 axes, with values ranging from negative four to positive three on the Pin3 axis and negative one to positive one on the Pin4 axis. Clones are similarly marked with numbers, including 10(17), 7 and others, with some clusters and isolated points. Below the plots, a list of clone labels is provided, numbered from 1 to 40, with names like P.eura.vernirubensis:1(16), P. eura, I-214:2(36) and others, indicating different species or varieties.
Percentage of variance and coefficients of the eigenvectors for the four principal components derived from principal component analysis (PCA)
Table 4 Long description
The table presents the coefficients of eigenvectors and the percentage of variance for four principal components from a PCA. The first component accounts for 54.66% of the variance, with CI (0.353) and %TF (0.342) having the highest positive coefficients. The second component explains 20.20% of the variance, with LA (0.547) and TL (0.475) as significant contributors. The third component accounts for 13.33% of the variance, with DRL (0.608) and A2 (0.581) having the highest coefficients. The fourth component explains 7.05% of the variance, with Ploidy (0.917) as the dominant factor. Cumulatively, the four components explain 95.25% of the total variance. The data suggests that the first component is the most significant in explaining variance, while the fourth is the least, with Ploidy being a unique contributor in the fourth component.
Quantitative genomic size comparison: Diploids vs. triploids
Based on precise karyometric measurements, the volume of individual chromosomes in diploid clones ranged from 0.52 to 0.81 µm3, whereas this range was narrower in triploid clones, spanning 0.56–0.65 µm3. Calculation of the total metaphase chromosomal volume per cell revealed a distinct increase in triploids. The total metaphase chromosome per cell was significantly greater in triploids (31.92–37.05 µm3) than in diploids (19.76–30.80 µm3). Notably, the minimum recorded volume in triploids exceeded the maximum volume observed in diploids. The ratio of the mean total volume of triploids to that of diploids was approximately 1.4, which is close to the theoretical ratio of 1.5 (based on chromosome number increase). This suggests a nearly proportional and additive increase in genomic content (Table 5). To quantitatively assess the genomic consequences of triploidy, the total haploid chromosome length (THL) and mean chromosome length were calculated for representative diploid and triploid clones. For instance, the mean chromosome length for the diploid clone P. caspica (36) (2n = 2x = 38) was 1.27 ± 0.2 µm (THL = 24.13 µm), while for the superior triploid clone P. canadensis 45/51: (7) (2n = 3x = 57), it was 1.27 ± 0.3 µm (THL = 24.13 µm) (Table 2). Consequently, the estimated DNA content per somatic cell scaled directly with chromosome number. The DNA content index for triploids was approximately 1.5 (i.e., 57/38), confirming a purely additive genome doubling event without significant changes in chromatin condensation or chromosome architecture. This precise additivity underscores the structural fidelity of the genome following polyploidization in these clones.
Estimation of chromosomal volume and nuclear DNA content in two selected clones from two ploidy levels
Table 5 Long description
The table compares chromosomal volume and nuclear DNA content between two clones, P. caspica (2x) and P. canadensis (3x), across different ploidy levels. P. canadensis (3x) exhibits a higher mean chromosomal volume (∼0.56–0.65 µm3) and total metaphase volume (∼31.92–37.05 µm3) compared to P. caspica (2x), which has a mean chromosomal volume of ∼0.52–0.81 µm3 and a total metaphase volume of ∼19.8–30.80 µm3. P. canadensis also has a greater total chromosome number (57) and a relative DNA content index ranging from ∼1.2 to 1.62, indicating a higher DNA content than P. caspica, which has 38 chromosomes and a baseline DNA content index of 1.0. These differences highlight the impact of ploidy level on chromosomal and DNA characteristics.
Cluster analysis and intra-specific diversity
Cluster analysis using the average method grouped the 40 clones into 19 distinct classes at a metric distance of 2.49 (cophenetic correlation coefficient r = 0.92) (Fig. 3). Accordingly, clones P. euramericana (canadensis) var. vernirubensis: 1(16), P. euramericana (canadensis) var. I-154: 4(3-37), P. euramericana (canadensis) var. 455: (9), P. euramericana (canadensis) var. 92/40: 8(22), P. euramericana (canadensis) var. triplo: (7) and P. euramericana (canadensis) var. 45/51: 10(17), all belonging to the same species (P. euramericana [canadensis]), were each placed in separate classes. The different clones of P. nigra were grouped into six distinct classes, indicating very high intra-specific diversity within this species. Inter-chromosomal symmetry parameters were effective in contributing to the diversity among clones of this species. The two clones (35) and (36) of P. caspica were grouped in the same class as clones from P. alba, P. nigra and P. euramericana (canadensis), suggesting chromosomal homology between P. caspica and these species. The two clones (34) and 30(6) of P. euphratica, despite belonging to the same species, were assigned to different classes, indicating intra-specific diversity within this species. Inter-chromosomal symmetry indices appear to have played an important role in this diversity, and the placement of these two clones in different classes in Stebbins’ two-way table confirms this. The clone P. tremula: (29) was grouped with clones from P. alba and P. nigra, indicating similarity in chromosomal characteristics among these species.
Dendrogram generated from cluster analysis (average) based on karyotypic traits and r = 0.92.
Figure 3 Long description
The dendrogram displays the hierarchical clustering of various plant clones based on karyotypic traits. The left side lists the names of the clones, such as P. euramericana, P. deltoides, P. alba, P. caspica, P. nigra and others, each followed by specific identifiers in parentheses. The branching structure on the right visually represents the relationships and distances between these clones, indicating how they are grouped into distinct classes. The vertical lines and horizontal branches illustrate the metric distances and cophenetic correlations among the clones, showing how closely related they are in terms of chromosomal characteristics.
Discussion
Extensive karyotypic diversity and its implications
The findings of this research confirm the existence of extensive karyotypic diversity at both intra- and interspecific levels within the genus Populus. Previous reports by researchers such as Gong et al. (Reference Gong, Bin, Ying, Lan, Zhong, Quan, Qin, Yang and Wang2005), Peto (Reference Peto1938) and Anamthawat-Jónsson and Sigurdsson (Reference Anamthawat-Jónsson and Sigurdsson2008) also support the presence of diploid and triploid ploidy levels and a base chromosome number of x = 19 in this genus. Asadi-Corom et al. (Reference Asadi-Corom, Mirzaie-Nodoushan and Calagari2025), in a karyological study of Populus euphratica populations in Iran, reported that chromosome sizes in the studied populations varied between 0.69 and 3.38 µm. Asadi-Corom et al. (Reference Asadi-Corom, Asadi and Mirzaie-Nodoushan2023), in an investigation of the evolutionary trend in the Caspian poplar (Populus caspica) using karyotype analysis, reported that the Mazandaran-konesi2 population, with a karyotype formula of 21m + 14sm + 3st and classification in Stebbins’ class 2B, exhibited the most asymmetric karyotype (considering both intra- and inter-chromosomal asymmetry). The cluster analysis results clearly demonstrate that genomic diversity is not confined to the interspecific level but is also highly significant intra-specifically. The dispersion of clones from the same species (e.g., P. euramericana (canadensis) and P. nigra) across different classes could stem from specific genomic adaptations of these clones to varying environmental conditions or different reproductive mechanisms. This level of diversity is highly valuable for breeding programs and the conservation of genetic resources. The clustering of P. caspica clones with clones from other species may indicate chromosomal homology and genetic affinity, potentially facilitating interspecific hybridization and the transfer of desirable genes. Furthermore, the karyotypic similarity between the P. tremula clone and some clones of P. alba and P. nigra might suggest a shared evolutionary history or the possibility of natural hybridization among these species. The separation of the two P. euphratica clones into different groups, which was associated with inter-chromosomal asymmetry indices, indicates that even within a single species, populations with varying degrees of karyotypic evolution can be found. This aligns with the findings of Asadi-Corom et al. (Reference Asadi-Corom, Asadi and Mirzaie-Nodoushan2023) on the presence of karyotypic asymmetry in different populations of a species.
Genomic additivity and structural stability in triploids: A key finding
The quantitative findings of this study provide a clear picture of structural stability in the superior triploid clones. First, the overlap in the range of individual chromosome volumes between ploidy levels, coupled with its narrower distribution in triploids, suggests greater uniformity and standardization in chromatin organization. This may indicate more efficient regulatory mechanisms for maintaining chromosomal structural integrity following genome duplication. Second, the increase in total chromosomal volume, which closely corresponds to the chromosome number ratio (∼1.5×), supports the concept of whole genome duplication (WGD) without major structural alterations.
This type of stable polyploidy, characterized by the preservation of near-theoretical volumetric ratios, reduces the risk of meiotic instability and genomic abnormalities. Consequently, the observed presumed or reported phenotypic superiority based on prior literature in these triploids can be attributed primarily to a stable gene dosage effect and enhanced cellular efficiency, rather than to complex chromosomal rearrangements.
This characteristic renders them ideal and predictable candidates for sustainable breeding programs, offering the benefits of polyploidy with minimized genetic risk. Beyond quantifying the chromosomal parameters, our analysis of genomic additivity leads to a significant insight regarding the mechanism and stability of polyploidy in these clones. A pivotal and novel finding of this study is the demonstration of strict genomic additivity in the analysed triploid poplar clones. Unlike some polyploid systems where chromosome elongation or restructuring accompanies ploidy increase (e.g., Leitch and Bennett Reference Leitch and Bennett2004), our karyometric data reveal that triploidy here results from what appears to be a precise WGD event, preserving the original diploid chromosome morphology. This maintenance of chromosomal dimensions suggests exceptionally high structural stability and balanced chromatin packaging post-duplication, a factor critical for mitotic and meiotic fidelity (Comai Reference Comai2005).
This finding has two significant implications. First, it indicates that the observed possible phenotypic vigour (e.g., rapid growth) in these triploids is likely driven primarily by a direct gene dosage effect and the inherent buffering capacity of polyploidy, rather than by concomitant changes in nuclear architecture or large-scale genomic rearrangements. Second, and more importantly for applied breeding, it reveals these clones as inherently stable genetic resources. The predictable 1.5-fold genomic increase, coupled with conserved karyotype symmetry, minimizes risks of meiotic irregularity and genomic shock often associated with nascent polyploids. Therefore, these triploids represent not merely plants with more DNA but rather stabilized genomic amplifiers of a proven diploid architecture. This makes them exceptionally valuable for clonal propagation and future hybridization, as their genetic behaviour is more predictable and their superior traits are underpinned by a robust and consistent genomic foundation. Our work thus shifts the selection paradigm from seeking polyploids per se to identifying those, like our candidates, that have achieved polyploidy without compromising karyotypic stability, a key factor for long-term commercial deployment.
Toward applied cytogenetic selection markers
By presenting a quantitative and analytical cytogenetic framework, this study not only describes existing diversity but also provides a solid scientific basis for the informed selection of possibly superior clones particularly triploids with high structural stability for sustainable breeding and rapid afforestation programs. Our findings emphasize the selection of clones that, in addition to higher ploidy, have also maintained their genomic efficiency and stability at the level of chromosomal structure. In conclusion, the results of this study demonstrate that karyotype analysis can serve as a powerful and quantitative tool for distinguishing and differentiating various poplar clones and species, investigating phylogenetic relationships and determining evolutionary trends within this important genus.
General conclusion
This comprehensive cytogenetic study on 40 clones from 10 species within the genus Populus revealed a remarkable degree of karyotypic diversity, underpinning the significant genetic variation present in this economically and ecologically important genus. The investigation confirmed a conserved base chromosome number of x = 19, with both diploid (2n = 38) and triploid (3n = 57) ploidy levels observed, consistent with previous scientific literature. A detailed analysis of karyotypic symmetry indices demonstrated a wide spectrum of chromosomal organization, from highly symmetric karyotypes, characterized by a predominance of metacentric chromosomes and high %TF values, to highly asymmetric ones with significant intra-chromosomal (A1) and inter-chromosomal (A2, DRL) variation. Statistical ANOVA confirmed that significant differences existed among clones for the vast majority of karyotypic parameters, firmly establishing the presence of substantial karyotypic diversity. Multivariate analyses provided profound insights into the patterns of this diversity. PCA successfully quantified the major sources of variation, attributing them to four distinct components: intra-chromosomal symmetry, chromosome size, inter-chromosomal symmetry and ploidy level. Furthermore, average cluster analysis distinctly classified the 40 clones into 19 discrete groups. This clustering pattern was highly informative, revealing not only expected interspecific differentiation but also pronounced intraspecific variation. The fact that different clones from the same species (e.g., P. euramericana (canadensis), P. nigra, P. euphratica) were frequently separated into distinct classes highlights the extensive genomic plasticity and adaptive potential within these species. Conversely, the grouping of clones from different species (e.g., P. caspica with P. alba, P. nigra and P. euramericana (canadensis); P. tremula with P. alba and P. nigra) suggests underlying chromosomal homologies and potential phylogenetic affinities, which could be leveraged in future hybridization and breeding programs. In essence, this research underscores the critical role of cytogenetic and karyotypic studies as powerful, quantitative tools for the precise diagnosis and differentiation of poplar clones and species. The findings provide a robust chromosomal framework that is essential for informing strategic conservation efforts, guiding advanced breeding initiatives for the selection of superior clones and elucidating evolutionary relationships within the genus Populus. The significant intraspecific diversity observed also emphasizes the importance of conserving individual clones, not just species, to maintain the full spectrum of genetic resources necessary for adaptation and resilience in the face of environmental change.
A key finding of our study is that the triploid clones exhibited a strictly additive genome size with no concomitant increase in individual chromosome length. This indicates that the triploid condition in these poplar clones likely arose through whole-genome duplication (e.g., unreduced gamete fusion) and is maintained with remarkable structural fidelity. The preservation of original chromosome dimensions suggests a high degree of genomic stability and balanced chromatin packaging, even after a major ploidy shift. This structural conservation is crucial, as aberrant chromosome condensation or elongation can disrupt gene regulation and meiotic pairing. Therefore, the possible phenotypic superiority observed in these triploids (e.g., rapid growth) may be primarily attributed to a simple ‘gene dosage’ effect and the buffering capacity of polyploidy, rather than to changes in nuclear architecture. This makes them particularly valuable for breeding they offer the benefits of polyploidy (vigour, stress tolerance) without the genomic instability often associated with karyotypic rearrangements. Their stable, predictable karyotype simplifies their use in further hybridization programs. The advancement of existing knowledge through this research occurs on several fronts:
1. Provision of New Quantitative Metrics for Stability: This study demonstrates that uniformity in chromosome volume (narrow range in triploids) coupled with a more symmetric karyotype can serve as a quantitative, cost-effective cellular marker for the initial screening of clones with high potential for genomic stability.
2. Elucidating the Mechanism of Triploid Superiority with a Focus on Stability: Beyond chromosome counting, our data indicate that triploidy in these clones has resulted in stable and additive genome duplication, whereby the basic chromosomal structure and DNA packaging efficiency are preserved. This structural stability may underlie possible phenotypic superiority (such as more rapid growth) through a stable gene dosage effect, without the common instabilities associated with nascent polyploids. This hypothesis generated from our data, which should be tested in future integrated phenotyping studies.
3. Discovery of Valuable Intra-Species Diversity at the Karyotype Level: Cluster analysis grouped clones from the same species into different karyotypic clusters. This important discovery shows that diversity in genomic structure (karyotype and ploidy) within a single species can be as valuable as diversity between species, opening a new path for selecting specific genotypes with desirable karyotypic profiles in breeding programs.
4. Our study underscores that detailed cytogenetic analysis is not a substitute for modern genomic sequencing or molecular marker techniques but serves as a fundamental and complementary framework. The integration of karyological data with high-throughput genomic, transcriptomic and phenomic information will be crucial for a systems-level understanding of genome structure and function in Populus and its application in breeding programs.
