Genomic organization and dynamics of repetitive ...

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Genomic organization and dynamics of repetitive DNA sequences in representatives of three Fagaceae genera Sofia Alves, Teresa Ribeiro, Vera Inácio, Margarida Rocheta, and Leonor Morais-Cecílio

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Abstract: Oaks, chestnuts, and beeches are economically important species of the Fagaceae. To understand the relationship between these members of this family, a deep knowledge of their genome composition and organization is needed. In this work, we have isolated and characterized several AFLP fragments obtained from Quercus rotundifolia Lam. through homology searches in available databases. Genomic polymorphisms involving some of these sequences were evaluated in two species of Quercus, one of Castanea, and one of Fagus with specific primers. Comparative FISH analysis with generated sequences was performed in interphase nuclei of the four species, and the co-immunolocalization of 5-methylcytosine was also studied. Some of the sequences isolated proved to be genus-specific, while others were present in all the genera. Retroelements, either gypsy-like of the Tat/Athila clade or copia-like, are well represented, and most are dispersed in euchromatic regions of these species with no DNA methylation associated, pointing to an interspersed arrangement of these retroelements with potential gene-rich regions. A particular gypsy-sequence is dispersed in oaks and chestnut nuclei, but its confinement to chromocenters in beech evidences genome restructuring events during evolution of Fagaceae. Several sequences generated in this study proved to be good tools to comparatively study Fagaceae genome organization. Key words: Fagaceae, Quercus, Castanea, Fagus, retroelements, euchromatin, AFLP. Résumé : Les chênes, les châtaigniers et les hêtres sont des espèces importantes de fagacées au plan économique. Afin de comprendre les relations entre les membres de cette famille, une connaissance approfondie de la composition et de l’organisation de leurs génomes est nécessaire. Dans ce travail, les auteurs ont isolé et caractérisé plusieurs fragments AFLP obtenus chez le Quercus rotundifolia Lam. via des recherches d’homologie dans des bases de données. Les polymorphismes génomiques impliquant certaines de ces séquences ont été évalués chez deux espèces du genre Quercus, une du genre Castanea et une du genre Fagus à l’aide d’amorces spécifiques. Des analyses FISH comparatives ont été réalisées en employant certaines de ces séquences comme sondes sur des noyaux en interphase chez quatre de ces espèces et pour étudier la co-immunolocalisation avec la 5-méthylcytosine. Certaines des séquences isolées se sont avérées spécifiques d’un genre, tandis que d’autres étaient présentes chez tous les genres. Les rétroélément, soit de type gypsy du clade Tat/Athila ou de type copia, sont bien représentés et la plupart sont dispersés au sein de régions euchromatiques chez ces espèces, sans association avec la méthylation de l’ADN, ce qui suggère une dispersion des rétroéléments au sein des régions riches en gènes. Une séquence particulière de type gypsy était dispersée au sein des noyaux chez les chênes et les châtaigniers, tandis qu’elle était confinée aux chromocentres chez les hêtres, ce qui suggère des évènements de restructuration génomique au cours de l’évolution chez les fagacées. Plusieurs séquences générées au cours de cette étude constituent de bons outils pour comparer l’organisation du génome chez les fagacées. Mots‐clés : fagacées, Quercus, Castanea, Fagus, rétroéléments, euchromatine, AFLP. [Traduit par la Rédaction]

Received 20 December 2011. Accepted 10 April 2012. Published at www.nrcresearchpress.com/gen on 20 April 2012. Paper handled by Associate Editor G. Jenkins. S. Alves*, V. Inácio, M. Rocheta, and L. Morais-Cecílio. Centro de Botânica Aplicada à Agricultura, Instituto Superior de Agronomia, Technical University of Lisbon, Tapada da Ajuda, 1349–017 Lisboa, Portugal. T. Ribeiro.* Centro de Botânica Aplicada à Agricultura, Instituto Superior de Agronomia, Technical University of Lisbon, Tapada da Ajuda, 1349–017 Lisboa, Portugal; Centro de Biotecnologia Agrícola e Agro-Alimentar do Baixo Alentejo e Litoral, Escola Superior Agrária, Rua Pedro Soares, 7801-908 Beja, Portugal; Centre for Research in Ceramics & Composite Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal. Corresponding author: Leonor Morais-Cecílio (e-mail: [email protected]). *These

authors contributed equally to this work.

Genome 55: 348–359 (2012)

doi:10.1139/G2012-020

Published by NRC Research Press

Alves et al.


Introduction The Fagaceae family, including oaks (Quercus spp.), chestnuts (Castanea spp.), and beeches (Fagus spp.) among other genera of trees, comprises many of the major hardwoods of the temperate forests of the Northern Hemisphere, and they are the main forest genera in Europe and North America (Kremer et al. 2010). Fagaceae trees are extremely important in their ecosystems, providing environmental functions such as carbon sequestration, energy production, and water cycle regulation, as well as having utility as sources of high quality timber, plywood, charcoal, and cork (Neale and Kremer 2011). The species of these three genera all have the same number of chromosomes (2n = 24) (Ribeiro et al. 2011) and high levels of macrosynteny, as was shown for Castanea sativa and Quercus robur (Casasoli et al. 2006), although their genome sizes vary almost twofold from 544 Mb/1C in Fagus sylvatica to 941 Mb/1C and 980 Mb/1C in C. sativa and Quercus ilex, respectively (Zoldos et al. 1998; Gallois et al. 1999; Barow and Meister 2002). Genome size is associated with specific interphase chromatin organization at least in plants (Dong and Jiang 1998), with the proportion and distribution of different DNA sequences in chromosomes and chromatin domains being directly related with size variation. In plants with small genomes and small chromosomes, such as Arabidopsis thaliana, most of the compact heterochromatin fraction, which maintains its high levels of condensation throughout the cell cycle, is preferentially localized in centromeric regions (Fransz et al. 2002), whereas in plants with large genomes this fraction can be dispersed along the chromosome arms or accumulated in subtelomeric regions (Guerra 2000). Regions of the genome associated with heterochromatin organization are mainly composed of repetitive DNA sequences organized in tandem, in which one copy follows the other in arrays of many tens or even thousands of copies, or which can also be dispersed in the genome (Schmidt and Heslop-Harrison 1998). The dispersed repetitive sequences are likely to be, or have evolved from, transposable elements, especially from retrotransposons with long terminal repeats (LTR-retrotransposons), which are the predominant transposable elements in plant genomes (Zhang and Wessler 2004). LTR-retrotransposons in plants include Ty1-copia and Ty3-gypsy elements, which differ according to the relative position of their domains (Bennetzen 2000) and are differently represented according to the species. Owing to their ‘‘copy and paste’’ mechanism of propagation transposable elements can rapidly increase genome size and accumulate in specific genome locations (Kidwell 2002; Morse et al. 2009; Zedek et al. 2010), resulting in great differences in heterochromatin distribution along chromosomes (Heslop-Harrison and Schwarzacher 2011). While the Ty3-gypsy group is normally found in plant centromeric regions (reviewed in Jiang et al. 2003), Ty1-copia elements accumulate at the heterochromatin of the telomeric position in Allium cepa chromosomes (Pearce et al. 1996). Repetitive DNA therefore varies extensively from species to species in absolute amount, sequence variation, and dispersion pattern. The study of genome organization in terms of repetitive sequences can be made either by molecular or cytological

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techniques, or by a combination of both. DNA based marker techniques such as RFLP (restriction fragment length polymorphism), SSR (simple sequence repeats), and AFLP (amplified fragment length polymorphism) are frequently used for this purpose (Reinisch et al. 1994; Acheré et al. 2005; Legesse et al. 2007). The most widely used repetitive sequences are the rRNA genes, used as cytological fluorescence in situ hybridization (FISH) markers, owing to their high abundance and their tandemly repeated nature. The evolutionary high conservation of rRNA genes make them easily detectable in a wide range of species by use of heterologous rDNA probes (Ribeiro et al. 2008). Nevertheless, other repetitive sequences can also be physically mapped, such as SSRs (Cuadrado et al. 2008) and AFLPs (Reamon-Büttner et al. 1999) among others. Retroelements can also be used for distinguishing between parental genomes in hybrids (Lamb and Birchler 2006). The organization of rDNA sequences (Zoldos et al. 1999; Chokchaichamnankit et al. 2008; Ribeiro et al. 2011), as well as the distribution of heterochromatin (Zoldos et al. 1999), including seven Q. robur-specific sequences (Zoldos et al. 2001), have been studied in several species of the genus Quercus. To our knowledge, apart from these studies, there are no data that describe the composition of repetitive sequences and their relevance for genome differentiation and evolution in the Fagaceae. In this work, we have isolated and characterized several AFLP-derived sequences to study the genomic organization of several Fagaceae species belonging to genera Quercus, Castanea, and Fagus at the molecular and cytogenetic levels. This study will shed light into the genome evolution in the Fagaceae.

Materials and methods Plant material Seeds of Quercus rotundifolia Lam. (Holm oak) and Quercus suber L. (cork oak) both from Alcácer-do-Sal, Portugal, Castanea sativa Mill. (chestnut) from Trás-os-Montes, Portugal, and Fagus sylvatica L. (beech) provided by St. Isidro nursery, Portugal, were germinated and the seedlings grown in a greenhouse (22 ± 2 °C and photoperiod of 16 h) for the collection of root tips and leaves. Root tips were fixed in a fresh ethanol:glacial acetic acid mixture of 3:1 (v/v) and kept at –20 °C; leaves were frozen and kept at –80 °C until used. For meiotic pachytene isolation, immature male flowers of Q. rotundifolia were collected in the field near Lisbon, from several trees, and anthers selected for the desired stage and further fixed in a fresh ethanol:glacial acetic acid mixture of 3:1 (v/v). Genomic DNA extraction Genomic DNA extraction from several Q. rotundifolia and C. sativa trees was performed either according the DNA isolation protocol from small amounts of plant tissue developed by Doyle and Doyle (1990) or using the Dneasy plant mini kit (QIAGEN, Hilden, Germany) for Q. suber and F. sylvatica. Amplified fragment length polymorphism (AFLP) The AFLP technique was performed using the AFLP ligaPublished by NRC Research Press


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tion and preselective amplification kit for regular plant genomes (500–6000 Mb) (Invitrogen, Alcobendas, Spain), according to manufacturer’s instructions. Quercus rotundifolia DNA (250 ng) was digested in duplicate samples for 2 h at 37 °C with 2.5 U of EcoRI and MseI enzymes. Ligation of EcoRI and MseI adaptors was followed by preamplification and then a selective amplification was performed with the following combination of primers: MseI-CAA/EcoRI-AA; MseI-CAG/EcoRI-AC; and MseI-CAT/EcoRI-AG (see Supplementary data,1 Table S1). The amplified fragments from both samples were separated in contiguous lanes on a 6% denaturing polyacrylamide gel. The gel was transferred to the staining solution (2 g of AgNO3, 3 mL of 37% formaldehyde, distilled water up to 2 L) and shaken for 20 min. Two litres of developing solution (0.3% w/v Na2CO3) was then poured into the tray, with agitation, until bands started to appear. The reaction was stopped with 10% acetic acid. The AFLP fragments (Table S2) were excised from the dry gel, placed for 5 min in boiling water, and centrifuged at 16 000g for 10 min. The resulting supernatant was used as a template for DNA reamplification with the same set of primers used to generate them prior to cloning with NZY-PCR cloning kit (NZYTech, Lisbon, Portugal) and sequencing. In silico analysis The homology search was performed using the BLASTn and BLASTx algorithms (Altschul et al. 1997) against NCBI and Gypsy databases (Llorens et al. 2011). Similarities to known sequences were considered significant when the probability values were less than 1 × 10–4 and 1 × 10–2, respectively. Genomic comparative analysis Knowledge of the AFLP fragments sequences allowed us to design primers for those regions (Table 1). Assuming that those amplified sequences are present in other Fagaceae species, the primers designed for Q. rotundifolia were used for comparative analysis in other Fagaceae species: Q. suber, C. sativa, and F. sylvatica. Single bands were recovered from Q. rotundifolia amplifications, cloned, and sequenced. DNA in situ hybridization and immunolocalization of 5methylcytosine Roots from at least three seeds of each species, and anthers from distinct trees were digested with an enzymatic mixture according to Ribeiro et al. (2011). Root meristematic nuclei preparations were obtained by the drop technique, also described in Ribeiro et al. (2011), and pachytenes were obtained by the spreading technique. The DNA FISH technique was adapted from Schwarzacher and Heslop-Harrison (2000), with a stringency of 75% and post-hybridization washes with a stringency of 84%. FISH probes were produced either through the reamplification of the isolated AFLP fragments, with the appropriate primers shown in Table S1 (Rot8, Rot9, Rot10, Rot18, and Rot20), or from the genomic regions amplified with primers designed for the AFLP fragments and presented in Table 1 (IAS-Rot8.24, IAS-Rot20, and IAS-Rot23). Simultaneously, a highly repeated sequence containing a 9-kb EcoRI fragment 1Supplementary

of the ribosomal DNA isolated from Triticum aestivum (Gerlach and Bedbrook 1979), pTa71, was used as a control . All the probes were labeled by PCR or by nick translation either with digoxigenin-dUTP or biotin-dUTP (Roche, Gipf-Oberfrick, Switzerland) for detection with anti-dig-FITC conjugate (Roche, Indianapolis, USA) or streptavidin-Cy3 conjugate (Sigma, Madrid, Spain), respectively. Immunodetection of 5-methylcytosine (5-mC) was performed after FISH, according to Castilho et al. (1999). Briefly, the slides were blocked for 30 min in 1% (w/v) BSA in 1× PBST (PBS supplemented with 0.5% (v/v) Tween 20) at room temperature. After washing in 1× PBST, the slides were incubated for 1 h at 37 °C in a humid chamber with anti-5-mC (Abcam, Cambridge, UK) diluted 1:200 in 1% (w/v) BSA, 1× PBST. The slides were further washed in 1× PBST and then incubated in Cy3-conjugated anti-mouse IgG diluted 1:100 in 1× PBS, 1% BSA for 1h at 37 °C. All cytological preparations were counterstained with 4′,6diamidino-2-phenylindole (DAPI) in Citifluor antifade mounting medium (AF1; Agar Scientific, Essex, UK). Preparations were observed using a Zeiss epifluorescence microscope with 100× objective and a set of appropriate filters to detect FITC, Cy3, and DAPI. The images were captured using an AxioCam digital camera and the Axiovision software. The images were then processed with Adobe Photoshop version 7.0.

Results Isolation and characterization of repetitive sequences from the Q. rotundifolia genome Seventeen AFLP strongly staining fragments, with lengths from 83 to 525 bp, were chosen for sequencing which revealed that all fragments had CG contents varying between 29% and 48% (Table S3). BLAST2 sequence analysis, involving all the AFLPderived sequences, revealed no homologous regions between any of them. Nucleotide and amino acid homology searches were performed with all the sequences against entries in the NCBI GenBank nonredundant nucleic acid sequence database, GSS, and GyDB (Gypsy database 2.0), using the BLASTn and BLASTx algorithms (Tables S3–S5). BLASTn searches against all the databases showed several sequences with high similarity with Vitaceae, Salicaceae, and Fabaceae species, four with BAC-end sequences of Q. robur, and six were identified with AFLP-derived sequences from Q. ilex subsp. rotundifolia (syn. Q. rotundifolia), previously published in GenBank (FJ656199, FJ656200, FJ656201, FJ656202, FJ656203), and two sequences failed to match with any database (Rot8.24 and Rot23) (Fig. 1). BLASTx was also performed against GenBank databases and GyDB, with default parameters. A summary of the protein homologies is present in Tables S4 and S5: only eight clones carried open reading frames (ORFs), with significant e-values between 1 × 10–26 and 8 × 10–7. Among them there was a mechanosensitive ion channel protein (Rot2.1), a stearoyl-acyl carrier protein desaturase (Rot22), and five retroelements portions: two copia-like (Rot6 and Rot9) and three gypsy-like (Rot10, Rot20, Rot27) (Tables S4 and S5). Rot6 and Rot9

data are available with the article through the journal Web site (http://nrcresearchpress.com/doi/suppl/10.1139/g2012-020). Published by NRC Research Press

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Table 1. Primers designed for the AFLP fragments. Oligo name Rot4 Rot6 Rot8.11 Rot8.24 Rot9 Rot20


Rot23 Rot27

Sequence 5′-AACAAGTTTAGCATCCAAGTCC-3′ 5′-GGATGAGATGGATTCCTTGTTGG-3′ 5′-GTTTGTTGTGGTTTTGTCACGGATT-3′ 5′-GGGAGGGAGCGGAGTTGGAG-3′ 5′-TTCTATGGGTCCTCAGTGTTTTGG-3′ 5′-ATGGGGAACTCTCTGAGGATGTAGA-3′ 5′-AGGCAGCCGGGAACTCATGC-3′ 5′-GCATGGACCACCAGCCTTGGA-3′ 5′-ACAGGAGGCAGAGTAGGTAAGG-3′ 5′-AGATTCTGCCTTACTCTTTACCAC-3′

Nucleotide position in accession 21→42 30→52 57→81 171→190 48→72 265←289 16→35 265←285 3→24 11→34

Fig. 1. AFLP sequences homology. Half of the sequences with homology in the databases are putatively translated.

showed similarity with Ty1-copia LTR retroelements Tto1, described in Nicotiana tabacum (Hirochika et al. 1996), and with SIRE1, described in the genome of Glycine max (Laten 1999), respectively, whereas Rot10, Rot20, and Rot27 are gypsy-like retroelements, all of them belonging to the Tat/ Athila clade. To study the genomic variability of regions from retroelements present in Q. rotundifolia and in other Fagaceae species, primers were designed to amplify the inter-AFLP sequences (IAS) for Rot9, the homologues of GAG_SIRE1–4 (copia-like), and Rot20, the homologues of RT_RIRE2 (gypsy-like) (Table 1), in Q. rotundifolia, Q. suber, C. sativa, and F. sylvatica. Rot9 primers amplified a fragment of an expected ∼250 bp length with the same size in Q. suber and C. sativa, but it failed to amplify any product in F. sylvatica (Fig. 2A). Conversely, Rot20 primers produced several bands resulting in different patterns for each species. In Q. rotundifolia, a strong band of the expected size (270 bp) was generated along with three larger ones. Quercus suber and F. sylvatica also presented a band of similar size (∼270 bp), although it was completely absent in C. sativa (Fig. 2A). The same approach was used to identify genomic regions surrounding these AFLP-derived sequences, and also to characterize the variability of these regions, in Q. suber, C. sativa, and F. sylvatica. For such an approach, primers to AFLPs homologous to retroelements copia-like (Rot6) and gypsy-like (Rot20 and Rot27) (Fig. 2B) and fragments with homology only to genomic DNA of Quercus spp. (Rot4 and Rot8.11) (Fig. 2C) were used. The amplification patterns obtained with only one primer for the RT of the copia-like, Tto1

Identity No homology RT_Tto1 (copia-like) No homology No homology

PCR product size, nt

GAG_SIRE1–4 (copia-like) RT_RIRE2 (gypsy-like)

242 270

No homology RT_Tat4 (gypsy-like)

(Rot6), and for both RTs of the gypsy-like, RIRE2 (Rot20) and Tat4 (Rot27) (Table 1), are different in all the species. However, a band with similar size (∼650 bp) was visible in all species for the amplification obtained with the Rot20 forward (F) primer itself, and a band with ∼750 bp was detected in both Quercus spp. and in C. sativa with the Rot20 reverse (R) primer itself (Fig. 2B). The amplification with Rot6 F primer and with Rot27 F primer itself produced different banding profiles in all species, although similar bands are shared by Q. rotundifolia and Q. suber, revealing the similarity between these two genomes and accentuating their differences from C. sativa and F. sylvatica. Amplification with only one primer designed from the AFLP fragments with high homology with genomic DNA of Quercus spp. (Rot4 and Rot8.11), revealed one discrete and strong band with ∼500 bp and ∼400 bp, respectively, in the four Fagaceae genomes. However, in the F. sylvatica genome a marked difference was detected for each sequence: a stronger band was produced with Rot4 F, while a faint one was obtained with Rot8.11 F, demonstrating the different representation of these two sequences in the four genomes. Finally, two sequences that failed to show homology in all the databases (Rot8.24 and Rot23) produced multiple bands of different lengths from 200 bp to ∼3 Kb, with fewer bands in F. sylvatica and a large number in C. sativa (Fig. 2C), indicating different insertion sites and number of copies of these sequences in the Fagaceae genomes studied. PCR products from Q. rotundifolia IAS-Rot8.24, IASRot20, and IAS-Rot23 were cloned and sequenced, revealing that all sequences had CG contents varying between 30% and 60% (Table S6). After nucleotide and amino acid homology searches (Tables S6–S8) all the clones revealed to be mainly composed of sequences with no homology to proteins (Fig. 3), although they all show nucleotide matches especially with Fagaceae (Table S6). Five of the six sequences analyzed were retroelements, and only one was homologous to the Ricinus communis phosphatidylcholine-sterol O-acyltransferase gene. Gypsy-like sequences accounted for the majority of the retroelements identified in this work, while only two belong to the copia-like class (Fig. 4). Genome organization and chromosome mapping of AFLP-derived sequences in Quercus, Castanea, and Fagus To study the structure and organization of Fagaceae genomes, the individual AFLP bands or the complete IAS amPublished by NRC Research Press

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Fig. 2. Banding profiles in Quercus rotundifolia (R), Quercus suber (S), Castanea sativa (C), and Fagus sylvatica (F) genomes obtained with (A) primers Rot9 FR (forward, reverse) and Rot20 FR; (B) Rot6 F, Rot20 F, Rot20 R, Rot27 F; (C) Rot4 F, Rot8.11 F, Rot23 F, and Rot8.24 R. MW, molecular weight marker 1 kb plus.

Fig. 3. Inter-AFLP sequences (IAS) homology. Most of the IAS are nonprotein homologous sequences.

Fig. 4. Retroelements composition in the AFLP-derived sequences.

plifications from IAS-Rot8.24, IAS-Rot23, and IAS-Rot20 (Figs. 2A, 2C), as well as the rDNA unit from T. aestivum (pTa71), were used as probes to perform FISH on Q. rotundifolia, Q. suber, C. sativa, and F. sylvatica interphase somatic nuclei or at pachytene. Drop slides of meristematic root-tip cells allowed for the visualization of specific chromatin organization through DAPI staining. Detailed analysis of Q. rotundifolia, Q. suber, and C. sativa interphase nuclei showed several heterochromatic DAPI positive domains evenly distributed over the entire nucleus, interspersed with clear euchromatic regions (Figs. 5A–5C). Conversely, F. sylvatica meristematic interphase nuclei presented well defined chromocenters (Fig. 5D). AFLP sequences Rot8, Rot10, and Rot20 with CG contents of 43%, 48%, and 45%, respectively, presented numerous dotlike signals dispersed throughout the nucleus, but mainly located in the euchromatic region (Figs. 6A–6C). On the con-

trary, Rot18, a fragment with higher AT content (63%), was present in the DAPI positive domains (Fig. 6D). To study the chromosome distribution of a copia-like related retroelement sequence at high resolution, FISH with Rot9 fragment (copia-like GAG_SIRE1–4, Table S4) was performed on Q. rotundifolia meristematic interphase and pachytene chromosomes (Fig. 7). The nucleus and chromosome distribution of this sequence proved to be of a repetitive nature, with several discrete signals dispersed more or less evenly all over the nucleus and chromosomes but being absent from the more heterochromatic regions (Fig. 7A, inset). It was also possible to distinguish two signals on both homologous in some interstitial domains, as well as in some telomere regions (Fig. 7D, inset). To evaluate the methylation state of this retroelement, in a genomic context, simultaneous immunodetection of 5-mC residues was performed with the same nuclei. This detection revealed a dispersed 5-mC patPublished by NRC Research Press

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Fig. 5. Meristematic root-tip interphase nuclei of (A) Quercus rotundifolia, (B) Quercus suber, (C) Castanea sativa, and (D) Fagus sylvatica stained with DAPI. Heterochromatin is visible as bright blue. Chromocenters are more evident in F. sylvatica (D). Scale bar = 10 mm.

Fig. 6. FISH with AFLP sequences (A) Rot8, (B) Rot10, (C) Rot20, and (D) Rot18 in meristematic interphase nuclei of Quercus rotundifolia detected in green. DNA is counterstained with DAPI. Rot18 is exclusively over heterochromatin blocks (D). Scale bar = 5 mm.

Fig. 7. Simultaneous FISH with AFLP sequence Rot9 (green; A, D) and 5-mC immunolocalization (red; B, E) in meristematic root-tip interphase nuclei (A–C) and pachytene chromosomes (D–F) of Quercus rotundifolia. Almost no superimposition of Rot9 signals and DNA methylation are detected (insets C, F). Chromatin is counterstained with DAPI. n denotes the nucleolus. Scale bar = 10 mm.

tern all over the interphase nucleus (Fig. 7B) and along the bivalent arms at pachytene (Fig. 7E), although not coincident with the pattern produced by Rot9 fragment (Figs. 7C, 7F).

In addition, to characterize the genomic distribution of inter-AFLP regions, the complete amplification reactions of IAS-Rot8.24, IAS-Rot20, and IAS-Rot23, and the rDNA unit Published by NRC Research Press

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Fig. 8. FISH with 18S–25S rDNA genes (green; B, F, J, N) and inter-AFLP sequence IAS-Rot8.24 (red; C, G, K, O) in meristematic root-tip interphase nuclei of Quercus rotundifolia (A–D), Quercus suber (E–H), Castanea sativa (I–L), and Fagus sylvatica (M–P). Absence of IASRot8.24 in C. sativa (K) and F. sylvatica (O) indicates that this probe is genus specific. Nuclei are counterstained with DAPI (blue; A, E, I, M). The fourth column shows the merged images of both signals and DAPI counterstaining (D, H, L, P). Scale bar = 10 mm.

from T. aestivum (pTa71), were used as probes to perform FISH. The analysis performed on interphase nuclei with IAS-Rot8.24 probe, composed of at least one genic sequence and several sequences with no homology in the databases, showed numerous dot-like signals dispersed throughout the entire nuclei of Q. rotundifolia and Q. suber (Figs. 8A–8H) and localized mostly in nonheterochromatic DAPI positive domains. In C. sativa and F. sylvatica nuclei (Figs. 8I–8P) no signal was visible, except for the rDNA loci, pointing to a specificity of this probe for the Quercus spp. studied. FISH with IAS-Rot20 and IAS-Rot23 (data not shown),

mainly composed of gypsy-like retroelements of the Tat4 element (nonchromodomain retroviruses), exhibited repetitive and dispersed patterns in the euchromatic regions, with no visible clusters (Fig. 9). In addition, the chromocenters of F. sylvatica interphase nuclei showed intense labelling with IAS-Rot20 (Figs. 9O, 9P).

Discussion Although there are a number of comprehensive genetic studies revealing some genomic resources in the Fagaceae, Published by NRC Research Press

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Fig. 9. FISH with 18S–25S rDNA genes (green; B, F, J, N) and inter-AFLP sequence IAS-Rot20 (red; C, G, K, O) in meristematic root-tip interphase nuclei of Quercus rotundifolia (A–D), Quercus suber (E–H), Castanea sativa (I–L), and Fagus sylvatica (M–P). Nuclei are counterstained with DAPI (blue; A, E, I, M). The fourth column shows the merged images of both signals and DAPI counterstaining (D, H, L, P). IAS-Rot20 is confined to chromocenters in F. sylvatica (P). Scale bar = 10 mm.

there is a lack of knowledge of their genome organization at the molecular and cytogenetic levels. The present work aimed to isolate a set of fragments potentially representative of the most abundant repeats of the Q. rotundifolia genome, to gain a better understanding of the composition and molecular organization of this fraction and their organization in other Fagaceae genera. Emphasis was placed on the characterization of a number of relatively short sequences that could serve as future molecular probes for taxonomic or phylogenetic studies.

All the sequences obtained either by AFLP or by AFLPderived PCR (IAS sequences) showed a CG content between 39% and 60%, which includes the average value calculated for the Q. ilex genome, 39.8% (Zoldos et al. 1998), which is a very close species of Q. rotundifolia (Romane and Terradas 1992). Different CG contents can be correlated with different types of sequences: coding sequences generally present high GC content, although variations have been detected within genes (Yu et al. 2002). Our results indicate that we have acPublished by NRC Research Press

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cessed both coding and noncoding regions of the Q. rotundifolia genome. Specific sequences of Fagaceae genera Homology searches in nucleotide databases revealed no hits for 6 of the 17 AFLP fragments and 10 of the 17 IAS fragments. Further searches in GSS and ESTs databases gave positive results with Fagaceae entries, indicating the presence of elements not yet identified in other plants and potentially specific for the Fagaceae genomes, as already proposed by Rampant et al. (2011). Moreover, FISH with sequences with no homology in the databases except for the gene for phosphatidylcholine-sterol O-acyltransferase (IAS-Rot8.24), conspicuously marked the Quercus spp. nuclei with several dots, although it failed to hybridize with C. sativa and F. sylvatica nuclei. This indicates that we are probably dealing with a set of genera-specific sequences, although more species should be studied from this genus to support this. Representation of Ty1-copia-like and Ty3-gypsy-like retroelements in Fagaceae genomes Retroelements constitute the majority of chromosomal DNA in many plants, and therefore have an important role in the evolution of plant genomes. The AFLP analysis of Q. rotundifolia produced several fragments belonging to Ty1-copia and Ty3-gypsy families of retroelements, evidencing the contribution of these elements to the structure of the Q. rotundifolia genome. In our study more sequences belonging to gypsy-like elements than to copia-like were found, in contrast to results from BAC-end sequencing of Q. robur where a major presence of copia-like elements has been detected (Rampant et al. 2011). This discrepancy could be due to the limited number of sequences in our sample and to the restriction enzymes used in the AFLP procedure that can bias the result, rather than to a difference in the composition of these two Quercus genomes. Indeed both Q. robur and Q. ilex (a very close Q. rotundifolia taxon, sometimes considered as two subspecies) genomes are very similar, namely in the number of chromosomes, number and location of ribosomal loci, and heterochromatin distribution (Zoldos et al. 1999), although with slight differences in genome size and base composition (Zoldos et al. 1998). It is reasonable to believe that these differences are mainly owing to different numbers of retroelements. All the gypsy-like retroelements found are homologous of retroelements belonging to the Tat/Athila clade, such as Tat4 from A. thaliana (Wright and Voytas 1998) and Ogre which is a giant LTR retrotransposon described in the genome of Pisum sativum (Neumann et al. 2003). An important characteristic of this lineage of plant LTR retrotransposons, as well as the isolated copia-like one, like the homologous of Tto1 from N. tabacum (Hirochika et al. 1996), is the lack of a chromodomain in their constitution. The chromodomain like the heterochromatin protein 1 (HP1) of Drosphila melanogaster, functions as a chromatin organization modifier responsible for chromatin targeting and recognition, namely of the heterochromatic regions (Eissenberg 2001). Chromodomains have recently been associated with the integration of retroelements into heterochromatin (Gao et al. 2008), a highly condensed chromatin fraction enriched in silent marks such as DNA methylation (Fransz et al. 2006). In Q. rotundi-


folia, the pattern of retroelements distribution, apart from Rot18, is uniform over the entire nucleus, with a marked preference for the euchromatic regions. Simultaneous FISH with the copia-like SIRE retroelement as a probe, and 5-mC immunodetection, shows that both signals do not coincide, evidencing the euchromatic preferential location of these retroelements. A similar situation was described in the pepper genome (Park et al. 2011), where a huge number of LTR retroelements insertions were detected in the euchromatin. Also, the probes IAS-Rot20 and IAS-Rot23, enriched in gypsy-like elements Tat4 and RIRE2 from the Tat/Athila clade, respectively, showed strong hybridization with all four species, despite the target nuclear domain being different for IAS-Rot20 in F. sylvatica. While IAS-Rot23 has a similar euchromatin dispersion in all species, IAS-Rot20 in Quercus spp. and C. sativa is markedly euchromatic, but in F. sylvatica, interestingly, it has a heterochromatic location, showing a strong signal in the chromocenters. The F. sylvatica genome has the smallest DNA amount of the Fagaceae studied (Kremer et al. 2007), and it is also the basal genus of the family (Manos et al. 2001), presenting the chromatin topology typical of plants with small genomes with conspicuous heterochromatic regions. All the other Fagaceae studied have genomes with the heterochromatin fraction distributed throughout the genome. Some retroelements tend to accumulate in the heterochromatic regions of plant chromosomes, as in barley, soybean, and maize, among others (Presting et al. 1998; Lin et al. 2005; Wolfgruber et al. 2009). However, retroelements are not only heterochromatic but can also insert in euchromatin regions, as the case of the gypsy-like Gret1 in Vitis vinifera (Pereira et al. 2005). The difference in IASRot20 location, either in the heterochromatin in F. sylvatica or in the euchromatic portion in the other Fagaceae, is evidence for the dynamic events that have occurred during the evolution of this family, since Quercus spp. and C. sativa genomes have twice the DNA amount of F. sylvatica (Kremer et al. 2007). The enlargement of genome size through the accumulation of LTR retrotransposons is well documented among flowering plants (Piegu et al. 2006), and it seems that this also happened during the evolution of the Fagaceae. The distribution of retroelements without a chromodomain is consistent with their accumulation in the Quercus and Castanea genomes, pointing to the expansion and reallocation into the euchromatic portion of LTR-type retroelement families, like the gypsy-like Tat/Athila clade during Fagaceae evolution. Alternatively, if a reduction of the genome size occurred during F. sylvatica evolution, as hypothesized by Kremer et al. (2007), a preferential maintenance of these elements in the heterochromatic region of F. sylvatica is evident, as happened during the evolutionary history of the Solanaceae family (Park et al. 2011). Assuming that each retroelement was inserted many times during evolution, and that its copies were positioned in a direct and (or) inverse orientation, we studied the genome evolution of three Fagaceae genera, Quercus, Castanea, and Fagus, separated at different periods of time through the variation of these potential insertions sites. The PCR patterns correlates well with the distances between species: both Quercus spp. share the most patterns, while C. sativa, and specially F. sylvatica, show major differences. Comparative mapping between Quercus and Castanea suggests that there Published by NRC Research Press


Alves et al.

is a strong macrosynteny between them (reviewed in Kremer et al. 2007), while in Fagus this comparative genomic analysis is more difficult, and for that reason this study makes an important contribution to the comparative analysis of Fagaceae genomes. Amplification of the GAG region of the SIRE copia-like element gives a strong band of approximately the same size in Quercus and Castanea and no amplification in F. sylvatica DNA as also testified by FISH. Our results corroborate the low success of genetic markers transferability between F. sylvatica and the other Fagaceae. From dozens of SSR markers studied, only one marker from Q. rubra and one from C. sativa could be placed on the F. sylvatica map (other examples reviewed in Kremer et al. 2007). Also, Zoldos et al. (2001) failed to detect any homologues of this element in the genome of several Quercus spp., although homologues of this type of retrotransposon had already been described in Q. suber (Carvalho et al. 2010), there being an expectation that these elements are well represented in some Quercus genomes. In summary, this retroelement could be a good candidate for use as a marker in Fagaceae comparative genomic studies.

Concluding remarks Our study sheds light on the evolution at the genomic level of the Fagaceae. It shows that besides a strong genomic similarity between Quercus and Castanea, with reduced genetic divergence, some differences can be found that have utility for studying the evolutionary history of this family. Here we have isolated and characterized several sequences that make good tools to study genome evolution in the Fagaceae family, namely sequences specific for Mediterranean oaks (Q. rotundifolia and Q. suber) but absent in the other genera; and sequences that, although present in the three genera, showed different locations and are therefore useful for studying dynamic processes that occurred during the evolution of this family.

Acknowledgements We thank W. Viegas for providing lab conditions for this work and S. Pereira for helpful comments and technical assistance. We are also grateful to R.N. Jones for his critical revision of the manuscript and editing of English. T.R. and M.R. were supported by Fundação Ciência e Tecnologia, Portugal, with grants SFRH/BD/13319/2003 and SFRH/BPD/64905/ 2009, respectively. This research was funded by Fundação para a Ciência e Tecnologia (PTDC/AGR-GFL/104197/ 2008).

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