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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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Level of DNA methylation at 25S and 18S Bam HI restriction sites in Fagus, Castanea and Quercus spp.
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pone-0098678-g006: Level of DNA methylation at 25S and 18S Bam HI restriction sites in Fagus, Castanea and Quercus spp.

Mentions: In Q. suber blots, it would be expected a fragment around 4 kb corresponding to the Qs2 variant isolated by PCR, however this fragment was not detected. In order to investigate the possible methylation in the Bam HI sites [56] that would be responsible for the absence of this variant, two methylation-specific quantitative PCR with primers flanking the 25S and the18S Bam HI restriction sites (B2 and B3, Figure 1) were performed in digested and undigested genomic DNA of all species and the percent of methylation was determined (Figure 6) [12]. Regarding the 25S Bam HI restriction site F. sylvatica, Q. faginea, Q. rubra, and C. mollissima showed high levels of methylation (≥80%) suggesting that this Bam HI restriction site is mainly methylated. In Q. suber, Q. pyrenaica, and C. sativa the levels of methylation were lower, ranging from 30% up to 60% in Q. suber. Concerning the 18S gene, only Q. suber, and Q. faginea showed higher levels of methylation: 30% and 15%, respectively.


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)

Level of DNA methylation at 25S and 18S Bam HI restriction sites in Fagus, Castanea and Quercus spp.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098678-g006: Level of DNA methylation at 25S and 18S Bam HI restriction sites in Fagus, Castanea and Quercus spp.
Mentions: In Q. suber blots, it would be expected a fragment around 4 kb corresponding to the Qs2 variant isolated by PCR, however this fragment was not detected. In order to investigate the possible methylation in the Bam HI sites [56] that would be responsible for the absence of this variant, two methylation-specific quantitative PCR with primers flanking the 25S and the18S Bam HI restriction sites (B2 and B3, Figure 1) were performed in digested and undigested genomic DNA of all species and the percent of methylation was determined (Figure 6) [12]. Regarding the 25S Bam HI restriction site F. sylvatica, Q. faginea, Q. rubra, and C. mollissima showed high levels of methylation (≥80%) suggesting that this Bam HI restriction site is mainly methylated. In Q. suber, Q. pyrenaica, and C. sativa the levels of methylation were lower, ranging from 30% up to 60% in Q. suber. Concerning the 18S gene, only Q. suber, and Q. faginea showed higher levels of methylation: 30% and 15%, respectively.

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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