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Phenotypic instability and epigenetic variability in a diploid potato of hybrid origin, Solanum ruiz-lealii.

Marfil CF, Camadro EL, Masuelli RW - BMC Plant Biol. (2009)

Bottom Line: AFLP markers and restriction patterns of mitochondrial DNA did not allow the differentiation of normal from abnormal flower phenotypes.However, methylation patterns of nuclear DNA discriminated normal and abnormal flower phenotypes into two different groups, indicating that abnormal phenotypes have a similar methylation status which, in turn, was different from the methylation patterns of normal phenotypes.In addition, the variability detected for DNA methylation was greater than the detected for nucleotide sequence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratorio de Biología Molecular, EEA La Consulta INTA, Facultad de Ciencias Agrarias, Universidad Nacional de Cuyo and CONICET, Chacras de Coria Mendoza, Argentina.

ABSTRACT

Background: The wild potato Solanum ruiz-lealii Brüch. (2n = 2x = 24), a species of hybrid origin, is endemic to Mendoza province, Argentina. Recurrent flower malformations, which varied among inflorescences of the same plant, were observed in a natural population. These abnormalities could be the result of genomic instabilities, nucleus-cytoplasmic incompatibility or epigenetic changes. To shed some light on their origin, nuclear and mitochondrial DNA of plants with normal and plants with both normal and malformed flowers (from here on designated as plants with normal and plants with abnormal flower phenotypes, respectively) were analyzed by AFLP and restriction analyses, respectively. Also, the wide genome methylation status and the level of methylation of a repetitive sequence were studied by MSAP and Southern blots analyses, respectively.

Results: AFLP markers and restriction patterns of mitochondrial DNA did not allow the differentiation of normal from abnormal flower phenotypes. However, methylation patterns of nuclear DNA discriminated normal and abnormal flower phenotypes into two different groups, indicating that abnormal phenotypes have a similar methylation status which, in turn, was different from the methylation patterns of normal phenotypes. The abnormal flower phenotype was obtained by treating a normal plant with 5-Azacytidine, a demethylating agent, giving support to the idea of the role of DNA methylation in the origin of flower abnormalities. In addition, the variability detected for DNA methylation was greater than the detected for nucleotide sequence.

Conclusion: The epigenetic nature of the observed flower abnormalities is consistent with the results and indicates that in the diploid hybrid studied, natural variation in methylation profiles of anonymous DNA sequences could be of biological significance.

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MSAP analysis of eight S. ruiz-lealii plants. A, representative MSAP profiles of two EcoRI/HpaII (H) and EcoRI/MspI (M) digest of DNA extracted from eight S. ruiz-lealii plants. The primer combinations used were E-AGA/HM-TCAA (left panel) and E-AGA/HM-TCCA (right panel). The arrows indicate positions of size markers. B, detail of the primer combination E-AGA/HM-TCCA. Arrows heads, fragments analyzed as "methylation sensitive polymorphism". Arrow, fragment analyzed as "methylation insensitive polymorphism". C, graphical interpretation of methylation sensitive fragments. The boxes represent the double-stranded recognition site (CCGG) of the HpaII-MspI isoschizomer. Black boxes indicate methylated cytosine. Fragments 1 and 2 epialleles present in plants with normal and intermediate flower phenotype. Fragments 3 and 4, specific epialleles of plants with abnormal flower phenotype. Fragments 5 and 6, methylated epialleles present in three plants with abnormal flower phenotype and in plant 6, with intermediate flower phenotype; and demethylated epialleles specific of plants with normal flower phenotype. aMethylation patterns not determined, because the absence of a MSAP fragment can result from either a full methylation of cytosines on both strands or the absence of the restriction sites.
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Figure 7: MSAP analysis of eight S. ruiz-lealii plants. A, representative MSAP profiles of two EcoRI/HpaII (H) and EcoRI/MspI (M) digest of DNA extracted from eight S. ruiz-lealii plants. The primer combinations used were E-AGA/HM-TCAA (left panel) and E-AGA/HM-TCCA (right panel). The arrows indicate positions of size markers. B, detail of the primer combination E-AGA/HM-TCCA. Arrows heads, fragments analyzed as "methylation sensitive polymorphism". Arrow, fragment analyzed as "methylation insensitive polymorphism". C, graphical interpretation of methylation sensitive fragments. The boxes represent the double-stranded recognition site (CCGG) of the HpaII-MspI isoschizomer. Black boxes indicate methylated cytosine. Fragments 1 and 2 epialleles present in plants with normal and intermediate flower phenotype. Fragments 3 and 4, specific epialleles of plants with abnormal flower phenotype. Fragments 5 and 6, methylated epialleles present in three plants with abnormal flower phenotype and in plant 6, with intermediate flower phenotype; and demethylated epialleles specific of plants with normal flower phenotype. aMethylation patterns not determined, because the absence of a MSAP fragment can result from either a full methylation of cytosines on both strands or the absence of the restriction sites.

Mentions: To test if the methylation patterns were correlated with flower abnormalities, the global methylation status of the same eight genotypes was analyzed. For MSAP analysis, six pairs of primers were used and a total of 338 fragments were analyzed (Figure 7). The MSAP bands were separated as methylation-sensitive and methylation-insensitive, to ensure that the scored epigenetic polymorphism was due to alterations in methylation and not to genetic changes at the CCGG sites.


Phenotypic instability and epigenetic variability in a diploid potato of hybrid origin, Solanum ruiz-lealii.

Marfil CF, Camadro EL, Masuelli RW - BMC Plant Biol. (2009)

MSAP analysis of eight S. ruiz-lealii plants. A, representative MSAP profiles of two EcoRI/HpaII (H) and EcoRI/MspI (M) digest of DNA extracted from eight S. ruiz-lealii plants. The primer combinations used were E-AGA/HM-TCAA (left panel) and E-AGA/HM-TCCA (right panel). The arrows indicate positions of size markers. B, detail of the primer combination E-AGA/HM-TCCA. Arrows heads, fragments analyzed as "methylation sensitive polymorphism". Arrow, fragment analyzed as "methylation insensitive polymorphism". C, graphical interpretation of methylation sensitive fragments. The boxes represent the double-stranded recognition site (CCGG) of the HpaII-MspI isoschizomer. Black boxes indicate methylated cytosine. Fragments 1 and 2 epialleles present in plants with normal and intermediate flower phenotype. Fragments 3 and 4, specific epialleles of plants with abnormal flower phenotype. Fragments 5 and 6, methylated epialleles present in three plants with abnormal flower phenotype and in plant 6, with intermediate flower phenotype; and demethylated epialleles specific of plants with normal flower phenotype. aMethylation patterns not determined, because the absence of a MSAP fragment can result from either a full methylation of cytosines on both strands or the absence of the restriction sites.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2656509&req=5

Figure 7: MSAP analysis of eight S. ruiz-lealii plants. A, representative MSAP profiles of two EcoRI/HpaII (H) and EcoRI/MspI (M) digest of DNA extracted from eight S. ruiz-lealii plants. The primer combinations used were E-AGA/HM-TCAA (left panel) and E-AGA/HM-TCCA (right panel). The arrows indicate positions of size markers. B, detail of the primer combination E-AGA/HM-TCCA. Arrows heads, fragments analyzed as "methylation sensitive polymorphism". Arrow, fragment analyzed as "methylation insensitive polymorphism". C, graphical interpretation of methylation sensitive fragments. The boxes represent the double-stranded recognition site (CCGG) of the HpaII-MspI isoschizomer. Black boxes indicate methylated cytosine. Fragments 1 and 2 epialleles present in plants with normal and intermediate flower phenotype. Fragments 3 and 4, specific epialleles of plants with abnormal flower phenotype. Fragments 5 and 6, methylated epialleles present in three plants with abnormal flower phenotype and in plant 6, with intermediate flower phenotype; and demethylated epialleles specific of plants with normal flower phenotype. aMethylation patterns not determined, because the absence of a MSAP fragment can result from either a full methylation of cytosines on both strands or the absence of the restriction sites.
Mentions: To test if the methylation patterns were correlated with flower abnormalities, the global methylation status of the same eight genotypes was analyzed. For MSAP analysis, six pairs of primers were used and a total of 338 fragments were analyzed (Figure 7). The MSAP bands were separated as methylation-sensitive and methylation-insensitive, to ensure that the scored epigenetic polymorphism was due to alterations in methylation and not to genetic changes at the CCGG sites.

Bottom Line: AFLP markers and restriction patterns of mitochondrial DNA did not allow the differentiation of normal from abnormal flower phenotypes.However, methylation patterns of nuclear DNA discriminated normal and abnormal flower phenotypes into two different groups, indicating that abnormal phenotypes have a similar methylation status which, in turn, was different from the methylation patterns of normal phenotypes.In addition, the variability detected for DNA methylation was greater than the detected for nucleotide sequence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratorio de Biología Molecular, EEA La Consulta INTA, Facultad de Ciencias Agrarias, Universidad Nacional de Cuyo and CONICET, Chacras de Coria Mendoza, Argentina.

ABSTRACT

Background: The wild potato Solanum ruiz-lealii Brüch. (2n = 2x = 24), a species of hybrid origin, is endemic to Mendoza province, Argentina. Recurrent flower malformations, which varied among inflorescences of the same plant, were observed in a natural population. These abnormalities could be the result of genomic instabilities, nucleus-cytoplasmic incompatibility or epigenetic changes. To shed some light on their origin, nuclear and mitochondrial DNA of plants with normal and plants with both normal and malformed flowers (from here on designated as plants with normal and plants with abnormal flower phenotypes, respectively) were analyzed by AFLP and restriction analyses, respectively. Also, the wide genome methylation status and the level of methylation of a repetitive sequence were studied by MSAP and Southern blots analyses, respectively.

Results: AFLP markers and restriction patterns of mitochondrial DNA did not allow the differentiation of normal from abnormal flower phenotypes. However, methylation patterns of nuclear DNA discriminated normal and abnormal flower phenotypes into two different groups, indicating that abnormal phenotypes have a similar methylation status which, in turn, was different from the methylation patterns of normal phenotypes. The abnormal flower phenotype was obtained by treating a normal plant with 5-Azacytidine, a demethylating agent, giving support to the idea of the role of DNA methylation in the origin of flower abnormalities. In addition, the variability detected for DNA methylation was greater than the detected for nucleotide sequence.

Conclusion: The epigenetic nature of the observed flower abnormalities is consistent with the results and indicates that in the diploid hybrid studied, natural variation in methylation profiles of anonymous DNA sequences could be of biological significance.

Show MeSH
Related in: MedlinePlus