Abstract
Alteration of chromosomal dosage severely affects the expressions of cis and trans genes in human and fruit fly. However, its effect on gene expression in plants needs to be further explored. Here, we generated 84 aneuploid lines with extra 1 to 16 chromosomes by crossing a female diploid poplar and a male triploid poplar. The chromosome compositions of the aneuploid population were dissected by DNA sequencing and cytogenetics. An RNA-seq analysis showed that cis genes were affected by both dosage and inverse effects, while trans genes were mainly affected by inverse effect. When the chromosomal dosage in an aneuploid line exceeds a threshold, the inverse effect may exceed the dosage effect on cis genes, causing dosage overcompensation. Interestingly, we found that even the genes located on the same chromosome may be subjected to different types of effect. These findings provided an important theoretical basis for analyzing the gene expression from the perspective of chromosomal dosage level.
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Introduction
Aneuploidy refers to an individual that contain an abnormal number of chromosomes in somatic cells1. Genomic imbalance in aneuploids often causes detrimental effect on growth and development in animal2. However, plants are more tolerant to genomic imbalance than animals3, which is attributed to their relative redundant genome originated from the paleopolyploidy event4. For aneuploids, the changed chromosomal dosages often cause extensive genetic variations, which directly affected their phenotypes5. In Arabidopsis, the trisomy of chromosome 2 (Chr 2) reduces rosette leaf size, whereas in tomato, the additional Chr 2 increase fruit locule number and soluble sugar content by 15%2,6,7. Dosage alterations across multiple chromosomes increase phenotypic complexity relative to aneuploids with single chromosome changes8. The double trisomic Chr 1 and Chr 3 in Arabidopsis simultaneously reduce rosette leaf size and plant height2. Thus, generation of artificial aneuploid is considered as a novel way to increase species diversity at chromosome level in plants9.
Previous studies in aneuploid maize and Drosophila showed that genomic imbalance can influence the varied (cis genes) and unvaried region (trans genes) of genome jointly7,10,11. Aneuploids, including haploidy, trisomy and tetrasomy, show a wide range of distinct effects on cis and trans genes compared with diploid11. In general, the mRNA amounts produced by cis genes are proportional to the varied chromosomal dosage, known as dosage effect12. For trans genes located on the unvaried chromosomes, their expressions are inversely regulated by varied chromosomal dosage, known as inverse effect13,14. In other cases, although a few chromosomes in aneuploids have added copies, the mRNA amounts produced by cis genes on these chromosomes are similar to that of diploid level, known as dosage compensation15,16. Dosage compensation is attributed to the fact that the expressions of cis genes are also affected by inverse effect, which offsets the dosage effect15. The unique gene expression profiles observed in aneuploids can be interpreted as positive response, which is helpful to maintain their genomes within a tolerable range when faced with genomic imbalance17. However, it remains unclear whether the magnitude of dosage effects and inverse effects on cis genes and trans genes is influenced by the varied chromosomal dosages. Here, the term “varied chromosomal dosages” refers not only to the increase of a single chromosome but also to concurrent dosage alterations involving multiple chromosomes in one aneuploidy. Studies in aneuploid population reveal that tetrasomies have more differentially expressed genes (DEGs) than trisomies owing to large genomic imbalance in tetrasomy11,18. In poplar, chromosomes 1 to 19 exhibit substantial variations in chromosomal dosage. Alterations in the dosages of multiple chromosomes often lead to more intricate gene expression profiles, which in turn contribute to enhanced phenotypic diversity19.
Poplars are fast-growing tree species with significant economic value20. Populus trichocarpa has been used as a model species for tree biology research due to its relatively small genome size and ease of genetic manipulation21. The research of aneuploids in plants mainly focuses on Arabidopsis, maize and wheat7,18,22, while in perennial tree species, related research is still neglected. In this study, we obtained 84 aneuploid seedlings with different chromosome compositions by crossing a female diploid poplar and a male triploid poplar. We provided a comprehensive analysis of the expression of cis and trans genes in the aneuploid population. Benefit from the sufficient chromosome compositions (extra 1 to 16 chromosomes) in the aneuploid seedlings, we observed a significant negative correlation between chromosomal dosages and inverse effects on cis and trans genes. The expressions of cis genes in aneuploid lines with extra 12 or more chromosomes decreased below the diploid level. We defined this phenomenon as dosage overcompensation. These findings revealed not only common and unique effects on expressions of cis and trans genes but also relationship between inverse effect and chromosomal dosage.
Results
Generation of an aneuploid poplar population by crossing a diploid poplar with a triploid poplar
Populus alba ‘Berolinensis’ (2n = 3x = 57), a male triploid poplar widely cultivated in Northeast China21, was used as the male parent. We first confirmed of its ploidy level by fluorescence in situ hybridization (FISH) (Fig. 1A) and flow cytometry (FCM) (Fig. 1B), and then investigated if the triploid poplar can generate viable male gametophytes for fertilization. We observed a complete developmental process from microsporocyte to microspore cells in the triploid poplar (Fig. 1C). We also observed the process from microspore cells to male gametophytes (Fig. 1D). The branches of the triploid poplar were collected for hydroponic culture, and pollens were obtained after 14 days of the culture (Supplementary Fig. 1A–F). Although the shapes of the pollen were irregular, some of them were viable and pollen tubes germinated normally in vitro (Supplementary Fig. 1G–I). All these results indicated that the pollens of the triploid poplar were partially fertile.
A The chromosome composition of P. alba ‘Berolinensis’ by FISH. Blue bars represent chromosomes. The green dots represent the telomere probe at 494 nm. B Ploidy analysis of the triploid poplar by FCM. C Meiosis period of P. alba ‘Berolinensis’, including microsporocyte; leptotene; pachytene; metaphase I; anaphase I; prophase II; metaphase II; anaphase II; telophase II (tetrad). D Mitosis period of the microspore cell in the triploid poplar. Mid-single nucleus represents the period of middle single nucleus; Mar single nucleus represents the period of Marginal single nucleus; Germination represents the period of pollen tube germination.
Since P. alba ‘Berolinensis’ was reported to contain three subgenomes of Prunus davidiana, P. alba, and P. alba var. pyramidalis21, we selected a diploid poplar of P. davidiana as the female parent for the hybridization. The pollens from the triploid poplar were fertilized on the female inflorescence of the diploid poplar, and pollen tubes were observed under microscope after 24 h of pollination (Supplementary Fig. 1J). 5 weeks after pollination, a total of 214 seeds were obtained, 84 of which developed into seedlings, with a survival rate of 39.25%. It is interested that extensive genetic variations were observed in the seedlings, including plant heights, leaf colors and leaf shapes (Fig. 2A).
A Phenotypic of seedlings, including 1A, 12A, 50A, 54A, 59A, 60A, 74A, 86-1A, 110A and Y3A. B FCM of diploid, aneuploid and triploid 103A. X axis represent fluorescence intensities and Y axis represent the cell number of detected samples. C The box-plot summarizing the fluorescence intensities of 84 seedlings (n = 84 biologically independent samples). The box-plot spans the first through third quartile values, the line inside each box indicates the median, the solid black square designates the mean, and the whiskers mark the range of the fluorescence intensities. The error bars represent mean ± SD.
The cellular DNA contents, which can reflect the ploidy level23, were measured for the seedlings using FCM (Fig. 2B, C). We found that the fluorescence intensities of the majority of the aneuploid lines were higher than the diploid poplar, but lower than the triploid poplar. The fluorescence intensities of the diploid and triploid poplars were 6252 and 9337, respectively (Supplementary Table 1). While the fluorescence intensities of the seedlings ranged from 6676 to 9676, with a median of 7936.5 (Fig. 2C and Table S1). However, the fluorescence intensities of line 107A reached to 9676, which exceeded the cellular DNA contents of the triploid (Supplementary Table 1). These results indicated that the seedlings we obtained were aneuploid poplar.
Chromosomal compositions of the aneuploid poplar population
It is reported that hybridization between diploids and triploids can produce offspring with different ploidies, including diploids, aneuploids and triploids24. We sequenced the genomic DNA of the seedlings to further dissect their chromosomal compositions. For an euploid individual, the sequencing depths of different chromosomes are equal. While for an aneuploid individual, the sequencing depths of chromosomes with changed copy number will differ from the unchanged chromosomes (Fig. 3A). We calculated sequencing depths for chromosomes in each aneuploid individual and examined if the sequencing depths of the varied chromosomes were significantly different from the unvaried chromosomes by Z-scores (Supplementary Table 2). For example, in 63A, the sequencing depth of chromosome (Chr) 19 was 122 with a Z score of 38.83, while the sequencing depths of other chromosomes were 85 with a Z score of 1.02. In 68-1A, the sequencing depth of Chr 7 was 142 with a Z score of 63.11, while the sequencing depths of the other chromosomes were 94 with a Z score of 0.52. These results indicated that 63A and 68-1A were trisomic poplars with additional Chr 19 and Chr 7, respectively (Fig. 3B, C and Supplementary Fig. 1K). The chromosomal compositions of 63A and 68-1A were further verified by FISH using centromere probes that were specific to Chr 19 and Chr 7 (Fig. 3D and Supplementary Table 3).
A The profile of resequencing protocol. Blue bars represent chromosomes with unvaried copies, red and green bars represent chromosomes 1 and 19 with varied copies. The reference genome used the A genome of P. alba ‘Berolinensis’. B, C The boxplots of 63A and 68-1A, the X-axis represents the chromosomes, and the Y-axis represents the mode of sequencing depth. D The karyotype analysis of 63A and 68-1A by FISH. The green dots represent the telomere probe at 494 nm. The pink dots represent the fluorescence signals of centromere probes that were specific to Chr 19 and Chr 7 in poplar at 510 nm, respectively. Bar = 5 μm. E The aneuploid population showed a wide range of variation in chromosome number. F The probabilities of different chromosomes being allocated into aneuploid seedlings.
We found that most of the seedings were aneuploid plants with extra chromosomes. We defined their chromosome numbers as 2n+x, where x is between 1 and 18. We also identified three putative triploid seedlings (3A, 4A and 98-1A) that contained a complete set of extra chromosomes (Supplementary Table 2). We found that 86-1A and 107A may contain two copies of extra chromosomes. The aneuploid line 86-1A contained two additional copies of Chr 15 (Z = 7.97), while 107A contained two additional copies of Chr 6, Chr 8, Chr 9, Chr 11 and Chr 13 (Z scores > 3) (Supplementary Table 2). In contrast, 56A contained only one copy of Chr 19 (Z = −5.37). These results indicated that hybridization between diploid and triploid plants may generate tetrasomy and monosomy. Further analysis revealed that most of the aneuploid lines contained 44 to 52 chromosomes (2n+6 to 2n+14), with a peak of 50 chromosomes (2n+12=50) (Fig. 3E). The chromosomal compositions of all the 84 aneuploid seedlings are displayed in Fig. 4 and Supplementary Table 4. Statistical analysis on the chromosomal compositions of the aneuploid population showed that different chromosomes have various probabilities being allocated into aneuploid gametocytes. As shown in Fig. 3F, 62.82%, 61.54%, 60.26% and 60.26% of the aneuploid seedlings contained extra Chr 8, 1, 4 and 19, respectively. While less than 40% of the aneuploid seedlings contained extra chromosomes of Chr 3, 9, 11 and 16. Only 24.36% of the aneuploid seedlings contained an extra Chr 3.
The Fig. 4 displays the copy number of each chromosome across all 84 aneuploid lines. The rows represent chromosomes (Chr) 1 to 19, and the columns represent different aneuploid lines. Light yellow squares represent chromosomes with two copies, red square represents chromosomes with three copies, blue squares represent chromosomes with one copy, and green squares represent chromosomes with four copies.
We also used 45S and 5S ribosomal DNA (45S and 5S rDNA) probes to verify the chromosomal compositions of the aneuploid population. 45S and 5S rDNA are located on Chr 14 and Chr 17 of the section Leuce in Populus, respectively25. The FISH results showed that these probes generated two and three chromosome-specific signals in the diploid and triploid poplars, respectively (Supplementary Fig. 2). Telomere probes and 4′,6-diamidino-2-phenylindole (DAPI) were used to indicate the chromosomes. Six aneuploid lines with different chromosome compositions were selected for the FISH confirmation. The aneuploid lines 110A and 40A contained three copies of Chr 14, and three fluorescence signals from 45S rDNA were observed. While 70-1A and 103A contained three copies of Chr 17. As expected, three fluorescence signals from 5S rDNA were observed in this two aneuploid lines. The aneuploid line 66-1A contained three copies of both Chr 14 and Chr 17, and three fluorescence signals from both 45S rDNA and 5S rDNA were observed (Supplementary Fig. 2). In contrast, the copy numbers of Chr 14 and Chr 17 were not changed in Y3A, and two fluorescence signals from each probe of 45S rDNA and 5S rDNA were detected (Supplementary Fig. 2).
The effect of genomic imbalance on the expression of cis genes
The genes located on varied chromosomes were defined as cis genes, and the genes located on unvaried chromosomes were defined as trans genes11. An RNA-seq analysis was performed on 69 lines to explore the impact of genomic imbalance on gene expression in the aneuploid population. We first calculated the total expression of genes on each chromosome, and then calculated the ratios of each aneuploid line to the diploid poplar. We found that genomic imbalance generally led to a dosage effect on cis genes in the aneuploid population (Fig. 5 and Supplementary Table 5). The relative expression levels of cis genes were generally higher than the diploid level (1.0), which was indicated by red dotted lines in Fig. 5. In a trisomy, 68-1A, which contained an extra copy of Chr 7, the relative expression of cis genes was increased to 1.53 (P < 0.05) compared to the diploid level (Fig. 5A and Supplementary Table 6). We identified 248 differentially expressed cis genes (cis DEGs) in 68-1A compared to the diploid poplar and found that 211 (85.1%) of them were up-regulated (Supplementary Table 7). The aneuploid line 81A contains three copies of Chr 13 and Chr 16, and the relative expression levels of genes on the two chromosomes were increased to 1.46 and 1.36 (P < 0.05), respectively (Fig. 5B). We identified 528 cis DEGs in 81A, 395 (74.8%) of which were up-regulated (Supplementary Table 7). The aneuploid line 50A contains three copies of Chr 1, 3, 7, 8, 13, 15 and 18, and genes located on these chromosomes were increased to 1.3, 1.37, 1.37, 1.3, 1.35, 1.35 and 1.36 (P < 0.05), respectively (Fig. 5C). A total of 2221 cis DEGs were identified in 50A, and 67.9% of them were upregulated.
A–I The X-axis represent chromosome 1 to 19, the Y-axis represent the ratio of aneuploid with extra chromosomes to diploid. Orange dots represent chromosomes with normal (two) copies, while green dots represent chromosomes with varied (three) copies. Compared with diploid level (red dotted line, with value of 1.0), the dosage effect was referred to expression of cis genes were higher than the diploid level, while the dosage compensation was referred to expression of cis genes were similar or lower than the diploid level. The inverse effect was referred to expression of trans genes were lower than the diploid level, while the positive inverse effect was referred to expression of trans genes were higher than the diploid level.
Previous studies reported that inverse effects on cis genes were also widespread in aneuploid population causing a phenomenon known as dosage compensation7,10. In this study, we found that the mRNA abundances of the cis genes were not proportional to the chromosomal dosages in the aneuploid population (Supplementary Tables 5 and 6), implying that the dosage effects may be offset by the inverse effects to a certain degree (dosage compensaion). The aneuploid line 39A contained three copies of Chr 1, 4, 5, 6, 7, 8, 10, 11, 13, 14, 15 and 19. The relative expressions of cis genes on these chromosomes exceeded the diploid level (Fig. 5D). However, only the expressions of the cis genes on Chr 6, 7, 8 and 10 were significantly altered (P < 0.05) (Supplementary Table 7). The aneuploid line 34A contained 12 chromosomes with three copies, and the expressions of the cis genes on six chromosomes were significantly altered (Fig. 5E). Similarly, the aneuploid line 66-1A contained 12 triplicate chromosomes and the expressions of the cis genes on these chromosomes were higher than diploid level but lower than triploid level (Fig. 5F), Only the expressions of the cis genes on five chromosomes (Chr 2, 10, 12, 15 and 17) were significantly altered (Supplementary Table 6). However, the proportion of the upregulated cis DEGs located on these five chromosomes were similar to the other seven varied chromosomes (Supplementary Table 7). The above results indicated that the expressions of the cis genes in the aneuploid population may be regulated by both dosage effect and inverse effect, ranged from dosage compensation to dosage effect.
The effect of genomic imbalance on the expression of trans gene
For trans genes located on unvaried chromosomes, their expressions were inversely regulated by increasing chromosomal dosage10,11. In this study, three effects on trans genes were observed in the aneuploid seedlings, including significant or non-significant inverse effect and positive inverse-effect (Supplementary Tables 5 and 6). As shown in Fig. 5G, 66–2A contained 13 unvaried chromosomes, the expressions of the trans genes located on 12 out of the 13 chromosomes were lower than the diploid level except for the genes located on the Chr 1, which were maintained at the diploid level (expression value was 1.01, P > 0.05). The expressions of the trans genes on Chr 5, 6, 13 and 14 in 66-2A were 0.80, 0.81, 0.94 and 0.81 (P < 0.05), respectively, while the expressions of trans genes on Chr 2, 4, 10, 12, 16, 17, 18 and 19 were non-significantly decreased to the diploid level (P > 0.05). There were 656 differentially expressed trans genes (trans DEGs) on Chr 1, of which 239 (36.4%) were upregulated, and 417 (63.5%) were downregulated (Supplementary Table 7). The proportions of trans DEGs located on remaining unvaried chromosomes were similar (Supplementary Table 7).
As shown in Fig. 5H, 12A contained 12 unvaried chromosomes and the trans genes located on these chromosomes displayed decreased expression levels. The expressions of trans genes on unvaried Chr 3, 4, 5, 6, 7, 9, 10, 11, 13, 14, 15 and 16 of 12A dropped to 0.86, 0.90, 0.81, 0.77, 0.86, 0.83, 0.72, 0.81, 0.87, 0.76, 0.83, and 0.75 (P < 0.05), respectively (Fig. 5H). We identified 2627 trans DEGs in 12A, and 60.4% of them were down-regulated (Supplementary Table 7). Similar trends were also observed in a majority of trans genes located on unvaried chromosomes in the aneuploid population (Supplementary Tables 6 and 7). In contrast to inverse effect, we found that the expressions of some trans genes in aneuploid lines were elevated. For example, the expressions of trans genes on three out of the 16 unvaried chromosomes (including Chr 12, 15 and 18) in 74A were higher than the diploid level. The relative expressions of trans genes located on Chr 12, 15 and 18 were 1.28, 1.21 and 1.15 (P < 0.05), respectively (Fig. 5I and Supplementary Table 6). A total of 817 trans DEGs were identified on these three chromosomes, of which 433 (52.9%) were down-regulated, respectively (Supplementary Table 7). These results showed that the expressions of trans genes may be also positively regulated in the aneuploid population.
The genome wide effect of genomic imbalance on the expressions of genes
To investigate the genome wide effect of genomic imbalance on the expressions of genes, we calculated the Pearson correlation coefficients (r) between the mRNA abundances of each DEG with the chromosomal dosages (Supplementary Tables 9 and 10). Based on the values of the correlations, we classified the genes into four groups: genes with predominant dosage effect (r > 0.5), genes with partial dosage compensation effect (0.3 ≤ r ≤ 0.5), genes with dosage compensation effect (0 ≤ r ≤ 0.3), and genes with dosage overcompensation effect (r < 0). The results revealed that the genes subjected to different effects were stochastically distributed on a chromosome to a certain degree.
In the aneuploid population, 74.51% of the DEGs were subjected to inverse effect and exhibited dosage compensation or partial compensation, especially for Chr 2. A total of 23.61% and 68.43% of the DEGs on Chr 2 were subjected to partial dosage compensation effect and dosage compensation effect, respectively. We found that ~17.81% of the DEGs were subjected to dosage overcompensation effect. On Chr 7, 25.8% of the DEGs were subjected to dosage overcompensation effect. While none of the DEGs on Chr 2 were negativity related to the chromosomal dosages. Approximately 9.23% of the DEGs were subjected to dosage effect. We observed that 20.3% of the DEGs on Chr 7 were subjected to dosage effect, whereas only 1.36% of the DEGs on Chr 19 were subjected to dosage effect. These results indicated that although different types of differential gene expression were distributed on a chromosome, some genes on specific chromosomes show expression sensitive to dosage effects, overcompensation and dosage overcompensation.
Larger genomic imbalance resulted in greater inverse effects on both cis and trans genes
Previous reports indicated that greater inverse effects on cis and trans genes were observed in tetrasomy than in trisomy, implying that the magnitude of inverse effects may be correlated to the chromosomal dosage of aneuploid genome11,16. To examine whether larger chromosomal dosages produce greater inverse effects in the aneuploid population, we further calculated the expression levels of cis and trans genes in aneuploid lines with extra chromosomes ranged from 1 to 16. With the increasing of chromosomal dosage, the expressions of cis genes were closed to dosage compensation (1.0), while the expressions of trans genes were decreased to 0.5 (Fig. 6). Significant negative correlations between chromosomal dosages and inverse effects on cis gene (r = −0.974) and trans gene (r = −0.967) were found as the number of varied chromosomes increasing (Fig. 6). The relative expression of cis genes in aneuploid lines with extra 1 to 16 chromosomes were gradually decreased from 1.527 to 1.038 compared to the diploid (Fig. 6A), while the relative expression of trans genes in these lines were gradually decreased from 0.999 to 0.591 (Fig. 6B). The aneuploid lines containing extra 16 and 15 chromosomes exhibited the largest inverse effects on cis and trans genes compared to the diploid, respectively (Supplementary Tables 11 and 12). In addition, more cis and trans genes were downregulated in the aneuploid population with increasing number of varied chromosomes (Fig. 6 and Supplementary Table 7). The proportion of up-regulated cis genes in aneuploid groups containing extra 1 to 16 chromosomes were gradually decreased from 80.4% to 53.2% (Fig. 6C), while the proportion of downregulated trans genes in these groups were gradually increased from 51.5% to 72.3% (Fig. 6D). These results showed that the aneuploid lines with greater chromosomal dosages may produce greater inverse effects on both cis and trans genes.
A The relative expression values on cis dosage effect in aneuploid line with increasing chromosomal dosage. B The relative expression values on trans dosage effect in aneuploid line with increasing chromosomal dosage. For each group contain different chromosomal dosages, the number of samples corresponds to the number of RNA-seq samples available for that group, with details provided in Tables S4 and S5. The error bars represent mean ± SD. C The percentage of up and down-regulated cis genes in aneuploid line with increasing chromosomal dosage. D The percentage of up and down-regulated trans genes in aneuploid line with increasing chromosomal dosage.
Further analysis showed that the cis genes in 12 aneuploid lines exhibited similar or lower expressional levels with the diploid. Notably, all these lines contained at least 12 extra chromosomes (Fig. 7). Take 59A as an example, which contained three copies of 15 chromosomes and the expression of cis genes located on Chr 4, Chr 5, Chr 10 and Chr 17 were 0.919, 0.899, 0.915 and 0.925, respectively (p > 0.05). While the p-value indicated no significant difference, we further calculated Cohen’s d value to assess the effect size of the observed differences in 12 aneuploid lines exhibit a larger inverse effect. The Cohen’s d value was −1.14, with a 95% confidence interval ranging from 0.959 to 0.957. This negative effect size, which is considered the inverse effect on cis gene expression is significant and outweighs the dosage effect. These results showed when the chromosomal dosage in an aneuploid line exceed a threshold, inverse effect may exceed dosage effect on cis genes. We defined this phenomenon as dosage overcompensation. It is interested that the expressions of cis genes located on Chr 10 may be more likely affected by varied chromosomal dosages compared to other chromosomes in the aneuploid population. In 9 out of the 12 aneuploid lines, the cis genes located on Chr 10 exhibited dosage overcompensation (Fig. 7). GO analysis revealed that a significant number of annotated functional genes on Chr 10 of poplar enriched in key metabolic pathways such as ribose phosphate metabolic process, purine nucleotide metabolic process and nucleoside phosphate metabolic process (each of fold enrichmentå 2) (Supplementary Table 8). The proteins encoded by these genes are linked to DNA repair and nucleotide metabolism and display increased susceptibility to methylation in polyploid organisms, which consequently impacts their expression levels26.
The X-axis represent chromosome 1 to 19, the Y-axis represent the ratio of aneuploid with extra 1 to 16 chromosomes to diploid. Orange dots represent chromosomes with two copies, green dots represent chromosomes with three copies. Compared with diploid level (red dotted line, as 1.0). The blue arrows were indicated that the expression level of the cis genes located on this chromosome was lower than the diploid level, defined as dosage overcompensated (expression value ≤ 1.0 and Cohen’s d value > 0.08).
Discussion
Although most organisms exist in nature as form of euploid, natural aneuploid individuals were found in some species such as wheat, corn, and loquat27. However, no naturally occurring Populus aneuploids have been identified in wild populations or germplasm repositories (Global Biodiversity Information Facility, 2023). Poplar breeding has a long history, yet the absence of naturally occurring aneuploidy remains intriguing. It is widely recognized that natural aneuploids are produced by crossing allopolyploid28. Triploid plant often expected to be meiotically irregular, resulting in unequal number of chromosomes distributed into the microspore cells29. However, studies in Arabidopsis, Maize and Wheat reported that triploid plants are semi-fertile and may contribute to formation of allopolyploid during population evolution30,31. In this study, we demonstrated that triploid poplar (2n = 3X = 57) has complete meiosis and mitosis phases, and the microspore cells developed normally into male gametophytes. During metaphase I and II of meiosis, we observed abnormal behavior that lagging chromosomes did not reach the cell poles as expected (Fig. 1C). It is reported that these lagging chromosomes exists in the form of micronucleus during telophase II and eventually disappear in the cytoplasm30. Our results indicated that the microspore cells of triploid poplar divided into trinuclear pollen, and sperm cells subsequently moved in the germinated pollen tubes. Although pollen with aneuploid chromosomes exhibits significantly reduced viability compared to euploid pollen, a subset of abnormal pollen grains can develop into viable progeny.
The size and shape of pollen are key factors for the success of hybridization32. The aneuploid pollens of the triploid poplar are larger and more irregular than euploid pollen (Supplementary Fig. 1G). Although irregular pollens tend to increase the probability of abortion during the process of hybridization, the dense tapetum on the surface of triploid pollens produce more sporopollenins and recognition proteins, which enhance the stability of hybridization33. In this study, only a small percentage (less than 30%) of the triploid pollen germinated pollen tubes in vitro (Supplementary Fig. 1H), and this percentage increased significantly (more than 70%) when was fertilized on the stigmas of diploid poplar (Supplementary Fig. 1J). Given that aneuploid pollen is capable of completing hybridization, flowering asynchronism may be the primary factor contributing to the rarity of natural aneuploid plants. In Northeast China, only the artificially triploid P. alba ‘Berolinensis’ is utilized for aneuploid breeding34. However, this triploid poplar is primarily utilized as a street tree, and its main hybridization partners (diploid polar) are predominantly thrive in mountainous regions35. The pollen dispersal time of the triploid poplar typically occurs in mid-March, whereas the flowering time of diploid poplar usually falls in early April. In addition, aneuploid populations generally display poor growth state and often difficult to survive through the winter34. Thus, the scarcity of aneuploid poplar varieties in natural environment arises from two primary factors: absence of naturally established triploid populations and the genetic instability of triploid progeny, which compromises their ability to withstand the combined pressures of natural and artificial selection throughout their extended life cycles. Nevertheless, both natural and synthetic fertile triploids remain valuable for generating aneuploid plants.
For aneuploidy, the behavior of chromosomes allocated into gametocytes is incompletely equal36. Some factors may affect the probability of homologous chromosome pairing, including chromosome structure37, chromosomal dosage, and the degree of damage from DNA replication38. Aneuploid seedlings with different chromosome compositions have always been considered as suitable material for studying the specify behavior in meiosis39. In aneuploid seedlings, we observed an interesting phenomenon that different chromosomes have various probabilities being allocated into aneuploid gametocytes. Among them, the probability of Chr 8 being assigned to an aneuploid gamete was 62.82%, and the probability of Chr 3 being assigned to an aneuploid gamete was 24.36% (Fig. 3F). Upon analyzing the distribution of extra chromosomes in aneuploids, we observed that Chr 1 was more likely to be co-segregated into gamete cells along with other chromosomes, whereas Chr 3 exhibited the lowest probability of forming pairings. These results implied that the two homologous chromosomes paired during meiosis may experience differential traction forces or exhibit distinct capacities to form bivalent.
During meiosis, the primary exchange of DNA information occurs during the pairing of homologous chromosomes, a process known as synapsis40. The region where homologous chromosomes form a synaptonemal complex, which is composed of central proteins. The function of synaptonemal complex is hold homologous chromosomes together through the formation of threads that extend along both sides41. Previous studies showed ZEP1 and ZYP1 encode central proteins that form a complex in plants, which plays a crucial role in influencing the process of homologous recombination. Mutant lines zep1 and zyp1 showed that homologous chromosomes can pair normally during meiosis, but they are unable to form stable bivalent structures42. The chromosomes 1 to 19 of poplar exhibit significant difference in the distribution of DNA exchange sites43,44. In poplar, the DNA exchange sites on Chr 1 are uniformly and densely distributed, whereas those on Chr 3, Chr 9, and Chr 16 are unevenly distributed. Inadequate exchange sites on Chr 3, Chr 9, and Chr 16 may result in the premature disassembly of the bivalent, thereby leading to aneuploid chromosomes allocated to the gametocytes43. Moreover, poplar chromosomes 1 to 19 exhibit distinct chromosomal dosage, with Chr 1 containing the highest chromosomal dosage45. Previous studies indicated that DNA replication is frequently associated with mutations, which in turn trigger DNA self-repair mechanisms. Both animal and plant organisms tend to allocate chromosomes with higher chromosomal dosage and a greater number of self-repairs to gametes46. In summary, knocking out the gene encoding the central protein and introducing incorrect bases during DNA replication may increase the likelihood of generating aneuploid offspring.
Research on aneuploidy can be traced back to the 1920s, originating from Blakeslee’s studies of Datura and parallel work on fruit flies by Bridges47. Initially, aneuploid phenotypes were attributed solely to dosage effects of varied chromosomal. However, decades of in-depth research have gradually revealed that cis genes located on varied chromosomes are influenced not only by dosage effects but also by inverse effects, resulting in gene expression remain unchanged or elevated in both animals and plants48. To date, the sole evidence linking inverse effect to chromosomal dosage is that tetrasomy produce a higher number of DEGs compared to trisomy, which is considered the primary driver of enhanced inverse effect49. In this study, we further proved that increased chromosomal dosage (ranging from extra 1 to 16 chromosomes) in aneuploids leads to the greater inverse effect on both cis and trans genes. Meanwhile, more down-regulated cis genes and trans genes were identified as the increasing chromosomal dosage (Supplementary Table 7). However, we found that the number of DEGs does not directly correlate with the total expression level of all cis or trans genes on that varied or unvaried chromosome. Take 33A as example, which contained an extra copy of Chr 4, Chr 12, Chr 13 and Chr 15. The total expression of cis genes on Chr 4 was 1.22 and no significant increase with elevated chromosomal dosage (Fig. 5E). However, the proportion of upregulated cis genes on Chr 4 is the highest of these four chromosomes. These results implied that larger chromosomal dosage resulted in wider effect on both cis and trans genes expression.
Significant alterations in chromosomal dosage not only leads to an increase in DEGs but also trigger epigenetic modifications, chromosome rearrangements, and the inactivation of certain key enzymes43. Aneuploidy may decrease the methylation levels in transposon regions within plant genomes. During this process, the expressions of CMT2, HEN1 and AGO4 that regulate methylation levels are significantly downregulated. Alterations in chromosomal dosage can also result in chromosomal inversions and translocations50. Following such rearrangements, chromosomes may cause certain key genes that control plant growth and development to be transferred from the varied chromosome to the unvaried chromosome, or even from an autosome to a non-homologous chromosome51. Further research is required to explore whether increased chromosomal dosage leads to a greater incidence of chromosomal structural alterations. In general, cis genes expression ranged from dosage effect to dosage compensation7. We observed that the expression of cis genes located on Chr 10 more likely to exhibit compensation effects, or even expression levels that decreased below diploid (Fig. 7). GO enrichment analysis revealed that Chr 10 of poplar is highly enriched with numerous dosage-sensitive factors, which linking to DNA repair and nucleotide metabolism process. In the context of polyploidy, these genes are more susceptible to methylation26. In addition, dosage-sensitive factors may produce more protein in the aneuploids. However, an excess of protein products could disrupt the structure of protein complexes due to subunit mismatches, thereby negatively impacting the growth and development of the plant52. Ultimately, no individuals carrying extra 17 or 18 chromosomes were identified in the aneuploid population derived from hybridization. We believe that two primary factors led to this phenomenon. First, the limited size of aneuploid population results in a reduced likelihood of obtaining individuals with more than 16 chromosomes. Second, when aneuploid offspring already possess 16 chromosomes, they exhibit the larger inverse effect that can cause severely growth defect than aneuploids with smaller chromosomal dosages. In summary, increased chromosomal dosage of varied chromosomes results in more extensive genetic variation, and the judicious utilization of this variation can enhance the efficiency of poplar breeding.
Materials and methods
Plant material
To generate aneuploid population, crosses were performed between the diploid poplar species P. davidiana (2n = 2x = 38) and the triploid poplar species P. alba ‘Berolinensis’ (2n = 3x = 57). Both the parental species belong to the same genus and are not reproductively isolated. The diploid P. davidiana is one of the parental species of the triploid P. alba ‘Berolinensis’. The genome of the diploid species P. davidiana exhibits high nucleotide sequence identity to the homologous chromosomes within the triploid genome21. Triploid pollen was collected in mid-March using the cut-branch hydroponic method, and subsequently applied to female inflorescences in early April to complete the pollination process. 1 month after pollination, 214 seeds were harvested from the dehiscent capsules and sown in seed trays to initiate seedling cultivation. Of these 214 seeds, 84 seedlings ultimately survived and were used for FCM analysis and DNA sequencing. The seedlings were cultivated in a greenhouse under a 16 h/8 h photoperiod, with day/night temperatures of 26/22 °C and relative humidity maintained between 65 and 95%. After 60 days of growth, measurements of plant height and leaf area were taken to assess the extensive phenotypic variation present in the progeny. Among the 84 aneuploid seedlings that underwent FCM analysis and DNA sequencing, three triploids (3A, 4A, and 98-1A) and 15 individuals (21A, 26A, 41A, 42A, 43A, 44A, 58A, 62A, 64A, 69-2A, 75-1A, 86-2A, 88A, 98-2A and 107A) exhibited extremely poor growth status and were ultimately not used for RNA-seq analysis.
Cytogenetic analysis of meiotic and mitotic phases
The male inflorescences of the triploid poplar were fixed with Carnoy’s fixative for 24 h. Anthers were isolated from the fixed male inflorescences and subsequently washed with 10 mM citrate buffer (pH 4.5). The fixed anthers were treated in a digestion mixture containing 0.3% (w/v) cellulase RS, 0.3% (w/v) Pectoylase Y23 and 0.3% (w/v) cell gyrase at 37 °C for 3 h53. Each digested anther was placed on a glass slide and heated in a 45 °C oven for 5 min. During this process, 20 μL of acetic acid was added to the slide and stirred to remove the cytoplasm. After staining with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min, metaphase chromosomes were observed under a fluorescence microscope. To observe the complete mitotic period, the collected pollens were incubated in a liquid Murashige and Skoog (MS) medium containing 20 g/L sugar, 0.03 g/L boric acid and 0.02 g/L calcium chloride54. The pollens were then transferred to a plastic culture dish, stained with 20 μL DAPI for 5 min, and observed under a fluorescence microscope (Zeiss Axio Imager A2). The staining process was monitored every 3 h in a completely dark environment.
Detection of morphology and fertility of the triploid pollens
The collected pollens were dusted onto a platform coated with conductive adhesive, and gold powder was sprayed onto the pollen surface at a voltage of 25 KV. The gold-sprayed pollens were then observed under a scanning electron microscope (SEM2000) with a separation rate of 0.2 μm. The collected pollens were stained with a 0.1% 2,3,5-Triphenyltetrazolium Chloride (TTC) solution and incubated at 25 °C for 30 min. The stained pollens were subsequently observed under a light microscope (Leica DM750P). Fifty microspore mother cells were selected from different fields of view for counting. Mature pollens were dusted into a 4 cm × 6 cm plastic culture dish and incubated at 25 °C for 3, 6, 9, 12, and 15 h. At each time point, the suspension from the liquid medium was transferred to a glass slide, and the germination of pollen tubes was observed under a microscope (Leica DM750P).
FCM analysis
FCM (Sysmex, Cube 6) was employed to confirm ploidy levels in both parents and progeny seedlings55. Leaf tissue samples (0.5 cm2) were chopped and immersed in Sysmex CyStain UV Precise P Nuclei Extraction Buffer for 45 s, followed by the addition of 1 mL of Sysmex CyStain UV Precise DAPI staining solution for 30 s. The stained solution was then filtered through a 30 μm filter and analyzed using FCM equipped with a 365 nm UV mercury arc lamp. The flow rate was set to 10 m/s, and the spectral response interval was configured to 435–500 nm. Using the DNA values of the parents as parameter controls, the aneuploid samples were analyzed sequentially at a low flow rate. A minimum of 3000 nuclei were measured per sample. The FCM data were analyzed using FCM Express software (version 3.0), with the X-axis representing relative fluorescence intensity and the Y-axis representing the number of nuclei. The ploidy levels of different aneuploid lines were determined based on the measured peak channel of fluorescence intensity corresponding to the aneuploid samples. The peak channel of fluorescence intensities across aneuploid population are presented in Supplementary Table 1 and Supplementary Data 2. We provided a box-plot summarizing the peaks of fluorescence intensities across diploid, triploid and aneuploids. Subsequent FCM analysis employed the quartile method to calculate DNA contents within the aneuploid population, revealing distinct distribution properties56.
DNA extraction, sequencing and chromosome copy number identification
DNA extraction was performed using the TaKaRA DNA extraction kit (9765A). The preparation of sequencing libraries (P-1051 DNA Library Preparation Kit) were conducted in accordance with the BGI platform sequencing guidelines57,58. DNA sequencing was performed on the MGI DNBSEQ T7 sequencing platform (BGI, Shenzhen, China) using 150-bp paired-end libraries and an average sequencing depth of ~50× for each aneuploid sample. The sequencing reads were aligned to the subgenome A reference of P. alba ‘Berolinensis’ using Bowie2 (version 2.5.4) with the parameters of “-D 15 -R 2 -N 0 -L 22 -i S,1,1.15” 21. The sequencing depths of each nucleotide on the reference genome was calculated individually by using samtools (1.19.2) with the parameters of “samtools depth aligned.sorted.bam”. The mode of each sequencing depth was determined on a per-chromosome basis. The mode of each chromosome in aneuploids is presented in the Supplementary Table 2 and Supplementary Data 3. Supplementary Tables 2 and 4 are provided in supplementary data 1 file.
Statistical definition of extra chromosomes
Statistical analyzes for DNA sequencing data were performed using IBM SPSS (Version 26.0). In the process of defining chromosomal copy number using the Z-score method, the selected variables were the mode and standard deviation of observed values. The resulting values were automatically calculated using the normalization settings (The default of Z-score point >±3 is True, while the default for Z-score point < ±3 is False) provided by the SPSS software. We employed a Z-score based method to examine if a chromosome in an aneuploid seedling is trisomic59. We sequenced the genomic DNA extracted from each aneuploid seedling (Supplementary Table 2) and then used k-mer distribution to estimate the expected sequencing depth (μe) for the cellular genomic DNA, μe = D*L/(L-K + 1). Where D is the k-mer depth of haploid genome, L and K are the lengths of sequencing reads and k-mer, respectively60. In an aneuploid individual, the expected sequencing depths for unvaried chromosomes were expected to be 2μe. The expected sequencing depth for the cellular genomic DNA across aneuploid seedlings are presented in Supplementary Data 4.
The sequencing reads were then mapped to the reference genome21 using BWA with default parameters61. We calculated the observed sequencing depths (μo) for each chromosome in an aneuploid seedling. The mapping depths at each position along chromosomes of each sample were extracted using SAMtools62. Because short reads were reported to be unevenly distributed across the genomic regions such as GC enrich and repetitive sequences63, we used modes rather than means to represent the μo for chromosomes of each individual.
For an aneuploid seedling, the chromosomes can be divided into two groups according to the copy numbers. The first group included chromosomes with two copies (unvaried copy number) and the second group included chromosomes with three copies (varied copy number). We used k-means method64 to cluster the chromosomes of each individual into two groups according to the values of μo and then calculated the intragroup standard deviations for each group (Sintra).
Finally, we calculated the Z-score values for each chromosome in an aneuploid seedling, Z-score = (μo-2μe)/Sintra. If the Z-score value of a chromosome was larger than three, the chromosome was defined as varied chromosome.
Fluorescence in situ hybridization (FISH)
To dissect the chromosome composition of aneuploids during mitotic metaphase, root tips were obtained from aneuploid seedlings, and fixed in Carnoy’s fixative at 4 °C for 24 h. Telomere probes are used to mark the number of chromosomes under 494 nm wavelength. Centromere probes of Chr 7 and Chr 19, 45S rDNA and 5S rDNA were used to detect the copy number. The DNA sequences of above probes are provided in Supplementary Table 3. The FISH experiment was performed as described by Wang et al.65. Briefly, root tips are broken into mist in 45% acetic acid and to fix the DNA at −80 °C refrigerator. The hybridization mixture (100 ng of chromosome painting probes, 50% formamide, 10% dextran sulfate, 20% 2×SSC and 10% salmon DNA) was applied to denatured chromosomes and incubated at 37 °C for 24 h. The slides after incubating were washed thrice in 2×SSC at 42 °C for 5 min and observed under fluorescence microscope. Telomere probes were labeled with 5-Carboxyfluorescein modification (5-FAM), while centromere of Chr 7 and 19, 5S rDNA and 45S rDNA probes were labeled with 5-Carboxy-tetra-methyl-rhodamine (5-TAMRA).
RNA-seq
Mature leaves from 2-month-old euploid and aneuploid plants that were grown in a greenhouse were collected20 and frozen in liquid nitrogen. Two biological replicates were performed for each sample66. Total RNA was extraced using Takara RNAiso Plus 9108 kit67. The construction and quality control of cDNA libraries were performed by Annoroad Gene Technology (Beijing, China) as follows. Messenger RNA (mRNA) was enriched using eukaryotic mRNA by Oligo(dT) magnetic beads (Vazyme, RN702). The pre-chilled Fragmentation Buffer was added and then incubated at 94 °C for 10 min. One strand of cDNA was synthesised using a six-base random primer using mRNA as a template, followed by the synthesis of the second strand of cDNA using buffer, dNTPs, DNA polymerase I and RNase H, and cDNA purification. Purified double-stranded cDNA was subjected to end repair, base A was added, sequencing junctions were added, and fragments were screened to recover cDNA around 350 bp. The cDNA library was generated by performing enrichment of the above Polymerase Chain Reaction (PCR) products. The sequences for the final cDNA library amplification are 5′-AATGATACGGCGACCACCGAGATCTACAC-3′ and 5′-ATCTCGTATGCCGTCTTCTGCTTG-3′. Using Qubit 3.0 to initially quantify concentrations, the Agilent 2100 system initially assessed library fragment size. After meeting expectations, the effective concentration of the library was accurately quantified using the Bio-RAD CFX 96 Fluorescent Quantitative PCR Instrument.
The sequencing of the cDNA libraries was performed on the MGI DNBSEQ T7 platform (BGI, Shenzhen, China) using 150-bp paired-end model with an average sequencing depth of ~70× for each sample. For gene expression analysis, the nf-core/rnaseq pipeline within nextflow (Version 22.10.7)68,69 was utilized. Quality control of the raw reads was performed using FastQC (Version 0.12.1)70. Trimmomatic (Version 1.0.0)71 was employed to remove adapter-containing and low-quality reads. Spliced Transcripts Alignment to a Reference (STAR, Version 2.7.10) was used for mapping the sequencing reads to the P. alba ‘Berolinensis’ subgenome A72. Gene transcript levels were quantified using RNA-Seq by Expectation-Maximization (RSEM, Version 1.3.3) and normalized via the Trimmed Mean of M-value method73.
Ratio distributions of cis and trans gene expression were generated as previously described7,67. Normalized counts were calculated for each sample, including diploid, triploid, and aneuploid samples, by averaging the values across biological replicates. The ratios were computed by dividing the average count of aneuploids by that of the diploid control, and these ratios were then depicted in the form of histograms. All histograms of the ratio are presented in Supplementary Fig. 3.
EdgeR was utilized for DEGs identification74. Supplementary Table 7 presents the number and percentage of up and down-regulated DEGs for each chromosome across all aneuploid lines. Supplementary Tables 5–7, 9 and 10–12 are provided in supplementary data 1 file. DEGs with a false discovery rate (FDR) of less than 0.05 and a log2 fold-change (log2FC) greater than 1 were considered significantly differentially expressed. Statistical significance relative to the leave groups was determined by a two-sided quasi-likelihood F-test with Benjamini–Hochberg FDR correction. Significant expression change were defined as p value <0.05 in varied chromosomes relative to unvaried regions. To further verify whether cis genes are subject to dosage overcompensation, we conduct a Cohen’s d analysis75 to calculate the effect size of the cis gene expressions of the aneuploid seedlings in Fig. 7. This negative effect size (Cohen’s d value > ±0.08), coupled with the confidence interval that does not include zero or one, supports the conclusion that the observed gene expression changes are statistically significant. GO enrichment analysis of genes on Chr 10 of poplar was performed using TAIR Blast67.
Statistics and reproducibility
In this study, cytogenetic analyzes (meiotic observation, pollen viability assays, and FISH) comprised a minimum of three independent biological replicates. A total of 84 aneuploid samples were detected for FCM. Each experiment used to DNA and RNA sequencing was conducted with two independent biological replicates, which were subsequently analyzed. Statistical analyses and data visualization were carried out SPSS (Version 26.0). Statistical significance relative to the diploid groups was determined by a two-sided quasi-likelihood F-test with Benjamini–Hochberg FDR correction. The observed value of Chr 1 to Chr 19 in aneuploids was calucated as mode, with significance levels defined as follows: Z > ± 3 was significantly value. The T-test was employed to compare the expression ratio between genes on different chromosomes, with significance levels defined as follows: p < 0.05 was significantly value. When the p-value was no significant difference in some chromosomes, Cohen’s d value to further assess the effect size of the observed differences, Cohen’s d value > ±0.8 was significantly value.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
All sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number PRJNA1204477. DNA sequencing data can be found in Supplementary data 1, 3 and 4. RNA sequencing data can be found in Supplementary data 1. The source data for Fig. 2C can be found in Table S1. The source data for Fig. 3B can be found in Table S2. The source data for Fig. 3E and F can be found in Table S4. The source data for Fig. 4 can be found in Table S4. The source data for Figs. 5, 7 can be found in Table S5. The source data for Fig. 6A, B can be found in Tables S11 and S12. The source data for Fig. 6C, D can be found in Table S7. Supplementary Tables 2, 4-7, 9, 11, and 12 are provided in the Supplementary Data 1 file. All data are available from the corresponding author (or other sources, as applicable) on reasonable request.
References
Henry, I. M., Dilkes, B. P., Miller, E. S., Burkart-Waco, D. & Comai, L. Phenotypic consequences of aneuploidy in Arabidopsis thaliana. Genetics 186, 1231–1245 (2010).
Williams, B. R. et al. Aneuploidy affects proliferation and spontaneous immortalization in mammalian cells. Science 233, 703–109 (2008).
Orr, B., Godek, K. M. & Compton, D. Aneuploidy. Curr. Biol. 25, R538–R542 (2015).
Cenci, A. et al. Effect of paleopolyploidy and allopolyploidy on gene expression in banana. BMC Genomics 20, 1–12 (2019).
Tan, Z. et al. Aneuploidy underlies a multicellular phenotypic switch. Proc. Natl Acad. Sci. USA 110, 12367–12372 (2013).
Li, X. et al. The genetic control of leaf and petal allometric variations in Arabidopsis thaliana. BMC Plant Biol. 20, 1–11 (2020).
Hou, J. et al. Global impacts of chromosomal imbalance on gene expression in Arabidopsis and other taxa. Proc. Natl Acad. Sci. USA 115, E11321–E11330 (2018).
Li, Y. et al. Genome dosage alteration caused by chromosome pyramiding and shuffling effects on karyotypic heterogeneity, reproductive diversity, and phenotypic variation in Zea–Tripsacum allopolyploids. Theor. Appl. Genet. 137, 1–16 (2024).
Church, S. A. Molecular phylogenetics of Houstonia (Rubiaceae): descending aneuploidy and breeding system evolution in the radiation of the lineage across North America. Mol. Phylogenet. Evol. 27, 223–238 (2003).
Johnson, A. F. et al. Magnitude of modulation of gene expression in aneuploid maize depends on the extent of genomic imbalance. J. Genet. Genomics 47, 93–103 (2020).
Shi, X. et al. Genomic imbalance determines positive and negative modulation of gene expression in diploid maize. Plant Cell 33, 917–939 (2021).
Birchler, J. A., Bhadra, U., Bhadra, M. P. & Auger, D. L. Dosage-dependent gene regulation in multicellular eukaryotes: Implications for dosage compensation, aneuploid syndromes, and quantitative traits. Dev. Biol. 234, 275–288 (2001).
Sun, L. et al. Differential effect of aneuploidy on the X chromosome and genes with sex-biased expression in Drosophila. Proc. Natl Acad. Sci. USA 110, 16514–16519 (2013).
Birchler, J. A. A study of enzyme activities in a dosage series of the long arm of chromosome one in maize. Genetics 92, 1211–1229 (1979).
Birchler, J. amesA. The genetic basis of dosage compensation of alcohol dehydrogenase-1 in maize. Genetics 97, 625–637 (1981).
Scandalios, J. G. Alcohol dehydrogenase in maize: Genetic basis for isozymes. Science 166, 623–624 (1969).
Sheltzer, J. M., Torres, E. M., Dunham, M. J. & Amon, A. Transcriptional consequences of aneuploidy. Proc. Natl Acad. Sci. USA109, 12644–12649 (2012).
Zhang, A. et al. Global analysis of gene expression in response to whole-chromosome aneuploidy in hexaploid wheat. Plant Physiol. 175, 828–847 (2017).
Dang, J. et al. Identification and characterization of a loquat aneuploid with novel leaf phenotypes. HortScience 54, 808–804 (2019).
Yu, Y. et al. The B-box transcription factor PabBBX27 in the regulation of chlorophyll biosynthesis and photosynthesis in poplar (Populus alba × P. Berolinensis). Ind. Crops Prod. 203, 117159 (2023).
Chen, S. et al. Chromosome-level genome assembly of a triploid poplar Populus alba ‘Berolinensis’. Mol. Ecol. Resour. 1–16 https://doi.org/10.1111/1755-0998.13770 (2023).
Birchler, J. A., Riddle, N. C., Auger, D. L. & Veitia, R. A. Dosage balance in gene regulation: Biological implications. Trends Genet 21, 219–226 (2005).
Liu, Y. et al. Analysis of changes in morphological characters and drought resistance of tetraploid Populus alba L. Plant Cell. Tissue Organ Cult. 162, 27 (2025).
Henry, I. M. et al. Aneuploidy and genetic variation in the Arabidopsis thaliana triploid response. Genetics 170, 1979–1988 (2005).
Xin, H. et al. An extraordinarily stable karyotype of the woody Populus species revealed by chromosome painting. Plant J. 101, 253–264 (2020).
Yin, L. et al. DNA repair- and nucleotide metabolism-related genes exhibit differential CHG methylation patterns in natural and synthetic polyploids (Brassica napus L.). Hortic. Res. 8, 1–17 (2021).
Antunovics, Z., Szabo, A., Heistinger, L., Mattanovich, D. & Sipiczki, M. Synthetic two-species allodiploid and three-species allotetraploid Saccharomyces hybrids with euploid (complete) parental subgenomes. Sci. Rep. 13, 1–13 (2023).
Franasiak, J. M. et al. The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening. Fertil. Steril. 101, 656–663.e1 (2014).
Bradshaw, H. D. & Stettler, R. F. Molecular genetics of growth and development in Populus. I. Triploidy in hybrid poplars. Theor. Appl. Genet. 86, 301–307 (1993).
Wang, B. et al. Cytological and transcriptomic analyses provide insights into the pollen fertility of synthetic allodiploid Brassica juncea hybrids. Plant Cell Rep. 43, 1–19 (2024).
Fradkin, M. et al. Cytogenetic analysis of meiotic behaviour and stability in a trigeneric hybrid (triticale x trigopiro). Protoplasma 261, 1221–1231 (2024).
Hao, K., Tian, Z. X., Wang, Z. C. & Huang, S. Q. Pollen grain size associated with pollinator feeding strategy. Proc. R. Soc. B Biol. Sci. 287, 1933 (2020).
Lora, J., Garcia-Lor, A. & Aleza, P. Pollen development and viability in diploid and doubled diploid citrus species. Front. Plant Sci. 13, 1–18 (2022).
Li, Y. et al. Generation of a golden leaf triploid poplar by repressing the expression of GLK genes. For. Res. 1, 1–7 (2021).
Hou, Z., Li, A. & Zhang, J. Genetic architecture, demographic history, and genomic differentiation of Populus davidiana revealed by whole-genome resequencing. Evol. Appl. 13, 2582–2596 (2020).
Acquaviva, L. et al. Ensuring meiotic DNA break formation in the mouse pseudoautosomal region. Nature 582, 426–431 (2020).
Nicodemi, M. & Pombo, A. Models of chromosome structure. Curr. Opin. Cell Biol. 28, 90–95 (2014).
Yi, Q. et al. HP 1 links centromeric heterochromatin to centromere cohesion in mammals. EMBO Rep. 19, 1–13 (2018).
Compton, D. A. Mechanisms of aneuploidy. Curr. Opin. Cell Biol. 23, 109–113 (2011).
Hotta, Y., Ito, M. & Stern, H. Synthesis of DNA during meiosis. Proc. Natl Acad. Sci. Usa. 56, 1184–1191 (1966).
Del Priore, L. & Pigozzi, M. I. Dna organization along pachytene chromosome axes and its relationship with crossover frequencies. Int. J. Mol. Sci. 22, 1–12 (2021).
Wang, M. et al. The central element protein ZEP1 of the synaptonemal complex regulates the number of crossovers during meiosis in rice. Plant Cell 22, 417–430 (2010).
Hou, J. et al. Major chromosomal rearrangements distinguish willow and poplar after the ancestral ‘salicoid’ genome duplication. Genome Biol. Evol. 8, 1868–1875 (2016).
Geng, X. et al. High-frequency homologous recombination occurred preferentially in Populus. Front. Genet. 12, 1–12 (2021).
Wang, J. et al. Conversion between duplicated genes generated by polyploidization contributes to the divergence of poplar and willow. BMC Plant Biol. 22, 1–15 (2022).
Liu, W. et al. RAD51 bypasses the CMG helicase to promote replication fork reversal. Science 380, 382–387 (2023).
Mohammad, K. A., Rahim, W. A., Abdul, H. A. H., Foong, E. & Lim, Y. S. Double autosomal aneuploidy: a case of trisomy 18 and 21 mosaicism in a neonate with clinical Down Syndrome. Malays. J. Paediatr. Child Heal. 30, 37–43 (2024).
Zhang, S., Wang, R., Zhang, L., Birchler, J. A. & Sun, L. Inverse and proportional trans modulation of gene expression in human aneuploidies. Genes 15, 637 (2024).
Shi, X. et al. Dosage-sensitive miRNAs trigger modulation of gene expression during genomic imbalance in maize. Nat. Commun. 13, 1–15 (2022).
Tolmacheva, E. N., Vasilyev, S. A. & Lebedev, I. N. Aneuploidy and DNA methylation as mirrored features of early human embryo development. Genes 11, 1–21 (2020).
Tian, S. et al. A 2.09 Mb fragment translocation on chromosome 6 causes abnormalities during meiosis and leads to less seed watermelon. Hortic. Res. 8, 256 (2021).
Zhang, S., Wang, R., Huang, C., Zhang, L. & Sun, L. Modulation of global gene expression by aneuploidy and cnv of dosage sensitive regulatory genes. Genes 12, 1606 (2021).
Gonzalo, A. et al. Reducing MSH4 copy number prevents meiotic crossovers between non-homologous chromosomes in Brassica napus. Nat. Commun. 10, 1–9 (2019).
Brandoli, C., Cristofori, V., Silvestri, C., Todeschini, C. & Sgarbi, E. The development of an improved medium for the in vitro germination of Corylus avellana L. Pollen. Forests 15, 1–17 (2024).
Doležel, J., Greilhuber, J. & Suda, J. Estimation of nuclear DNA content in plants using flow cytometry. Nat. Protoc. 2, 2233–2244 (2007).
Darcy, D. P. et al. A. A practical guide to cancer subclonal reconstruction from DNA sequencing. Nat. Methods 19, 1208–12188 (2022).
Mardis, E. R. Next-generation DNA sequencing methods. Annu. Rev. Genomics Hum. Genet. 9, 387–402 (2008).
Belova, V. et al. System analysis of the sequencing quality of human whole exome samples on BGI NGS platform. Sci. Rep. 12, 1–15 (2022).
Kim, D. et al. KF-NIPT: K-mer and fetal fraction-based estimation of chromosomal anomaly from NIPT data. BMC Bioinform. 26, 133 (2025).
Ranallo-Benavidez, T. R., Jaron, K. S. & Schatz, M. C. GenomeScope 2.0 and Smudgeplot for reference-free profiling of polyploid genomes. Nat. Commun. 11,1432 (2020).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Danecek, P. et al. Twelve years of SAMtools and BCFtools. Gigascience 10, 1–4 (2021).
Wang, P., Meng, F., Moore, B. M. & Shiu, S. H. Impact of short-read sequencing on the misassembly of a plant genome. BMC Genomics 22, 1–18 (2021).
Boleda, G., Schulte im Walde, S. & Badia, T. Modeling regular polysemy: a study on the semantic classification of catalan adjectives. Comput. Linguist. 38, 575–616 (2012).
Wang, X. et al. Generation and characterization of a tetraploid Populus davidiana. Plant Cell. Tissue Organ Cult. 160, 1–14 (2025).
Denyer, T. et al. Spatiotemporal developmental trajectories in the Arabidopsis root revealed using high-throughput single-cell RNA sequencing. Dev. Cell 48, 840–852.e5 (2019).
Yu, Y. et al. Transcriptomic profiles of poplar (Populus simonii × P. nigra) cuttings during adventitious root formation. Front. Genet. 13, 1–16 (2022).
Ewels, P. et al. The nf-core framework for community-curated bioinformatics pipelines. Nat. Biotechnol. 38, 276–278 (2020).
DI Tommaso, P. et al. Nextflow enables reproducible computational workflows. Nat. Biotechnol. 35, 316–319 (2017).
Wei, S. et al. Chromosome-level genome assembly of the yellow boxfish (Ostracion cubicus) provides insights into the evolution of bone plates and ostracitoxin secretion. Front. Mar. Sci. 10, 1–13 (2023).
Pérez-rubio, P., Lottaz, C. & Engelmann, J. C. FastqPuri: high-performance preprocessing of RNA-seq data. BMC Bioinformatics 20, 226 (2019).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Parrish, N., Hormozdiari, F. & Eskin, E. Assembly of non-unique insertion content using next-generation sequencing. Bioinform. Impact Accurate Quantif. Proteomic Genet. Anal. Res. 21–40 https://doi.org/10.1201/b16589 (2014).
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2009).
Lakens, D. Calculating and reporting effect sizes to facilitate cumulative science: a practical primer for t-tests and ANOVAs. Front. Psychol. 4, 1–12 (2013).
Acknowledgements
This work was supported by the National Key R&D Program of China (2021YFD2200800), Natural Science Foundation of Heilongjiang Province of China (LH2024C051) and China Postdoctoral Science Foundation (2025M771924). We thank the reviewers and editors for their work on this manuscript. We thank Annoroad Gene Technology (Beijing) Co., Ltd. (Beijing, China) for performing the DNA/RNA sequencing.
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Y.Y. contributed to Data curation, Formal analysis, Visualization, Writing original draft. S.C. and K.L. contributed to DNA sequencing and RNA-seq data processing. X.W. and H.L. contributed to FISH and FCM. Y.W., X.L., W.L., and F.L. contributed to Hybridization and Phenotypic observation. Q.L., X.L., and G.Q. contributed to Experimental design. S.C. contributed to Conceptualization, Supervision, Writing – review & editing, Resources, Funding acquisition.
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Communications Biology thanks Laura Gonzalez-Garcia, Paulo Izquierdo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Jorge Duitama and David Favero. A peer review file is available.
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Yu, Y., Chen, S., Li, K. et al. A comprehensive analysis of the impact of genomic imbalance on gene expression in a poplar aneuploidy population. Commun Biol 9, 54 (2026). https://doi.org/10.1038/s42003-025-09318-8
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DOI: https://doi.org/10.1038/s42003-025-09318-8
