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

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.

Figure 1

Diagram showing the experimental plan and the hypothesis tested to explain the phenotypic abnormalities observed.

Figure 2

Flower phenotypes of eight S. ruiz-lealii plants. A, B and C, normal flowers from plants 13.4, V0 and 17.1, respectively. D, E and F, flowers from plant 6. D, intermediate flower phenotype with twisted anthers and normal petals and style. E, twin flower. F, flower with twisted style. G and H, flowers from plant 13.2. G, flower with bilateral symmetry. H, staminoid petals (sepals and style removed). I, flower from plant 19.3 that resembles normal flower, but with dissected petals. J, carpelloid stamens from plant 19.3. K, extreme flower phenotype from plant 19.3, with rudimentary stamens and petals. L, M, N and O, flowers from plant 03. L, normal flower. M, flower with staminoid petals and rudimentary stamens. N, flower with reduced number of stamens, dissected petals and fusion between a stamen and the pistil. O, flower with extra number of petals (dissected) and reduced number of stamens. P, Q and R, flowers from plant 9. P, normal flower. Q, flower with staminoid petals, rudimentary stamens and bifurcated stigma. R, flower with dissected petals and twisted anthers.

Figure 3

Meiosis in S. ruiz-lealii. A, metaphase I showing heteromorphic bivalents (arrowheads). B, chromosomal bridges in metaphase-anaphase I (arrow). C, metaphase I with heteromorphic bivalents outside the equatorial plate (arrowheads) and early separation of univalent (arrow). D, meiocytes at telophase I and telophase II with one or two lagging chromosomes (arrowheads).

Figure 4

RFLP analysis of mtDNA of S. ruiz-lealii plants with normal and abnormal phenotypes. Polymorphic mitochondrial sequences. The molecular weights of bands are indicated. a, i and n, abnormal, intermediate and normal flower phenotypes, respectively, of the analyzed plants.

Figure 5

Model of gene-cytoplasmic interaction in S. ruiz-lealii. Nuclear restorer genes (Df) in Solanum chacoense. The nuclear recessive (df) gene conditions malformed flowers in interaction with the sensible [df^s] cytoplasm.

Figure 6

Cluster analysis based on molecular markers of eight S. ruiz-lealii plants. Dice similarity matrices and dendrograms obtained by cluster analysis based on presence/absence of AFLP (A), MSAP – methylation insensitive polymorphism (B) and MSAP – methylation sensitive polymorphism (C). a, i and n, abnormal, intermediate and normal flower phenotypes, respectively, of the analyzed plants.

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.

Figure 8

Southern analysis of nine S. ruiz-lealii plants. DNA was digested with HpaII and probed with the 0.6 kb PCR product of the repetitive sequence 2D8. The molecular weights of fragments are indicated. a, i and n, abnormal, intermediate and normal flower phenotypes, respectively, of the analyzed plants.

Figure 9

Novel flower phenotypes observed in a S. ruiz-lealii plant treated with the demethylating agent AzaC. A, flower from an untreated control plant. B, flower with overdeveloped sepals and light purple petals. C, flower with fused sepals and purple petals. D, flower with fused sepals and fused and dissected petals. E, flower with dissected petals and twisted anthers. F, two flowers from a single inflorescence, both with dissected petals. G, three flowers from a single inflorescence, two of which were normal and the remaining one presented dissected petals. H, two flowers from a single inflorescence. Upper, flower with dissected and extra number petals and twisted anthers. Lower, detail of a flower with fused stamens and petals. I, MSAP patterns observed in two independent experiments of the treated plant (T1 and T2) respect to the control untreated plant (C1 and C2). Arrowheads, hypermethylation of the treated plant respect to the control plant. Arrows, demethylation of the treated plant respect to the control plant. (-), control of the PCR experiment.