Concerted Evolution of Rdna in Recently Formed Tragopogon Allotetraploids Is Typically Associated with an Inverse Correlation Between Gene Copy Number and Expression

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Concerted Evolution of rDNA in Recently Formed Tragopogon Allotetraploids Is Typically Associated With an Inverse Correlation Between Gene Copy Number and Expression

Roman Matya´sˇek,* Jennifer A. Tate,† Yoong K. Lim,‡ Hana Sˇrubarˇova´,* Jin Koh,† Andrew R. Leitch,‡ Douglas E. Soltis,† Pamela S. Soltis§ and Alesˇ Kovarˇ´ık*,1 *Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i, Laboratory of Molecular Epigenetics, Kra´lovopolska´ 135, CZ-61265 Brno, Czech Republic, †Department of Botany, University of Florida, Gainesville, Florida 32611, ‡School of Biological Sciences, Queen Mary, University of London, E1 4NS, UK and §Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611 Manuscript received March 1, 2007 Accepted for publication May 23, 2007

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).

PIGENETIC silencing of parental genes is an im- DNA methylation and histone acetylation (for review E portant feature of newly established allopolyploids see Preuss and Pikaard 2007). Other factors may also and is believed to reconcile regulatory incompatibilities play a role in ND, e.g., the position of rRNA genes in the between parental subgenomes (Adams and Wendel nucleus (Shaw and Jordan 1995), the relative amount 2005; Birchler et al., 2005). Nucleolus organizer re- of heterochromatin (Viegas et al. 2002), and the activity gions (NORs) contain tandemly arranged highly reit- of unlinked genes (Lewis et al. 2004). Despite consid- erated ribosomal rRNA genes coding for 18S-5.8S-26S erable progress in our understanding of ND, little is rRNA whose expression is under epigenetic control known about the relationship between ND and sequence (Pikaard 2000; for review see Neves et al. 2005). homogenization, particularly in young populations of Indeed nucleolar dominance (ND) was one of the ﬁrst newly formed polyploidy species. The goal of this article examples of differential gene expression discovered in is to study this relationship and the inheritance patterns plant hybrids nearly a century ago (Navashin 1934). of ND and sequence homogenization at individual Numerous studies in both plants and animals support and population levels in recently formed allotetraploid the view that ND/rRNA gene silencing may be stable species. or unstable, partial, or reversible (Volkov et al. 2007). Besides epigenetic silencing homogenization of re- In Arabidopsis and Brassica biochemical (Chen and peats seems to be another interesting aspect of rDNA Pikaard 1997a) and genetic (Lawrence et al. 2004; biology. The consequence of homogenization is little Probst et al. 2004) studies provided evidence that rRNA sequence variability among the units within and across silencing depends on epigenetic factors, including the arrays in genome. In many allopolyploids’ rDNA one particular unit overwrites preexisting units, probably mediated by recombination or copy number expansion/ Sequence data from this article have been deposited with the EMBL/ contraction mechanism that leads to the fast evolu- GenBank Data Libraries under accession nos. AM493990 and AM493995. tion of novel families, a process called concerted evo- 1Corresponding author: Institute of Biophysics, Academy of Sciences of lvarez endel the Czech Republic, v.v.i., Laboratory of Molecular Epigenetics, Kra´lovo- lution (for review see A and W , 2003; polska´ 135, CZ-612 65 Brno, Czech Republic. E-mail: [email protected] Volkov et al. 2007). However, the frequency/occurrence

Genetics 176: 2509–2519 (August 2007) 2510 R. Matya´sˇek et al. of homogenization does vary between species. Allopo- TABLE 1 lyploids of Arabidopsis and Brassica apparently have no Summary of collections of Tragopogon used rDNA unit loss or concerted evolution, despite an evolutionary history lasting several thousands of gen- No. of erations (Bennett and Smith 1991; O’kane et al. 1996). Collection individuals In contrast studies in some recently formed allopoly- Species no. Locality analyzed ovarik ploids (K et al. 2005), synthetic interspecific T. dubius Population 2613 Pullman, WA 3 in luster hybrids and polyploids (L et al. 1985; C et al. Population 2614 Rosalia, WA 3 1996; Weiss and Maluszynska, 2000; Skalicka´ et al. Population 2615 Spokane, WA 3 2003; Pontes et al. 2004; Lim et al. 2006) reveal evidence T. porrifolius Population 2607 Troy, ID 3 of astonishingly fast genetic change influencing thou- Population 2611 Pullman, WA 3 sands of rDNA units, perhaps in just a few generations Population 2612 Troy, ID 3 T. pratensis Population 2598 Colton, WA 3 following allopolyploidy. The reasons for interspecies Population 2608 Moscow, ID 3 differences remain enigmatic, but evolutionary change Population 2609 Spangle, WA 3 probably occurs through a range of processes including T. mirus Population 2601 Pullman, WA 17 locus loss/gain, amplification/reduction in repeat copy Population 2602 Palouse, WA 29 number, gene conversion, and recombination. Population 2603 Rosalia, WA 10 In nature there are several examples of allopolyploids Population 2690 Tekoa, WA 6 of recent (,150 years) origin, including in the genera Population 2673 Oaksdalle, WA 6 T. miscellus Population 2604 Moscow, ID 10 Senecio (Abbott and Lowe 2004), Spartina (Ainouche oltis Population 2605 Pullman, WA 7 et al. 2004), and Tragopogon (S et al. 2004), en- Population 2606 Spangle, WA 14 abling direct comparison of expression patterns between parents and polyploids. However, in none of these systems has the question of parental rRNA genes expres- these questions we have analyzed rDNA copy numbers, sion been addressed so far. Tragopogon mirus Ownbey rRNA gene expression, and rDNA chromatin conden- and T. miscellus Ownbey are allotetraploids (2n ¼ 24) sation at interphase and metaphase in several popula- that formed during the past 80 years following the tions each of T. mirus and T. miscellus. introduction of three diploids ½T. dubius, T. pratensis, T. porrifolius (2n ¼ 12) from Europe to western North America (Ownbey 1950; Soltis et al. 2004). T. mirus and MATERIALS AND METHODS T. miscellus originated by hybridization between T. dubius Plant material: Seeds of the three diploid and two allote- and T. porrifolius, and T. dubius and T. pratensis, respec- traploid species of Tragopogon were collected from natural tively. Both allotetraploids have formed repeatedly— populations in Idaho and Washington (Table 1). For each there may be as many as 21 lineages of separate origin of population sampled, we collected seeds from 10 to 20 individual plants; the seeds from each plant were kept separate. T. miscellus and 11 of T. mirus in the Palouse region of These seeds were subsequently planted in the greenhouses at Washington and Idaho (Soltis and Soltis 1989; Cook the University of Florida; plants were grown to maturity and et al. 1998). We previously reported little or no deviation allowed to self-pollinate. The resultant selfed seeds (S1) were from additivity for chromosome numbers, satellite se- collected from individual plants (Kovarik et al. 2005). Some of quences, and genome size (Pires et al. 2004). However, a these seeds, representing one generation of selfing (S1) in the greenhouse, were later germinated in a greenhouse in the quantitative study of the inheritance of rRNA genes Institute of Biophysics, Brno for use in this investigation. among several populations of T. mirus and T. miscellus Collected leaf samples were dipped into RNAlater solution revealed up to 70–95% reduction in the number of (Ambion, Austin, TX), stored at 20°, and analyzed within 12 T. dubius repeats, likely by deletion or recombination months after harvest. Root tips for cytogenetic studies and leaf events. The genetic lability of the T. dubius genome tissue for DNA isolation were harvested from 5- to 8-week-old plants. Plants were sampled in duplicate. In some cases, plants component in the allopolyploids was recently demon- were sampled during both the first and second year of the life strated by Tate et al. (2006), who found stochastic gene span of these biennials. All individuals were genotyped using loss at several low-copy loci in T. miscellus individuals PCR-CAPS analysis of the PP2C gene (Tate et al. 2006) and from two populations. their diploid parentage was confirmed. Here, we addressed the following questions. Is there Reverse transcription-cleaved amplified polymorphic sequence (RT-CAPS) assay: This assay was used to determine the any association between: (i) epigenetic silencing and relative abundance and parental origin of pre-rRNA tran- sequence elimination, (ii) rDNA copy number and gene scripts. Total cellular RNA from fresh leaves was isolated using expression, and (iii) rDNA chromatin condensation TRIzol Reagent (Invitrogen, Carlsbad, CA) as recommended and rDNA copy number? Do populations of T. mirus and by the supplier. RNA quantity was measured using a spectro- T. miscellus have the same patterns of rDNA evolution, photometer, and its quality was checked by agarose-formalde- hyde gel electrophoresis. Total RNA was treated with and do individuals within a population/species become RNase-free DNase I RQ1 (Promega, Madison, WI) or DNA fixed for epigenetic or genetic characteristics after at free kit (Ambion) and checked for the presence of genomic most 80 years (40 generations) of evolution? To address DNA using PCR. rDNA Transcription in Allotetraploids 2511

To study the transcription of parental rDNA, we analyzed contained a binding site for T7 polymerase at its 59 end. The the ITS1 and, in some plants, the ITS2 regions of the rRNA gel-purified DNA template was transcribed with T7 polymerase precursors, using two-step RT-PCR. Reverse transcription re- using a nonradioactive biotinylated precursor (MAXIscript; actions (20 ml) typically contained 1 mg of total RNA, 2 pmol of Ambion). The antisense 168-nt band was purified by elec- either 5.8Srev primer (for the ITS1 region) or 26Srev primer trophoresis in a polyacrylamide gel, eluted, and used in (for the ITS2 region) (for sequences see Kovarik et al. 2005), downstream procedures. 10 nmol of each dNTP, and 20 units of reverse transcriptase Homeologous primary rRNA transcripts were identified (Superscript II RNAse H; Invitrogen) using the conditions by hybridization of RNAs with a riboprobe derived from the recommended by the supplier. After reverse transcription, the T. dubius unit using a RPA III kit (Ambion). Briefly, 5–10 mgof RNA was digested using RNase A (Sigma, St. Louis). total RNA was mixed with 1 ng of biotinylated RNA probe, For PCR (10 ml) amplification we used 0.5 ml of RT mixture lyophilized, resuspended in hybridization buffer, and dena- as template, 4 pmol of each primer, 2.4 nmol of each dNTP, tured for 15 min at 95° in a hybridization oven. The probe was and 0.4 units of DyNAzyme II DNA polymerase (Finnzymes, hybridized to RNA samples at 68° overnight followed by in- Epsoo, Finland). Cycling conditions were as follows: initial cubation at 42° for 3 hr. Resulting RNA:RNA hybrid was denaturation step (94°, 180 sec) and 15–35 cycles (94°, 20 sec; treated with RNase A/RNase T1 mix for 30 min at 37° using a 57°, 30 sec; 72°, 30 sec) with 18S-FOR and 5.8S-REV primers 1:100 enzyme dilution. After enzyme inactivation the samples for ITS1 or 35 cycles (94°, 20 sec; 60°, 30 sec; 72°, 30 sec) with were precipitated, vacuum dried, resuspended in gel loading 5.8-FOR and 26S-REV primers for ITS2, followed by a final 72° buffer, denatured for 5 min at 94°, placed on ice, and elec- extension for 10 min. To discriminate among ITS variants trophoresed in 7% (44:1, 48% urea) denaturing polyacryl- originating from the diploid progenitors, the PCR products amide gel at 22V/cm. After electrophoresis, the urea was washed were digested with restriction enzymes that produce locus- out and RNA was blotted onto nylon membrane (Hybond XL; specific restriction patterns. Mapping of locus-specific sites was GE Healthcare) using semi-dry transfer (TRANS-BLOT SD; BIO- carried out on sequences in the GenBank/NCBI database RAD, USA). The protected probe fragments were visualized ½AY645813, AY508169, AY508167 (Mavrodiev et al. 2004) us- by chemiluminiscence (BrightStar BioDetect Nonisotopic ing Wisconsin GCG package software. The ITS1 PCR prod- Detection Kit; Ambion) using a luminescent image analyzer uct was digested with BstNI (Figure 1) or MspI (New England (LAS-3000; FUJIFILM, Japan). Biolabs, Beverly, MA). The ITS2 region was analyzed by double- Single-strand conformation polymorphism (SSCP): The digestion with Fnu4HI and Tsp509I, and the resulting frag- species-specific polymorphisms in rDNA units were studied ments were separated on a 7% polyacrylamide gel. by SSCP (Sambrook and Russel 2001). Briefly, ITS1 sequen- Quantification of expression phenotypes and statistical ces generated by PCR were digested with Fnu4HI, isopropanol procedures: After RT-CAPS analysis, the gel was stained with precipitated, resuspended in 30% dimethyl sulfoxide, heat ethidium bromide and the resulting DNA bands in the gel denatured (10 min, 99°), immediately cooled on ice, and were visualized using ultraviolet light transillumination (CCD loaded onto a pre-electrophoresed and pre-cooled (5°) 10% camera was a Discovery model; Ultra-Lum, Claremont, CA). polyacrylamide gel. After electrophoresis (Tris-borate buffer, Images were processed by UltraQuant molecular imaging and pH 8.0, 5°, 8 V/cm), the gels were silver stained (Sambrook analysis software (Ultra-Lum) fluorescent signals quantified by and Russel 2001). a rectangle integration method. In some PCR experiments Chromosome preparation and fluorescent in situ hybrid- radioactive incorporation of a-½32PdCTP (ICN, Irvine, CA) ization (FISH): Chromosome preparations and FISH were precursor was used to monitor linearity of amplification. Both performed according to Leitch et al. (2001) and Pires et al. radioactive and fluorescence quantification methods were in a (2004). The rDNA probe pTa71 (Gerlach and Bedbrook good agreement. The fluorescence integer of both T. dubius- 1979) was labeled with biotin-16-dUTP (Sigma-Aldrich Com- specific bands was divided by the intensity of all bands and pany, Poole, Dorset, UK), and hybridization was detected with expressed as a percentage of the total expression. avidin-Cy3 (GE Healthcare). The 5S probe was an 120-bp Restriction site analysis and Southern blot hybridization: coding region of the Nicotiana tabacum gene (Fulnecek et al. Genomic DNA was isolated from fresh young leaves according 1998). Images were taken with a Leica CTRMIC epifluorescent to the modified cetylammonium bromide (CTAB) method of microscope with an Orca ER camera, and images were pro- Saghai-Maroof et al. (1984). For Southern blot hybridization, cessed using Openlab software (Improvision). Only those func- purified DNAs were digested with restriction enzymes, sepa- tions that apply equally to all pixels in the image were used. For rated by gel electrophoresis, and blotted onto nylon mem- every set of observations, 5 to 10 metaphases were examined branes (GE Healthcare). The Southern blot hybridization was from each of one to three slides. carried out in a modified Church–Gilbert buffer (Lim et al. 2000) using the 26S rDNA labeled with a-½32PdCTP (ICN). The probe was an 220-bp PCR product derived from the 39 RESULTS end of the 26S rRNA tobacco gene (Lim et al. 2000). The bands were visualized by PhosphorImaging (GE Healthcare). rRNA gene expression in diploid and tetraploid Genomic-cleaved amplified polymorphic sequence (genomic- Tragopogon: To examine expression of progenitor CAPS)assay:This assay was used to determine the relative abun- rDNA homeologues, we isolated RNA and DNA from dance and parental origin of rDNA sequences. The protocol leaf tissue of 10–30 individuals from each population followed the RT-CAPS assay except that 0.5 ng of genomic DNA was used as template in the PCR step rather than the analyzed of the two allotetraploids. The parental origin products of reverse transcription. and relative abundance of the rRNA genes themselves Ribonuclease protection assay: A riboprobe was prepared were previously studied by Southern blot hybridization from a DNA template covering a polymorphic ITS2 region (Kovarik et al. 2005). Here, we analyzed parental genes downstream from the 5.8S gene. Briefly, the 138-bp fragment by genomic-CAPS assay allowing a direct comparison of was amplified using ITS2FORdub 59-GCA TCGCGTCGCCC CCCCCCAT and ITS2T7REV 59-TAATACGACTCACTATAGG copy number and expression profiles. To determine GAGAACAAACACAACCACC ACTAGCCGTGCGTCAA primers relative abundance of rRNA primary transcripts (pre- and a cloned ITS2 from T. dubius. The ITS2T7REV primer rRNA), we used the RT-CAPS assay, taking advantage of 2512 R. Matya´sˇek et al.

CAPS were expected to generate bands at 700, 500, and 200 bp. However, in many populations this was not al- ways the case (see below). RT-CAPS data were compared with the following controls: (i) no ampliﬁed products resulted when RNA was used alone, indicating an absence of contamination with DNA (not shown), (ii) DNA igure mixing experiments showed that the primers do not F 1.—Restriction enzyme maps of the most abundant ovarik T. dubius, T. pratensis, and T. porrifolius rDNA units. The posi- discriminate between the parental unit types (K tion of a conserved BstNI site in the T. dubius units is indi- et al. 2005), and (iii) selected RNA samples were analyzed cated. Large arrows indicate positions of primers. Distances by nuclease protection assay. are approximately to scale. Expression patterns in T. mirus: At the DNA level, the 700-bp bands inherited from T. porrifolius were stronger a BstNI restriction polymorphism in the ITS1 region (Figure 2, lanes ‘‘DNA’’) than those inherited from T. (Figure 1). There is a single BstNI site in the ITS1 of T. dubius (500 and 200 bp), which is consistent with re- dubius, but not in the units of T. porrifolius and duced copy numbers of the T. dubius-origin units in most T. pratensis. Therefore, RT-CAPS and genomic-CAPS Tragopogon allotetraploids. The quantitative propor- in T. porrifolius and T. pratensis generate 700-bp bands, tions of parental genes determined by genomic-CAPS while in T. dubius there are two bands at 500 bp and 200 were in a good agreement with those obtained by the bp (Figures 2 and 3). Consequently, in T. mirus (Figure Southern blot analysis (Kovarik et al. 2005). In lanes 2) and T. miscellus (Figure 3) RT-CAPS and genomic- loaded with RT-CAPS products (lanes ‘‘RNA’’) from

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). rDNA Transcription in Allotetraploids 2513

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). individuals of T. mirus (populations 2601 and 2603), the characteristic for each population is summarized in 700-bp band was absent or weak, suggesting strong Figure 2B. The meiotic stability of nucleolar dominance silencing of T. porrifolius-origin rDNA expression (Fig- was studied in the progeny of 2601 and 2602 individuals. ure 2). Quantitative measurements using densitometry While all 2601 plants showed stable silencing there was scanning of ethidium bromide gels, or incorporated up to fivefold variation in the expression of parental radioactivity, indicated that 95% of transcripts in T. rRNA genes among the 2602 individuals (Figure S1 at mirus are derived from T. dubius-origin units. This result http://www.genetics.org/supplemental/). holds true even in cases where the total number of genes The nuclease protection assay was further carried out of T. dubius origin is extremely small (individual 33 from to confirm the RT-PCR results. The assay is highly spe- population 2603). Similar results were obtained by cific and quantitative due to the RNase sensitivity of reverse transcription of the ITS2 subregion (not shown). mismatched base pairs and the use of solution-phase In contrast to populations 2601 and 2603, T. mirus pop- hybridization driven toward completion by excess of probe. ulation 2602 had a more balanced number of genes The sequence alignment revealed several mismatches inherited from each parent and this was usually re- and indels between the T. dubius and T. porrifolius ITS2 flected in transcript abundance (Figure 2). However, (Figure 4) forming potential targets for enzyme digestion two individuals (1, 49) of this population showed only of RNA–RNA heteroduplexes. First, we carried out a trace amounts of T. dubius-origin rRNA, suggesting recip- control experiment by mixing of T. dubius and T. porrifolius rocal silencing as compared to individuals of popula- RNA at different ratios (left) and hybridizing these with tion 2601 and 2603. The mean rRNA expression pattern a 168-bp probe derived from T. dubius ITS2. It is evident 2514 R. Matya´sˇek et al.

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. Figure 4.—rDNA transcription in T. mirus determined by Diagnostic conformers: D, T. dubius origin; Po, T. porrifolius RNAse protection assay. (A) Experimental strategy and probe origin; Pr, T. pratensis origin. U is a unique conformer specific design. Mismatched nucleotides between T. dubius (accession to T. mirus population 2602. The gels show analysis of individ- no. AM 493990) and T. porrifolius (AM 493995) sequences are uals from populations of T. mirus (pops. 2601-0-10 and 2602-7) indicated. The probe was designed according the T. dubius and T. miscellus (2604-4 and 2606-15). 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 Expression patterns in T. miscellus: We analyzed ex- (left). There was a progressive digestion of probe with increas- pression patterns in three populations of T. miscellus ing concentration of T. porrifolius RNA due to mismatched (Figure 3). Genomic-CAPS revealed genes of both T. nucleotidesinanRNA–RNAheteroduplex.Right:RNAseprotec- tion analysis of representative samples of T. mirus RNA showing dubius and T. pratensis origin in all populations. RT-CAPS nucleolar dominance of T. porrifolius (2602-1) and T. dubius of individuals of populations 2606 and 2604 revealed (2601-7) unit types and a codominance (2602-7). prominent bands of T. dubius-origin transcripts. In population 2605, the ratio of transcripts of T. dubius and the probe remained fully protected to digestion when T. pratensis origin was more balanced. The mean rRNA mixed with fully homologous T. dubius RNA only. expression pattern characteristic for each population is However, increased amounts of T. porrifolius RNA in a summarized in Figure 3B. hybridization reaction resulted in an enhanced diges- SSCP analysis: Compared to RT-CAPS, SSCP exhibits tion of the probe forming distinct low-molecular-weight higher sensitivity, and alteration of the nucleotide se- bands on the gel. Thus, the parental ITS2 could be dis- quence of the molecule by as little as a single base can tinguished based on the differential sensitivity of RNA– reshape the secondary structure, with consequent changes RNA homo- and heteroduplexes. Next we selected RNA in electrophoretic mobilities through native gels. We samples to map all three expression phenotypes in allo- carried out SSCP on selected samples (Figure 5) from tetraploids: the T. dubius expression dominance (2601-7), diploid and tetraploid species. In parental diploids, four a codominance (2602-7) and T. porrifolius dominance or five species-specific bands (conformers) could be (2602-1). The T. dubius probe was fully protected from distinguished. At the DNA level, all four conformers of digestion with a nuclease when hybridized to RNA from T. dubius origin (Du1-4) were significantly reduced in an individual with a typical T. dubius nucleolar domi- three allotetraploid individuals (T. mirus 2601 and both nance (2601-7). In contrast, the probe was partially or T. miscellus 2604 and 2606) while they clearly dominated completely digested when hybridized with RNA from the cDNA (‘‘RNA’’) profiles. No parental transcripts of individuals with codominant and T. porrifolius pheno- T. porrifolius and T. pratensis origin could be detected in types. The expression phenotypes in allotetraploids were 2601 and 2606 individuals, respectively. The sample thus confirmed by both RT-CAPS and nuclease protec- from population 2602 was again exceptional in having tion methods. relatively equal proportion of bands inherited from rDNA Transcription in Allotetraploids 2515

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.

both parents and no apparent differences between the nucleus and its condensation state in individuals cDNA and DNA profiles. In general, the SSCP analysis with silenced (displaying nucleolar dominance) and was in good agreement with the RT-CAPS data. In- non-silenced (codominant) phenotypes. In T. dubius terestingly, some conformers were more pronounced at (Figure 7A) and T. pratensis (Figure 7G) there was a the DNA level while others were more prominent at the single rDNA locus (NOR) that localizes to chromosome RNA level. For example, the D4 conformer of T. dubius A as described by Pires et al. (2004). In T. porrifolius, one origin was barely expressed in T. miscellus 2604, whereas NOR occurs on chromosome A, while a second NOR is it was highly expressed in other accessions. The Pr5 localized to chromosome D (Figure 7B). In this species, conformer was transcriptionally enhanced in T. miscellus chromosome A, unlike chromosome D, carries a 5S 2604. One interpretation is that only a subset of genes rDNA locus, allowing discrimination of both NORs after characterized by distinct ITS1 conformers is expressed double labeling with the 35S and 5S rDNA probes within the array. (Pires et al. 2004). In most cases, only the NOR on Structural polymorphism of rDNA among tetraploid chromosome D shows 35S rDNA decondensation (Fig- populations: To determine if there were polymorphisms ure 7B), suggesting its dominance over the NOR on in IGS, we digested genomic DNA with restriction en- chromosome A. zymes and hybridized the blots with rDNA probes. An individual from T. mirus population 2601 (0-10), Several enzymes, including combined BstYI/SspI (Fig- representing a plant with strong silencing of T. porrifolius- ure 6) digestion, revealed IGS polymorphisms among origin rDNA (Figure 2A), had two partially decondensed diploid species. We analyzed individuals from three and four highly condensed rDNA signals at metaphase different populations of diploids collected in the Pa- (Figure 7C). The locus on chromosome D of T. porrifolius louse region and found little or no variability. In the origin was identified by an absence of a linked 5S rDNA allotetraploid species, most parental BstYI/SspI bands site. These sites were highly condensed at metaphase. were inherited. There were, however, several distinct The chromosome A homeologue of T. dubius origin, deviations from band additivity. First, the intensity of characterized by a strong DAPI band in the centromeric the upper doublet band inherited from T. dubius was position and relatively strong 5S signal, was partially significantly reduced in the 2601 and 2603 individuals, decondensed at metaphase. At interphase, the con- consistent with the previous observation (Figure 2). densed loci remained condensed while the rDNA units Second, the 2602 population of T. mirus differed from on chromosome A of T. dubius origin formed a dispersed the other two populations by having an extra doublet of array of signals within the nucleolus (Figure 7D). These 3–4 kb that did not occur in either of the diploid data indicate that only the latter locus is decondensed progenitors or was present in low amounts beyond the and active. sensitivity limit. Further, the band positions are slightly The rDNA condensation patterns were also analyzed shifted in the 10-kb region. Within-population variation in a plant of T. mirus population 2602 (7) that had a in the structure of the rDNA units was low, and the codominant rDNA expression profile (Figure 2A). At hybridization profiles of several T. mirus individuals metaphase, four of the six rDNA sites showed secondary were identical. constrictions (Figure 7E). The two condensed sites were Condensation patterns in rDNA chromatin: We used from chromosome A of T. porrifolius origin. At inter- FISH to compare the distribution of rDNA chromatin in phase (Figure 7F), the pattern of rDNA decondensation 2516 R. Matya´sˇek et al.

DISCUSSION Nucleolar dominance occurs in some populations of Tragopogon allotetraploids: We analyzed here expression of rRNA genes and rDNA loci in T. mirus and T. miscellus, two recently and recurrently formed allotetraploids that originated naturally within the past 80 years. Among several populations of independent origin, we found evidence for complete dominance (4 populations), partial dominance (2), and codominance (2). In T. mirus both NORs inherited from T. porrifolius were frequently silenced and their expression negligi- ble. At interphase, units of T. porrifolius origin were often condensed and extranucleolar in location. These data indicate that a single T. dubius-origin locus can effi- ciently dominate expression over two other loci. The ability of a single master locus to dominate supernumerary loci has been noted before, e.g., a single NOR- bearing chromosome from Aegilops umbellutata can suppress all remaining NORs in hexaploid wheat (Martini et al. 1982), and in Solanum somatic hybrids, a single tomato locus can dominate NORs from potato (Komarova et al. 2004). But in Arabidopsis suecica, six A. arenosa-origin NORs dominated expression over two NORs inherited from the A. thaliana parent (Pontes et al. 2003). In general, variation among individuals for expression ratios of the progenitor rDNA types was low, whereas differences between populations were significant. Phe- notypes were usually stably inherited in progeny (with the Figure 7.—rDNA chromatin condensation patterns in pop- exception of one population of T. mirus), suggesting that ulations of T. mirus with dominant (C, D, population 2601 plant interspecific hybridization and subsequent polyploidiza- no. 0-10) and codominant (E, F, population 2602 plant no. 7) phenotypes. T. miscellus (H) was an individual from population tion have generated distinct rDNA epiallelic states, each 2604 (plant no. 4). Diploid T. dubius, T. porrifolius, and T. praten- being typical for a particular population. In synthetic A. sis progenitors are in A, B, and G, respectively. rDNA was visu- suecica, the direction of silencing was concordant with alized by FISH to root-tip metaphase (A–C, E, G, H) and natural A. suecica, suggesting that there might be a pre- interphase (D, F) using FITC-labeled 35S rDNA probe (green disposition toward silencing of parental genomes. Nev- fluorescence)andbiotin-labeled5SrDNAdetected withavidin- Cy3 (red fluorescence). Nuclei were counterstained with DAPI ertheless, one strain of A. suecica showed codominance for DNA (blue fluorescence). Note: in C, the rDNA sites that (Pontes et al. 2003), and only certain inter-ecotype show some decondensation have large, linked 5S rDNA sites diploid Arabidopsis hybrids exhibited NOR silencing (arrows); in F and D, note the condensed extranucleolar rDNA (Lewis et al. 2004). These data indicate that ND may sites (arrows). depend on the particular genotype or combination of alleles. Two T. mirus individuals from population 2602 had reflected the secondary constrictions at metaphase. The strongly dominant T. porrifolius rRNA genes, suggesting inactive loci from chromosome A of T. porrifolius origin that the general trend in expression patterns can be formed a condensed, extranucleolar cluster of genes. reversed. Stochastic losses of low- and single-copy genes Thus, at least one rDNA locus from T. porrifolius was have been observed in populations 2605 and 2604 of active in this T. mirus individual. T. miscellus at 5% frequency (Tateet al. 2006). Perhaps In T. miscellus we analyzed population 2604 that show there is a stochastic factor influencing which rDNA loci nearly complete silencing of T. pratensis units (Figure are silenced. Experiments with synthetic allotetraploids 7H). At metaphase of individual 4, two of the four rDNA of Arabidopsis suggested that rRNA gene silencing is sites showed secondary constriction. Relatively small size stochastic in the early generations, but becomes fixed in of decondensed signals on both homologs suggests that later generations (Chen et al. 1998). Perhaps T. mirus the active NOR in T. miscellus is formed from low copy population 2602 remains unstable because it is relatively T. dubius units. young (recently formed) with incompletely established rDNA Transcription in Allotetraploids 2517 ´ˇ epigenetic patterns. However, Ownbey collected plants origin units in synthetic diploid F1 hybrids (R. Matyasek, of T. mirus in Palouse, WA (the collection site of 2602) in unpublished data). While dominance was nearly com- 1949 (Ownbey 1950; Novak et al. 1991). plete in four populations of T. mirus, it was partial in Previous allozymic (Soltis et al. 1995) and AFLP most individuals of T. miscellus, suggesting that the partner (Tateet al. 2006) analysis indicated little or no inter- and genome may modulate the magnitude of silencing. One intrapopulation genetic variability among the diploids, T. mirus individual (33), belonging to the population with T. pratensis and T. porrifolius, while there is some var- nucleolar dominance (2603), had extremely few T. dubius- iability among populations of the widespread T. dubius origin units, perhaps only 200–300 copies per diploid consistent with expectations based on the introduction cell, yet these units were highly transcribed, while .3000 of a limited number of individuals initially introduced units of T. porrifolius origin were completely suppressed. into the Palouse region. Similarly, there were some In A. thaliana, which has 800 rRNA genes per diploid polymorphisms in the ITS (Kovarik et al. 2005) and IGS cell, 50% of the units are estimated to be active. Electron (Figure 6) regions among populations of T. dubius and microscopy observations indicate that there are 300 T. mirus suggesting that T. dubius subgenome is a major units active in pea nucleoli (Gonzalez-Melendi et al. source of genetic variability in Tragopogon tetraploids. 2001). By extension, nearly all units of T. dubius origin The influence of genetic diversity within or outside of might be active in the 2603-33 individual. The situa- rDNA locus on rRNA silencing remains to be deter- tion here is clearly different from Crepis, for example, in mined. IGS sequence might not, however, play a major which low-copy genes on supernumerary B-chromosomes role in nucleolar dominance in allotetraploid Tragopo- do not seem to significantly contribute to the total rRNA gon because rDNA units in populations 2605 and 2606 pool despite their low-level transcription (Leach et al. are identical or near identical (based on ITS sequencing 2005). and blot hybridization data with 10 restriction enzymes; T. mirus population 2602 had a more balanced ratio of data not shown) but show different expression patterns. parental genes that were equally expressed. This result It is more likely that epigenetic factors are important. indicated that expression of T. dubius units themselves Variation in rDNA methylation levels occurs among does not suppress rDNA units on partner chromosomes. ecotypes of A. thaliana, and this has been related to NOR Perhaps elimination of a part of the array might have activity (Riddle and Richards 2002). In Tragopogon, it contributed to elevated transcriptional activity in pop- is likely that rapid epigenetic changes following poly- ulations with a strongly silenced phenotype (2601 and ploidization are the likely cause of expression variability 2603). Alternatively, there might have been a recombi- between populations. nation event between homeologous chromosomes re- Frequent dominance of low-copy rDNA: In many sulting in formation of recombinant NORs and perhaps allopolyploids, concerted evolution has homogenized novel units. Appearance of unique SSCP conformers rDNA repeats to variable degrees and directions, alter- (Figure 5) and restriction bands (Figure 6) in popula- ing the copy number of inherited genes (Alvarez and tion 2602 samples is in accord with this hypothesis. Wendel 2003; Kovarik et al. 2004; Volkov et al. 2007). Chromosomal rearrangements are thought to alter In most cases, expression is dominated by a high-copy expression patterns in wheat (Schubert and Kunzel rRNA gene family (Flavell 1986; Lim et al. 2000; Joly 1990), and deletions of heterochromatin adjacent to et al. 2004). Nevertheless, certain incongruence exists the rDNA locus resulted in the loss of suppression at between copy number and expression. For example, a the underdominant NOR in Drosophila (Durica and less abundant unit was expressed in one accession of Krider 1978). Interestingly, loss of suppression did not Glycine ( Joly et al. 2004) and a NOR in wheat with fewer prevent rDNA amplification in polytene tissues (termed repeats was more active than another with more repeats replicative dominance) indicating that nucleolar dom- (Flavell and O’Dell 1976). inance and developmentally directed genetic changes A previous study of Tragopogon allotetraploids in- might be independent processes (Goodrich-Young and dicated that concerted evolution has resulted in the Krider 1989). Nevertheless, rDNA copy number at indi- partial homogenization of rDNA units, typically involv- vidual NORs did influence both nucleolar and replica- ing a reduction in the number of T. dubius-origin repeats tive dominance in Drosophila interspecific hybrids. In (Kovarik et al. 2005). Surprisingly, in Tragopogon yeast, spontaneous deletions of rDNA repeats released allopolyploids, the less common T. dubius-origin units silencing factors, including histone deacetylases that were expressed and, in most cases (all except 3 indi- acted in trans to silence distal heterochromatic regions viduals out of .100 tested), dominated overall rDNA (Michel et al. 2005). Interestingly, histone acetylation expression. It is significant that both tetraploid species, and DNA methylation seem to be in the center of reg- T. mirus and T. miscellus, combine the T. dubius genome ulation of nucleolar dominance in Arabidopsis and with different partner genomes, indicating that the Brassica allopolyploids (Earley et al. 2006). Perhaps the capacity to dominate rDNA expression might be en- reduction of rDNA repeats at the T. dubius NOR could coded, in part, by the T. dubius-origin subgenome. This have released putative silencing factors (e.g., histone hypothesis is supported by higher expression of T. dubius- deacetylases or even RNA signals (Mayer et al. 2006) 2518 R. Matya´sˇek et al. that might have silenced the NOR activity on the partner Adams, K. L., and J. F. Wendel, 2005 Novel patterns of gene expres- chromosome. sion in polyploid plants. Trends Genet. 21: 539–543. Ainouche, M. L., A. Baumel and A. Salmon, 2004 Spartina anglica The question arises as to how rDNA expression and Schreb.: a natural model system for analysing evolutionary copy number will evolve over longer time frames. Be- changes that affect allopolyploid genomes. Biol. J. Linn. Soc. cause it is unlikely that the most active, decondensed 82: 475–484. Alvarez, I., and J. W. Wendel, 2003 Ribosomal ITS sequences and units will in the long run be lost, we might expect one or plant phylogenetic inference. Mol. Phylogenet. 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