Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(2): 69-79, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/cayologia-232 Citation: A. Vangelisti, G. Usai, F. Mascagni, L. Natali, T. Giordani, A. Cavallini (2019) A whole genome anal- ysis of long-terminal-repeat retrotrans- poson transcription in leaves of Popu- lus trichocarpa L. subjected to different stresses. Caryologia 72(2): 69-79. doi: 10.13128/cayologia-232 Published: December 5, 2019 Copyright: © 2019 A. Vangelisti, G. Usai, F. Mascagni, L. Natali, T. Giorda- ni, A. Cavallini. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. A whole genome analysis of long-terminal- repeat retrotransposon transcription in leaves of Populus trichocarpa L. subjected to different stresses Alberto Vangelisti#, Gabriele Usai#, Flavia Mascagni#, Lucia Natali, Tommaso Giordani*, Andrea Cavallini Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, I-56124 Pisa, Italy # These authors contributed equally to this study * Corresponding author: tommaso.giordani@unipi.it Abstract. Long terminal repeat retrotransposons have a main role in shaping the struc- ture of plant genomes. We used available genomic resources to study as several factors affect the expression of long terminal repeat retrotransposons in Populus trichocarpa. Such factors included redundancy of a retrotransposon in the genome, chromosomal localization, “genotype” of the retrotransposon, and changes in the environment. Over- all, we identified and annotated 828 full-length retrotransposons, and analyzed their abundance in the genome. Then, we measured their expression in leaves of plants subjected to several stresses (drought, cold, heat, and salt) as well as in control plants. Our analyses showed that the expression of retrotransposons was generally low, espe- cially that of abundant elements. The transcription of an element was found to be only slightly dependent on its chromosomal localization, rather it depended on the super- family and the lineage to which the retrotransposon belonged. Finally, some retrotrans- posons were specifically activated by different environmental stresses. Keywords. LTR-retrotransposons, retrotransposon expression, retrotransposon abun- dance, Illumina cDNA libraries, Populus trichocarpa. INTRODUCTION Transposable elements are mobile DNA sequences, which are abundant and widespread in all eukaryotic genomes. They can change their position on chromosomes by a mechanism, called transposition, driven by enzymes encoded by the element itself. Transposable elements can be divided between retrotransposons (REs, Class I) and DNA transposons (Class II), according to their transposition mechanism (Wicker et al. 2007). The transposition of REs occurs through a “copy and paste” replica- tive mechanism that includes the transcription of an RNA intermediate fol- lowed by its retro-transcription and insertion into the genome (Wicker et al. 70 Alberto Vangelisti et al. 2007). This transposition mechanism has allowed REs to become the largest portion of most eukaryotic genomes, often represented by many thousands of copies (San- Miguel et al. 1998; Vicient et al. 1999). A retrotransposon can be classified as LTR- or not LTR-RE, according to the presence of long termi- nal repeats (LTRs) at its ends. As for the LTR-REs, the promoter elements, the polyadenylation signals and the expression enhancers are found in the LTRs. These domains regulate the transcription of the element (Ben- netzen 2000). In the coding portion of the LTR-REs, Gag and Pol domains can be found. Gag encodes virus-like particles, Pol encodes the enzymes necessary to produce new cDNA molecules from the RE transcripts and to integrate them into new sites in the host genome (Ben- netzen 2000). Other structural features involved in the RE replication process include a primer binding site and a poly-purine tract (Bennetzen 2000). The LTR-REs are essentially subdivided into two superfamilies, Gypsy and Copia (Wicker et al. 2007), according to the order of gene sequences within the Pol domain. Superfamilies, in turn, are distinguished into several lineages in relation to sequence conservation and structure (Barghini et al. 2015a; Usai et al. 2017; Buti et al. 2018; Mascagni et al. 2017; 2018a). The replicative activity of LTR-REs can determine large variations in the genome structureof eukaryotic species (Springer et al. 2009; Vitte et al. 2014). Among the effects of retrotransposition, besides determining changes in genome size, RE-related structural variations can modify the regulation patterns of protein-encoding genes and, consequently, their activity, influencing the phenotype (Slotkin and Martienssen 2007; Butelli et al. 2012; Falchi et al. 2013; Lisch 2013). The first phase of retrotransposition is represented by the transcription of the element. The RE transcripts can be capped and polyadenylated or not. In the former case, transcripts should be destined to be translated into the enzymes for retrotransposition, in the latter case, transcripts should be reverse-transcribed (Chang et al. 2013; Meignin et al. 2003). Transcription of REs has been described in several plant species (Grandbastien 2015). In some grass species LTR-REs are poorly constitutively transcribed (Vicient et al. 2001; Ishiguro et al. 2014). In other species, for example in Populus x canadensis, certain LTR-REs are expressed constitutively, without apparent induction conditions (Giordani et al. 2016). Retrotransposition is completed when a new copy of the element is inserted into the genome. This has been reported for Tnt1 and Tto1 elements of Nicotiana and for Tos17 of rice, induced by tissue culture (Grandbastien 1998). Complete retro- transposition of a Copia RE has been described also in sunflower seedlings, grown under standard conditions (Vukich et al. 2009). Retrotransposition is generally limited by the host genome due to its potentially mutagenic action. A major mechanism to inactivate mobile elements involves the methylation of histones and cytosine residues with con- sequent silencing of chromatin (Dieguez et al. 1998). Post-transcriptional silencing by RNA degradation also plays an important role in the epigenetic control of RE activity (Slotkin and Martienssen 2007; Lisch 2013; Ito 2013). In recent years, many studies have been carried on the LTR-REs of the genus Populus and in particular on P. trichocarpa, which is considered a model species for forest trees. The P. trichocarpa genome was the first genome to have been sequenced for a forest species (Tus- kan et al. 2006) and has been recently updated (Zeng et al. 2017). This species has a relatively small genome (550 Mbp) and REs cover approximately 176 Mbp (32% of the genome), with a prevalence of Gypsy over Copia RE sequences (Tuskan et al. 2006). Populus trichocarpa REs have been identified and annotated according to their superfamily and lineage, and LTR-RE genomic abun- dance and age of insertion were analyzed as well (Natali et al. 2015; Mascagni et al. 2018b). P. trichocarpa LTR- REs have been also used as a reference for several analy- ses related to the repetitive component in other species of the genus Populus (Giordani et al. 2016; Usai et al. 2017). The transcription of REs is only the first step for ret- rotransposition and insertion of new copies of the ele- ment in the genome. For this reason, analyses on LTR- RE activity should include searching for new insertion events. However, an overall study of factors potentially able to influence the transcription of these elements is not yet available for poplar. We therefore decided to perform a meta-analysis of LTR-RE expression by using publicly available genomic DNA and cDNA libraries obtained from leaves of plants cultivated under stand- ard conditions or subjected to four types of abiotic stress (cold, drought, heat, and salt). The objectives of this work were to evaluate i) the expression level of REs under standard and stress conditions; ii) the correlation between abundance of REs and their expression level; iii) the possibility that different LTR-REs are induced by dif- ferent (and specific) stresses; iv) the possibility that the expression of a RE is related to the “genotype” of the RE itself, i.e., to the lineage to which it belongs; v) the pos- sibility that the chromosomal localization of a RE can influence its expression. 71Genome analysis of long-terminal-repeat retrotransposon transcription in leaves of Populus trichocarpa METHODS Isolation of full-length LTR-REs of P. trichocarpa Putative full-length LTR-REs were isolated from the GCA_000002775.3 version (Zeng et al. 2017) of the P. trichocarpa genome sequence (Tuskan et al. 2006; Sla- vov et al. 2012), deposited at the NCBI site (WGS pro- ject number AARH02, http://www.ncbi.nlm.nih.gov/ assembly/GCF_000002775.3). Full-length LTR-REs were isolated by using: i) LTRharvest (Ellinghaus et al. 2008) with the following parameters: minlenltr=100, maxlen- ltr=6000, mindistltr=1500, maxdistltr=25000, mintsd=5, maxtsd=5, similar=85, vic=10, including the presence of TG and CA dinucleotides at 5’ and 3’-ends, respectively; ii) LTR-FINDER (Xu et al. 2007), under default. A random sample of putative LTR-REs (around 20% of the isolated elements) were manually validated using DOTTER (Sonnhammer and Durbin 1995) to verify the occurrence of the two LTRs, of dinucleotides TG and CA at the respective 5’ and 3’ ends, and of the tandem site duplications. All LTR-REs were annotated by using BLASTN search against plant RE datasets (Barghini et al. 2015b; Natali et al. 2015; Usai et al. 2017; Buti et al. 2018) and by using the Domain Search tool of RepeatEx- plorer (Novak et al. 2013). Whenever possible, the full- length LTR-REs were identified as belonging to Gypsy or Copia superfamilies and to the respective lineages. A multi-FASTA file with the sequences of identified full-length LTR-REs is available at the sequence reposi- tory site of the Department of Agriculture, Food and Environment of the University of Pisa (http://pgagl.agr. unipi.it/sequence-repository/). Illumina cDNA libraries collection The expression of LTR-REs was analyzed using Illu- mina cDNA paired-end libraries publicly available at the NCBI SRA (https://www.ncbi.nlm.nih.gov/sra/, BioPro- ject accession PRJEB19784) (Filichkin et al. 2018). Such cDNA libraries were obtained from RNAs from leaves of P. trichocarpa (clone Nisqually 1) plants exposed to different stresses, i.e., heat, cold, drought, and salt. All cultivation conditions are described by Filichkin et al. (2018). In brief, for heat stress, plants were treated at 39°C for 12 h (short treatment) or 7 days (prolonged treatment). For cold stress, plants were exposed to cycles of 4°C (night)/12°C (day) for 24 h (short treatment) or 7 days (prolonged treatment). For drought treatment, watering was withheld until soil moisture reached 0.1 m3/m3 and maintained at the level of 0.06 –0.1 m3/m3 for 5 days (short treatment) or for 12 days after water withholding (prolonged treatment). For salt stress, plants were irrigated with 100 mM NaCl solution for 24 h (short treatment) or for 7 days (prolonged treatment). Three replicate libraries were downloaded for each stress and control plants. Illumina genomic DNA sequences of the same clone of P. trichocarpa were retrieved from the NCBI Sequence Read Archive (NCBI, Washington, USA, https://www. ncbi.nlm.nih.gov/sra, SRA ID SRR1801106). The quality of the cDNA and genomic DNA reads was checked using FastQC (v. 0.11.3) (http://www.bio- informatics.babraham.ac.uk/projects/fastqc/), and the overall quality was improved by removing Illumina adapters and trimming the reads using Trimmomatic (v. 0.38) (Bolger et al., 2014) with different param- eters for cDNA (ILLUMINACLIP:2:30:10, SLIDING- WINDOW:4:20, CROP:96, HEADCROP:12 and MIN- LEN:90) and genomic DNA (ILLUMINACLIP:2:30:10, SLIDI NGW I N DOW:4:15, CROP:85, M I N L EN:85). Organellar sequences were removed from the Illumina libraries by mapping against a database of chloroplast genomes of poplar species (Usai et al. 2017) using CLC- BIO GenomicWorkbench (v. 9.5.3, CLC-BIO, Aarhus, Denmark) with the following parameters: mismatch cost 2, insertion cost 3, deletion cost 3, length fraction 0.5, similarity fraction 0.8. All matching reads were considered putatively belonging to organellar genomes and removed. Estimation of retrotransposon expression and abundance in the genome The expression of LTR-REs was measured map- ping cDNA sequence reads of control and cold-, drought-, heat-, or salt-exposed leaves onto the library of P. trichocarpa full-length LTR-REs, using CLC-BIO Genomic Workbench with the following parameters: mismatch cost 1, deletion cost 1, insertion cost 1, simi- larity 0.9 and length fraction 0.9. The expression level of each sequence was calculated and converted both to mapped reads per million (MRpM) and to RPKM (Mor- tazavi et al. 2008). LTR-REs mapped by 1 to 10 reads per million of reads in at least one sample were considered as expressed (Lu et al. 2013), those mapped by at least 10 reads per million were considered as highly expressed. Expression values were compared, using Baggerley’s test (Baggerley et al. 2003), considering RPKM values in the short and prolonged stage of each treatment in com- parison to control leaves. The weighted proportion fold changes between a treatment and controls were consid- ered significant when the weight of a sample was at least two-fold higher or lower than another, with a false dis- 72 Alberto Vangelisti et al. covery rate (FDR; Benjamini and Hochberg, 1995) cor- rected p-value ≤ 0.05. In order to assess genomic abundance of REs, genomic DNA reads of P. trichocarpa were mapped onto reference retrotransposon domains library using CLC- BIO Genomics Workbench with the same parameters described above. For each LTR-RE the average coverage was calculated. The average coverage is the sum of the bases of the aligned parts of all the reads divided by the length of the reference sequence. Localization of expressed REs along the poplar genome Each of the 19 linkage groups (LGs) of the cur- rently available P. trichocarpa genome sequence (version GCA_000002775.3, Zeng et al. 2017) were subdivided into 3-Mbp-long genome regions. Then, in order to local- ize LTR-RE sequences in the genome, the LTR-REs were used for masking the 3-Mbp-long fragments of the pop- lar genome using RepeatMasker (http://www.repeatmas- ker.org) with the following parameters: s, no-is, no-low. Masking was performed using i) all isolated full-length elements; ii) all Chromovirus LTR-REs; iii) a putative poplar centromeric sequence (Islam-Faridi et al. 2009; Cossu et al. 2012); iv) all LTR-REs expressed in control leaves (mapped by more than ten reads per million). The number of masked bases was then counted for each of the 3 Mbp fragment using an in-house perl script. RESULTS Identification of full-length LTR-REs of P. trichocarpa The full-length LTR-REs used in this study were iso- lated from the updated genome sequence of P. trichocar- pa (Zeng et al. 2017), by performing a complete genome scan with LTRharvest and LTRFinder. Besides using these tools with stringent parameters, a sample of isolat- ed elements were manually validated at structural level and all were confirmed as LTR-REs. The dataset includes 828 full-length LTR-REs. Table 1 reports the number of LTR-REs belonging to the Gyp- sy and Copia superfamilies, subdivided according to the lineage to which they belong, i.e. Athila, Ogre and Chromovirus for Gypsy elements and Ale (distinguished into AleI and AleII), Angela, Bianca, Ivana/Oryco, SIRE and TAR/Tork for Copia elements. For each lineage the mean average coverage is also reported, calculated after mapping elements with Illumina gDNA reads, which represents the mean abundance of that lineage in the P. trichocarpa genome. Transcription of LTR-REs The expression of 828 full-length LTR-REs was measured by mapping the elements with Illumina cDNA reads obtained from leaves of plants of P. trichocarpa cultivated in standard conditions (controls) and under different stress (drought, heat, cold, or salt). In the con- trol leaves, only 0.47% of the cDNA reads mapped the library, hence, in general, LTR-REs are barely expressed (Fig. 1). The expression level of LTR-REs decreased with stress, in the decreasing order drought-cold-heat-salt (Fig. 1). No significant difference was observed between short and prolonged treatments, with the exception of cold treatment, where expression decreased reduced in prolonged exposition. According to Lu et al. (2013), we considered as expressed those LTR-REs mapped by more than one read per million. The number of expressed LTR-REs in controls and in drought-, cold-, heat- and salt-exposed leaves is reported in Fig. 2. The number of LTR-REs expressed in drought-treated leaves is similar to that of control leaves. On the contrary, this number is strongly reduced after the other treatments (Fig. 2). However, the Table 1. Number and mean average coverage of full-length LTR-REs collected in the P. trichocarpa genome (version GCA_000002775.3) and separated according to their superfamily and lineage. Super-family Lineage Nr. of elements Mean average coverage Copia AleI 42 14.04 AleII 122 22.33 Angela 2 64.13 Bianca 1 28.24 Ivana/Oryco 104 19.06 SIRE 7 42.11 TAR/Tork 90 17.45 Total 368 19.89 Gypsy Athila 126 57.37 Chromovirus 174 40.20 Ogre 67 41.16 Unknown 50 13.46 Total 417 42.34 Unknown 43 22.65 Total 828 31.34 73Genome analysis of long-terminal-repeat retrotransposon transcription in leaves of Populus trichocarpa number of expressed LTR-REs increased after prolonged salt treatment. The relationship between the abundance of a retrotranspo- son in the genome and its expression In another analysis we measured the relationship between the abundance of a LTR-RE in the genome and the corresponding expression level. Such data are report- ed for control leaves in Fig. 3. It can be observed that abundant LTR-REs (average coverage > 100) are lowly expressed. Similar results were also found in leaves of plants exposed to different stresses (not reported). Since assessing the expression of a LTR-RE is based on the occurrence of LTR-RE sequences in cDNA librar- ies, one might ascribe such occurrence to genomic DNA contamination. Actually, since the most abundant LTR- REs resulted slightly or even not expressed, contami- nation by genomic DNA in the cDNA libraries can be largely ruled out. Influence of chromosomal localization on retrotransposons expression In order to verify whether active LTR-REs were localized at specific chromosomal sites, we aligned LTR-RE sequences, highly expressed (MRpM > 10) in the control leaves, to the genome of P. trichocarpa (Fig. 4). For comparison, we separately aligned the genome with all the 828 LTR-REs; furthermore we determined the putative position of the centromere on each linkage group (LG) by aligning a tandemly repeated centromeric sequence of P. trichocarpa (Islam-Faridi et al. 2009) and Chromovirus elements (which preferentially localize at centromeres, Neumann et al. 2011). The chromosomal profiles of all the LTR-REs and highly expressed LTR- REs were substantially similar (Fig. 4). In some cases, minor peaks in the general LTR-REs profiles were appar- ently absent in the expressed LTR-REs profiles, suggest- ing that elements at those loci were generally inactive. Fig. 1. Total number of mapped reads (per million of reads) on the 828 full-length LTR-REs of P. trichocarpa, in leaves of control and stress-exposed plants. Stresses included cold, drought, heat and salt treatments, for short and prolonged times. The differences between control and each treatment and between short and prolonged stress treatments were significant at p<0.001 (***), p<0.01 (**), p<0.05 (*), or not significant (n.s.) according to Tukey’s test. Fig. 2. Number of expressed (MRpM > 1) LTR-REs in leaves of control and stress-exposed plants. Stresses included cold, drought, heat and salt treatments, for short and prolonged times. 74 Alberto Vangelisti et al. Only two peaks (within LG I and LG XIX) were appar- ently more evident in the expressed LTR-REs profiles, indicating that REs at these loci were particularly active. In general, it can be observed that peaks in putative centromere positions were less evident in the expressed LTR-RE profiles, suggesting that centromere LTR-REs were less active than elements lying at other loci (Fig. 4). Influence of the superfamily/lineage of the retrotransposon on its expression In order to assess whether the expression of LTR- REs was related to the superfamily/lineage to which the element belonged, LTR-REs were subdivided into line- ages and separated among highly expressed (i.e., mapped by more than 10 reads per million), expressed (1-10 mapped reads per million) and not expressed (less than 1 mapped read per million). Gypsy elements resulted more expressed than Copia, in fact 48 out of 417 Gypsy REs (11.5%) were expressed, compared to 16 out of 368 Copia REs (4.4%). In general, most lineages showed low percentages of highly expressed or expressed LTR-REs (Fig. 5). However, for two Copia lineages (SIRE and TAR/Tork) and one Gypsy lineage (Ogre) the majority of LTR-REs resulted highly expressed or expressed (Fig. 5). In particular, the Ogre lineage showed the highest per- centage of expressed elements. Diffused LTR-RE expres- sion was also observed for those elements belonging to the Gypsy superfamily, but for which the lineage could not be determined. Fig. 3. Relationship between average coverage and RPKM for each of 828 full-length LTR-REs of P. trichocarpa. Fig. 4. Percentage of aligned nucleotides along P. trichocarpa LGs using all the full-length LTR-REs expressed in control leaves, all isolated full-length LTR-REs, all isolated Chromovirus REs and a putative centromeric sequence (in black). Red arrows indicate the putative position of the centromeres. Black arrows indicate minor peaks which are especially evident in the expressed LTR-REs pro- files or in the profiles of all LTR-REs. Fig. 5. Percentages of highly expressed (MRpM > 10), expressed (MRpM ranging from 1 to 10) and unexpressed (MRpM < 1) LTR- REs, distinguished among LTR-RE superfamilies and lineages. 75Genome analysis of long-terminal-repeat retrotransposon transcription in leaves of Populus trichocarpa Stress-specific induction of retrotransposons expression Most LTR-REs which were expressed in control leaves were also expressed in leaves of stress exposed plants. Of 313 LTR-REs expressed (MRpM > 1) in con- trols and/or in different stresses, only 4 (1.3%) were expressed only in controls and 81 (25.9%) were specifi- cally activated by one or more stresses. Figure 6 reports the number of LTR-REs expressed (i.e. with more than one mapped read per million) after different stresses (in both short and prolonged treatments). Of 264 LTR- REs expressed during one or more stresses, 87 (33.0%) were active under each stress. Fifty-six elements (21.2%) were specifically active during drought treatments, 30 (11.4%) during salt treatments and 35 (13.3%) during both drought and salt stresses, indicating that these treatments were the most effective in inducing LTR- RE expression. On the contrary, cold and heat stresses induced only a limited number of LTR-REs (Fig. 6). We also analysed differential expression of LTR- REs during the different stresses compared to the con- trols. Considering only the 72 highly expressed elements (MRpM > 10), 70 showed differential expression (fold change > 2, FDR-corrected p < 0.05) in at least one treat- ment (Fig. 7). No elements were differentially expressed along all treatments. In most cases, the same LTR-RE was under-expressed (blue in Fig. 7) or unaffected (white in Fig. 7) during the different stresses. Only 7 LTR-REs were induced (red in Fig. 7) or unaffected. Thirteen ele- ments were repressed by certain treatments, activated by other, or unaffected (blue, red, or white in Fig. 7). DISCUSSION Availability of the updated sequence of the P. trichocarpa genome and of genomic DNA and cDNA libraries obtained from plants of the same genotype and subjected to different treatments, allowed us to evalu- ate several factors which can influence the expression of LTR-REs in this species. In general, our data confirmed that the expression of retrotransposons is generally limited: only 72 out of 828 LTR-REs were mapped by more than ten reads per million. The transcription of REs have been reported in tissues and organs of many plant species (Grandbastien 2015), related to biotic and abiotic stresses or even with- out apparent induction. In rice, sunflower, Citrus sin- ensis, and even in poplars certain LTR-REs are actively transcribed (Rico-Cabanas and Martínez-Izquierdo Fig. 6. Venn diagram of expressed (MRpM > 1) LTR-REs in the four stresses used in these experiments (D = drought; H = heat; C = cold; S = salt). Results of short and prolonged treatments were cumulated for each stress. Fig. 7. Differential expression (fold change > 2, FDR-corrected p < 0.05) of LTR-REs after short (S) or prolonged (P) treatments with different stresses compared to controls. Blue cells refer to under- expression, red cells to over-expression, white cells indicate no effect of the treatment in comparison to control. 76 Alberto Vangelisti et al. 2007; Vukich et al. 2009; Gao et al. 2015; Giordani et al. 2016). However, the majority of LTR-REs are barely expressed (Vicient et al. 2001; Ishiguro et al. 2014; Van- gelisti et al. 2019). In some cases, specif ic LTR-R E sublineages have been shown to be activated and possibly over- expressed by different culture conditions, as tissue cul- ture (Kashkush et al. 2003; Liu et al. 2004), wounding, methyl jasmonate and fungal elicitors (Takeda et al. 1999), various phytohormones and cold stress (He et al. 2010, 2012), heat stress (Ito et al. 2013). Hormones, and biotic/abiotic stresses induced a general LTR-RE activa- tion in pine (Voronova et al. 2014; Fan et al. 2014) and in sunflower (Vangelisti et al. 2019). In the present study, as in all previous works, the same treatment up-regulated certain LTR-REs and repressed or unaffected other ele- ments. In P. trichocarpa, the overall RE expression level was higher in leaves of control plants than in those of plants exposed to different stresses, suggesting that plants responded to stresses increasing defence mecha- nisms related to REs. This is different from what found by Vangelisti et al (2019) in roots of sunflower: in this species, the expression level of LTR-REs remained sub- stantially very low but it slightly increased after differ- ent stresses. Although the generally low level of LTR-REs expression, more than 40 elements showed a significant activity (more than 10 mapped reads x million), either in controls and stressed plants, suggesting that they are not silenced and hence may still have mutagenic potential, if retrotranscription and insertion in new sites would occur after expression. The comparison, for each LTR-RE, of the abundance in the genome and its expression in leaves of control or stressed plants, showed that most expressed elements are generally lowly abundant. Such lack of correlation between LTR-RE abundance and transcription is not surprising: other studies showed that the more an ele- ment is repeated the more it is recognized by the RNA silencing machinery (Meyers et al. 2001; Yamazaki et al. 2001; Lisch 2009). Low levels of transcription of repeated sequences are often attributed to DNA contamination of RNA samples. The low expression level of most abundant LTR-REs sug- gested also that the occurrence of retrotransposon-relat- ed reads in the cDNA libraries was not due to DNA con- tamination. Genome localization of highly expressed (MRpM > 10) LTR-REs indicated that, in poplar, the expression of an element is only slightly related to its chromosomal localization, because the profiles of expressed LTR-REs parallels those of all LTR-REs. However, we observed a few specific chromosome regions showing differences between profiles of all the LTR-REs and expressed LTR- REs, suggesting that some regions are specifically acti- vated or repressed. In species with much larger genomes than poplar, as the sunflower, LTR-RE expression was observed in specific genomic regions, relatively distant from putative centromeres, and preferentially located at chromosome ends (Mascagni et al. 2019). Concerning the relationship between expression and superfamily/lineage of the elements, our results showed that expression of Gypsy REs was higher than expres- sion of Copia elements. At lineage level, Ogre LTR-REs were by far the most transcribed elements. Among Copia lineages, the most expressed were SIRE and TAR/Tork, indicating that, besides chromosomal localization and genome abundance, also the “genotype” of the LTR-RE may play a role in its activation. Our results confirmed what previously shown in other studies, since many of the LTR-REs expressed in other species are actually of the Copia superfamily (Ma et al. 2008). In the case of tobacco, both Tnt1 and Tto1 (which are induced by tis- sue culture) belong to the TAR/Tork lineage (Neumann et al. 2019). Gypsy LTR-RE induction was reported in cotton (Hawkins et al. 2006), one of the families ana- lyzed in that study belonged to the Ogre lineage. It can be concluded that, probably, different LTR-RE lineages are specifically activated in different species. It can be assumed that young LTR-REs are more prone to be expressed than older elements, prob- ably because the host needs time to develop defence mechanisms against new elements. Ogre and TAR/Tork elements are the youngest LTR-REs in P. trichocar- pa (Mascagni et al. 2018b): this could explain why these two lineages showed the highest percentages of expressed elements. Although most LTR-REs were expressed at the same level in plants subjected to different treatments, two stresses (salt and drought) specifically induced a num- ber of LTR-REs. No elements were always induced or always repressed by every stress. In some cases, the same element was up-regulated by one stress and repressed by another, probably because of the occurrence, within the LTRs, of cis-regulatory motifs recognized in specific stresses, as those identified in the LTR of the HaCRE1 element of sunflower (Buti et al. 2009). In conclusion, this study outlines a general picture of LTR-RE activity in leaves of poplar plants treated with different stresses. Results allowed us to have a global insight on the features that affect LTR-RE expression. 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Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 72, Issue 2 - 2019 Firenze University Press Karyotype analysis of a natural Lycoris double-flowered hybrid Jin-Xia Wang1, Yuan-Jin Cao1, Yu-Chun Han1, Shou-Biao Zhou1,2, Kun Liu1,* Insights on cytogenetic of the only strict African representative of genus Prunus (P. africana): first genome size assessment, heterochromatin and rDNA chromosome pattern Justine Germo Nzweundji1, Marie Florence Sandrine Ngo Ngwe2, Sonja Siljak-Yakovlev3,* Assessment of cytotoxicity and mutagenicity of insecticide Demond EC25 in Allium cepa and Ames Test Arzu Özkara Cytogenetic effects of Fulvic acid on Allium cepa L. root tip meristem cells Özlem Sultan Aslantürk Evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of Allium test Ioan Sarac1, Elena Bonciu2,*, Monica Butnariu1, Irina Petrescu1, Emilian Madosa1 Fluorescence In Situ Hybridisation Study of Micronuclei in C3A Cells Following Exposure to ELF-Magnetic Fields Luc Verschaeve1,2,*, Roel Antonissen1, Ans Baeyens3, Anne Vral3, Annemarie Maes1 Phytochemical analysis and in vitro assessment of Polystichum setiferum extracts for their cytotoxic and antimicrobial activities Nicoleta Anca Şuţan1,*, Irina Fierăscu2, Radu Fierăscu2, Deliu Ionica1, Liliana Cristina Soare1 Telomeric heterochromatin and meiotic recombination in three species of Coleoptera (Dorcadion olympicum Ganglebauer, Stephanorrhina princeps Oberthür and Macraspis tristis Laporte) Anne-Marie Dutrillaux, Bernard Dutrillaux* A whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of Populus trichocarpa L. subjected to different stresses Alberto Vangelisti#, Gabriele Usai#, Flavia Mascagni#, Lucia Natali, Tommaso Giordani*, Andrea Cavallini Differences in C-band patterns between the Japanese house mice (Mus musculus) in Hokkaido and eastern Honshu Hikari Myoshu, Masahiro A. Iwasa* Karyotypic description and repetitive DNA chromosome mapping of Melipona interrupta Latreille, 1811 (Hymenoptera: Meliponini) Natália Martins Travenzoli1, Ingrid Cândido de Oliveira Barbosa2, Gislene Almeida Carvalho-Zilse2, Tânia Maria Fernandes Salomão3, Denilce Meneses Lopes1,*