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    Topic review

    TERT Gene in Polyploid Plants

    Subjects: Plant Sciences
    View times: 5
    Submitted by: Eva Sykorova

    Definition

    The gene coding for the telomerase reverse transcriptase (TERT) is essential for the maintenance of telomeres. Previously we described the presence of three TERT paralogs in the allotetraploid plant Nicotiana tabacum, while a single TERT copy was identified in the paleopolyploid model plant Arabidopsis thaliana. Here we examine the presence, origin and functional status of TERT variants in allotetraploid Nicotiana species of diverse evolutionary ages and their parental genome donors, as well as in other diploid and polyploid plant species. A combination of experimental and in silico bottom-up analyses of TERT gene copies in Nicotiana polyploids revealed various patterns of retention or loss of parental TERT variants and divergence in their functions. RT–qPCR results confirmed the expression of all the identified TERT variants. In representative plant and green algal genomes, our synteny analyses show that their TERT genes were located in a conserved locus that became advantageous after the divergence of eudicots, and the gene was later translocated in several plant groups. In various diploid and polyploid species, translocation of TERT became fixed in target loci that show ancient synapomorphy.

    1. Introduction

    Flowering plants (angiosperms) are important for the existence of many terrestrial organisms, including humans, and a long history of plant breeding has taught us that polyploidization can be advantageous in terms of quantitative traits of crops. Gains and losses of paralogs, their neofunctionalization and sub-functionalization, have all been associated with the generation of duplicate gene copies, e.g., by whole-genome duplications (WGDs) and further rounds of genome duplication/reduction, resulting in genetic diversity upon which the fittest combinations thrived in a competitive environment [1][2][3][4]. An ancient WGD has been reconstructed at the base of seed plants, another at the base of angiosperms [5][6][7] and numerous additional, subsequent WGD events were associated with the divergence of many angiosperm lineages [3]. Polyploidy is usually associated with many genetic and epigenetic changes, including chromosomal rearrangements, expansions of transposable elements and changes in gene expression [8][9]. At the gene level, polyploids can tolerate the presence of paralogs or eliminate a copy of the spare gene. Thus, evolutionary forces result in an equilibrium defined by gene dosage [10]. Studies of model plants have mostly focused on genes important for crop production; however, genes that are critical for genome stability are extremely important for understanding repeated polyploidization events during natural selection, and these remain underexplored.
    Telomerase reverse transcriptase (TERT) is involved in the maintenance of telomeres, nucleoprotein structures that are essential for genome stability [11][12][13]. Telomerase adds telomere repeats to the ends of eukaryotic chromosomes, thereby elongating telomeres and compensating for their shortening due to incomplete end-replication. When telomerase is not active, telomeres become shortened, and their function in the protection of chromosomes is disrupted. The extreme evolutionary success of telomerase-based mechanisms of telomere maintenance is illustrated by current findings in plants (reviewed in [14]). Even among apparent exceptions in telomere sequences, in plant genera Allium (Asparagales) and Cestrum (Solanales) [15][16][17][18], recent research has revealed that novel, unusual telomere DNA sequences are synthesized by telomerase [16][18][19] and not by alternative mechanisms as had been suggested previously (reviewed in [20]). Moreover, we recently demonstrated that changes in the template region of the telomerase RNA subunit directed the observed evolutionary transitions in telomere DNA sequences [14][21][22]. In contrast to the RNA subunit, the protein subunit TERT is evolutionary well conserved and possesses a central reverse transcriptase domain essential for its catalytic function [23][24]. Plant TERTs are structurally similar to human, ciliate or yeast TERTs with a telomerase-specific T motif [25][26][27][28][29]. The gene encoding TERT is usually expressed at low mRNA levels even in telomerase-positive tissues and is maintained as a single copy gene in most eukaryotic genomes. However, the natural allotetraploid Nicotiana tabacum possesses three sequence variants of the TERT gene [30]. Various allopolyploidization events among closely and distantly related diploid parental species (Figure 1) in Nicotiana make the genus an ideal experimental model system to study the long-term evolution of TERT following natural gene duplication. The increasing number of publicly available assembled plant genomes enables the exploration of TERT genomic loci, gene copy numbers and gene synteny in diverse plant species for comparisons with the data from Nicotiana polyploids and the diploid species most closely related to their progenitors (hereafter called progenitor diploids). The Nicotiana genus [31][32][33][34][35] comprises relatively young polyploids (i) N. tabacum (section Nicotianae), N. rustica (sect. Rusticae), N. arentsii (sect. Undulatae) that formed approx. 0.4–0.6 million years ago, (ii) N. clevelandii and N. quadrivalvis (ca. 1.5 million years ago, sect. Polydicliae), (iii) four species from the 4–5 million years old section Repandae (N. nudicaulisN. repandaN. nesophila and N. stocktonii), and (iv) ~35 species including the model N. benthamiana from the oldest section Suaveolentes formed about 6 million years ago [31]. Among these species, members of sections Suaveolentes and Repandae are of interest because, with N. tabacum, they share an ancient genome donor, N. sylvestris, and these speciation events happened at different times. In N. tabacum, two TERT variants originated from the maternal N. sylvestris genome (TERT_Cs, TERT_D) and one from the N. tomentosiformis paternal genome (TERT_Ct). Variants TERT_Cs and TERT_Ct code for a full-length functional protein, while the TERT_D variant is truncated and contains several indels resulting in premature stop codons, suggesting that it is a pseudogene [30]. All three variants are nevertheless transcribed and show distinct, tissue-dependent levels of mRNA transcripts, indicating a sub-functionalization of TERT variants [30][36].
    Figure 1. Overview of experimental results and illustration of phylogenetic relationships of Nicotiana species used in this study. Phylogeny and the proposed origin of polyploids were adapted from [31][32]. An uncertain parental genome donor for section Suaveolentes is indicated by a question mark. Summary of experimental and in silico results (squares, analyses of genomic DNA; circles, expression of TERT variants investigated by RT–qPCR) is shown in boxes of Nicotiana sections, the origin of TERT variant in polyploids is depicted by color of respective parental diploids, and variants that were not identified are depicted with open squares. Nicotiana accessions used in the experimental analyses are listed in Table S1, genomic assemblies and genomic/transcriptomic SRA data used for in silico analyses are listed in Material and Methods. For the purposes of this paper, we refer to a TERT copy that does not code for a catalytically active protein as a putative pseudogene (dashed symbols) in contrast to a functional TERT gene copy (open symbols), n.a. not analyzed.
    Based on previously described TERT variants in N. tabacum, we explored the fate of TERT paralogs in other Nicotiana polyploids to determine whether both parental TERT genes are conserved in allotetraploid genomes, whether they are transcribed, present in syntenic, collinear arrays with their progenitor diploids, and whether any relationship exists between telomere lengths in polyploids and their progenitor diploids. Of particular interest in this study was to clarify the origin of the presumed pseudogene variant TERT_D in N. sylvestris, a diploid genome donor of N. tabacum, as well as of even older species from sections Repandae and Suaveolentes. In addition, we investigated in silico whether diploid and polyploid plants outside of the family Solanaceae sustained TERT paralogs/pseudogenes in their genomes, and we explored syntenic relationships of genes adjacent to TERT to interpret the evolutionary success of TERT copies after translocation.

    2. Number of TERT Variants in Nicotiana Polyploids as a Case Study

    At the beginning of this project, there was limited genomic sequence data available for the majority of Nicotiana allopolyploids and their parents. To characterize experimentally the number, identity and origin of TERT copies in genomes of polyploid Nicotiana species and representatives of their diploid progenitors, we employed several primer combinations derived from conserved TERT regions of the evolutionarily distant relatives N. sylvestris and N. tomentosiformis (Figure 1), designed originally for amplification of N. tabacum TERT variants [30][36]. These PCR primers (Figure 2A, Table S2) amplify TERT regions nonspecifically, i.e., all variants are produced in a single PCR. Sequencing of PCR products then identifies single nucleotide polymorphisms (SNPs) and/or indels evidencing the presence of multiple TERT variants. Primer positions were with respect to Nicotiana TERT gene structure with 13 exons (Figure 2A), which differed from the prevalent 12-exon structure of plant TERTs [23]. As expected, a successful amplification was achieved mostly using primers derived from the more conserved sequences at the 3′ end of TERT genes (Table S3). As the first screening experiment, we applied this approach to six diploid Nicotiana species investigated as representatives of parental genome donors, including N. sylvestris as a control, and to nine polyploid Nicotiana species (Figure 1). Among parental diploids, we detected one TERT variant in N. alataN. attenuataN. undulataN. wigandoidesN. paniculata and N. obtusifolia (Supplementary A1), and two TERT variants (TERT_C and TERT_D) in N. sylvestris [30]. In the case of N. attenuata and N. obtusifolia, species representing parents of polyploid sections Polydicliae and Repandae, we further confirmed our results by in silico analysis using genome assemblies (GenBank accessions: GCA_001879085.1 and GCA_002018475.1, respectively). To complete the set of representative parental species, we assembled available transcriptomic SRA data of N. noctiflora (GenBank accession: SRR2106514) and identified one TERT variant. In conclusion, our results show the presence of more than one TERT variant in diploid N. sylvestris [30], an exception among parental species of Nicotiana polyploids.
    Figure 2. Experimental identification of TERT variants and analysis of gene expression in Nicotiana polyploids. (A) Conserved telomerase specific motifs (T2, NLS, CP, QFP, T) and reverse transcriptase motifs (1, 2, A–E) are highlighted in protein and mRNA of Nicotiana TERT (modified from [30]). Positions of primers used for screening experiments (blue arrows) and TERT-variant-specific primers (black arrows) are indicated at corresponding TERT mRNA regions (primers are listed in Table S2). The triangle within exon 9 shows the position of a 102 nt long deletion that was identified in N. repandaN. nesophila and N. stocktonii and represents a specific TERT-variant of N. obtusifolia origin. (B,C) Validation of primer specificity for TERT variants in N. repanda (B) and N. nudicaulis (C). PCR products amplified with primers 9exF1 and 9exR1 show two bands corresponding to TERT_O and TERT_Cs variants that differ by a 102 bp long deletion. Specific amplification of TERT_O and TERT_Cs variants was demonstrated using the 9exF1 primer in combination with variant-specific reverse primers 9exR_Nrepa/O and 9exR_Nrepa/S, respectively. (C) For validation of qPCR primers and to distinguish three TERT variants in N. nudicaulis, the PCR products amplified with indicated qPCR primer combinations were digested with MseI. A specific cut of the TERT_O variant that possesses the restriction site for MseI within the amplified region confirmed the specificity of amplified TERT-variants. (D) Relative mRNA levels of specific TERT variants were determined by RT–qPCR in N. nudicaulisN. repanda and N. rustica. Relative mRNA abundance of particular parental TERT variants (in %) was calculated by the delta Ct method [37]. Ct values were normalized using the reaction efficiency calculated from a standard curve analysis (Table S3).
    The same experimental approach applied to representative Nicotiana polyploids detected variant-specific SNPs and/or indels, demonstrating the presence of two TERT variants in 5 of 9 polyploid species investigated (N. arentsiiN. rusticaN. repandaN. nesophilaN. stocktonii) and three variants were identified in N. nudicaulis (summarized in Figure 1Table 1, see below for details). While PCR products obtained from N. clevelandii, N. quadrivalvis and N. benthamiana genomic DNA revealed the presence of a single copy of the TERT gene, our search for TERT variants in raw transcriptomic data from N. clevelandii showed the occurrence of two gene variants. To avoid possible errors in comparison of experimental and in silico data that could be caused, e.g., by possible incorrect mapping of TERT reads to the raw genome/transcriptome data, assembly version or allele sequence, we analyzed in detail individual SNPs in sequences from each polyploid species and its progenitor diploids (see Supplemental Text S1Figure S1Table S4). Results deduced from sequence similarity (in %, Table 1) and individual SNPs (Table S4) were in agreement in all cases analyzed.
    Table 1. Origin of telomerase reverse transcriptase (TERT) variants in polyploid Nicotiana species determined by sequence similarity with representative progenitor diploids.
    Allopolyploids GeneBank
    Accessions
    Sequence Similarity [%] Analyzed Region 1
    Maternal Parent Paternal Parent
    SUAVEOLENTES   N. alata N. noctiflora2 N. syl. C var. N. syl. D var.  
    N. benthamiana NbS000104 96.3 n.a. 97.5 n.a. exon 4 to 5
    27g0116.1 n.a. 96.1 97.6 93.4 exons 10, 11, 12
    REPANDAE   N. syl. C var. N. syl. D var. N. obtusifolia  
    N. repanda MG242402 1 95.9 91.6 97.4 exon 9
    MG242403 1 97.9 92.4 96.4 exon 9
    N. stocktonii MG242407 1 95.6 91.7 97.6 exon 9
    MG242408 1 98.6 93.1 97.0 exon 9
    N. nesophila MG242405 1 95.2 91.6 97.0 exon 9
    MG242406 1 98.5 92.9 96.9 exon 9
    N. nudicaulis MG242409 1 98.6 94.3 94.4 exon 10 to 12
    MG545647 1 92.8 94.8 91.6 exon 10 to 12
    MG242410 1 94.2 93.3 96.3 exon 10 to 12
    POLYDICLIAE   N. obtusifolia N. attenuata  
      MG242422 1 94.3 99.3 exon 4 to 5
    N. clevelandii var1 2 97.3 98.9 exon 9 2
      var2 2 99.2 97.3 exon 9 2
    N. quadrivalvis MG242423 1 94.9 98.6 exon 4 to 5
    ARENTSII   N. undulata N. wigandiodes  
    N. arentsii MG242418 1 99.5 98.4 exon 9
    MG242419 1 98.8 99.8 exon 9
    RUSTICA   N. paniculata N. undulata  
    N. rustica MG242413 1 100.0 98.2 exon 9
    MG242414 1 98.2 99.8 exon 9
    1 all sequences cloned in this work are in Supplementary A1, including corresponding sequences cloned from progenitor diploids; 2 regions mapped to raw RNAseq data or extracted from genome assembly (Supplementary A1).

    3. Origin of TERT Genes in Polyploids with the Ancestral N. sylvestris Donor Genome

    An N. sylvestris progenitor is considered to be a progenitor diploid of the allopolyploid sections Suaveolentes, Repandae and Nicotianae (Figure 1). The evolutionary history of TERT associated with allopolyploidy is inferred for each of these sections.

    3.1. Suaveolentes

    In the section Suaveolentes, we used the model plant N. benthamiana as a representative species of the section, and N. alata and N. noctiflora as recent relatives of the putative maternal lineage originating from sections Alatae or Noctiflorae, respectively (Figure 1, [31][32][38]). We detected a single copy of the N. benthamiana TERT experimentally, and this result was confirmed in silico using (i) an N. benthamiana genome assembly based on deep sequencing (N. benthamiana Genome v1.0.1) and (ii) analysis of raw genomic NGS reads [39] by BLAST followed by read-mapping back to the query. Comparison of corresponding regions of N. benthamiana TERT and representative parental TERT sequences (Table 1Table S4) revealed that the N. benthamiana TERT sequence (accession number NbS00010427g0116.1) was more similar to N. sylvestris TERT_C variant than to the TERT sequence cloned from N. alata (GenBank accession MG242421) or deduced from N. noctiflora SRA data (Supplementary A1). Thus, we conclude an N. sylvestris origin of N. benthamiana TERT and a loss of the second parental TERT copy during the evolution of N. benthamiana.

    4. Discussion

    To test experimentally and in silico TERT gene balance following ancient polyploidization events, we identified and characterized TERT copies in genomes of polyploid Nicotiana species and representatives of their diploid progenitors. We also investigated the expression of TERT variants identified in the polyploids using RT–qPCR. We found that the N. sylvestris progenitor was a very successful parent of sections Suaveolentes, Repandae and Nicotianae because the TERT_Cs variant of N. sylvestris origin was identified in all polyploid genomes investigated (Figure 1), and high levels of its transcripts were detected. Moreover, an additive occurrence of TERT copies observed in N. tabacum and N. nudicaulis suggests that gene/genome duplication resulting in the formation of TERT_C and TERT_D variants in N. sylvestris had occurred at least before the formation of the section Repandae. The TERT_D transcripts were detectable but heavily under-represented in N. nudicaulis (Figure 2D), similar to TERT_D expression in N. sylvestris and N. tabacum [30][36]. In contrast to the success of the N. sylvestris progenitor, the TERT_O variant of N. obtusifolia origin was pseudogenized in all four polyploid species from Repandae. A 102-nt-long in-frame deletion within exon 9 would shorten the linker region between motif 2 and motif A, including protein motif GSSVF that is well-conserved in plant TERTs. This region, termed as motif 3 in human TERT, was found to be crucial for telomerase catalytic functions [40]; however, its absence is not the only problem in TERT_O variants. Various indels found across TERT_O variants from Repandae would result in out-of-frame mutations, and interestingly, a nucleotide transition found within motif C of N. nudicaulis TERT_O would disrupt one of three Asp residues that are essential for the catalytic function of any telomerase [28][29]. However, mRNA levels of the TERT_O variant revealed expression comparable to the TERT_Cs variant in N. nudicaulis and N. repanda (Figure 2D). Comparable transcript levels of parental TERT variants coding for the functional TERT protein were detected in the relatively young polyploid, N. rustica (TERT_P and TERT_U, Figure 2D), and similarly in N. tabacum [36].
    Our experimental analyses were accompanied by an in silico approach to answering the question on the origin and fate of the TERT_D variant in the N. sylvestris genome and, for a wider perspective, in other polyploid plant genomes. Our experimentally estimated ratio 1:1 of TERT_C and TERT_D gene copies in five N. sylvestris accessions was confirmed by in silico analysis of raw data from the N. sylvestris genome sequencing project (Table 2). Moreover, we identified a part of the TERT_D variant sequence (TERT_12D) associated with high-copy repetitive sequences, and the MtATPO gene, within a novel genomic locus in the N. sylvestris genome. An unplaced genomic scaffold arranged similarly to the TERT_12D locus was identified in N. tabacum, suggesting that an ancestral split of the TERT_D copy had occurred at least before the formation of N. tabacum. There is no information about a species-specific WGD event or an additional genome donor in N. sylvestris, but the increase in transposable elements and repeats was reported [41]. Moreover, activation of transposable elements was observed as a stress response to genome instability that may have been caused by a polyploidization event or environmental stress [41][42]. We presume, therefore, that the ancestral TERT_D locus (including TERT_12exD and MtATPO) originated as a result of gene/segment duplication of the TERT_C (plus MtATPO) locus or vice versa (Supplemental Text S1Figure S2). Both loci were pseudogenized—the TERT_C locus within the MtATPO region and the TERT_D locus within the TERT region—and later, the TERT_D locus was split and translocated by Ogre/SD1-I. Currently, the mutual positioning of TERT_C, TERT_D and TERT_12exD within the genome of N. sylvestris is not known; however, similar scenarios could have resulted in pseudogenization and/or neofunctionalization of an additional TERT gene copy that we found in diploid species Populus trichocarpaVigna radiata and Mimulus guttatus. These TERT-like sequences may illustrate possible scenarios leading to the formation of TERT pseudogene variants in Nicotiana and the progression of gene elimination after gene/genome duplication: (i) A large-scale segment/genome duplication event had created an additional TERT locus, presumably encoding a TERT pseudogene on chromosome 1 in Populus, (ii) two TERT copies placed on the same scaffold in Mimulus. (iii) A completely different arrangement comprising an additional TERT variant of Vigna radiata that is formed by two adjacent inverted copies of exon 9 of TERT, and this TERT-like sequence was annotated as ncRNA (summarized in Figure S2). Multiple TERT copies were present in some, but not all polyploid species investigated, and toleration of more TERT copies after young polyploidization events is obvious (Figure 4).
    Table 2. Number of TERT gene copies in Nicotiana species determined in silico.
    Species/Genome Dataset Accession Total No. of TERT Reads Expected Genome Coverage (Depth) No. of Detected TERT Variants Read Counts Corresponding to Known TERT Variants Ratio of TERT Variants in Genome
    N. tabacum SRX338107 1259 35× 3 NtTERT_Cs NtTERT_D NtTERT_Ct 1:1:1
    425 424 410
    N. sylvestris ERX248848 644 26× 2 NsTERT_C NsTERT_D 1:1
    332 312
    N. tomentosiformis ERX248865 203 15× 1 NtomTERT   -
    203  
    N. benthamiana (raw data from [39]) 286 20× 1 NbenTERT   -
    Regarding the origin and evolution of the TERT loci in eudicots, comparison of eudicot phylogeny relationships [43] with the occurrence of syntenic loci that adopted the TERT gene demonstrated ancient synapomorphies, i.e., loci preserved in current genomes are assumed to have been present in their most recent common ancestor (nodes are depicted in Figure 4). The eudicot-like synteny locus emerged in early eudicots (Amborella) and adopted the TERT gene later in the ancestral parent of Nelumbo. The original Amborella TERT locus was probably fragmented. Another translocation of the TERT gene into novel loci grouped in all investigated malvids and Malphigiales (in fabids), and further translocations to other loci, took place later on. Interestingly, in several cases, we detected a translocation into loci that had already existed in ancestral genomes for a long time, e.g., the locus with the Citrus-like synteny originated in early eudicots, as assumed from the locus synapomorphy. The first TERT translocation from a locus with eudicot-type synteny was not caused by locus fragmentation because these loci occur in current eudicots (Figure 3B), the only exception being A. thaliana. Moreover, destabilization of the TERT position within the eudicot-like synteny locus was probably not caused by gene rearrangement because the predicted TERT gene structure with 10 exons is specific for Populus and does not occur in other Malphigiales, and TERTs with 13 exons were found in Solanaceae [30] that share eudicot-like synteny. Thus, it could be speculated that the successful TERT translocation event was more likely into target loci that show ancient synapomorphy (Figure 4). The only exceptions from this observed pattern are the Theobroma and Gossypium loci that were not syntenic to other genomes. This could indicate that these species-specific translocations are relatively recent.
    Figure 3. Occurrence of TERT syntelogs in Angiosperms. (A) Detailed analysis of the eudicot-like type of synteny (represented by Vitis syntelog as a query) in indicated genomes shows the presence of syntenic regions with/without the TERT gene in the majority of investigated eudicots and Amborella. The number of syntelogs and synteny categories are shown for each species. (B) The occurrence of conserved syntenic regions corresponding to the species-specific TERT query was investigated in representative genomes. Co-occurrence of syntelogs in more species suggests an ancient origin of target loci that accommodated TERT in current species. Analyses were carried out using CoGe, GEvo and SynFind. SynFind parameters—algorithm: last; Gene window size: 30; minimum number of genes: 5.
    Figure 4. Origin of syntenic TERT loci in angiosperms. An ancestral locus with eudicot-like synteny that is present in the Amborella genome adopted the TERT gene in basal eudicots (Nelumbo). This ancestral locus with eudicot-like synteny occurs within the genomes of eudicots with the exception of the model plant Arabidopsis thaliana (for simplicity, diagrams on right panels show occurrence and arrangement of loci with eudicot-type synteny and with a specific synteny if present). The TERT gene was translocated several times into novel loci with a conserved synteny observed in current species (termed here as Citrus-like, Populus-like, Eucalyptus-like, Ricinus-like, Arabidopsis-like syntelogs) that had already occurred in ancestors (nodes depicting synapomorphic relationships of specific synteny-types and thus the putative origin of ancestral syntenic loci are shown above respective phylogeny nodes). As an exception, Malpighiales (TheobromaGossypium) show the TERT gene translocated into novel species-specific loci. These genomes nevertheless still contain the ancient loci with conserved synteny (details in Figure 3). TERT is mostly maintained as a single copy gene, but polyploid species can tolerate more copies (M. luteusG. maxG. hirsutum are shown as representatives). Copies of genomic loci with the original synteny remain present after TERT gene elimination, e.g., in Actinidia chinensis, where it is difficult to distinguish which of the ohnologous loci (ohnologs = paralogs derived by WGD) have lost their TERT gene copy (see Supplemental Text S1Figure S2). Phylogeny was adapted from APG IV [43], WGDs were mapped according to [7] in eudicots, and according to [44] in Actinidia.
    In conclusion, our results show that natural Nicotiana polyploids tolerate more TERT copies and, similarly to other polyploid genomes investigated, retention of various copies is obvious in species formed by young polyploidization events. A comparison of TERT locus arrangement in current genomes suggests that the TERT gene was placed in a conserved locus that became advantageous following the emergence of basal eudicots (Figure 4). The gene was relocated later in several plant groups where only a narrow syntenic relationship restricted to closely related species could be found. Various evolutionary scenarios took place in ancestral genomes with multiple TERT copies resulting in elimination, pseudogenization and/or fragmentation, and neofunctionalization of novel TERT copies that could also illustrate the origin and fate of N. sylvestris and polyploid Nicotiana TERT variants (Figure S2).

    This entry is adapted from 10.3390/ijms22041783

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