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Molecular organization of the 25S-18S rDNA IGS of Fagus sylvatica and Quercus suber: a comparative analysis.

Inácio V, Rocheta M, Morais-Cecílio L - PLoS ONE (2014)

Bottom Line: In the NTS: the sub-repeats structure in Beech is more organized than in Cork Oak, sharing some short motifs which results in the lowest sequence similarity of the entire IGS; the AT-rich region differed in both spacers by a GC-rich block inserted in Cork Oak.Methylation of 25S Bam HI site was confirmed in all species and detected for the first time in the 18S of Q. suber and Q. faginea.These results provide important clues for the evolutionary trends of the rDNA 25S-18S IGS in the Fagaceae family.

View Article: PubMed Central - PubMed

Affiliation: Centre for Botany Applied to Agriculture (CBAA), Instituto Superior de Agronomia, University of Lisbon, Lisbon, Portugal.

ABSTRACT
The 35S ribosomal DNA (rDNA) units, repeated in tandem at one or more chromosomal loci, are separated by an intergenic spacer (IGS) containing functional elements involved in the regulation of transcription of downstream rRNA genes. In the present work, we have compared the IGS molecular organizations in two divergent species of Fagaceae, Fagus sylvatica and Quercus suber, aiming to comprehend the evolution of the IGS sequences within the family. Self- and cross-hybridization FISH was done on representative species of the Fagaceae. The IGS length variability and the methylation level of 18 and 25S rRNA genes were assessed in representatives of three genera of this family: Fagus, Quercus and Castanea. The intergenic spacers in Beech and Cork Oak showed similar overall organizations comprising putative functional elements needed for rRNA gene activity and containing a non-transcribed spacer (NTS), a promoter region, and a 5'-external transcribed spacer. In the NTS: the sub-repeats structure in Beech is more organized than in Cork Oak, sharing some short motifs which results in the lowest sequence similarity of the entire IGS; the AT-rich region differed in both spacers by a GC-rich block inserted in Cork Oak. The 5'-ETS is the region with the higher similarity, having nonetheless different lengths. FISH with the NTS-5'-ETS revealed fainter signals in cross-hybridization in agreement with the divergence between genera. The diversity of IGS lengths revealed variants from ∼ 2 kb in Fagus, and Quercus up to 5.3 kb in Castanea, and a lack of correlation between the number of variants and the number of rDNA loci in several species. Methylation of 25S Bam HI site was confirmed in all species and detected for the first time in the 18S of Q. suber and Q. faginea. These results provide important clues for the evolutionary trends of the rDNA 25S-18S IGS in the Fagaceae family.

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Physical mapping of Fagus sylvatica and Quercus suber NTS-5′-ETS in Fagus, Castanea and Quercus spp.FISH with F. sylvatica NTS-5′-ETS (red- B,F,J), Q. suber NTS-5′-ETS (red - N,R,V), and wheat rDNA pTa71 probe (green - C,G,K,O,S,W) in meristematic root-tip metaphase chromosomes of F. sylvatica (A–D, U–X), Q. suber (E–H, M–P), Q. pyrenaica (I–L), and C. sativa (Q–T). DNA is counterstained with DAPI (blue - A,E,I,M,Q,U). The fourth column shows the merged images of both signals and DNA (D,H,L,P,T,X). Arrowheads indicate three overlapped NORs (B–D); arrows indicate small loci NORs (F–H; J–L; N–P; V–X).
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pone-0098678-g007: Physical mapping of Fagus sylvatica and Quercus suber NTS-5′-ETS in Fagus, Castanea and Quercus spp.FISH with F. sylvatica NTS-5′-ETS (red- B,F,J), Q. suber NTS-5′-ETS (red - N,R,V), and wheat rDNA pTa71 probe (green - C,G,K,O,S,W) in meristematic root-tip metaphase chromosomes of F. sylvatica (A–D, U–X), Q. suber (E–H, M–P), Q. pyrenaica (I–L), and C. sativa (Q–T). DNA is counterstained with DAPI (blue - A,E,I,M,Q,U). The fourth column shows the merged images of both signals and DNA (D,H,L,P,T,X). Arrowheads indicate three overlapped NORs (B–D); arrows indicate small loci NORs (F–H; J–L; N–P; V–X).

Mentions: In order to confirm the chromosomal location of the isolated IGSs, the NTS-5′-ETS from Beech and Cork Oak were used as FISH probes into metaphase chromosomes of both species, simultaneously with the wheat rDNA probe pTa71. FISH co-localization with the wheat rDNA probe confirmed the expected loci number and the location of the Beech and Cork Oak IGSs (Figure 7 D, P). Despite the confirmation of the maximum number of FISH signals in all specimens, the size and intensity of the FISH signals can differ between homologous loci and one site of the minor locus was not always detectable as already referred in several Quercus spp. [57]. Since Cork Oak bears two 18S–25S rDNA loci (one subterminal major loci with twice the size of the pericentromeric minor loci), using the same cell we measured the IGS and pTa71 fluorescent signal intensity within each NOR, in order to study the representativeness of the isolated IGS sequences in the rDNA units. The mean ratio between the fluorescent signal intensity of pTa71 probe, which in Fagaceae potentially labels only the rRNA genes, was 3.34±0.18 SE in the minor NOR and 3.08±0.19 SE in the major NOR. The differences detected were not significant, being the IGS probe equally represented in both rDNA loci (p-value  = 0.166). In order to study the similarity of the isolated IGS regions in other members of the Fagaceae we have first hybridized the Beech IGS with Cork Oak metaphase chromosomes and the other way around. Both IGSs have hybridized with major and minor rDNA loci, showing however less intensity than the self-hybridization, being these results consistent with the percentage of sequence identity previously detected (Figure 7 F, V). FISH with the Beech IGS in Q. pyrenaica and C. sativa, revealed a fainter signal in Q. pyrenaica (Figure 7 J) and in C. sativa (Figure S5) as expected when compared with the hybridization in F. sylvatica (Figure 7 B). The Cork Oak IGS was hybridized in C. sativa metaphase chromosomes, resulting in a weaker signal when compared with Q. suber (Figure 7 R, N). Since we have obtained hybridization signal using the Cork Oak IGS probe with the distant genera Castanea we have not performed FISH with the other Quercus spp.


Molecular organization of the 25S-18S rDNA IGS of Fagus sylvatica and Quercus suber: a comparative analysis.

Inácio V, Rocheta M, Morais-Cecílio L - PLoS ONE (2014)

Physical mapping of Fagus sylvatica and Quercus suber NTS-5′-ETS in Fagus, Castanea and Quercus spp.FISH with F. sylvatica NTS-5′-ETS (red- B,F,J), Q. suber NTS-5′-ETS (red - N,R,V), and wheat rDNA pTa71 probe (green - C,G,K,O,S,W) in meristematic root-tip metaphase chromosomes of F. sylvatica (A–D, U–X), Q. suber (E–H, M–P), Q. pyrenaica (I–L), and C. sativa (Q–T). DNA is counterstained with DAPI (blue - A,E,I,M,Q,U). The fourth column shows the merged images of both signals and DNA (D,H,L,P,T,X). Arrowheads indicate three overlapped NORs (B–D); arrows indicate small loci NORs (F–H; J–L; N–P; V–X).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043768&req=5

pone-0098678-g007: Physical mapping of Fagus sylvatica and Quercus suber NTS-5′-ETS in Fagus, Castanea and Quercus spp.FISH with F. sylvatica NTS-5′-ETS (red- B,F,J), Q. suber NTS-5′-ETS (red - N,R,V), and wheat rDNA pTa71 probe (green - C,G,K,O,S,W) in meristematic root-tip metaphase chromosomes of F. sylvatica (A–D, U–X), Q. suber (E–H, M–P), Q. pyrenaica (I–L), and C. sativa (Q–T). DNA is counterstained with DAPI (blue - A,E,I,M,Q,U). The fourth column shows the merged images of both signals and DNA (D,H,L,P,T,X). Arrowheads indicate three overlapped NORs (B–D); arrows indicate small loci NORs (F–H; J–L; N–P; V–X).
Mentions: In order to confirm the chromosomal location of the isolated IGSs, the NTS-5′-ETS from Beech and Cork Oak were used as FISH probes into metaphase chromosomes of both species, simultaneously with the wheat rDNA probe pTa71. FISH co-localization with the wheat rDNA probe confirmed the expected loci number and the location of the Beech and Cork Oak IGSs (Figure 7 D, P). Despite the confirmation of the maximum number of FISH signals in all specimens, the size and intensity of the FISH signals can differ between homologous loci and one site of the minor locus was not always detectable as already referred in several Quercus spp. [57]. Since Cork Oak bears two 18S–25S rDNA loci (one subterminal major loci with twice the size of the pericentromeric minor loci), using the same cell we measured the IGS and pTa71 fluorescent signal intensity within each NOR, in order to study the representativeness of the isolated IGS sequences in the rDNA units. The mean ratio between the fluorescent signal intensity of pTa71 probe, which in Fagaceae potentially labels only the rRNA genes, was 3.34±0.18 SE in the minor NOR and 3.08±0.19 SE in the major NOR. The differences detected were not significant, being the IGS probe equally represented in both rDNA loci (p-value  = 0.166). In order to study the similarity of the isolated IGS regions in other members of the Fagaceae we have first hybridized the Beech IGS with Cork Oak metaphase chromosomes and the other way around. Both IGSs have hybridized with major and minor rDNA loci, showing however less intensity than the self-hybridization, being these results consistent with the percentage of sequence identity previously detected (Figure 7 F, V). FISH with the Beech IGS in Q. pyrenaica and C. sativa, revealed a fainter signal in Q. pyrenaica (Figure 7 J) and in C. sativa (Figure S5) as expected when compared with the hybridization in F. sylvatica (Figure 7 B). The Cork Oak IGS was hybridized in C. sativa metaphase chromosomes, resulting in a weaker signal when compared with Q. suber (Figure 7 R, N). Since we have obtained hybridization signal using the Cork Oak IGS probe with the distant genera Castanea we have not performed FISH with the other Quercus spp.

Bottom Line: In the NTS: the sub-repeats structure in Beech is more organized than in Cork Oak, sharing some short motifs which results in the lowest sequence similarity of the entire IGS; the AT-rich region differed in both spacers by a GC-rich block inserted in Cork Oak.Methylation of 25S Bam HI site was confirmed in all species and detected for the first time in the 18S of Q. suber and Q. faginea.These results provide important clues for the evolutionary trends of the rDNA 25S-18S IGS in the Fagaceae family.

View Article: PubMed Central - PubMed

Affiliation: Centre for Botany Applied to Agriculture (CBAA), Instituto Superior de Agronomia, University of Lisbon, Lisbon, Portugal.

ABSTRACT
The 35S ribosomal DNA (rDNA) units, repeated in tandem at one or more chromosomal loci, are separated by an intergenic spacer (IGS) containing functional elements involved in the regulation of transcription of downstream rRNA genes. In the present work, we have compared the IGS molecular organizations in two divergent species of Fagaceae, Fagus sylvatica and Quercus suber, aiming to comprehend the evolution of the IGS sequences within the family. Self- and cross-hybridization FISH was done on representative species of the Fagaceae. The IGS length variability and the methylation level of 18 and 25S rRNA genes were assessed in representatives of three genera of this family: Fagus, Quercus and Castanea. The intergenic spacers in Beech and Cork Oak showed similar overall organizations comprising putative functional elements needed for rRNA gene activity and containing a non-transcribed spacer (NTS), a promoter region, and a 5'-external transcribed spacer. In the NTS: the sub-repeats structure in Beech is more organized than in Cork Oak, sharing some short motifs which results in the lowest sequence similarity of the entire IGS; the AT-rich region differed in both spacers by a GC-rich block inserted in Cork Oak. The 5'-ETS is the region with the higher similarity, having nonetheless different lengths. FISH with the NTS-5'-ETS revealed fainter signals in cross-hybridization in agreement with the divergence between genera. The diversity of IGS lengths revealed variants from ∼ 2 kb in Fagus, and Quercus up to 5.3 kb in Castanea, and a lack of correlation between the number of variants and the number of rDNA loci in several species. Methylation of 25S Bam HI site was confirmed in all species and detected for the first time in the 18S of Q. suber and Q. faginea. These results provide important clues for the evolutionary trends of the rDNA 25S-18S IGS in the Fagaceae family.

Show MeSH