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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 Quercus faginea rDNA loci.FISH with wheat rDNA probe in c-metaphase of Q. faginea with 24 chromosomes shows four rDNA loci. Chromosomes are counterstained with DAPI.
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pone-0098678-g008: Physical mapping of Quercus faginea rDNA loci.FISH with wheat rDNA probe in c-metaphase of Q. faginea with 24 chromosomes shows four rDNA loci. Chromosomes are counterstained with DAPI.

Mentions: Physical mapping of the 18S-5.8S-25S rDNA in Q. pyrenaica and Q. faginea revealed the same loci number and location as for the majority of Quercus spp. (Figures 7 K and 8) confirming the dominant rDNA FISH pattern present in European and Asian Quercus subgenus Quercus[22], [57]. FISH signal with self-IGS probe is weaker than with the wheat gene probe which can be explained by the length of sequences hybridized, since genes have around 5.3 Kb, and the length of the IGS vary between ∼2 Kb to ∼4 Kb. The similar ratio of fluorescence detected in both major and minor rDNA loci of Q. suber, indicate that similar sequences are present in both NOR loci. This result is expected, since Bauer and colleagues [8] found that the two IGS variants of the white oaks, although different in length, shared more than 95% of sequence identity. Cross hybridization of Beech and Cork Oak IGSs in different species gave a weaker signal than the self-hybridization what is consistent with the variability calculated for this region in Beech and Oak. Presumably, the positive cross hybridization is mainly due to the 5′-ETS that share more than 70% of identity between Beech and Oaks (Table S8). This is, to our knowledge, the first time that IGS sequences were physically mapped and quantified into the NOR loci of the Fagaceae species.


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 Quercus faginea rDNA loci.FISH with wheat rDNA probe in c-metaphase of Q. faginea with 24 chromosomes shows four rDNA loci. Chromosomes are counterstained with DAPI.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098678-g008: Physical mapping of Quercus faginea rDNA loci.FISH with wheat rDNA probe in c-metaphase of Q. faginea with 24 chromosomes shows four rDNA loci. Chromosomes are counterstained with DAPI.
Mentions: Physical mapping of the 18S-5.8S-25S rDNA in Q. pyrenaica and Q. faginea revealed the same loci number and location as for the majority of Quercus spp. (Figures 7 K and 8) confirming the dominant rDNA FISH pattern present in European and Asian Quercus subgenus Quercus[22], [57]. FISH signal with self-IGS probe is weaker than with the wheat gene probe which can be explained by the length of sequences hybridized, since genes have around 5.3 Kb, and the length of the IGS vary between ∼2 Kb to ∼4 Kb. The similar ratio of fluorescence detected in both major and minor rDNA loci of Q. suber, indicate that similar sequences are present in both NOR loci. This result is expected, since Bauer and colleagues [8] found that the two IGS variants of the white oaks, although different in length, shared more than 95% of sequence identity. Cross hybridization of Beech and Cork Oak IGSs in different species gave a weaker signal than the self-hybridization what is consistent with the variability calculated for this region in Beech and Oak. Presumably, the positive cross hybridization is mainly due to the 5′-ETS that share more than 70% of identity between Beech and Oaks (Table S8). This is, to our knowledge, the first time that IGS sequences were physically mapped and quantified into the NOR loci of the Fagaceae species.

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