Similar patterns of rDNA evolution in synthetic and recently formed natural populations of Tragopogon (Asteraceae) allot...

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Title:
Similar patterns of rDNA evolution in synthetic and recently formed natural populations of Tragopogon (Asteraceae) allotetraploids
Series Title:
BMC Evolutionary Biology
Physical Description:
Book
Language:
English
Creator:
Malinksa, Hana
Tate, Jennifer A.
Matyasek, Roman
Leitch, Andrew R.
Soltis, Douglas E.
Soltis, Pamela S.
Kovarik, Ales
Publisher:
BioMed Central
Publication Date:

Notes

Abstract:
Background: Tragopogon mirus and T. miscellus are allotetraploids (2n = 24) that formed repeatedly during the past 80 years in eastern Washington and adjacent Idaho (USA) following the introduction of the diploids T. dubius, T. porrifolius, and T. pratensis (2n = 12) from Europe. In most natural populations of T. mirus and T. miscellus, there are far fewer 35S rRNA genes (rDNA) of T. dubius than there are of the other diploid parent (T. porrifolius or T. pratensis). We studied the inheritance of parental rDNA loci in allotetraploids resynthesized from diploid accessions. We investigate the dynamics and directionality of these rDNA losses, as well as the contribution of gene copy number variation in the parental diploids to rDNA variation in the derived tetraploids. Results: Using Southern blot hybridization and fluorescent in situ hybridization (FISH), we analyzed copy numbers and distribution of these highly reiterated genes in seven lines of synthetic T. mirus (110 individuals) and four lines of synthetic T. miscellus (71 individuals). Variation among diploid parents accounted for most of the observed gene imbalances detected in F1 hybrids but cannot explain frequent deviations from repeat additivity seen in the allotetraploid lines. Polyploid lineages involving the same diploid parents differed in rDNA genotype, indicating that conditions immediately following genome doubling are crucial for rDNA changes. About 19% of the resynthesized allotetraploid individuals had equal rDNA contributions from the diploid parents, 74% were skewed towards either T. porrifolius or T. pratensis-type units, and only 7% had more rDNA copies of T. dubius-origin compared to the other two parents. Similar genotype frequencies were observed among natural populations. Despite directional reduction of units, the additivity of 35S rDNA locus number is maintained in 82% of the synthetic lines and in all natural allotetraploids. Conclusions: Uniparental reductions of homeologous rRNA gene copies occurred in both synthetic and natural populations of Tragopogon allopolyploids. The extent of these rDNA changes was generally higher in natural populations than in the synthetic lines. We hypothesize that locus-specific and chromosomal changes in early generations of allopolyploids may influence patterns of rDNA evolution in later generations.

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dochead Research article
bibl
title
p Similar patterns of rDNA evolution in synthetic and recently formed natural populations of it Tragopogon (Asteraceae) allotetraploids
aug
au id A1 snm Malinskafnm Hanainsr iid I1 email srubarova@ibp.cz
A2 Tatemi AJenniferI2 j.tate@massey.ac.nz
A3 MatyasekRomanmatyasek@ibp.cz
A4 LeitchRAndrewI3 a.r.leitch@qmul.ac.uk
A5 SoltisEDouglasI4 dsoltis@botany.ufl.edu
A6 SoltisSPamelaI5 psoltis@flmnh.ufl.edu
ca yes A7 KovarikAleskovarik@ibp.cz
insg
ins Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i, Laboratory of Molecular Epigenetics, Kralovopolska 135, CZ-61265 Brno, Czech Republic
Institute of Molecular BioSciences, Massey University, Palmerston North 4442, New Zealand
School of Biological Sciences, Queen Mary University of London, E1 4NS, UK
Department of Biology, University of Florida, Gainesville, FL 32611, USA
Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA
source BMC Evolutionary Biology
issn 1471-2148
pubdate 2010
volume 10
issue 1
fpage 291
url http://www.biomedcentral.com/1471-2148/10/291
xrefbib pubidlist pubid idtype pmpid 20858289doi 10.1186/1471-2148-10-291
history rec date day 16month 6year 2010acc 2292010pub 2292010
cpyrt 2010collab Malinska et al; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
abs
sec
st
Abstract
Background
Tragopogon mirus and T. miscellus are allotetraploids (2n = 24) that formed repeatedly during the past 80 years in eastern Washington and adjacent Idaho (USA) following the introduction of the diploids T. dubius, T. porrifolius, and T. pratensis (2n = 12) from Europe. In most natural populations of T. mirus and T. miscellus, there are far fewer 35S rRNA genes (rDNA) of T. dubius than there are of the other diploid parent (T. porrifolius or T. pratensis). We studied the inheritance of parental rDNA loci in allotetraploids resynthesized from diploid accessions. We investigate the dynamics and directionality of these rDNA losses, as well as the contribution of gene copy number variation in the parental diploids to rDNA variation in the derived tetraploids.
Results
Using Southern blot hybridization and fluorescent in situ hybridization (FISH), we analyzed copy numbers and distribution of these highly reiterated genes in seven lines of synthetic T. mirus (110 individuals) and four lines of synthetic T. miscellus (71 individuals). Variation among diploid parents accounted for most of the observed gene imbalances detected in Fsub 1 hybrids but cannot explain frequent deviations from repeat additivity seen in the allotetraploid lines. Polyploid lineages involving the same diploid parents differed in rDNA genotype, indicating that conditions immediately following genome doubling are crucial for rDNA changes. About 19% of the resynthesized allotetraploid individuals had equal rDNA contributions from the diploid parents, 74% were skewed towards either T. porrifolius or T. pratensis-type units, and only 7% had more rDNA copies of T. dubius-origin compared to the other two parents. Similar genotype frequencies were observed among natural populations. Despite directional reduction of units, the additivity of 35S rDNA locus number is maintained in 82% of the synthetic lines and in all natural allotetraploids.
Conclusions
Uniparental reductions of homeologous rRNA gene copies occurred in both synthetic and natural populations of Tragopogon allopolyploids. The extent of these rDNA changes was generally higher in natural populations than in the synthetic lines. We hypothesize that locus-specific and chromosomal changes in early generations of allopolyploids may influence patterns of rDNA evolution in later generations.
bdy
Background
Chromosome counts suggest that between 30 and 100% of angiosperm species are polyploids abbrgrp
abbr bid B1 1
, and Wood et al.
B2 2
propose that 15% of angiosperm speciation events are associated with polyploidy whereas recent genomic studies of selected model and crop species have revealed that all plant genomes sequenced to date have signatures of one or more whole-genome duplications in their evolutionary history
B3 3
B4 4
. The success of newly formed angiosperm polyploids is partly attributable to their highly plastic genome structure as manifested by deviations from Mendelian inheritance of genetic loci and chromosome aberrations
B5 5
. Indeed, there are numerous examples of intergenomic exchanges, chromosomal translocations, transposon proliferation, and sequence loss in both newly formed and ancient allopolyploid species (for review see
B6 6
B7 7
).
In plants, nuclear ribosomal DNA (rDNA) units occur in tandem arrays at one or several loci (for review see
B8 8
B9 9
). Each large 35S rDNA unit contains the 18S, 5.8S, and 26S rRNA genes, the internal transcribed spacers (ITS), and the intergenic spacer (IGS). The 5S genes encoding 120-nt transcripts are usually, but not always
B10 10
, located at different chromosomal loci than 35S rDNA. The genes are highly conserved even between eukaryotes and prokaryotes, whereas divergence of ITS is sufficient to resolve species relationships within most genera
B11 11
. The IGS, which contains the transcription start site and genetic and epigenetic features that influence the regulation of the downstream genes, diverges rapidly, and substantial differences in structure may occur even within a species
B12 12
B13 13
B14 14
. The number of gene copies may vary from 500 up to tens of thousands in certain plant species
B15 15
. Similar variation has been observed in locus number, with levels ranging from one to several loci per haploid set
B16 16
. Within species, the copy and locus number is usually stable, although intraindividual and intergenerational variation in copy number has been reported in some plants
B17 17
. As with other repeated sequences, rDNA can undergo concerted evolution involving sequence homogenization
B18 18
B19 19
. Such a process efficiently eliminates mutated copies maintaining long arrays of functional tandemly arranged genes.
The behavior of rDNA in allopolyploids has attracted considerable attention because it is used as a molecular and cytogenetic marker of allopolyploidy
B20 20
. Indeed, the hybrid origin of many species has been successfully deciphered using ITS sequences. Nevertheless, repeat and locus loss, and intra- and interlocus recombination seem to be ongoing evolutionary processes, potentially preventing identification of hybrids. In fact, many well-defined allopolyploid species have either lost one or several loci
B21 21
B22 22
B23 23
, eliminated or contracted parental arrays
B24 24
B25 25
B26 26
, recombined
B27 27
or replaced the units
B28 28
B29 29
. On the other hand, some polyploid species seem to maintain both parental copies in the genome long after allopolyploid formation
14
B30 30
B31 31
B32 32
B33 33
. These studies indicate that the process of rDNA evolution is complicated, and that no firm conclusion can be drawn on the tempo and direction of repeat homogenization. Nevertheless, there are examples of synthetic allopolyploid lines in which rDNAs have already undergone rearrangements at the chromosomal and unit levels
13
B34 34
B35 35
.
Recently formed allopolyploids represent unique natural systems in which to study the immediate consequences of allopolyploidy. Only a few polyploid plant species are known to have formed in the past 200 years: Cardamine schulzii
B36 36
, Spartina anglica
B37 37
, Senecio cambrensis and S. eboracensis
B38 38
, Tragopogon mirus and T. miscellus
B39 39
. Cardamine allopolyploid populations seem to evolve recombined ITS types
B40 40
. In Spartina anglica, the two individuals collected from different localities differed in the composition of rDNA units
B41 41
.
Allotetraploid Tragopogon mirus and T. miscellus formed in the Palouse region (eastern Washington, and western Idaho, USA) within the last 80 years
39
B42 42
and thus represent an excellent model for examining early events in allopolyploid evolution. Recent studies using different methodological approaches have shown frequent loss of homeologous sequences, including low-copy protein-coding genes
B43 43
B44 44
and high-copy rDNA
B45 45
. In the latter, a population-level analysis revealed reduction of rDNA arrays derived from the T. dubius diploid parent in all but one natural population examined (T. mirus). While the average magnitude of gene loss was about 50%, there were individuals that lost as many as 95% of all repeats. Cytogenetic studies confirmed that rearrangements were homologous and were not linked to chromosome loss
B46 46
.
The interpretation of genetic variation in natural polyploids is always complicated by the fact that genetic parents are unknown even in the case of recently formed species. That is, some allopolyploids may start out with far more rDNA copies of one parent than the other, simply because the diploid parents differ in copy number. On the other hand, genotypic variation may arise from genetic changes induced by stressful conditions experienced during allopolyploidy
5
. In this study we asked: (i) What is the contribution of parental diploids to copy number variation in newly formed allopolyploid T. mirus and T. miscellus? (ii) What are the dynamics of rDNA rearrangements, and do they occur suddenly or gradually? (iii) Is there a directionality and genetic predisposition for locus rearrangement? (iv) Are there parallels in the evolution of rDNA in natural and synthetic populations of the two allopolyploids? (v) What are the likely mechanisms of rDNA rearrangement? To address these questions we synthesized allotetraploid lines of T. mirus and T. miscellus
B47 47
using several different populations of parental accessions. We determined homeolog gene ratios by Southern blot and slot blot hybridization. The locus numbers were assessed using FISH. Evidence was obtained for repeat loss in early generations of synthetic allopolyploid lines at frequencies and directionality similar to those observed in natural situations.
Methods
Plant material
Field-collected seeds of three diploid Tragopogon species were planted and selfed for one generation. Then 103 different crosses were made between individuals from three populations of T. dubius (2613, 2615, 2616), two populations of T. porrifolius (2607, 2611), and two populations of T. pratensis (2608, 2609) from different localities (Table tblr tid T1 1, Table T2 2 and
47
). Seeds from successful crosses were treated with 0.1 or 0.25% (w/v, water solution) colchicine during germination (overnight), washed, and placed in pots with soil. Approximately 6 months after germination, young plants were genotyped to determine their parentage using a marker (TDF 85) specific for all three diploid species
43
47
. The non-treated F1 diploid hybrids were planted as controls. Detailed information about crosses is described in
44
47
.
tbl Table 1caption Parental origin of synthetic allotetraploids and direction of crossestblbdy cols 12
r
c
center cspan 7
b
T. mirus
4
T. miscellus
11
hr
left
Line
70
73
98
116
121
134
135
67
79
111
129
parents
♀ × ♂
2611 × 2613
2611 × 2613
2611 × 2613
2607 × 2615
2615 × 2607
2607 × 2613
2613 × 2607
2609 × 2616
2609 × 2616
2608 × 2613
2613 × 2608
sup 1N
9
48
7
28
8
14
16
15
22
39
3
2S0
2
3
1
1
0
1
1
2
1
4
1
2S1
7
34
6
27
8
13
15
13
21
35
2
2S2
0
11
0
0
0
0
0
0
0
0
0
tblfn
1total number of individuals analyzed by Southern blot hybridization.
2numbers in S0-S2 generations; the S2 was the progeny of selfed S1 parents (73-14, 73-1, 73-2) that have been analyzed in this study by molecular and cytogenetic approaches.
Table 2Characteristics of rDNA loci in populations of parental diploid species used for construction of synthetic lines
Species
1Collection no.
Location
2rDNA copies
rDNA
genotype
No. of 35S rDNA sites
No. of 5S rDNA sites
T. porrifolius
2607
Troy, ID
1.5 ± 0.3
High-copy
4
4
2611
Pullman, WA
1.0 ± 0.2
Low-copy
4
4
T. pratensis
2608
Moscow, ID
1.2 ± 0.1
Medium-copy
2
2
2609
Spangle, WA
1.5 ± 0.2
High-copy
2
2
T. dubius
2613
Pullman, WA
1.7 ± 0.4
High-copy
2
2
2615
Spokane, WA
0.9 ± 0.1
Low-copy
2
2
2616
Spangle, WA
1.0 ± 0.2
Low-copy
2
2
1Soltis & Soltis collection numbers; vouchers deposited at FLAS.
2DNA copies per haploid set in thousands.
Molecular cytogenetic analysis
Root tips cut from vigorously growing plants were pre-treated with 2 mM 8-hydroxyquinoline (Sigma-Aldrich Company Ltd, Poole, Dorset, UK) to obtain metaphase nuclei. After 2 hours of incubation on ice, root tips were fixed in ethanol: acetic acid (3: 1) at room temperature overnight, then stored in 70% ethanol at -20°C. Fixed root tips were digested in 0.3% (w/v) cellulase Onozuka R-10 (Apollo Scientific Ltd, Stockport, Cheshire, UK), 0.3% (w/v) pectolyase Y23 (MP Biomedicals, Solon, Ohio, USA), and 0.3% (w/v) drieselase (Sigma-Aldrich Company Ltd., Poole, Dorset, UK) for 27 min and transferred to 1% citrate buffer pH 4.8 and incubated for 1 hour. The meristematic cells behind the root cap were squashed onto a glass slide in a drop of 60% acetic acid. Coverslips were removed after freezing in liquid nitrogen.
Fluorescent in situ hybridization (FISH) of the diploids and polyploids followed standard protocols
B48 48
. The probe for 5S rDNA was prepared by PCR amplification of the cloned Nicotiana tabacum 5S rRNA gene
B49 49
followed by labeling with biotin-16-dUTP as described in
48
. The probe for 35S rDNA was a clone that includes part of the 18S rDNA isolated from Allium cernuum, which was labeled with digoxigenin-11-dUTP as described in
48
. Sites of probe hybridization were detected using 20 μg mL-1 fluorescein-conjugated anti-digoxigenin immunoglobulin (GE Healthcare, Chalfont St Giles, Buckinghamshire, UK) or 5 μg mL-1 Cy3-conjugated avidin (Roche Pharmaceuticals, Lewes, East Sussex, UK) in 4 × SSC containing 0.2% (v/v) Tween 20 and 5% (w/v) bovine serum albumin. Chromosomes were counterstained with 2 μg mL-1 DAPI (4',6-diamidino-2-phenylindole (Sigma-Aldrich Company Ltd. Dorset, UK) in 4 × SSC) and stabilized in Vectashield medium (Vector Laboratories Ltd., Peterborough, UK) prior to data acquisition using a Leica DMRA2 epifluorescent microscope fitted with an Orca ER camera and Open Lab software® (Improvision, Coventry, UK). The images were adjusted with Adobe Photoshop® version 7 and treated for color contrast and uniform brightness only. At least five mitotic cells per plant were scored with each probe used.
DNA isolation, Southern blotting
Genomic DNA was extracted either from fresh leaves or leaves preserved in RNAlater reagent (Applied Biosystems, Ambion, Warrington, UK), following the instructions of the manufacturer of RNAlater or by a standard CTAB method described in
B50 50
and modified in
B51 51
. DNA concentration was estimated by two independent methods: (i) a CYBR green fluorescence method following a protocol at http://www.dnagenotek.com/); the green fluorescence was measured on a Rotor gene thermocycler (Corbett Research, Brisbane, Australia) as recommended; and (ii) ethidium bromide fluorescence measured after gel electrophoresis using phage lambda DNA as a standard. The estimates obtained from both methods were concordant. Integrity of DNA was checked on gels.
Southern blotting followed the protocol described in
51
using rDNA probes labeled with [α-32P]dCTP (Izotop, Budapest, Hungary). The ITS-1 probe was a BstNI fragment from the cloned 18S-ITS-5.8S subregion of T. mirus rDNA (GenBank: ext-link ext-link-id AY458586 ext-link-type gen AY458586). The 18S rDNA probe was a cloned 1.7-kb fragment of the tomato 18S rRNA gene
B52 52
, and the 26S rDNA probe was a 280-bp PCR product derived from the 3' end of the tobacco 26S rRNA gene
B53 53
. Hybridization signals were visualized by phosphorimaging (Storm, Molecular Dynamics Sunnyvale, CA, USA).
For rDNA quantification, 17.5-200 ng of DNA were denatured in 0.2 M NaOH and loaded on a nylon membrane (Hybond XL, GE Healthcare, Little Chalfont, UK) using a slot blot apparatus (Schleicher & Schuell, Sigma-Aldrich, Dorset, UK). The membranes were hybridized with respective DNA probes in a Church Gilbert buffer
B54 54
. Radioactivity in each band was counted using a rectangle integration method (ImageQuant software, GE Healthcare, Little Chalfont, UK). A standard curve was constructed using a diluted plasmid carrying the 18S rDNA insert
52
. The experiments were repeated three times and data averaged.
Statistical calculations were carried out using a chi-square function implemented in MicroSoft Excel.
Results
Plants and scheme of experiments
The generation of allotetraploid lines and phenotypic and karyological analysis were described elsewhere
47
. Briefly, the allotetraploids were derived from independent crosses (Figure figr fid F1 1) involving two or three different accessions of diploid T. dubius, T. porrifolius, and T. pratensis (Table 2). In this study, we used seven lines of synthetic T. mirus, four lines of synthetic T. miscellus, and two lines of diploid F1 hybrids. The parental origin for each line is given in Table 1. A "line" was defined as the progeny originating from a single cross (S0, S1, S2...) and maintained through selfing. "Lineages" were obtained by selfing of plants from the same cross but from different F1 seeds.
fig Figure 1Scheme of crossing strategiestext
Scheme of crossing strategies. Tragopogon dubius and T. porrifolius (or T. pratensis) were used as either the maternal or paternal parent; each cross (line) gave rise to sterile diploid F1 progeny. To double chromosomes and restore fertility of these hybrids, seeds were treated with colchicine, obtaining fertile allotetraploid S0 plants. These individuals were selfed to produce lineages of fertile allotetraploid hybrids (generation S1, S2).
graphic file 1471-2148-10-291-1 hint_layout single
Copy number estimates in diploid genome donors
Because estimates of rDNA amounts may vary among populations of diploid species, we determined gene copy number of diploid parents using slot blot hybridization. Two to six individuals from each population were analyzed. The DNA was hybridized on blots with the 18S rDNA probe and the amount of radioactivity estimated. An example of our hybridization analysis is shown in Additional file supplr sid S1 1. It is evident that the strongest hybridization signals were obtained with DNA from T. dubius 2613 and T. porrifolius 2607; these were scored as "high-copy" accessions. On the other hand, T. dubius 2615 and T. porrifolius 2611 showed relatively weak hybridization signals and were scored as "low-copy" accessions. Variation within populations and among progeny was low (< 15%) or negligible. The copy number estimates for each population are given in Table 2.
suppl
Additional file 1
Slot blot quantification of gene copies in parental diploids. The DNA amounts are indicated above each lane. The blot was hybridized with the 32P labeled 18S rDNA probe. Experiments I and II were carried out in this study; experiment III is from
45
.
name 1471-2148-10-291-S1.PDF
Click here for file
Southern blot hybridization in parental plants
The ITS region was analyzed taking advantage of a diagnostic BstNI restriction site polymorphism that distinguishes the diploid parents. In T. dubius, ITS-1 contains a BstNI site close to the 5.8S gene while there is no BstNI site in the ITS-1 of T. porrifolius and T. pratensis
45
. Consistent with previous results, the ITS-1 probe hybridized to the ~ 700-bp fragment in T. pratensis and T. porrifolius and to the ~ 500-bp fragment in T. dubius (Figures F2 2D, F3 3D).
Figure 2rDNA repeat inheritance in T. mirus determined by Southern blot hybridization
rDNA repeat inheritance in T. mirus determined by Southern blot hybridization. (A) Synthetic allotetraploid lines of T. mirus. (B) Diploid T. porrifolius x T. dubius hybrids. (C) Natural populations of T. mirus. (D) Profiles of parental diploids. Representative individuals are shown in the panels; the rest of the analysis expressed as a ratio of parental bands is shown in Figure 5A. Each S0 and S1 generation of synthetic allopolyploid (A) is encoded as follows: e.g. in 70-1, the first number is the line number, the second number indicates a particular lineage (Figure 1 and Table 1). The DNAs were digested with BstNI and hybridized with the ITS-1 probe. The number below each lane indicates percentage of T. dubius units out of total rDNA. DU T. dubius units; PO T. porrifolius units.
1471-2148-10-291-2 double
Figure 3rDNA repeat inheritance in T. miscellus determined by Southern blot hybridization
rDNA repeat inheritance in T. miscellus determined by Southern blot hybridization. (A) Synthetic allotetraploid lines of T. miscellus. (B) Diploid T. pratensis x T. dubius hybrids. (C) Natural populations of T. miscellus. (D) Profiles of parental diploids. Representative individuals are shown in the panels; the rest of the analysis expressed as a ratio of parental bands is shown in Figure 5B. The digests and conditions of the blot and labels are the same as in Figure 2. DU T. dubius units; PR T. pratensis units.
1471-2148-10-291-3
The IGS region was analyzed using BstYI and SspI restriction enzymes (Additional file S2 2). There are more BstYI sites in the IGS of T. porrifolius than in T. dubius, based on the sequenced clones [GenBank: FN666261.1 FN666261.1 and FN645941.1 FN645941.1]. Consequently, the probe hybridized to low-molecular-weight fragments in T. porrifolius and to high-molecular-weight fragments in T. dubius.
Additional file 2
Analysis of intergenic rDNA spacer polymorphisms in DNA of synthetic T. mirus (S1 generation). Genomic DNA was digested with BstYI and SspI restriction enzymes. Southern blot hybridization was carried out using the 26S rDNA probe.
1471-2148-10-291-S2.PDF
Click here for file
FISH analysis in parental plants
To determine the number and chromosomal position of rRNA genes, we carried out rDNA-FISH on metaphase chromosomes. In T. dubius (Figure F4 4C), there were two sites of probe hybridization for both 35S and 5S rDNA, located on the largest chromosome pair (Adu; the nomenclature follows that of Pires et al.
B55 55
). T. pratensis also has two sites of 35S rDNA (both decondensed) and one pair of 5S rDNA loci at metaphase (Figure 4A). Both 35S and 5S rDNA loci occur on chromosomes Apr. T. porrifolius has four 35S and four 5S rDNA sites at metaphase (Figures 4B, D). Chromosome Apo carries both 35S and 5S rDNA loci, at homeologous loci to the other diploids, but there are also 35S rDNA loci on the two homologs of chromosome Dpo, and 5S rDNA loci on both homologs of chromosome Fpo. The 5S signal on Apo was much weaker in accession 2611 (Figure 4D) than in accession 2607 (Figure 4B). The FISH results are consistent with previous cytogenetic analysis carried out in the same diploid populations, but with different individuals
55
, indicating chromosomal stability of loci.
Figure 4FISH to metaphase spreads of parental diploid species (A-D) and synthetic T. miscellus lines (E-F)
FISH to metaphase spreads of parental diploid species (A-D) and synthetic T. miscellus lines (E-F). (A) T. pratensis 2608. (B) T. porrifolius 2607. (C) T. dubius 2613. (D) T. porrifolius 2611. (E) and (F) stand for the 111-1 and 111-4 synthetic individuals, respectively. Metaphases were hybridized with the 18S rDNA (painting 35S sites in green) and 5S rDNA (red fluorescence) probes. Note the discontinuous chromatin condensation along the loci. Regions of condensed and decondensed chromatin are interconnected with dotted lines in (A-C). Scale bar = 10 μm.
1471-2148-10-291-4
Molecular and cytogenetic analysis in synthetic hybrids and allotetraploids
We carried out a population-level study of 35S rRNA gene copy number using Southern blot hybridization using the ITS-1 probe (Figures 2, 3). The ratios of gene units in each sample are shown in Figures F5 5A (T. mirus) and 5B (T. miscellus); the averaged values for each line are summarized in Figure 5C, D. FISH with the 18S rDNA probe was then carried out on selected individuals (Figures 4, F6 6 and Table T3 3). In the following sections, we describe the results of both types of analysis for each line derived from diploid parents with high, medium, or low copy numbers at the 35S rDNA loci (called high-, medium-, or low-copy) parents.
Figure 5Summary of rDNA repeat ratios analysis estimated for individual synthetic T. mirus (A, C) and T. miscellus (B, D) plants
Summary of rDNA repeat ratios analysis estimated for individual synthetic T. mirus (A, C) and T. miscellus (B, D) plants. Homeolog gene ratios were determined from quantification of band signals after the Southern blot hybridization (Figures 2, 3). Each parental hybridization band was quantified using phosphorimaging, and gene ratios were expressed as a percentage of T. dubius units out of the total signal (A, B). The averaged values for each line are shown in graphs (C and D shaded bars); for F1 diploid hybrids the shaded bars are drawn at low contrast. Filled bars represent expected gene ratios based on strict Mendelian inheritance of gene copy numbers. The interindividual variability within the line is expressed by standard deviation from the mean. Differences between expected and observed ratios were highly significant (P < 0.001, standard chi-square test) in all lines except the diploid F1-47 and F1-2 hybrids and line 121.
1471-2148-10-291-5
Figure 6FISH to metaphase spreads of synthetic T. mirus
FISH to metaphase spreads of synthetic T. mirus. The probe labels and scale bar are as in Figure 4. Individuals 73-14 (A, B), 70-4 (C), and 73-13 (D) had four strong plus 0-2 very weak signals (dependent on particular metaphase and sample). (B) is an expanded region of (A) showing the large locus of T. dubius origin and the minute Dpo locus (arrowheads) left after the deletion of the majority of genes from the array. Lineages 98-1 (E), 134-15 (F), 116-1 (G), and 135-5 (H) showed regularly six 18S rDNA signals. The metaphase in (G) has largely decondensed Adu (arrows), partially decondensed Dpo (arrowheads), and fully condensed Apo loci.
1471-2148-10-291-6
Table 3Summary of cytogenetic analysis
Species
3N
35S rDNA sites
5S rDNA sites
Synthetic T. mirus1
17
4-6
6-7
Natural T. mirus2
11
64
6-7
Synthetic T. miscellus1
2
4
4
Natural T. miscellus2
6
4
4
1this study.
2 references 454655.
3number of individuals analyzed by rDNA-FISH.
4some individuals from population Rosalia 45 had a nearly deleted locus on chromosome Adu inherited from T. dubius (Figure 2C).
F1 diploid hybrids
We analyzed rRNA gene ratios using Southern blot hybridization with the ITS-1 probe against diploid F1 hybrid plants. In 11 F1 individuals (cross 47) from a single cross involving a "high-copy" T. dubius 2613 paternal parent and a "low-copy" T. porrifolius 2611 maternal parent (Figure 2B), the probe hybridized strongly to the T. dubius-origin rDNA units (DU) forming a lower molecular weight band, while the upper band of T. porrifolius-origin rDNA units (PO) was significantly weaker. Radioactivity scanning revealed that on average 60% of the total hybridization signal occurred in the lower molecular weight band, representing rDNA units of T. dubius origin. This value is close to the expected ratio considering the unit copy numbers in the diploid parents (Table 2 and Figure 5C, D). With the exception of one individual (47-8), plant to plant variation in gene ratios was low (Figure 2B). The second diploid hybrid (cross 2) examined was derived from a cross involving "low-copy" T. dubius 2615 and "medium copy" T. pratensis 2607 (Figure 3B). In this case, the upper molecular weight band of T. pratensis-origin rDNA units (PR) was stronger than the band inherited from T. dubius. Again, F1 hybrids generally showed the expected number of genes inherited from their parents (Figure 5C, D).
Synthetic T. mirus
Line 73
This line was derived from a cross involving "low-copy" 2611 T. porrifolius (maternally inherited) and "high-copy" T. dubius 2613 (paternally inherited). Thirty-seven individuals obtained from four lineages (73-1, 73-2, 73-13, and 73-14) were analyzed by Southern blot hybridization using the ITS-1 probe (examples are shown in Figure 2A). There was significant variation in rRNA gene ratios, and at least two distinct rDNA genotypes were distinguished: lineages 73-2 and 73-13 had nearly balanced DU/PO ratios, while lineages 73-1 and 73-14 had ratios skewed towards T. dubius-origin rDNA (61-75%) (Figures 5A and 5C). The 26S rDNA probe, which mapped polymorphisms in the intergenic spacer (IGS), showed similar results (Additional file 2). The homeolog gene ratios established in the S1 seem to be inherited among the S2 individuals (Additional file S3 3).
Additional file 3
Southern blot analysis of the S2 generation of synthetic T. mirus
. Individuals were the progenies of three lineages from line 73.
1471-2148-10-291-S3.PDF
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Representative individuals of both genotypes were analyzed by FISH (Figure 6A, B and 6D). The 18S rDNA probe hybridized strongly to terminal regions of both homeologs of chromosomes Apo and Adu. However, there was no, or very little, hybridization signal on chromosome Dpo (Figure 6B arrowheads), indicating locus loss or drastic elimination of units. This pattern appears to be typical for all S1 individuals and their progeny (Additional file S4 4). There were also quantitative differences in signal intensities. While lineages 73-2 and 73-13 had comparable sizes of all four 35S rDNA loci, lineages 73-1 and 73-14 had enlarged Adu loci. The number of 5S sites was additive (6 signals) in the S1 generation while one S2 individual gained a strong site likely on chromosome F (Additional file 4D). Thus, cytogenetic and molecular analysis both show considerable variability in rDNA locus sizes in these lineages.
Additional file 4
FISH analysis of the S2 generation of synthetic T. mirus
. The same plants as in Additional file 3 were analyzed. Most metaphases displayed aneuploid karyotypes (23 chromosomes). Arrowheads in (C, F) indicate a minute Dpo locus left after the deletion of the majority of genes. Note fusion of subtelomeric NORs at the chromatids (arrowheads, E) and considerable variability in condensation of rDNA chromatin among sister plants (A-C). The following individuals are shown: (A) 73-14-6A, (B) 73-14-6B, (C) 73-14-6C, (D, E) 73-1-3 D, (F) 73-2-8B.
1471-2148-10-291-S4.PDF
Click here for file
Line 70
These allopolyploids were derived from "low-copy" T. porrifolius 2611 (maternally inherited) and "high-copy" T. dubius 2613 (paternally inherited), but different individuals were used as parents than in crosses 73 and 98. On the Southern blots, the ratios of parental bands were mostly balanced (Figure 2A). As in line 73, there were only four 35S rDNA signals (some with secondary constrictions) instead of the expected six, indicating locus loss (Figure 6C).
Line 98
Only a single line, 98-1, was recovered from this cross, originating from populations of "low-copy" T. porrifolius 2611 (maternally inherited) and "high-copy" T. dubius 2613 (paternally inherited). Different individuals were used as parents than in the previous two lines. On blots, the probe showed a much stronger hybridization to the band corresponding to the T. porrifolius-origin homeolog than to the homeolog of T. dubius origin (Figure 2A). There were six 35S and six 5S rDNA chromosomal sites, indicating an additive number of loci from that expected of the diploid parents (Figure 6E).
Line 116
In this cross we used "high-copy" T. porrifolius 2607 as the maternal genome donor and "low-copy" T. dubius 2615 as the paternal genome donor. All individuals from eight lineages showed relatively uniform Southern hybridization profiles, with band ratios highly skewed towards the T. porrifolius-origin units (Figures 2A, 5). FISH showed the expected number of 35S signals, with signals on chromosomes Apo, Dpo, and Adu although the signals on chromosome Adu (arrows) are smaller and highly decondensed (Figure 6G). On the other hand, the 35S signals on T. porrifolius chromosomes were strong, and NORs on Dpo (arrowheads) showed only partial decondensation.
Line 121
This line originated from a cross involving "low-copy" T. dubius 2615 as the maternal parent and a "high-copy" T. porrifolius 2607 as a donor of the paternal genome. The gene ratio of all eight allotetraploid individuals was slightly shifted towards the T. porrifolius homeolog (Figure 5A). FISH was not conducted on this line.
Line 134
This line was derived from a "high-copy" T. porrifolius 2607 (maternally inherited) and a "high-copy" T. dubius 2613 (paternally inherited) and yielded two lineages (134-15 and 134-16). On the blots, individuals from both lines showed a relatively weak T. dubius-origin band and a strong T. porrifolius-origin band (Figures 2A, 5). At the cytogenetic level, the individuals inherited an additive number of 35S rDNA loci, four strong sites and two weaker ones, the latter likely of T. dubius origin, that are slightly remote from the bulk of the chromosomes due to secondary constriction (Figure 6F).
Line 135
This line originated from a cross reciprocal to that giving line 134 and comprised three lineages (1, 2, and 5). While both lineages 135-1 and 135-2 had gene ratios skewed towards T. porrifolius-origin units, the gene ratios were balanced in the 135-5 individuals (Figures 2A, 5). There was some variability among the 135-5 individuals (Figure 5A). However, the number of loci was additive in the individual examined using FISH (Figure 6H).
Synthetic T. miscellus
Line 67
In this cross, a "high-copy" T. pratensis 2609 was used as the maternal genome donor and "low-copy" T. dubius 2616 as the paternal genome donor. There was little or no variation in the gene ratios among the individuals analyzed, and all individuals had reduced Southern hybridization signal against the T. dubius-origin units (Figure 3A).
Line 79
This cross is a reciprocal cross to that which generated line 67, and it yielded 5 lineages. There was some, albeit little, intralineage variability in band hybridization signal intensities. For example, the S1 individual in the middle lane (Figure 3A) had a stronger T. dubius-origin band compared to that from the other parent (T. pratensis) while most other members of this line had a dominant band of T. pratensis origin (Figure 5B).
Line 111
In this cross a "medium-copy" T. pratensis 2608 (maternally inherited) and "high-copy" T. dubius 2613 (paternally inherited) were used as genome donors. This combination of parents yielded the largest number of allotetraploid individuals (35) out of the T. miscellus crosses attempted. Lineages 111-4, 111-5, and 111-7 had relatively uniform profiles of Southern hybridization bands with ratios skewed away from the units of T. dubius origin (Figure 3A). Lineage 111-1 differed in having relatively large intralineage variability (Figure 5B). There were individuals with signals skewed towards the T. pratensis type as well as individuals with balanced gene ratios. FISH was carried out on randomly selected individuals of lineages 111-1 (Figure 4E) and (Figure 4F). Both plants retained an additive number of 35S and 5S rDNA loci from that expected of the diploid parents (i.e., four 35S and four 5S rDNA sites). However, plant 111-4 had two larger and two smaller 35S rDNA signals, all with secondary constrictions, while individual 111-1 had signals of comparable sizes on all four chromosomes.
Line 129
Line 129 resulted from a cross reciprocal to that which generated line 111. Only two allotetraploid individuals were recovered from this cross, and both had gene ratios highly skewed away from T. dubius-origin units (Figure 5B).
Discussion
rRNA gene copy number variation in synthetic T. mirus and T. miscellus
We analyzed the inheritance of rRNA genes in seven lines of synthetic T. mirus and four lines of synthetic T. miscellus. In synthetic T. mirus, 32 individuals (29%) exhibited balanced rDNA genotypes, 69 individuals (63%) showed more 35S rDNA of T. porrifolius origin than expected, and only 9 plants (8%) had more T. dubius-origin rDNA (Table T4 4). In synthetic T. miscellus, three individuals (4%) had balanced gene ratios while 65 individuals (92%) inherited more T. pratensis-origin units than expected. The genetic variation in copy ratios among the progeny of a single cross ranged from low or negligible (5%, line 134) to high (40%, line 111) (Figure 5C). Some crosses involving the same parental accessions (lines 70, 73, and 98) gave rise to individuals with expected ratios of parental 35S rDNA units considering the copy numbers in the diploid parents, while others had rDNA genotypes balanced or skewed towards units derived from either of the diploid parents.
Table 4Comparison of rDNA ratios in synthetic and natural populations of allotetraploids5
Species
Genotype
Gene ratio, Du:Po/Pr
1N
% of individuals
Synthetic T. mirus
high Du
60%/p
/c
c ca="center"
p9/p
/c
c ca="center"
p8/p
/c
/r
r
c
p/
/c
c ca="left"
pbalanced/p
/c
c ca="left"
p40-60%/p
/c
c ca="center"
p32/p
/c
c ca="center"
p29/p
/c
/r
r
c
p/
/c
c ca="left"
phigh Po/p
/c
c ca="left"
p 60%p
c
c ca="center"
p69p
c
c ca="center"
p63p
c
r
r
c
p
c
c ca="left"
ptotalp
c
c
p
c
c ca="center"
p110p
c
c
p
c
r
r
c
p
c
c
p
c
c
p
c
c
p
c
c
p
c
r
r
c ca="left"
pNatural itT. mirusitp
c
c ca="left"
phigh Dup
c
c ca="left"
p 60%p
c
c ca="center"
p0p
c
c ca="center"
p0p
c
r
r
c
p
c
c ca="left"
pbalancedp
c
c ca="left"
p40-60%p
c
c ca="center"
p20p
c
c ca="center"
p29p
c
r
r
c
p
c
c ca="left"
phigh Pop
c
c ca="left"
p 60%p
c
c ca="center"
p48p
c
c ca="center"
p71p
c
r
r
c
p
c
c ca="left"
ptotalp
c
c
p
c
c ca="center"
p68p
c
c
p
c
r
r
c
p
c
c
p
c
c
p
c
c
p
c
c
p
c
r
r
c ca="left"
pSynthetic itT. miscellusitp
c
c ca="left"
phigh Dup
c
c ca="left"
p 60%p
c
c ca="center"
p3p
c
c ca="center"
p4p
c
r
r
c
p
c
c ca="left"
pbalancedp
c
c ca="left"
p40-60%p
c
c ca="center"
p3p
c
c ca="center"
p4p
c
r
r
c
p
c
c ca="left"
phigh Prp
c
c ca="left"
p 60%p
c
c ca="center"
p65p
c
c ca="center"
p92p
c
r
r
c
p
c
c ca="left"
ptotalp
c
c
p
c
c ca="center"
p71p
c
c
p
c
r
r
c
p
c
c
p
c
c
p
c
c
p
c
c
p
c
r
r
c ca="left"
pNatural itT. miscellusitp
c
c ca="left"
phigh Dup
c
c ca="left"
p 60%p
c
c ca="center"
p0p
c
c ca="center"
p0p
c
r
r
c
p
c
c ca="left"
pbalancedp
c
c ca="left"
p40-60%p
c
c ca="center"
p0p
c
c ca="center"
p0p
c
r
r
c
p
c
c ca="left"
phigh Prp
c
c ca="left"
p 60%p
c
c ca="center"
p31p
c
c ca="center"
p100p
c
r
r
c
p
c
c ca="left"
ptotalp
c
c
p
c
c ca="center"
p31p
c
c
p
c
r
tblbdytblfn
psup1supN number of individuals. The data were obtained from the Ssub1 subgeneration of synthetic lines (Table 1); natural populations were analyzed in abbrgrpabbr bid="B45"45abbrabbr bid="B60"60abbrabbrgrp.p
tblfntbl
sec
sec
st
pSources of rDNA copy number variability in allotetraploidsp
st
sec
st
p(i) Contribution of natural variation in parental accessionsp
st
pThere may be up to two-fold variation in 35S rDNA copy number between different accessions of the same itTragopogon itspecies. The differences in copy number were larger between populations of the same species than between species. These data indicate that shifts in rDNA array sizes occur at the lineage level. Similar levels of interpopulation variability were reported among itArabidopsis itaccessions abbrgrp
abbr bid="B56"56abbr
abbrgrp. As a consequence, hybridizing species may inherit a variable number of rRNA genes depending on the parental populations involved. This hypothesis has been tested in Fsub1 subdiploid hybrids. For example, a "low-copy" itT. dubius itaccession 2615 combined with a "high-copy" itT. porrifolius it2607 should generate skewed 1:2 DUPO ratios in a hybrid (lines 116 and 121, Figure figr fid="F5"5Cfigr). Conversely, a "high-copy" itT. dubius it2613 combined with a "low-copy" itT. porrifolius it2611 should result in a 2:1 DUPO gene ratio. Indeed, the analysis of Fsub1 subdiploid hybrids confirmed the unequal gene dosage inherited from both parents (Figures figr fid="F2"2figr, figr fid="F3"3figr, and figr fid="F5"5figr). However, many allopolyploid lines involving a "high-copy" itT. dubius itparent showed far fewer copies of this parental type than expected, suggesting that standing variation in diploids does not account for all observed rDNA imbalances in the derived polyploids.p
pAnother source of "inherited variation" may stem from heterozygosity in locus sizes in the parents. For example, in itStreptocarpus ita major rDNA locus occurs in a hemizygous condition, accounting for gene copy number variation in derived hybrids abbrgrp
abbr bid="B57"57abbr
abbrgrp. This sort of heterozygosity would be evident if there were a very low-copy array and a very high-copy array in parents. However, FISH analysis revealed no indication of rDNA heterozygosity in the diploid itTragopogon itindividuals investigated, a situation which might be expected for species that are largely selfing abbrgrp
abbr bid="B58"58abbr
abbr bid="B59"59abbr
abbrgrp, and in plants that were derived from inbred lines propagated in a greenhouse (at least one generation). In estimating rDNA locus sizes, and hence relative copy numbers at individual loci, FISH may be influenced by the condensation state of the chromatin. However, we examined many cells in making our assessments and did not observe heterozygozity even in the most condensed metaphases. Furthermore, our data revealed clear associations between the size of the FISH signal and the number of rRNA gene copies estimated by Southern hybridization. Most Fsub1 subdiploid hybrids showed gene copy number ratios that were close to expectation. However, one Fsub1 subindividual (47-8) resulting from the cross itT. dubius it2613 × itT. porrifolius it2611 showed altered homeolog gene ratios from Mendelian expectation (Figure figr fid="F2"2Bfigr). Further, segregation of IGS sequence polymorphisms upon selfing of parental diploids was noted in some cases (Hana Malinska unpublished). Whether these low-frequency events reflect heterozygosity in locus sizes, gametic variation or postzygotic changes is currently unknown.p
pIn short, the genetic variation among the parents contributed some, but certainly not a major portion of the rDNA variability seen in the allotetraploids.p
sec
sec
st
p(ii) Contribution of allopolyploidy-related factors to rDNA variabilityp
st
pWe observed deviations from expected gene ratios in most of the synthetic tetraploid individuals we examined (Figures figr fid="F5"5C, Dfigr). The Southern blot and FISH data further show that this was mainly caused by underrepresentation of itT. dubiusit-origin units. Directionality of the change is not influenced by the partner genome being either itT. pratensis itor itT. porrifolius itin origin, as similar patterns occurred in both synthetic itT. mirus itand itT. miscellusit. However, the extent and frequency of these changes differed among the lines of the same species. For example, the expected 2:1 DUPO ratio of units has been reversed into a 1:4 ratio in line 98 of synthetic itT. mirusit, whereas in other lines the ratios changed relatively little (line 121). In general, deviation from repeat additivity occurred more frequently in lines resulting from crosses involving itT. dubius it2613 as a parent than any other accession (Figure figr fid="F5"5figr). The contribution of parental cytoplasm to gene imbalances could be assessed from the analysis of reciprocally formed individuals. The average DUPO gene ratios in reciprocally formed lines (134 and 135) of itT. mirus itwere comparable (Figure figr fid="F5"5Cfigr) although variation was higher in line 135 that inherited itT. dubius it2613 as a mother genome donor. However, relatively few individuals (28) were sampled to allow firm conclusion on this topic. Variation within lines was generally lower than that between the lines. Nevertheless, members of lineage 111-1 (synthetic itT. miscellusit) did show much variation (Figure figr fid="F5"5Bfigr), and most segregating progeny differed from the parental genotype.p
pRepeat number variation was reflected by differences in 35S rDNA locus sizes. For example, line 116 of synthetic itT. mirusit, which has only 19% rDNA of itT. dubius itorigin, showed small FISH signals on both Asupdu suphomologs in contrast to signals on Asuppo supand Dsupposup; their small sizes are indicative of loss in rDNA repeats at this locus (Figure figr fid="F6"6Gfigr). However, the high level of decondensation occurring at one or both Asupdu supsites suggests a high level of transcriptional activity at the preceding interphase. Indeed, RT-PCR experiments confirmed strong expression dominance of itT. dubius itloci in this (Additional file supplr sid="S5"5supplr) and other lines of synthetic itT. mirus itand itT. miscellus it(Hana Malinska unpublished). High transcriptional activity from an rDNA locus with reduced rRNA gene copy numbers has also been reported previously for natural itT. mirus itand itT. miscellus itindividuals abbrgrp
abbr bid="B60"60abbr
abbrgrp. Similarly, FISH revealed that lineage 111-4 of itT. miscellus ithad two small and two large rDNA loci (Figure figr fid="F4"4Ffigr) while a sister lineage, 111-1, had four large sites at both rDNA loci on chromosomes Asuppr supand Asupdu sup(Figure figr fid="F4"4Efigr). Line 111-1 also had more itT. dubiusit-origin gene copies than individual 111-4 (Figure figr fid="F3"3Afigr). Thus, FISH analysis confirmed that the shifts in rRNA gene ratios were likely caused by contractions of repeats on the Asupdu suplocus. Synthetic itT. mirus itlineage 73-14 is a notable exception in having an extremely large Asupdu suplocus (Figures figr fid="F6"6A, Bfigr and Additional files supplr sid="S4"4A, B, Csupplr). This may have been caused by amplification of repeats within the locus, but the plants also have a smaller locus on the other Asupdu suphomolog, potentially indicating translocation of repeats between homologs, perhaps as a consequence of unequal recombination at meiosis.p
suppl id="S5"
title
pAdditional file 5p
title
text
p
bExpression analysis of rDNA in synthetic itT. mirus it(line 116)b. RNA isolation and RT-CAPS assay were carried out as described in abbrgrp
abbr bid="B60"60abbr
abbrgrp. Note typical inverse correlation between gene copy number (grey bars) and their expression (black bars).p
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file name="1471-2148-10-291-S5.PDF"
pClick here for filep
file
suppl
sec
sec
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st
pMechanisms of rDNA rearrangementsp
st
pMeiotic aberrations have been implicated in rRNA gene imbalances in natural populations of itTragopogon it(e.g., abbrgrp
abbr bid="B46"46abbr
abbrgrp). Indeed, meiotic analysis of eight Ssub1 subplants (same material as used in this study) revealed a number of abnormalities, including multivalent formation, lagging chromosomes, and aneuploidy abbrgrp
abbr bid="B47"47abbr
abbrgrp. Chromosomes bearing rDNA appeared to be frequently involved in the formation of bridge complexes. Interestingly, lines involving itT. dubius it2613 (70 and 98) showed higher frequencies of both meiotic irregularities and rDNA rearrangements than lines derived from other accessions. There seems to be a good correlation between meiotic pairing abnormalities and frequency of rDNA changes in itTragopogonit. Ribosomal RNA gene copy number losses may occur through unequal recombination (perhaps also recombination between non-homologous or homologous chromosomes).p
pShifts in gene ratios were also observed in some Ssub0 subplants (i.e., the premeiotic synthetic allotetraploids), arguing that meiotic irregularities may not be the only mechanism responsible for rDNA rearrangements, but mechanisms acting during or immediately after genome doubling could also be involved. Such mechanisms include recombination between rDNA loci (at homologous loci or otherwise), aneuploidy, and chromosome loss in somatic cells, the latter two having been reported in interspecific itArabidopsis ithybrids and allopolyploids abbrgrp
abbr bid="B61"61abbr
abbrgrp. Reciprocal aneuploidy (loss compensated by gain of another chromosome of the complement) might explain the occurrence of four major 35S rDNA sites instead of six in itT. mirus itsynthetic lines 70 and 73 (Figure figr fid="F6"6figr). However, most synthetic polyploid individuals had the expected number of rDNA loci, and significantly altered gene ratios are likely arising through rearrangements targeted at the locus itself. In yeast, recombination between sister chromatids of the same chromosomes were shown to be a major source of array contractions and expansions abbrgrp
abbr bid="B62"62abbr
abbr bid="B63"63abbr
abbrgrp. A similar mitotic driven mechanism seems to be responsible for changes in copy number during development of itVicia faba it
abbrgrp
abbr bid="B17"17abbr
abbrgrp and in flax genotrophs abbrgrp
abbr bid="B64"64abbr
abbrgrp. Occasionally prolonged treatment with mitotic inhibitors causes the sister chromatids to separate but not to segregate as the cell proceeds towards anaphase. In one synthetic itT. mirus itcell (Additional file supplr sid="S4"4Esupplr) we observed that the chromatids were held together only at the rDNA loci, perhaps indicating unresolved recombination sites with rDNA, as can occur at the anaphase checkpoint in yeast abbrgrp
abbr bid="B65"65abbr
abbr bid="B66"66abbr
abbrgrp. Mitotic problems may underlie the high mortality of Ssub0 subgeneration plants in which > 50% of individuals did not survive or were sterile abbrgrp
abbr bid="B47"47abbr
abbrgrp.p
pWe have recently proposed that the nucleolus could be a site of inter- and intralocus recombination abbrgrp
abbr bid="B67"67abbr
abbrgrp. This hypothesis is supported by observation of increased homologous pairing at NORs in interphase of itArabidopsis it
abbrgrp
abbr bid="B68"68abbr
abbrgrp. Perhaps the decondensed chromatin of highly active genes promotes genetic recombination during interphase, resulting in the contractionexpansion of arrays. Another possibility is that subrepeated regions of the IGS may stimulate recombinogenic activity of units abbrgrp
abbr bid="B17"17abbr
abbrgrp. The IGS subrepeats in the sequenced itT. dubius itunit are longer and more homogeneous than those present in the itT. porrifolius itunit (Additional file supplr sid="S6"6supplr). itT. dubius itarrays displayed greater level of variation than the itT. porrifolius itarrays in the allopolyploid lines.p
suppl id="S6"
title
pAdditional file 6p
title
text
p
bAnalysis of IGS subrepeats in itT. dubius it2613 and itT. porrifolius it2611b. We used a dot plot alignment tool at urlhttp:www.vivo.colostate.edumolkitdnadoturl, self (x-axis) to self (y-axis) alignment (Window size: 9. Mismatch limit: 0). The IGSs were amplified using primers designed to conserved regions in 26S rDNA and 18S rDNA abbrgrp
abbr bid="B10"10abbr
abbrgrp. Briefly, the ~3.5-kb PCR products obtained were cloned into pSC-B-ampkan vector using StrataClone Blunt PCR Cloning Kit (Stratagene, La Jolla, CA, USA). Clones bearing inserts of expected lengths were initially sequenced from both ends using universal M13 reverse and T7 primers. To obtain full-length sequence, the IGS-specific primers were designed based on the partial sequence. Five new primers were needed to cover the whole IGS region.p
text
file name="1471-2148-10-291-S6.PDF"
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suppl
sec
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st
pComparison of natural and synthetic populations of allotetraploidsp
st
p
itT. mirus itand itT. miscellus itin the wild formed repeatedly within the last century and hence represent a unique system for studying the early stages of genome evolution following interspecific hybridization and genome duplication. In some areas, the progenitor diploids still occur along with expanding populations of the allotetraploids, and the polyploids at those locations likely represent descendents of the nearby diploid populations abbrgrp
abbr bid="B69"69abbr
abbrgrp. This scenario would apply, for example, to the itT. mirus itcollections from Pullman, Washington. The collected itT. dubius itindividuals from Pullman (2613) have nearly two-fold higher rDNA copy number compared to itT. porrifolius it(2611) at the same location. Yet, in both natural populations of itT. mirus itallotetraploids sampled, the itT. dubius itrDNA represents only 20-25% of the total rDNA abbrgrp
abbr bid="B45"45abbr
abbrgrp perhaps suggesting that ~75% of Asupdu suprepeats have been lost in the approximately 30-40 generations since the polyploids (which are biennials) formed at these locations. We were surprised to discover that one (98) out of three lines of itT. mirus itsynthesized from Pullman parents showed a genotype that closely resembled that of both natural populations. The gene imbalances were, however, less pronounced in the other two lines (70, 73). In line 73 some individuals even had more itT. dubius itunits, indicating that array size could be both maintained and altered during allopolyploidy. Interestingly, one natural population of itT. mirus it(from Palouse, Washington) also contained individuals with ratios skewed away from itT. porrifolius it(Figure figr fid="F2"2figr and Table tblr tid="T4"4tblr). These examples illustrate that, as in other allopolyploid species abbrgrp
abbr bid="B24"24abbr
abbr bid="B25"25abbr
abbrgrp, concerted evolution may occur bidirectionally in itTragopogon itdespite the prevalent trend towards contraction of itT. dubius itarrays in most plants and populations. In contrast to other systems abbrgrp
abbr bid="B24"24abbr
abbr bid="B28"28abbr
abbr bid="B40"40abbr
abbr bid="B70"70abbr
abbr bid="B71"71abbr
abbrgrp, we have no molecular evidence for interlocus recombination of rDNA in either natural or synthetic populations of itTragopogon italthough sequence analysis of ITS clones has not been conducted in synthetic material.p
pNatural populations of allotetraploids of independent origin differ morphologically, biochemically, and genetically abbrgrp
abbr bid="B39"39abbr
abbrgrp although these allotetraploids apparently originated from a relatively narrow genetic pool of parental populations abbrgrp
abbr bid="B69"69abbr
abbrgrp. One explanation for interpopulational diversity in the allopolyploids is that genetic variation was triggered during the early generations post-allopolyploidization. This hypothesis is supported by an observation that the same parental accessions may give rise to lineages with differing rDNA genotypes. itBrassica napus itallotetraploids appear to share many features with the itTragopogon itsystem, including homeolog pairing abbrgrp
abbr bid="B72"72abbr
abbr bid="B73"73abbr
abbrgrp and preliminary data that indicate that some rDNA loci may be lost in early generations (Ales Kovarik unpublished). In contrast to rDNAs, low-copy, protein-coding sequences do not seem to be markedly altered in the early generations of the synthetic lines of itT. mirus itand itT. miscellus it
abbrgrp
abbr bid="B74"74abbr
abbrgrp. Perhaps rearrangements of low-copy genes lag behind changes in the highly repeated fraction of the genome.p
pIt is assumed that, as time passes, homogenization processes such as unequal crossing over continue to gradually replace parental rDNA arrays with novel allopolyploid species arrays. However, the time factor does not seem to be the only player. For example, while most Old World itTragopogon itallotetraploids (assumed to be of ancient origin) homogenized ITS nearly to completion another old allotetraploid, itTragopogon castellanusit, retained equivalent amounts of both parental ITS types abbrgrp
abbr bid="B75"75abbr
abbrgrp. Similarly, in rice abbrgrp
abbr bid="B26"26abbr
abbrgrp and itGlycine it
abbrgrp
abbr bid="B25"25abbr
abbrgrp most, but not all, populations homogenized parental rDNAs. We envisage that the extent and tempo of rDNA homogenization in older allopolyploids is largely influenced by genetic and epigenetic changes in the early generations of allopolyploids. The fact that some rDNA genotypes seen in 80-year-old allopolyploids are already evident in the first generation of synthetic lines supports this hypothesis. However, this does not exclude the possibility that other changes in rDNA loci can occur gradually and stochastically over extended periods of time.p
sec
sec
sec
st
pConclusionsp
st
pWe observed similar reductions of homeologous rRNA gene copies in both synthetic and natural, 80-year-old populations of itTragopogon itallopolyploids, indicating that some aspects of genome evolution might be repeatable. The biological significance of gene losses as well as their potential adaptive significance is unclear. Uniparental deletions (partial or complete) would not affect fitness because there is a large excess of genes in the partner genome. One possibility is that intralocus rearrangements (translocation, deletions, amplification) preclude interlocus homogenization in older allopolyploids. The latter process is frequently associated with reduction of loci and repeats abbrgrp
abbr bid="B67"67abbr
abbrgrp. It is therefore possible that gene eliminationshrinkage of arrays serves as an alternative regulatory mechanism to epigenetic silencing, reducing the number of functional genes in a cell. Recently, Hawkins et al. abbrgrp
abbr bid="B76"76abbr
abbrgrp proposed that DNA loss may counterbalance genome expansion through retrotransposon proliferation. Perhaps rRNA gene elimination may reflect general tandem repeat instability in the allopolyploid nucleus. Retroelement activity in itTragopogon itpopulations remains to be analyzed. Finally, rDNA arrays were recently shown to influence gene expression at ectopic positions in itDrosophila it
abbrgrp
abbr bid="B77"77abbr
abbrgrp, and locus size could potentially serve as an epigenetic regulator harmonizing the expression of subgenomes.p
sec
sec
st
pList of abbreviationsp
st
pITS: internal transcribed spacer; IGS: intergenic spacer; FISH: fluorescent itin situ ithybridization; NOR: nucleolar organizer region; Organisms: itT. dubiusit: itTragopogon dubiusit; itT. mirusit: itTragopogon mirusit; itT. miscellusit: itTragopogon miscellusit; itT. porrifoliusit: itTragopogon porrifoliusit; itT. pratensisit: itTragopogon pratensisit
p
sec
sec
st
pAuthors' contributionsp
st
pHM carried out most of the molecular biology and cytogenetic experiments. AK, DS, PS, and AL designed the study. AK wrote and drafted the paper. RM participated in the DNA analysis. AL carried out some FISH experiments. JT made the crosses and prepared synthetic lines. All authors read and approved the final manuscript.p
sec
bdybm
ack
sec
st
pAcknowledgementsp
st
pWe thank Dr Blazena Koukalova (Institute of Biophysics, ASCR) for helpful discussions and critical reading of the manuscript. Drs Simon Renny-Byfield and Michael Chester (University of London) are acknowledged for advice and expertise with the FISH method. This research was funded by the Grant Agency of the Czech Republic P501-10-0208, 206091751, the Academy of Sciences of the Czech Republic (AVOZ50040507 and AVOZ50040702) and EMBO fellowship to HM (224-2009). Additional support for this research was provided by NSF grants MCB-0346437 to DES, PSS, and JAT; DEB-0614421 to DES and PSS, and DEB-09192540919348 to DES, PSS, W. B. Barbazuk, and P. S. Schnable.p
sec
ack
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allopolyploidptitleaugausnmTatesnmfnmJAfnmauausnmNisnmfnmZfnmauausnmScheensnmfnmACfnmauausnmKohsnmfnmJfnmauausnmGilbertsnmfnmCAfnmauausnmLefkowitzsnmfnmDfnmauausnmChensnmfnmZJfnmauausnmSoltissnmfnmPSfnmauausnmSoltissnmfnmDEfnmauaugsourceGeneticssourcepubdate2006pubdatevolume173volumeissue3issuefpage1599fpagelpage1611lpagexrefbibpubidlistpubid idtype="doi"10.1534genetics.106.057646pubidpubid idtype="pmcid"1526671pubidpubid idtype="pmpid"16648586pubidpubidlistxrefbibbiblbibl id="B44"titlepGene loss and silencing in itTragopogon miscellus it(Asteraceae): comparison of natural and synthetic allotetraploidsptitleaugausnmBuggssnmfnmRJAfnmauausnmDoustsnmfnmANfnmauausnmTatesnmfnmJAfnmauausnmKohsnmfnmJfnmauausnmSoltissnmfnmKfnmauausnmFeltussnmfnmFAfnmauausnmPatersonsnmfnmAHfnmauausnmSoltissnmfnmPSfnmauausnmSoltissnmfnmDEfnmauaugsourceHereditysourcepubdate2009pubdatevolume103volumeissue1issuefpage73fpagelpage81lpagexrefbibpubidlistpubid idtype="doi"10.1038hdy.2009.24pubidpubid idtype="pmpid" 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Natural populationsptitleaugausnmCooksnmfnmLMfnmauausnmSoltissnmfnmPSfnmauaugsourceHereditysourcepubdate1999pubdatevolume82volumefpage237fpagelpage244lpagexrefbibpubidlistpubid idtype="doi"10.1038sj.hdy.6884620pubidpubid idtype="pmpid"10336697pubidpubidlistxrefbibbiblbibl id="B60"titlepConcerted evolution of rDNA in recently formed itTragopogon itallotetraploids is typically associated with an inverse correlation between gene copy number and expressionptitleaugausnmMatyaseksnmfnmRfnmauausnmTatesnmfnmJAfnmauausnmLimsnmfnmYKfnmauausnmSrubarovasnmfnmHfnmauausnmKohsnmfnmJfnmauausnmLeitchsnmfnmARfnmauausnmSoltissnmfnmDEfnmauausnmSoltissnmfnmPSfnmauausnmKovariksnmfnmAfnmauaugsourceGeneticssourcepubdate2007pubdatevolume176volumeissue4issuefpage2509fpagelpage2519lpagexrefbibpubidlistpubid idtype="doi"10.1534genetics.107.072751pubidpubid idtype="pmcid"1950650pubidpubid idtype="pmpid"17603114pubidpubidlistxrefbibbiblbibl id="B61"titlepMitotic instability in resynthesized and natural polyploids of the genus itArabidopsis it(Brassicaceae)ptitleaugausnmWrightsnmfnmKMfnmauausnmPiressnmfnmJCfnmauausnmMadlungsnmfnmAfnmauaugsourceAm J Botsourcepubdate2009pubdatevolume96volumeissue9issuefpage1656fpagelpage1664lpagexrefbibpubid idtype="doi"10.3732ajb.0800270pubidxrefbibbiblbibl id="B62"titlepUnequal meiotic recombination within tandem arrays of yeast ribosomal DNA genesptitleaugausnmPetessnmfnmTDfnmauaugsourceCellsourcepubdate1980pubdatevolume19volumeissue3issuefpage765fpagelpage774lpagexrefbibpubidlistpubid idtype="doi"10.1016S0092-8674(80)80052-3pubidpubid idtype="pmpid" link="fulltext"6988084pubidpubidlistxrefbibbiblbibl id="B63"titlepRecombination regulation by transcription-induced cohesin dissociation in rDNA repeatsptitleaugausnmKobayashisnmfnmTfnmauausnmGanleysnmfnmARDfnmauaugsourceSciencesourcepubdate2005pubdatevolume309volumeissue5740issuefpage1581fpagelpage1584lpagexrefbibpubidlistpubid idtype="doi"10.1126science.1116102pubidpubid idtype="pmpid" link="fulltext"16141077pubidpubidlistxrefbibbiblbibl id="B64"titlepQuantitative variation of ribosomal RNA genes in flax genotrophsptitleaugausnmCullissnmfnmCAfnmauaugsourceHereditysourcepubdate1979pubdatevolume42volumefpage237fpagelpage246lpagexrefbibpubid idtype="doi"10.1038hdy.1979.25pubidxrefbibbiblbibl id="B65"titlepA role for the fission yeast Rqh1 helicase in chromosome segregationptitleaugausnmWinsnmfnmTZfnmauausnmMankourisnmfnmHWfnmauausnmHicksonsnmfnmIDfnmauausnmWangsnmfnmSWfnmauaugsourceJ Cell Scisourcepubdate2005pubdatevolume118volumeissue24issuefpage5777fpagelpage5784lpagexrefbibpubidlistpubid idtype="doi"10.1242jcs.02694pubidpubid idtype="pmpid" link="fulltext"16303848pubidpubidlistxrefbibbiblbibl id="B66"titlepSMC5 and SMC6 genes are required for the segregation of repetitive chromosome 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it(Plumbaginaceae)ptitleaugausnmAguilarsnmfnmJFfnmauausnmRossellosnmfnmJAfnmauausnmFelinersnmfnmGNfnmauaugsourceMol Ecolsourcepubdate1999pubdatevolume8volumeissue8issuefpage1341fpagelpage1346lpagexrefbibpubidlistpubid idtype="doi"10.1046j.1365-294X.1999.00690.xpubidpubid idtype="pmpid" link="fulltext"10447874pubidpubidlistxrefbibbiblbibl id="B71"titlepRecent hybrid speciation in itCardamine it(Brassicacea)-conversion of nuclear ribosomal ITS sequences in statu nascendiptitleaugausnmMummenhoffsnmfnmKfnmauaugsourceTheor Appl Genetsourcepubdate1999pubdatevolume98volumefpage831fpagelpage834lpagexrefbibpubid idtype="doi"10.1007s001220051140pubidxrefbibbiblbibl id="B72"titlepHomoeologous recombination in allopolyploids: the polyploid ratchetptitleaugausnmGaetasnmfnmRTfnmauausnmChris PiressnmfnmJfnmauaugsourceNew Phytolsourcepubdate2010pubdatevolume186volumeissue1issuefpage18fpagelpage28lpagexrefbibpubidlistpubid idtype="doi"10.1111j.1469-8137.2009.03089.xpubidpubid idtype="pmpid"20002315pubidpubidlistxrefbibbiblbibl id="B73"titlepThe first meiosis of resynthesized itBrassica napusit, a genome blenderptitleaugausnmSzadkowskisnmfnmEfnmauausnmEbersnmfnmFfnmauausnmHuteausnmfnmVfnmauausnmLodesnmfnmMfnmauausnmHuneausnmfnmCfnmauausnmBelcramsnmfnmHfnmauausnmCoritonsnmfnmOfnmauausnmManzanares-DauleuxsnmfnmMJfnmauausnmDelourmesnmfnmRfnmauausnmKingsnmfnmGJfnmauetalaugsourceNew Phytolsourcepubdate2010pubdatevolume186volumeissue1issuefpage102fpagelpage112lpagexrefbibpubidlistpubid idtype="doi"10.1111j.1469-8137.2010.03182.xpubidpubid idtype="pmpid"20149113pubidpubidlistxrefbibbiblbibl id="B74"titlepOn the road to diploidization Homoeolog loss in independently formed populations of the allopolyploid itTragopogon miscellus it(Asteraceae)ptitleaugausnmTatesnmfnmJAfnmauausnmJoshisnmfnmPfnmauausnmSoltissnmfnmKAfnmauausnmSoltissnmfnmPSfnmauausnmSoltissnmfnmDEfnmauaugsourceBMC Plant Biolsourcepubdate2009pubdatevolume9volumefpage80fpagexrefbibpubidlistpubid idtype="doi"10.11861471-2229-9-80pubidpubid idtype="pmcid"2708164pubidpubid idtype="pmpid"19558696pubidpubidlistxrefbibbiblbibl id="B75"titlepPutative parentage of six Old World polyploids in itTragopogon itL. (Asteraceae: Scorzonerinae) based on ITS, ETS, and plastid sequence dataptitleaugausnmMavrodievsnmfnmEVfnmauausnmSoltissnmfnmPSfnmauausnmSoltissnmfnmDEfnmauaugsourceTaxonsourcepubdate2008pubdatevolume57volumeissue4issuefpage1215fpagelpage1232lpagebiblbibl id="B76"titlepRepeated big bangs and the expanding universe: Directionality in plant genome size evolutionptitleaugausnmHawkinssnmfnmJSfnmauausnmGroversnmfnmCEfnmauausnmWendelsnmfnmJFfnmauaugsourcePlant Scisourcepubdate2008pubdatevolume174volumeissue6issuefpage557fpagelpage562lpagexrefbibpubid idtype="doi"10.1016j.plantsci.2008.03.015pubidxrefbibbiblbibl id="B77"titlepRibosomal DNA contributes to global chromatin regulationptitleaugausnmParedessnmfnmSfnmauausnmMaggertsnmfnmKAfnmauaugsourceProc Natl Acad Sci USAsourcepubdate2009pubdatevolume106volumeissue42issuefpage17829fpagelpage17834lpagexrefbibpubidlistpubid idtype="doi"10.1073pnas.0906811106pubidpubid idtype="pmcid"2764911pubidpubid idtype="pmpid"19822756pubidpubidlistxrefbibbiblrefgrp
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epdcx:valueString Similar patterns of rDNA evolution in synthetic and recently formed natural populations of Tragopogon (Asteraceae) allotetraploids
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Abstract
Background
Tragopogon mirus and T. miscellus are allotetraploids (2n = 24) that formed repeatedly during the past 80 years in eastern Washington and adjacent Idaho (USA) following the introduction of the diploids T. dubius, T. porrifolius, and T. pratensis (2n = 12) from Europe. In most natural populations of T. mirus and T. miscellus, there are far fewer 35S rRNA genes (rDNA) of T. dubius than there are of the other diploid parent (T. porrifolius or T. pratensis). We studied the inheritance of parental rDNA loci in allotetraploids resynthesized from diploid accessions. We investigate the dynamics and directionality of these rDNA losses, as well as the contribution of gene copy number variation in the parental diploids to rDNA variation in the derived tetraploids.
Results
Using Southern blot hybridization and fluorescent in situ hybridization (FISH), we analyzed copy numbers and distribution of these highly reiterated genes in seven lines of synthetic T. mirus (110 individuals) and four lines of synthetic T. miscellus (71 individuals). Variation among diploid parents accounted for most of the observed gene imbalances detected in F1 hybrids but cannot explain frequent deviations from repeat additivity seen in the allotetraploid lines. Polyploid lineages involving the same diploid parents differed in rDNA genotype, indicating that conditions immediately following genome doubling are crucial for rDNA changes. About 19% of the resynthesized allotetraploid individuals had equal rDNA contributions from the diploid parents, 74% were skewed towards either T. porrifolius or T. pratensis-type units, and only 7% had more rDNA copies of T. dubius-origin compared to the other two parents. Similar genotype frequencies were observed among natural populations. Despite directional reduction of units, the additivity of 35S rDNA locus number is maintained in 82% of the synthetic lines and in all natural allotetraploids.
Conclusions
Uniparental reductions of homeologous rRNA gene copies occurred in both synthetic and natural populations of Tragopogon allopolyploids. The extent of these rDNA changes was generally higher in natural populations than in the synthetic lines. We hypothesize that locus-specific and chromosomal changes in early generations of allopolyploids may influence patterns of rDNA evolution in later generations.
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Malinska, Hana
Tate, Jennifer A
Matyasek, Roman
Leitch, Andrew R
Soltis, Douglas E
Soltis, Pamela S
Kovarik, Ales
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BioMed Central Ltd
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Malinska et al.; licensee BioMed Central Ltd.
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BMC Evolutionary Biology. 2010 Sep 22;10(1):291
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RESEARCHARTICLEOpenAccess SimilarpatternsofrDNAevolutioninsynthetic andrecentlyformednaturalpopulationsof Tragopogon (Asteraceae)allotetraploids HanaMalinska 1 ,JenniferATate 2 ,RomanMatyasek 1 ,AndrewRLeitch 3 ,DouglasESoltis 4 ,PamelaSSoltis 5 AlesKovarik 1* Abstract Background: Tragopogonmirus and T.miscellus areallotetraploids(2 n =24)thatformedrepeatedlyduringthe past80yearsineasternWashingtonandadjacentIdaho(USA)followingtheintroductionofthediploids T.dubius T.porrifolius ,and T.pratensis (2 n =12)fromEurope.Inmostnaturalpopulationsof T.mirus and T.miscellus ,there arefarfewer35SrRNAgenes(rDNA)of T.dubius thanthereareoftheotherdiploidparent( T.porrifolius or T. pratensis ).WestudiedtheinheritanceofparentalrDNAlociinallotetraploidsresynthesizedfromdiploidaccessions. WeinvestigatethedynamicsanddirectionalityoftheserDNAlosses,aswellasthecontributionofgenecopy numbervariationintheparentaldiploidstorDNAvariationinthederivedtetraploids. Results: UsingSouthernblothybridizationandfluorescent insitu hybridization(FISH),weanalyzedcopynumbers anddistributionofthesehighlyreiteratedgenesinsevenlinesofsynthetic T.mirus (110individuals)andfourlines ofsynthetic T.miscellus (71individuals).Variationamongdiploidparentsaccountedformostoftheobservedgene imbalancesdetectedinF 1 hybridsbutcannotexplainfrequentdeviationsfromrepeatadditivityseeninthe allotetraploidlines.PolyploidlineagesinvolvingthesamediploidparentsdifferedinrDNAgenotype,indicating thatconditionsimmediatelyfollowinggenomedoublingarecrucialforrDNAchanges.About19%ofthe resynthesizedallotetraploidindividualshadequalrDNAcontributionsfromthediploidparents,74%wereskewed towardseither T.porrifolius or T.pratensis -typeunits,andonly7%hadmorerDNAcopiesof T.dubius -origin comparedtotheothertwoparents.Similargenotypefrequencieswereobservedamongnaturalpopulations. Despitedirectionalreductionofunits,theadditivityof35SrDNAlocusnumberismaintainedin82%ofthe syntheticlinesandinallnaturalallotetraploids. Conclusions: UniparentalreductionsofhomeologousrRNAgenecopiesoccurredinbothsyntheticandnatural populationsof Tragopogon allopolyploids.TheextentoftheserDNAchangeswasgenerallyhigherinnatural populationsthaninthesyntheticlines.Wehypothesizethatlocus-specificandchromosomalchangesinearly generationsofallopolyploidsmayinfluencepatternsofrDNAevolutioninlatergenerations. Background Chromosomecountssuggestthatbetween30and100% ofangiospermspeciesarepolyploids[1],andWood etal.[2]proposethat15%ofangiospermspeciation eventsareassociatedwithpolyploidywhereasrecent genomicstudiesofselectedmodelandcropspecies haverevealedthatallplantgenomessequencedtodate havesignaturesofoneormorewhole-genomeduplicationsintheirevolutionaryhistory[3,4].Thesuccessof newlyformedangiospermpolyploidsispartlyattributabletotheirhighlyplasticg enomestructureasmanifestedbydeviationsfromMendelianinheritanceof geneticlociandchromosomeaberrations[5].Indeed, therearenumerousexamplesofintergenomic exchanges,chromosomaltranslocations,transposonproliferation,andsequencelossinbothnewlyformedand ancientallopolyploidspecies(forreviewsee[6,7]). *Correspondence:kovarik@ibp.cz 1 InstituteofBiophysics,AcademyofSciencesoftheCzechRepublic,v.v.i, LaboratoryofMolecularEpigenetics,Kralovopolska135,CZ-61265Brno, CzechRepublic Fulllistofauthorinformationisavailableattheendofthearticle Malinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 2010Malinskaetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.

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Inplants,nuclearribosomalDNA(rDNA)unitsoccur intandemarraysatoneorseveralloci(forreview see[8,9]).Eachlarge35SrDNAunitcontainsthe18S, 5.8S,and26SrRNAgenes,theinternaltranscribed spacers(ITS),andtheinter genicspacer(IGS).The5S genesencoding120-nttranscriptsareusually,butnot always[10],locatedatdifferentchromosomallocithan 35SrDNA.Thegenesarehighlyconservedeven betweeneukaryotesandprokaryotes,whereasdivergence ofITSissufficienttoresolvespeciesrelationshipswithin mostgenera[11].TheIGS,whichcontainsthetranscriptionstartsiteandgeneticandepigeneticfeatures thatinfluencetheregulationofthedownstreamgenes, divergesrapidly,andsubstantialdifferencesinstructure mayoccurevenwithinaspecies[12-14].Thenumberof genecopiesmayvaryfrom500uptotensofthousands incertainplantspecies[15].Similarvariationhasbeen observedinlocusnumber,withlevelsrangingfromone toseverallociperhaploidset[16].Withinspecies,the copyandlocusnumberisusuallystable,although intraindividualandintergenerationalvariationincopy numberhasbeenreportedinsomeplants[17].Aswith otherrepeatedsequences,rDNAcanundergoconcerted evolutioninvolvingsequencehomogenization[18,19]. Suchaprocessefficientlyeliminatesmutatedcopies maintaininglongarraysoffunctionaltandemlyarranged genes. ThebehaviorofrDNAinallopolyploidshasattracted considerableattentionbecauseitisusedasamolecular andcytogeneticmarkerofa llopolyploidy[20].Indeed, thehybridoriginofmanyspecieshasbeensuccessfully decipheredusingITSsequen ces.Nevertheless,repeat andlocusloss,andintra-andinterlocusrecombination seemtobeongoingevolution aryprocesses,potentially preventingidentificationo fhybrids.Infact,manywelldefinedallopolyploidspecieshaveeitherlostoneorseveralloci[21-23],eliminatedorcontractedparental arrays[24-26],recombined[27]orreplacedtheunits [28,29].Ontheotherhand,somepolyploidspecies seemtomaintainbothparentalcopiesinthegenome longafterallopolyploidformation[14,30-33].ThesestudiesindicatethattheprocessofrDNAevolutioniscomplicated,andthatnofirmconclusioncanbedrawnon thetempoanddirectionofrepeathomogenization. Nevertheless,thereareexamp lesofsyntheticallopolyploidlinesinwhichrDNAshavealreadyundergone rearrangementsatthechromosomalandunitlevels [13,34,35]. Recentlyformedallopolyploidsrepresentuniquenaturalsystemsinwhichtostudytheimmediateconsequencesofallopolyploidy.Onlyafewpolyploidplant speciesareknowntohaveformedinthepast200years: Cardamineschulzii [36], Spartinaanglica [37], Senecio cambrensis and S.eboracensis [38], Tragopogonmirus and T.miscellus [39]. Cardamine allopolyploidpopulationsseemtoevolverecombinedITStypes[40].In Spartinaanglica ,thetwoindividualscollectedfromdifferentlocalitiesdifferedinthecompositionofrDNA units[41]. Allotetraploid Tragopogonmirus and T.miscellus formedinthePalouseregion(easternWashington,and westernIdaho,USA)withinthelast80years[39,42] andthusrepresentanexcellentmodelforexamining earlyeventsinallopolyploidevolution.Recentstudies usingdifferentmethodologicalapproacheshaveshown frequentlossofhomeologoussequences,includinglowcopyprotein-codinggenes[43,44]andhigh-copyrDNA [45].Inthelatter,apopulation-levelanalysisrevealed reductionofrDNAarraysderivedfromthe T.dubius diploidparentinallbutonenaturalpopulationexamined( T.mirus ).Whiletheaveragemagnitudeofgene losswasabout50%,therewereindividualsthatlostas manyas95%ofallrepeats.Cytogeneticstudiesconfirmedthatrearrangementswerehomologousandwere notlinkedtochromosomeloss[46]. Theinterpretationofgeneticvariationinnaturalpolyploidsisalwayscomplicatedbythefactthatgeneticparentsareunknowneveninthecaseofrecentlyformed species.Thatis,someallopolyploidsmaystartoutwith farmorerDNAcopiesofoneparentthantheother,simplybecausethediploidparentsdifferincopynumber. Ontheotherhand,genotypicvariationmayarisefrom geneticchangesinducedbystressfulconditionsexperiencedduringallopolyploidy[5].Inthisstudyweasked: (i)Whatisthecontributionofparentaldiploidstocopy numbervariationinnewlyformedallopolyploid T.mirus and T.miscellus ?(ii)WhatarethedynamicsofrDNA rearrangements,anddotheyoccursuddenlyorgradually?(iii)Isthereadirectionalityandgeneticpredispositionforlocusrearrangement?(iv)Arethereparallelsin theevolutionofrDNAinnaturalandsyntheticpopulationsofthetwoallopolyploids?(v)Whatarethelikely mechanismsofrDNArearra ngement?Toaddressthese questionswesynthesized allotetraploidlinesof T.mirus and T.miscellus [47]usingseveraldifferentpopulations ofparentalaccessions.Wedeterminedhomeologgene ratiosbySouthernblotandslotblothybridization.The locusnumberswereassessedusingFISH.Evidencewas obtainedforrepeatlossinearlygenerationsofsynthetic allopolyploidlinesatfrequenciesanddirectionalitysimilartothoseobservedinnaturalsituations.MethodsPlantmaterialField-collectedseedsofthreediploid Tragopogon species wereplantedandselfedforonegeneration.Then103 differentcrossesweremadebetweenindividualsfrom threepopulationsof T.dubius (2613,2615,2616),twoMalinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 Page2of17

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populationsof T.porrifolius (2607,2611),andtwo populationsof T.pratensis (2608,2609)fromdifferent localities(Table1,Table2and[47]).Seedsfromsuccessfulcrossesweretreatedwith0.1or0.25%(w/v, watersolution)colchicine duringgermination(overnight),washed,andplacedinpotswithsoil.Approximately6monthsaftergermination,youngplantswere genotypedtodeterminetheirparentageusingamarker (TDF85)specificforallthreediploidspecies[43,47]. Thenon-treatedF1diploidhybridswereplantedascontrols.Detailedinformationaboutcrossesisdescribedin [44,47].MolecularcytogeneticanalysisRoottipscutfromvigorouslygrowingplantswerepretreatedwith2mM8-hydroxyqu inoline(Sigma-Aldrich CompanyLtd,Poole,Dorset,UK)toobtainmetaphase nuclei.After2hoursofincubationonice,roottips werefixedinethanol:aceticacid(3:1)atroomtemperatureovernight,thenstoredin70%ethanolat-20C. Fixedroottipsweredigestedin0.3%(w/v)cellulase OnozukaR-10(ApolloScientificLtd,Stockport,Cheshire,UK),0.3%(w/v)pectolyaseY23(MPBiomedicals, Solon,Ohio,USA),and0.3%(w/v)drieselase(SigmaAldrichCompanyLtd.,Poole,Dorset,UK)for27min andtransferredto1%citratebufferpH4.8andincubatedfor1hour.Themeristematiccellsbehindtheroot capweresquashedontoaglassslideinadropof60% aceticacid.Coverslipswereremovedafterfreezingin liquidnitrogen. Fluorescent insitu hybridization(FISH)ofthediploids andpolyploidsfollowedstandardprotocols[48].The probefor5SrDNAwasprepar edbyPCRamplification ofthecloned Nicotianatabacum 5SrRNAgene[49] followedbylabelingwithbio tin-16-dUTPasdescribed in[48].Theprobefor35SrDNAwasaclonethat includespartofthe18SrDNAisolatedfrom Allium cernuum ,whichwaslabeledwithdigoxigenin-11-dUTP asdescribedin[48].Sitesofprobehybridizationwere detectedusing20 gmL-1fluorescein-conjugatedantidigoxigeninimmunoglobulin(GEHealthcare,Chalfont StGiles,Buckinghamshire,UK)or5 gmL-1Cy3-conjugatedavidin(RochePharmaceuticals,Lewes,EastSussex,UK)in4SSCcontaining0.2%(v/v)Tween20 and5%(w/v)bovineserumalbumin.Chromosomes werecounterstainedwith2 gmL-1DAPI(4 ’ ,6-diamidino-2-phenylindole(Sigma-AldrichCompanyLtd.Dorset,UK)in4SSC)andstabilizedinVectashield medium(VectorLaboratoriesLtd.,Peterborough,UK) priortodataacquisitionusingaLeicaDMRA2epifluorescentmicroscopefittedwithanOrcaERcameraand OpenLabsoftware(Improvision,Coventry,UK).The imageswereadjustedwithAdobePhotoshopversion7 andtreatedforcolorcontrastanduniformbrightness only.Atleastfivemitoticcellsperplantwerescored witheachprobeused. Table1ParentaloriginofsyntheticallotetraploidsanddirectionofcrossesT.mirusT.miscellus Line7073981161211341356779111129 parents 2611 2613 2611 2613 2611 2613 2607 2615 2615 2607 2607 2613 2613 2607 2609 2616 2609 2616 2608 2613 2613 26081N9487288141615223932S0231101121412S17346278131513213522S20110000000001totalnumberofindividualsanalyzedbySouthernblothybridization.2numbersinS0-S2generations;theS2wastheprogenyofselfedS1parents(73-14,73-1,73-2)thathavebeenanalyzedinthisstudybymolecularand cytogeneticapproaches. Table2CharacteristicsofrDNAlociinpopulationsofparentaldiploidspeciesusedforconstructionofsyntheticlinesSpecies1Collectionno Location2rDNAcopiesrDNA genotype No.of35SrDNAsitesNo.of5SrDNAsites T.porrifolius 2607Troy,ID1.50.3High-copy44 2611Pullman,WA1.00.2Low-copy44 T.pratensis 2608Moscow,ID1.20.1Medium-copy22 2609Spangle,WA1.50.2High-copy22 T.dubius 2613Pullman,WA1.70.4High-copy22 2615Spokane,WA0.90.1Low-copy22 2616Spangle,WA1.00.2Low-copy221Soltis&Soltiscollectionnumbers;vouchersdepositedatFLAS.2DNAcopiesperhaploidsetinthousands.Malinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 Page3of17

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DNAisolation,SouthernblottingGenomicDNAwasextractedeitherfromfreshleavesor leavespreservedinRNA later reagent(AppliedBiosystems,Ambion,Warrington,UK),followingtheinstructionsofthemanufacturerofRNA later orbyastandard CTABmethoddescribedin[50]andmodifiedin[51]. DNAconcentrationwasestimatedbytwoindependent methods:(i)aCYBRgreenfluorescencemethodfollowingaprotocolathttp:// www.dnagenotek.com/);the greenfluorescencewasmeasuredonaRotorgenethermocycler(CorbettResearch,Brisbane,Australia)as recommended;and(ii)ethidiumbromidefluorescence measuredaftergelelectrophoresisusingphagelambda DNAasastandard.Theestimatesobtainedfromboth methodswereconcordant.IntegrityofDNAwas checkedongels. Southernblottingfollowedtheprotocoldescribedin [51]usingrDNAprobeslabeledwith[ a -32P]dCTP(Izotop,Budapest,Hungary).TheITS-1probewasa Bst NI fragmentfromthecloned18S-ITS-5.8Ssubregionof T.mirus rDNA(GenBank:AY458586).The18SrDNA probewasacloned1.7-kbfragmentofthetomato18S rRNAgene[52],andthe26SrDNAprobewasa280-bp PCRproductderivedfromthe3 ’ endofthetobacco26S rRNAgene[53].Hybridizationsignalswerevisualized byphosphorimaging(Storm,MolecularDynamicsSunnyvale,CA,USA). ForrDNAquantification,17.5-200ngofDNAwere denaturedin0.2MNaOHandloadedonanylonmembrane(HybondXL,GEHealthcare,LittleChalfont,UK) usingaslotblotapparatus(Schleicher&Schuell,SigmaAldrich,Dorset,UK).Themembraneswerehybridized withrespectiveDNAprobesinaChurchGilbertbuffer [54].Radioactivityineachbandwascountedusinga rectangleintegrationmethod(ImageQuantsoftware,GE Healthcare,LittleChalfont,UK).Astandardcurvewas constructedusingadilutedplasmidcarryingthe18S rDNAinsert[52].Theexperimentswererepeatedthree timesanddataaveraged. StatisticalcalculationswerecarriedoutusingachisquarefunctionimplementedinMicroSoftExcel.ResultsPlantsandschemeofexperimentsThegenerationofallotetraploidlinesandphenotypic andkaryologicalanalysisweredescribedelsewhere [47].Briefly,theallotetraploidswerederivedfrom independentcrosses(Figure1)involvingtwoorthree differentaccessionsofdiploid T.dubius T.porrifolius and T.pratensis (Table2).Inthisstudy,weusedseven linesofsynthetic T.mirus ,fourlinesofsynthetic T. miscellus ,andtwolinesofdiploidF1hybrids.TheparentaloriginforeachlineisgiveninTable1.A “ line ” wasdefinedastheprogenyoriginatingfromasingle cross(S0,S1,S2...)andmaintainedthroughselfing. “ Lineages ” wereobtainedbyselfingofplantsfromthe samecrossbutfromdifferentF1seeds.CopynumberestimatesindiploidgenomedonorsBecauseestimatesofrDNAamountsmayvaryamong populationsofdiploidspecies,wedeterminedgenecopy numberofdiploidparentsusingslotblothybridization. Twotosixindividualsfromeachpopulationwereanalyzed.TheDNAwashybridizedonblotswiththe18S rDNAprobeandtheamountofradioactivityestimated. Anexampleofourhybridizationanalysisisshownin Additionalfile1.ItisevidentthatthestrongesthybridizationsignalswereobtainedwithDNAfrom T.dubius 2613and T.porrifolius 2607;thesewerescoredas “ high-copy ” accessions.Ontheotherhand, T.dubius 2615and T.porrifolius 2611showedrelativelyweak hybridizationsignalsandwerescoredas “ low-copy ” accessions.Variationwithinpopulationsandamong progenywaslow(<15%)ornegligible.ThecopynumberestimatesforeachpopulationaregiveninTable2.SouthernblothybridizationinparentalplantsTheITSregionwasanalyzedtakingadvantageofadiagnostic Bst NIrestrictionsitepolymorphismthatdistinguishesthediploidparents.In T.dubius ,ITS-1contains a Bst NIsiteclosetothe5.8Sgenewhilethereisno Bst NIsiteintheITS-1of T.porrifolius and T.pratensis [45].Consistentwithpreviousresults,theITS-1probe hybridizedtothe~700-bpfragmentin T.pratensis and T.porrifolius andtothe~500-bpfragmentin T.dubius (Figures2D,3D). TheIGSregionwasanalyzedusing Bst YIand Ssp I restrictionenzymes(Additionalfile2).Therearemore Bst YIsitesintheIGSof T.porrifolius thanin T.dubius basedonthesequencedclones[GenBank:FN666261.1 andFN645941.1].Consequently,theprobehybridizedto low-molecular-weightfragmentsin T.porrifolius andto high-molecular-weightfragmentsin T.dubius .FISHanalysisinparentalplantsTodeterminethenumberandchromosomalpositionof rRNAgenes,wecarriedoutrDNA-FISHonmetaphase chromosomes.In T.dubius (Figure4C),thereweretwo sitesofprobehybridizationforboth35Sand5SrDNA, locatedonthelargestchromosomepair(Adu;the nomenclaturefollowsthatofPires etal. [55]). T.pratensis alsohastwositesof35SrDNA(bothdecondensed) andonepairof5SrDNAlociatmetaphase(Figure4A). Both35Sand5SrDNAlocioccuronchromosomesApr. T.porrifolius hasfour35Sandfour5SrDNAsitesat metaphase(Figures4B,D).ChromosomeApocarries both35Sand5SrDNAloci,athomeologouslocitothe otherdiploids,buttherearealso35SrDNAlociontheMalinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 Page4of17

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twohomologsofchromosomeDpo,and5SrDNAloci onbothhomologsofchromosomeFpo.The5Ssignalon Apowasmuchweakerinaccession2611(Figure4D) thaninaccession2607(Figure4B).TheFISHresultsare consistentwithpreviouscytogeneticanalysiscarriedout inthesamediploidpopulations,butwithdifferentindividuals[55],indicatingchromosomalstabilityofloci.Molecularandcytogeneticanalysisinsynthetichybrids andallotetraploidsWecarriedoutapopulatio n-levelstudyof35SrRNA genecopynumberusingSou thernblothybridization usingtheITS-1probe(Figures2,3).Theratiosofgene unitsineachsampleareshowninFigures5A( T.mirus ) and5B( T.miscellus );theaveragedvaluesforeachline aresummarizedinFigure5C,D.FISHwiththe18S rDNAprobewasthencarriedoutonselectedindividuals(Figures4,6andTable3).Inthefollowingsections,wedescribetheresultsofbothtypesofanalysis foreachlinederivedfromdiploidparentswithhigh, medium,orlowcopynumbersatthe35SrDNAloci (calledhigh-,medium-,orlow-copy)parents.F1diploidhybridsWeanalyzedrRNAgeneratiosusingSouthernblot hybridizationwiththeITS1probeagainstdiploidF1hybridplants.In11F1individuals(cross47)fromasinglecrossinvolvinga “ high-copy ” T.dubius 2613paternalparentanda “ low-copy ” T.porrifolius 2611maternal parent(Figure2B),theprobehybridizedstronglytothe Figure1 Schemeofcrossingstrategies Tragopogondubius and T.porrifolius (or T.pratensis )wereusedaseitherthematernalorpaternal parent;eachcross(line)gaverisetosterilediploidF1progeny.Todoublechromosomesandrestorefertilityofthesehybrids,seedsweretreated withcolchicine,obtainingfertileallotetraploidS0plants.Theseindividualswereselfedtoproducelineagesoffertileallotetraploidhybrids (generationS1,S2). Malinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 Page5of17

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T.dubius -originrDNAunits(DU)formingalower molecularweightband,whiletheupperbandof T. porrifolius -originrDNAunits(PO)wassignificantly weaker.Radioactivityscanningrevealedthatonaverage 60%ofthetotalhybridizationsignaloccurredinthe lowermolecularweightband,representingrDNAunits of T.dubius origin.Thisvalueisclosetotheexpected ratioconsideringtheunitcopynumbersinthediploid parents(Table2andFigure5C,D).Withtheexception ofoneindividual(47-8),planttoplantvariationingene ratioswaslow(Figure2B).Theseconddiploidhybrid (cross2)examinedwasderivedfromacrossinvolving “ low-copy ” T.dubius 2615and “ mediumcopy ” T.pratensis 2607(Figure3B).Inthiscase,theupper molecularweightbandof T.pratensisoriginrDNA units(PR)wasstrongerthanthebandinherited from T.dubius .Again,F1hybridsgenerallyshowedthe expectednumberofgenesinheritedfromtheirparents (Figure5C,D).Synthetic T.mirus Line73Thislinewasderivedfromacrossinvolving “ lowcopy ” 2611 T.porrifolius (maternallyinherited)and “ high-copy ” T.dubius 2613(paternallyinherited). Thirty-sevenindividualsobtainedfromfourlineages (73-1,73-2,73-13,and73-14)wereanalyzedbySouthernblothybridizationusingtheITS-1probe(examples Figure2 rDNArepeatinheritancein T.mirus determinedbySouthernblothybridization .(A)Syntheticallotetraploidlinesof T.mirus .(B) Diploid T.porrifolius x T.dubius hybrids.(C)Naturalpopulationsof T.mirus .(D)Profilesofparentaldiploids.Representativeindividualsareshown inthepanels;therestoftheanalysisexpressedasaratioofparentalbandsisshowninFigure5A.EachS0andS1generationofsynthetic allopolyploid(A)isencodedasfollows:e.g.in70-1,thefirstnumberisthelinenumber,thesecondnumberindicatesaparticularlineage(Figure 1andTable1).TheDNAsweredigestedwith Bst NIandhybridizedwiththeITS-1probe.Thenumberbeloweachlaneindicatespercentageof T.dubius unitsoutoftotalrDNA.DUT.dubius units;POT.porrifolius units. Malinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 Page6of17

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areshowninFigure2A).TherewassignificantvariationinrRNAgeneratios,andatleasttwodistinct rDNAgenotypesweredistinguished:lineages73-2and 73-13hadnearlybalanc edDU/POratios,while lineages73-1and73-14hadratiosskewedtowards T.dubiusoriginrDNA(61-75%)(Figures5Aand5C). The26SrDNAprobe,whichmappedpolymorphisms intheintergenicspacer(IGS),showedsimilarresults (Additionalfile2).Thehom eologgeneratiosestablishedintheS1seemtobeinheritedamongtheS2individuals(Additionalfile3). RepresentativeindividualsofbothgenotypeswereanalyzedbyFISH(Figure6A,Band6D).The18SrDNA probehybridizedstronglytoterminalregionsofboth homeologsofchromosomesApoandAdu.However, therewasno,orverylittle,hybridizationsignalonchromosomeDpo(Figure6Barrowheads),indicatinglocus lossordrasticeliminationofunits.Thispatternappears tobetypicalforallS1individualsandtheirprogeny (Additionalfile4).Therewerealsoquantitativedifferencesinsignalintensities.Whilelineages73-2and7313hadcomparablesizesofallfour35SrDNAloci, lineages73-1and73-14hadenlargedAduloci.The numberof5Ssiteswasadditive(6signals)intheS1generationwhileoneS2individualgainedastrongsite likelyonchromosomeF(Add itionalfile4D).Thus, cytogeneticandmolecularanalysisbothshowconsiderablevariabilityinrDNAlocussizesintheselineages.Line70Theseallopolyploidswerederivedfrom “ low-copy ” T.porrifolius 2611(maternallyinherited)and “ highcopy ” T.dubius 2613(paternallyinherited),butdifferent individualswereusedasparentsthanincrosses73and 98.OntheSouthernblots,theratiosofparentalbands weremostlybalanced(Figure2A).Asinline73,there wereonlyfour35SrDNAsignals(somewithsecondary constrictions)insteadoftheexpectedsix,indicating locusloss(Figure6C).Line98Onlyasingleline,98-1,wasrecoveredfromthiscross, originatingfrompopulationsof “ low-copy ” T.porrifolius 2611(maternallyinherited)and “ high-copy ” T.dubius 2613(paternallyinherited). Differentindividualswere usedasparentsthanintheprevioustwolines.Onblots, theprobeshowedamuchstrongerhybridizationtothe bandcorrespondingtothe T.porrifolius -originhomeologthantothehomeologof T.dubius origin(Figure 2A).Thereweresix35Sandsix5SrDNAchromosomal Figure3 rDNArepeatinheritancein T.miscellus determinedbySouthernblothybridization .(A)Syntheticallotetraploidlinesof T.miscellus .(B)Diploid T.pratensis x T.dubius hybrids.(C)Naturalpopulationsof T.miscellus .(D)Profilesofparentaldiploids.Representative individualsareshowninthepanels;therestoftheanalysisexpressedasaratioofparentalbandsisshowninFigure5B.Thedigestsand conditionsoftheblotandlabelsarethesameasinFigure2.DUT.dubius units;PRT.pratensis units. Malinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 Page7of17

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sites,indicatinganadditivenumberoflocifromthat expectedofthediploidparents(Figure6E).Line116Inthiscrossweused “ high-copy ” T.porrifolius 2607as thematernalgenomedonorand “ low-copy ” T.dubius 2615asthepaternalgenomedonor.Allindividualsfrom eightlineagesshowedrelativelyuniformSouthernhybridizationprofiles,withbandratioshighlyskewedtowards the T.porrifolius -originunits(Figures2A,5).FISH showedtheexpectednumberof35Ssignals,withsignals onchromosomesApo,Dpo,andAdu,althoughthesignalsonchromosomeAdu(arrows)aresmallerand highlydecondensed(Figure6G).Ontheotherhand,the 35Ssignalson T.porrifolius chromosomeswerestrong, andNORsonDpo(arrowheads)showedonlypartial decondensation.Line121Thislineoriginatedfromacrossinvolving “ low-copy ” T.dubius 2615asthematernalparentanda “ highcopy ” T.porrifolius 2607asadonorofthepaternalgenome.Thegeneratioofalleightallotetraploidindividuals wasslightlyshiftedtowardsthe T.porrifolius homeolog (Figure5A).FISHwasnotconductedonthisline.Line134Thislinewasderivedfroma “ high-copy ” T.porrifolius 2607(maternallyinherited)anda “ high-copy ” T.dubius Figure4 FISHtometaphasespreadsofparentaldiploidspecies(A-D)andsynthetic T.miscellus lines(E-F) .(A) T.pratensis 2608.(B) T.porrifolius 2607.(C) T.dubius 2613.(D) T.porrifolius 2611.(E)and(F)standforthe111-1and111-4syntheticindividuals,respectively.Metaphases werehybridizedwiththe18SrDNA(painting35Ssitesingreen)and5SrDNA(redfluorescence)probes.Notethediscontinuouschromatin condensationalongtheloci.Regionsofcondensedanddecondensedchromatinareinterconnectedwithdottedlinesin(A-C).Scalebar=10 m. Malinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 Page8of17

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Figure5 SummaryofrDNArepeatratiosanalysisestimatedforindividualsynthetic T.mirus (A,C)and T.miscellus (B,D)plants HomeologgeneratiosweredeterminedfromquantificationofbandsignalsaftertheSouthernblothybridization(Figures2,3).Eachparental hybridizationbandwasquantifiedusingphosphorimaging,andgeneratioswereexpressedasapercentageof T.dubius unitsoutofthetotal signal(A,B).Theaveragedvaluesforeachlineareshowningraphs(CandD-shadedbars);forF1diploidhybridstheshadedbarsaredrawnat lowcontrast.FilledbarsrepresentexpectedgeneratiosbasedonstrictMendelianinheritanceofgenecopynumbers.Theinterindividual variabilitywithinthelineisexpressedbystandarddeviationfromthemean.Differencesbetweenexpectedandobservedratioswerehighly significant(P<0.001,standardchi-squaretest)inalllinesexceptthediploidF1-47andF1-2hybridsandline121. Malinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 Page9of17

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2613(paternallyinherited)andyieldedtwolineages (134-15and134-16).Ontheblots,individualsfrom bothlinesshowedarelativelyweak T.dubius -origin bandandastrong T.porrifolius -originband(Figures 2A,5).Atthecytogeneticlevel,theindividualsinherited anadditivenumberof35SrDNAloci,fourstrongsites andtwoweakerones,thelatterlikelyof T.dubius origin,thatareslightlyremotefromthebulkofthechromosomesduetosecondaryconstriction(Figure6F).Line135Thislineoriginatedfromac rossreciprocaltothatgivingline134andcomprisedthreelineages(1,2,and5). Figure6 FISHtometaphasespreadsofsynthetic T.mirus .TheprobelabelsandscalebarareasinFigure4.Individuals73-14(A,B),70-4(C), and73-13(D)hadfourstrongplus0-2veryweaksignals(dependentonparticularmetaphaseandsample).(B)isanexpandedregionof(A) showingthelargelocusof T.dubius originandtheminuteDpolocus(arrowheads)leftafterthedeletionofthemajorityofgenesfromthearray. Lineages98-1(E),134-15(F),116-1(G),and135-5(H)showedregularlysix18SrDNAsignals.Themetaphasein(G)haslargelydecondensedAdu(arrows),partiallydecondensedDpo(arrowheads),andfullycondensedApoloci. Malinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 Page10of17

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Whilebothlineages135-1and135-2hadgeneratios skewedtowards T.porrifolius -originunits,thegene ratioswerebalancedinthe1 35-5individuals(Figures 2A,5).Therewassomevariabilityamongthe135-5 individuals(Figure5A).Ho wever,thenumberofloci wasadditiveintheindividualexaminedusingFISH(Figure6H).Synthetic T.miscellus Line67Inthiscross,a “ high-copy ” T.pratensis 2609wasused asthematernalgenomedonorand “ low-copy ” T.dubius 2616asthepaternalgenomedonor.Therewaslittleor novariationinthegeneratiosamongtheindividuals analyzed,andallindividualshadreducedSouthern hybridizationsignalagainstthe T.dubius -originunits (Figure3A).Line79Thiscrossisareciprocalcrosstothatwhichgenerated line67,andityielded5lineages.Therewassome,albeit little,intralineagevariabilityinbandhybridizationsignal intensities.Forexample,theS1individualinthemiddle lane(Figure3A)hadastronger T.dubius -originband comparedtothatfromtheotherparent( T.pratensis ) whilemostothermembersofthislinehadadominant bandof T.pratensis origin(Figure5B).Line111Inthiscrossa “ medium-copy ” T.pratensis 2608(maternallyinherited)and “ high-copy ” T.dubius 2613(paternallyinherited)wereusedasgenomedonors.This combinationofparentsyieldedthelargestnumberof allotetraploidindividuals(35)outofthe T.miscellus crossesattempted.Lineages111-4,111-5,and111-7had relativelyuniformprofilesofSouthernhybridization bandswithratiosskewedawayfromtheunitsof T.dubius origin(Figure3A).Lineage111-1differedin havingrelativelylargeintralineagevariability(Figure5B). Therewereindividualswithsignalsskewedtowardsthe T.pratensis typeaswellasindividualswithbalanced generatios.FISHwascarriedoutonrandomlyselected individualsoflineages111-1(Figure4E)and(Figure4F). Bothplantsretainedanadditivenumberof35Sand5S rDNAlocifromthatexpectedofthediploidparents(i.e., four35Sandfour5SrDNAsites).However,plant111-4 hadtwolargerandtwosmaller35SrDNAsignals,all withsecondaryconstrictio ns,whileindividual111-1had signalsofcomparablesizesonallfourchromosomes.Line129Line129resultedfromacross reciprocaltothatwhich generatedline111.Onlytwoa llotetraploidindividuals wererecoveredfromthiscross,andbothhadgene ratioshighlyskewedawayfrom T.dubius -originunits (Figure5B).DiscussionrRNAgenecopynumbervariationinsynthetic T.mirus and T.miscellusWeanalyzedtheinheritanceofrRNAgenesinseven linesofsynthetic T.mirus andfourlinesofsynthetic T. miscellus .Insynthetic T.mirus ,32individuals(29%) exhibitedbalancedrDNAgenotypes,69individuals (63%)showedmore35SrDNAof T.porrifolius origin thanexpected,andonly9plants(8%)hadmore T. dubiusoriginrDNA(Table4).Insynthetic T.miscellus threeindividuals(4%)hadbalancedgeneratioswhile65 individuals(92%)inheritedmore T.pratensis -origin unitsthanexpected.Thegeneticvariationincopyratios amongtheprogenyofasinglecrossrangedfromlowor Table3SummaryofcytogeneticanalysisSpecies3N35SrDNAsites5SrDNAsites Synthetic T.mirus1174-66-7 Natural T.mirus211646-7 Synthetic T.miscellus1244 Natural T.miscellus26441thisstudy.2references[45,46,55].3numberofindividualsanalyzedbyrDNA-FISH.4someindividualsfrompopulationRosalia[45]hadanearlydeletedlocuson chromosomeAduinheritedfrom T.dubius (Figure2C). Table4ComparisonofrDNAratiosinsyntheticand naturalpopulationsofallotetraploidsSpeciesGenotypeGeneratio Du: Po/Pr1N%of individuals Synthetic T.mirus highDu>60%98 balanced40-60%3229 highPo>60%6963 total110 Natural T.mirus highDu>60%00 balanced40-60%2029 highPo>60%4871 total68 Synthetic T. miscellus highDu>60%34 balanced40-60%34 highPr>60%6592 total71 Natural T. miscellus highDu>60%00 balanced40-60%00 highPr>60%31100 total311N-numberofindividuals.ThedatawereobtainedfromtheS1generationof syntheticlines(Table1);naturalpopulationswereanalyzedin[45,60].Malinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 Page11of17

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negligible(5%,line134)tohigh(40%,line111)(Figure 5C).Somecrossesinvolvingthesameparentalaccessions(lines70,73,and98)gaverisetoindividualswith expectedratiosofparental35SrDNAunitsconsidering thecopynumbersinthedip loidparents,whileothers hadrDNAgenotypesbalancedorskewedtowardsunits derivedfromeitherofthediploidparents.SourcesofrDNAcopynumbervariabilityin allotetraploids (i)ContributionofnaturalvariationinparentalaccessionsTheremaybeuptotwo-foldvariationin35SrDNA copynumberbetweendifferentaccessionsofthesame Tragopogon species.Thediffere ncesincopynumber werelargerbetweenpopulationsofthesamespecies thanbetweenspecies.These dataindicatethatshiftsin rDNAarraysizesoccuratthelineagelevel.Similar levelsofinterpopulationvariabilitywerereportedamong Arabidopsis accessions[56].Asaconsequence,hybridizingspeciesmayinheritavariablenumberofrRNA genesdependingontheparentalpopulationsinvolved. ThishypothesishasbeentestedinF1diploidhybrids. Forexample,a “ low-copy ” T.dubius accession2615 combinedwitha “ high-copy ” T.porrifolius 2607should generateskewed1:2DU/POratiosinahybrid(lines116 and121,Figure5C).Conversely,a “ high-copy ” T.dubius 2613combinedwitha “ low-copy ” T.porrifolius 2611 shouldresultina2:1DU/POgeneratio.Indeed,the analysisofF1diploidhybridsconfirmedtheunequal genedosageinheritedfrombothparents(Figures2,3, and5).However,manyallopolyploidlinesinvolvinga “ high-copy ” T.dubius parentshowedfarfewercopiesof thisparentaltypethanexpected,suggestingthatstandingvariationindiploidsdoesnotaccountforall observedrDNAimbalancesinthederivedpolyploids. Anothersourceof “ inheritedvariation ” maystemfrom heterozygosityinlocussizesintheparents.Forexample, in Streptocarpus amajorrDNAlocusoccursinahemizygouscondition,accountingforgenecopynumber variationinderivedhybrids[57].Thissortofheterozygositywouldbeevidentiftherewereaverylow-copy arrayandaveryhigh-copyarrayinparents.However, FISHanalysisrevealednoindicationofrDNAheterozygosityinthediploid Tragopogon individualsinvestigated,asituationwhichmightbeexpectedforspecies thatarelargelyselfing[58 ,59],andinplantsthatwere derivedfrominbredlinespropagatedinagreenhouse (atleastonegeneration).InestimatingrDNAlocus sizes,andhencerelativecopynumbersatindividual loci,FISHmaybeinfluencedbythecondensationstate ofthechromatin.However,weexaminedmanycellsin makingourassessmentsanddidnotobserveheterozygozityeveninthemostcondensedmetaphases.Furthermore,ourdatarevealedclearassociationsbetweenthe sizeoftheFISHsignalandthenumberofrRNAgene copiesestimatedbySouthernhybridization.MostF1diploidhybridsshowedgenecopynumberratiosthat wereclosetoexpectation.However,oneF1individual (47-8)resultingfromthecross T.dubius 2613 T.porrifolius 2611showedalteredhomeologgeneratiosfrom Mendelianexpectation(Figure2B).Further,segregation ofIGSsequencepolymorphismsuponselfingofparental diploidswasnotedinsomecases(HanaMalinskaunpublished).Whethertheselow-frequencyevents reflectheterozygosityinlocussizes,gameticvariationor postzygoticchangesiscurrentlyunknown. Inshort,thegeneticvariationamongtheparentscontributedsome,butcertainlynotamajorportionofthe rDNAvariabilityseenintheallotetraploids.(ii)Contributionofallopolyploidy-relatedfactorstorDNA variabilityWeobserveddeviationsfromexpectedgeneratiosin mostofthesynthetictetraploidindividualsweexamined(Figures5C,D).TheSouthernblotandFISHdata furthershowthatthiswasmainlycausedbyunderrepresentationof T.dubius -originunits.Directionalityof thechangeisnotinfluencedbythepartnergenome beingeither T.pratensis or T.porrifolius inorigin,as similarpatternsoccurredinbothsynthetic T.mirus and T.miscellus .However,theextentandfrequencyof thesechangesdifferedamongthelinesofthesamespecies.Forexample,theexpected2:1DU/POratioof unitshasbeenreversedintoa1:4ratioinline98of synthetic T.mirus ,whereasinotherlinestheratios changedrelativelylittle(lin e121).Ingeneral,deviation fromrepeatadditivityoccurredmorefrequentlyinlines resultingfromcrossesinvolving T.dubius 2613asa parentthananyotheraccession(Figure5).Thecontributionofparentalcytoplasmtogeneimbalancescould beassessedfromtheanalysisofreciprocallyformed individuals.TheaverageDU/POgeneratiosinreciprocallyformedlines(134and135)of T.mirus werecomparable(Figure5C)althoughvariationwashigherin line135thatinherited T.dubius 2613asamothergenomedonor.However,relativelyfewindividuals(28) weresampledtoallowfirmconclusiononthistopic. Variationwithinlineswasgenerallylowerthanthat betweenthelines.Nevertheless,membersoflineage 111-1(synthetic T.miscellus )didshowmuchvariation (Figure5B),andmostsegregatingprogenydifferedfrom theparentalgenotype. Repeatnumbervariationwasreflectedbydifferences in35SrDNAlocussizes.For example,line116ofsynthetic T.mirus ,whichhasonly19%rDNAof T.dubius origin,showedsmallFISHsignalsonbothAduhomologsincontrasttosignalsonApoandDpo;theirsmall sizesareindicativeoflossinrDNArepeatsatthislocus (Figure6G).However,thehighlevelofdecondensationMalinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 Page12of17

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occurringatoneorbothAdusitessuggestsahighlevel oftranscriptionalactivityattheprecedinginterphase. Indeed,RT-PCRexperimentsconfirmedstrongexpressiondominanceof T.dubius lociinthis(Additionalfile 5)andotherlinesofsynthetic T.mirus and T.miscellus (HanaMalinska-unpublished).Hightranscriptional activityfromanrDNAlocuswithreducedrRNAgene copynumbershasalsobeenreportedpreviouslyfornatural T.mirus and T.miscellus individuals[60].Similarly, FISHrevealedthatlineage111-4of T.miscellus hadtwo smallandtwolargerDNAloci(Figure4F)whileasister lineage,111-1,hadfourlargesitesatbothrDNAlocion chromosomesAprandAdu(Figure4E).Line111-1also hadmore T.dubius -origingenecopiesthanindividual 111-4(Figure3A).Thus,FISHanalysisconfirmedthat theshiftsinrRNAgeneratioswerelikelycausedby contractionsofrepeatsontheAdulocus.Synthetic T. mirus lineage73-14isanotableexceptioninhavingan extremelylargeAdulocus(Figures6A,BandAdditional files4A,B,C).Thismayhavebeencausedbyamplificationofrepeatswithinthelocus,buttheplantsalsohave asmallerlocusontheotherAduhomolog,potentially indicatingtranslocationofrepeatsbetweenhomologs, perhapsasaconsequenceofunequalrecombinationat meiosis.MechanismsofrDNArearrangementsMeioticaberrationshavebeenimplicatedinrRNAgene imbalancesinnaturalpopulationsof Tragopogon (e.g., [46]).Indeed,meioticanalysisofeightS1plants(same materialasusedinthisstudy)revealedanumberof abnormalities,includingmultivalentformation,lagging chromosomes,andaneuploidy[47].ChromosomesbearingrDNAappearedtobefrequentlyinvolvedintheformationofbridgecomplexes.Interestingly,lines involving T.dubius 2613(70and98)showedhigherfrequenciesofbothmeioticirregularitiesandrDNArearrangementsthanlinesderivedfromotheraccessions. Thereseemstobeagoodcorrelationbetweenmeiotic pairingabnormalitiesandfrequencyofrDNAchanges in Tragopogon .RibosomalRNAgenecopynumber lossesmayoccurthroughunequalrecombination(perhapsalsorecombinationbetweennon-homologousor homologouschromosomes). ShiftsingeneratioswerealsoobservedinsomeS0plants(i.e.,thepremeiotics yntheticallotetraploids), arguingthatmeioticirregularitiesmaynotbetheonly mechanismresponsibleforrDNArearrangements,but mechanismsactingduringorimmediatelyaftergenome doublingcouldalsobeinvolved.Suchmechanisms includerecombinationbetweenrDNAloci(athomologouslociorotherwise),ane uploidy,andchromosome lossinsomaticcells,thelattertwohavingbeenreported ininterspecific Arabidopsis hybridsandallopolyploids [61].Reciprocalaneuploidy (losscompensatedbygain ofanotherchromosomeofthecomplement)might explaintheoccurrenceoffourmajor35SrDNAsites insteadofsixin T.mirus syntheticlines70and73(Figure6).However,mostsyntheticpolyploidindividuals hadtheexpectednumberofrDNAloci,andsignificantly alteredgeneratiosarelikelyarisingthroughrearrangementstargetedatthelocusi tself.Inyeast,recombinationbetweensisterchromatidsofthesame chromosomeswereshowntobeamajorsourceofarray contractionsandexpansions[62,63].Asimilarmitotic drivenmechanismseemstoberesponsibleforchanges incopynumberduringdevelopmentof Viciafaba [17] andinflaxgenotrophs[64].Occasionallyprolonged treatmentwithmitoticinhibitorscausesthesisterchromatidstoseparatebutnottosegregateasthecellproceedstowardsanaphase.Inonesynthetic T.mirus cell (Additionalfile4E)weobservedthatthechromatids wereheldtogetheronlyattherDNAloci,perhapsindicatingunresolvedrecombinationsiteswithrDNA,as canoccurattheanaphasecheckpointinyeast[65,66]. MitoticproblemsmayunderliethehighmortalityofS0generationplantsinwhich>50%ofindividualsdidnot surviveorweresterile[47]. Wehaverecentlyproposedthatthenucleoluscould beasiteofinter-andintralocusrecombination[67]. Thishypothesisissupportedbyobservationof increasedhomologouspairingatNORsininterphase of Arabidopsis [68].Perhapsthedecondensedchromatinofhighlyactivegenespromotesgeneticrecombinationduringinterphase,resultinginthecontraction/ expansionofarrays.Anothe rpossibilityisthatsubrepeatedregionsoftheIGSmaystimulaterecombinogenicactivityofunits[17].TheIGSsubrepeatsinthe sequenced T.dubius unitarelongerandmorehomogeneousthanthosepresentinthe T.porrifolius unit (Additionalfile6). T.dubius arraysdisplayedgreater levelofvariationthanthe T.porrifolius arraysinthe allopolyploidlines.Comparisonofnaturalandsyntheticpopulationsof allotetraploidsT.mirus and T.miscellus inthewildformedrepeatedly withinthelastcenturyandhencerepresentaunique systemforstudyingtheearlystagesofgenomeevolution followinginterspecifichybridizationandgenomeduplication.Insomeareas,theprogenitordiploidsstilloccur alongwithexpandingpopulationsoftheallotetraploids, andthepolyploidsatthoselocationslikelyrepresent descendentsofthenearbydiploidpopulations[69].This scenariowouldapply,forexample,tothe T.mirus collectionsfromPullman,Washington.Thecollected T. dubius individualsfromPullman(2613)havenearly two-foldhigherrDNAco pynumbercomparedto T.Malinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 Page13of17

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porrifolius (2611)atthesamelocation.Yet,inbothnaturalpopulationsof T.mirus allotetraploidssampled,the T.dubius rDNArepresentsonly20-25%ofthetotal rDNA[45]perhapssuggestingthat~75%ofAdurepeats havebeenlostintheapproximately30-40generations sincethepolyploids(whicharebiennials)formedat theselocations.Weweresurprisedtodiscoverthatone (98)outofthreelinesof T.mirus synthesizedfromPullmanparentsshowedagenotypethatcloselyresembled thatofbothnaturalpopulations.Thegeneimbalances were,however,lesspronouncedintheothertwolines (70,73).Inline73someindividualsevenhadmore T. dubius units,indicatingthatarraysizecouldbeboth maintainedandalteredduring allopolyploidy.Interestingly,onenaturalpopulationof T.mirus (fromPalouse, Washington)alsocontainedindividualswithratios skewedawayfrom T.porrifolius (Figure2andTable4). Theseexamplesillustratethat,asinotherallopolyploid species[24,25],concertedevolutionmayoccurbidirectionallyin Tragopogon despitetheprevalenttrend towardscontractionof T.dubius arraysinmostplants andpopulations.Incontrasttoothersystems [24,28,40,70,71],wehavenomolecularevidencefor interlocusrecombinationofrDNAineithernaturalor syntheticpopulationsof Tragopogon althoughsequence analysisofITScloneshasnotbeenconductedinsyntheticmaterial. Naturalpopulationsofallotetraploidsofindependent origindiffermorphologically,biochemically,andgenetically[39]althoughtheseallotetraploidsapparentlyoriginatedfromarelativelynarr owgeneticpoolofparental populations[69].Oneexplanationforinterpopulational diversityintheallopolyploidsisthatgeneticvariation wastriggeredduringtheearlygenerationspost-allopolyploidization.Thishypothesisissupportedbyanobservationthatthesameparentalaccessionsmaygiveriseto lineageswithdifferingrDNAgenotypes. Brassicanapus allotetraploidsappeartosharemanyfeatureswiththe Tragopogon system,includinghomeologpairing[72,73] andpreliminarydatathatindicatethatsomerDNAloci maybelostinearlygenerations(AlesKovarik-unpublished).IncontrasttorDNAs,low-copy,protein-coding sequencesdonotseemtobemarkedlyalteredinthe earlygenerationsofthesyntheticlinesof T.mirus and T.miscellus [74].Perhapsrearrangementsoflow-copy geneslagbehindchangesinthehighlyrepeatedfraction ofthegenome. Itisassumedthat,astimepasses,homogenization processessuchasunequalcrossingovercontinueto graduallyreplaceparentalrDNAarrayswithnovel allopolyploidspeciesarrays.However,thetimefactor doesnotseemtobetheonlyplayer.Forexample, whilemostOldWorld Tragopogon allotetraploids (assumedtobeofancientorigin)homogenizedITS nearlytocompletionanotheroldallotetraploid, Tragopogoncastellanus ,retainedequivalentamountsofboth parentalITStypes[75].Similarly,inrice[26]and Glycine [25]most,butnotall,populationshomogenized parentalrDNAs.Weenvisagethattheextentand tempoofrDNAhomogenizationinolderallopolyploids islargelyinfluencedbygeneticandepigeneticchanges intheearlygenerationsofallopolyploids.Thefactthat somerDNAgenotypesseenin80-year-oldallopolyploidsarealreadyevidentinthefirstgenerationof syntheticlinessupportsthishypothesis.However,this doesnotexcludethepossib ilitythatotherchangesin rDNAlocicanoccurgraduallyandstochasticallyover extendedperiodsoftime.ConclusionsWeobservedsimilarreductionsofhomeologousrRNA genecopiesinbothsyntheticandnatural,80-year-old populationsof Tragopogon allopolyploids,indicating thatsomeaspectsofgenomeevolutionmightbe repeatable.Thebiologicalsignificanceofgenelossesas wellastheirpotentialadaptivesignificanceisunclear. Uniparentaldeletions(partialorcomplete)wouldnot affectfitnessbecausethereisalargeexcessofgenesin thepartnergenome.Onepossibilityisthatintralocus rearrangements(translocatio n,deletions,amplification) precludeinterlocushomogenizationinolderallopolyploids.Thelatterprocessisfrequentlyassociatedwith reductionoflociandrepeats[67].Itisthereforepossiblethatgeneelimination/shrinkageofarraysservesas analternativeregulatorymechanismtoepigenetic silencing,reducingthenumberoffunctionalgenesin acell.Recently,Hawkinsetal.[76]proposedthat DNAlossmaycounterbalancegenomeexpansion throughretrotransposonproliferation.PerhapsrRNA geneeliminationmayreflectgeneraltandemrepeat instabilityintheallopolyploidnucleus.Retroelement activityin Tragopogon populationsremainstobeanalyzed.Finally,rDNAarrayswererecentlyshownto influencegeneexpressionatectopicpositionsin Drosophila [77],andlocussizecouldpotentiallyserveas anepigeneticregulatorharmonizingtheexpressionof subgenomes.AdditionalmaterialAdditionalfile1:Slotblotquantificationofgenecopiesinparental diploids .TheDNAamountsareindicatedaboveeachlane.Theblotwas hybridizedwiththe32P-labeled18SrDNAprobe.ExperimentsIandII werecarriedoutinthisstudy;experimentIIIisfrom[45]. Additionalfile2:AnalysisofintergenicrDNAspacerpolymorphisms inDNAofsynthetic T.mirus (S1generation) .GenomicDNAwas digestedwith Bst YIand Ssp Irestrictionenzymes.Southernblot hybridizationwascarriedoutusingthe26SrDNAprobe.Malinska etal BMCEvolutionaryBiology 2010, 10 :291 http://www.biomedcentral.com/1471-2148/10/291 Page14of17

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Additionalfile3:SouthernblotanalysisoftheS2generationof synthetic T.mirus .Individualsweretheprogeniesofthreelineages fromline73. Additionalfile4:FISHanalysisoftheS2generationofsynthetic T. mirus .ThesameplantsasinAdditionalfile3wereanalyzed.Most metaphasesdisplayedaneuploidkaryotypes(23chromosomes). Arrowheadsin(C,F)indicateaminuteDpolocusleftafterthedeletionof themajorityofgenes.NotefusionofsubtelomericNORsatthe chromatids(arrowheads,E)andconsiderablevariabilityincondensation ofrDNAchromatinamongsisterplants(A-C).Thefollowingindividuals areshown:(A)-73-14-6A,(B)-73-14-6B,(C)-73-14-6C,(D,E)-73-1-3D, (F)-73-2-8B. Additionalfile5:ExpressionanalysisofrDNAinsynthetic T.mirus (line116) .RNAisolationandRT-CAPSassaywerecarriedoutas describedin[60].Notetypicalinversecorrelationbetweengenecopy number(greybars)andtheirexpression(blackbars). Additionalfile6:AnalysisofIGSsubrepeatsin T.dubius 2613and T.porrifolius 2611 .Weusedadotplotalignmenttoolathttp://www. vivo.colostate.edu/molkit/dnadot/,self(x-axis)toself(y-axis)alignment (Windowsize:9.Mismatchlimit:0).TheIGSswereamplifiedusing primersdesignedtoconservedregionsin26SrDNAand18SrDNA[10]. Briefly,the~3.5-kbPCRproductsobtainedwereclonedintopSC-B-amp/ kanvectorusingStrataCloneBluntPCRCloningKit(Stratagene,LaJolla, CA,USA).Clonesbearinginsertsofexpectedlengthswereinitially sequencedfrombothendsusinguniversalM13reverseandT7primers. Toobtainfull-lengthsequence,theIGS-specificprimersweredesigned basedonthepartialsequence.Fivenewprimerswereneededtocover thewholeIGSregion. Listofabbreviations ITS:internaltranscribedspacer;IGS:intergenicspacer;FISH:fluorescent insitu hybridization;NOR:nucleolarorganizerregion;Organisms: T.dubius : Tragopogondubius ; T.mirus : Tragopogonmirus ; T.miscellus : Tragopogon miscellus ; T.porrifolius : Tragopogonporrifolius ; T.pratensis : Tragopogon pratensis Acknowledgements WethankDrBlazenaKoukalova(InstituteofBiophysics,ASCR)forhelpful discussionsandcriticalreadingofthemanuscript.DrsSimonRenny-Byfield andMichaelChester(UniversityofLondon)areacknowledgedforadvice andexpertisewiththeFISHmethod.ThisresearchwasfundedbytheGrant AgencyoftheCzechRepublicP501-10-0208,206/09/1751,theAcademyof SciencesoftheCzechRepublic(AVOZ50040507andAVOZ50040702)and EMBOfellowshiptoHM(224-2009).Additionalsupportforthisresearchwas providedbyNSFgrantsMCB-0346437toDES,PSS,andJAT;DEB-0614421to DESandPSS,andDEB-0919254/0919348toDES,PSS,W.B.Barbazuk,andP. S.Schnable. Authordetails1InstituteofBiophysics,AcademyofSciencesoftheCzechRepublic,v.v.i, LaboratoryofMolecularEpigenetics,Kralovopolska135,CZ-61265Brno, CzechRepublic.2InstituteofMolecularBioSciences,MasseyUniversity, PalmerstonNorth4442,NewZealand.3SchoolofBiologicalSciences,Queen MaryUniversityofLondon,E14NS,UK.4DepartmentofBiology,Universityof Florida,Gainesville,FL32611,USA.5FloridaMuseumofNaturalHistory, UniversityofFlorida,Gainesville,FL32611,USA. Authors ’ contributions HMcarriedoutmostofthemolecularbiologyandcytogeneticexperiments. AK,DS,PS,andALdesignedthestudy.AKwroteanddraftedthepaper.RM participatedintheDNAanalysis.ALcarriedoutsomeFISHexperiments.JT madethecrossesandpreparedsyntheticlines.Allauthorsreadand approvedthefinalmanuscript. Received:16June2010Accepted:22September2010 Published:22September2010 References1.SoltisPS,SoltisDE: Theroleofhybridizationinplantspeciation. 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0 10 20 30 40 50 60 70 80 90 100 116 1 7 116 3 2 116 5 2 116 7 8 116 8 6 116 14 1 116 15 1 %DU DNA %DU RNA


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