Concerted evolution of rDNA in recently formed Tragopogon allotetraploids is typically associated with an inverse correlation between gene copy number and expression

Abstract

We analyzed nuclear ribosomal DNA (rDNA) transcription and chromatin condensation in individuals from several populations of Tragopogon mirus and T. miscellus, allotetraploids that have formed repeatedly within only the last 80 years from T. dubius and T. porrifolius and T. dubius and T. pratensis, respectively. We identified populations with no (2), partial (2), and complete (4) nucleolar dominance. It is probable that epigenetic regulation following allopolyploidization varies between populations, with a tendency toward nucleolar dominance by one parental homeologue. Dominant rDNA loci are largely decondensed at interphase while silent loci formed condensed heterochromatic regions excluded from nucleoli. Those populations where nucleolar dominance is fixed are epigenetically more stable than those with partial or incomplete dominance. Previous studies indicated that concerted evolution has partially homogenized thousands of parental rDNA units typically reducing the copy numbers of those derived from the T. dubius diploid parent. Paradoxically, despite their low copy number, repeats of T. dubius origin dominate rDNA transcription in most populations studied, i.e., rDNA units that are genetic losers (copy numbers) are epigenetic winners (high expression).

Figure 1.—

Restriction enzyme maps of the most abundant T. dubius, T. pratensis, and T. porrifolius rDNA units. The position of a conserved BstNI site in the T. dubius units is indicated. Large arrows indicate positions of primers. Distances are approximately to scale.

Figure 2.—

Expression analysis of parental rRNA genes in natural populations of T. mirus. (A) Example of a representative polyacrylamide gel with genomic-CAPS (lanes “DNA”) and RT-CAPS (lanes “RNA”) assays. Primary RNA transcripts were analyzed in the ITS1 region by RT-PCR using BstNI restriction site polymorphisms (Figure 1). The digested PCR products were separated on a 7% polyacrylamide gel and stained with ethidium bromide. The sizes of the diagnostic bands were as follows: ∼200 bp (T. dubius), ∼500 bp (T. dubius), ∼700 bp (T. porrifolius). (B) Quantification of rDNA expression and parental genes ratios. Signals in individual locus-specific bands were counted by fluorescence scanning. Data were scored for several individuals per population (numbers are given in Table 1) and expressed as a percentage of T. dubius genes and transcripts out of total. Each sample was analyzed twice and values averaged. The expression patterns in population 2602 differed significantly from those in other four populations (paired t-test, P < 0.01). Differences between populations 2601, 2603, 2673, and 2690 were statistically insignificant (P > 0.1).

Figure 3.—

Expression analysis of parental rRNA genes in natural populations of T. miscellus. (A) Example of a representative polyacrylamide gel with genomic-CAPS (lanes “DNA”) and RT-CAPS (lanes “RNA”) assays. The methods were same as in Figure 2. (B) Quantification of rDNA expression and parental gene ratios among the populations. The expression patterns among populations differed significantly. Statistical analysis (paired t-test): 2604 vs. 2605, 2606, and 2605 vs. 2606, P < 0.01).

Figure 4.—

rDNA transcription in T. mirus determined by RNAse protection assay. (A) Experimental strategy and probe design. Mismatched nucleotides between T. dubius (accession no. AM 493990) and T. porrifolius (AM 493995) sequences are indicated. The probe was designed according the T. dubius sequence. (B) Polyacrylamide gel with probe digestion products. The specificity of a probe was confirmed by hybridization with variable amounts of RNA from T. dubius and T. porrifolius (left). There was a progressive digestion of probe with increasing concentration of T. porrifolius RNA due to mismatched nucleotides in an RNA–RNA heteroduplex. Right: RNAse protection analysis of representative samples of T. mirus RNA showing nucleolar dominance of T. porrifolius (2602-1) and T. dubius (2601-7) unit types and a codominance (2602-7).

Figure 5.—

SSCP analysis of ITS1 subregion in genomic DNA and cDNA. The high-resolution gels resolved the profiles in the progenitor species and respective allotetraploids. Diagnostic conformers: D, T. dubius origin; Po, T. porrifolius origin; Pr, T. pratensis origin. U is a unique conformer specific to T. mirus population 2602. The gels show analysis of individuals from populations of T. mirus (pops. 2601-0-10 and 2602-7) and T. miscellus (2604-4 and 2606-15).

Figure 6.—

Southern blot hybridization showing IGS restriction site polymorphisms. DNAs from several populations of the diploid progenitors, T. dubius and T. porrifolius, and three populations of the allotetraploid T. mirus were digested with BstYI /SspI restriction enzymes and hybridized with the 26S rDNA probe. Asterisks indicate bands unique to population 2602 of T. mirus. Note extremely weak T. dubius-specific bands in populations 2601 and 2603.

Figure 7.—

rDNA chromatin condensation patterns in populations of T. mirus with dominant (C, D, population 2601 plant no. 0-10) and codominant (E, F, population 2602 plant no. 7) phenotypes. T. miscellus (H) was an individual from population 2604 (plant no. 4). Diploid T. dubius, T. porrifolius, and T. pratensis progenitors are in A, B, and G, respectively. rDNA was visualized by FISH to root-tip metaphase (A–C, E, G, H) and interphase (D, F) using FITC-labeled 35S rDNA probe (green fluorescence) and biotin-labeled 5S rDNA detected with avidin-Cy3 (red fluorescence). Nuclei were counterstained with DAPI for DNA (blue fluorescence). Note: in C, the rDNA sites that show some decondensation have large, linked 5S rDNA sites (arrows); in F and D, note the condensed extranucleolar rDNA sites (arrows).