Differential rRNA genes expression in bread wheat and itsinheritance

Ana Carvalho • Carlos Polanco • Henrique Guedes-Pinto •

Jose Lima-Brito

Received: 26 April 2011 / Accepted: 15 August 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The expression of the ribosomal RNA (rRNA)

genes from rye, located within the nucleolus organizer

regions (NORs), is repressed by cytosine methylation in

wheat x rye hybrids and in triticale, as consequence of

nucleolar dominance. Our previous study revealed that

bread wheat cultivars with a maximum number of four Ag-

NORs presented high level of rDNA cytosine methylation

when compared to others with a maximum of six Ag-

NORs. In order to evaluate the inheritance of the Ag-NORs

number and NOR methylation patterns, we produced F1

hybrids between bread wheat cultivars with four Ag-NORs

and bread wheat cultivars with six Ag-NORs (in the direct

and reciprocal senses). The F2 progenies of these F1

hybrids were also evaluated for the NOR number and

methylation patterns. Parent bread wheat cultivars with a

maximum of four Ag-NORs after treated with 5-azacyti-

dine evidenced a maximum of six Ag-NORs per metaphase

cell and a maximum of six nucleoli per interphase nucleus,

confirming that the expression of the rRNA genes in bread

wheat is related to cytosine methylation. Most of the F1

hybrids showed a maximum number of four or six Ag-

NORs, similarly to that of the female parent suggesting a

non-mendelian inheritance, while other hybrids presented

four or six Ag-NORs in both senses of the cross. The F1

NOR methylation patterns showed some fragments com-

mon to their parents but also novel fragments suggesting

genomic and/or chromosome rearrangements after hybrid-

ization. Despite the different NOR patterns among the

parents, an invariable NOR pattern was found among the

F1 plants suggesting a tendency to stability, which was also

transmitted to the F2. The F2 progenies showed plants with

a maximum of four, five and/or six Ag-NORs. The ratio of

plants with four, five and/or six Ag-NORs per F2 progeny

was variable and did not follow any specific mendelian

proportion. These results allowed us to suggest that the

inheritance of the number of Ag-NORs by the F1 and F2

plants did not follow any mendelian inheritance and were

not correlated to NOR methylation patterns in contrast to

what was verified for their parents.

Keywords Ag-NORs � Bread wheat �Cytosine methylation � Differential expression

Introduction

Differential expression and ribosomal RNA (rDNA)

methylation has been widely studied in plant species,

including interspecific hybrids and allopolyploids (Lima-

Brito et al. 1998; Komarova et al. 2004; Carvalho et al.

2010; Riddle and Richards 2002, 2005; Volkov et al. 2007;

Woo and Richards 2008). The differential expression of the

rDNA could result from nucleolar dominance, an epige-

netic phenomenon firstly described by Navashin (1934)

that reflects the interaction between parental genomes in

interspecific hybrids and allopolyploids. The nucleolar

dominance consists in the formation of nucleoli by the

rRNA genes of only one parent in those plant materials.

The phenomenon has been widely correlated with DNA

A. Carvalho � H. Guedes-Pinto � J. Lima-Brito (&)

Institute of Biotechnology and Bioengineering (IBB), Centre of

Genomics and Biotechnology (CGB), University of Tras-os-

Montes and Alto Douro, P.O. Box 1013, 5001-801 Vila Real,

Portugal

e-mail: jbrito@utad.pt

C. Polanco

Department of Genetics and Molecular Biology, Faculty of

Biologic and Environmental Sciences, University of Leon,

24007 Leon, Spain

123

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DOI 10.1007/s10709-013-9731-8

methylation in plants (Reeder 1985; Neves et al. 1995;

Pikaard 2002). Nonetheless, after more than 70 years of

research, nucleolar dominance is still not well understood.

The Triticeae tribe has been considered an excellent bio-

logical system for the study of nucleolar dominance since it

includes natural and artificial allopolyploids. Additionally,

the nucleolar competition studies performed within this taxon

strongly contributed for the understanding of the equiva-

lence, dominance and suppression relationships among the

nucleolar chromosomes and genomes of several Triticeae

species (Cermeno et al. 1984; Lacadena et al. 1984a, b, 1988;

Lacadena and Cermeno 1985, and many others).

The DNA methylation patterns could be maintained

through the plant development cycle due to the action of

DNA methyltransferases (Neves et al. 1995). Recent

studies revealed the occurrence of de novo methylation

associated to the silencing of rRNA genes promoted by

small interference RNAs (siRNAs) that induced the

establishment and/or maintenance of nucleolar dominance

(Preuss et al. 2008). Chromatin modifications constitute

epigenetic marks that are transmitted to the progeny. In

plants, the absence of a global methylation resetting

between generations allows the inheritance of differential

degrees of methylation, as observed in natural populations

(Kakutani et al. 1996; Martienssen and Colot 2001; Shiba

and Takayama 2007).

Previous studies performed in natural accessions of

Arabidopsis thaliana indicated a spontaneous variation of

cytosine methylation, particularly at NOR region (Riddle

and Richards 2002, 2005; Woo and Richards 2008).

Additionally, the NOR methylation patterns were trans-

mitted in a stable way from the parents to the hybrids,

being maintained in the progeny as a result of a combi-

nation of genetic and epigenetic mechanisms (Riddle and

Richards 2005; Woo and Richards 2008).

Recently, we verified the occurrence of differential rRNA

genes expression in Old Portuguese bread wheat cultivars.

Bread wheat cultivars with a maximum of four Ag-NORs

showed higher levels of NOR methylation than those with a

maximum of six Ag-NORs (Carvalho et al. 2010).

In the present study, we aimed to evaluate the inheri-

tance of the number of Ag-NORs and the NOR methylation

patterns in F1 and F2 plants obtained from direct and

reciprocal crosses between bread wheat cultivars with a

maximum of four and six Ag-NORs.

Materials and methods

Plant material

Fifteen Old Portuguese bread wheat (2n = 6x = 42;

AABBDD) cultivars that were kindly given by the National

Plant Breeding Station (ENMP, Elvas, Portugal) were used

as parents in 60 direct and reciprocal intraspecific crosses.

These parent cultivars showed a maximum of four or six

Ag-NORs per metaphase cell in a previous work (see

Carvalho et al. 2010). Here, we assumed as: (1) direct

cross, 4 Ag-NORs 9 6 Ag-NORs; and as (2) reciprocal

cross, 6 Ag-NORs 9 4 Ag-NORs. The F2 generation was

obtained after self-pollination of the F1. All plants were

grown under greenhouse conditions.

Sequential silver nitrate staining and FISH

Seeds from parental cultivars with a maximum of four Ag-

NORs germinated in the presence of 8.2 9 10-5 M of

5-azacytidine that was daily renewed. Root-tips from these

plants were collected, treated in ice-cold water for 24 h,

fixed in ethanol: acetic acid (3:1), and used for the prepa-

ration of mitotic chromosome spreads which were further

stained with silver nitrate following the protocol of Stack

et al. (1991).

Seeds from the F1 and F2 individuals were allowed to

germinate in distilled water. The root-tips were treated with

ice-cold water for 24 h and fixed in ethanol:acetic acid

(3:1) and further used to prepare mitotic chromosome

spreads that were used in the sequential technique of silver

nitrate staining and fluorescence in situ hybridisation

(FISH). The FISH experiments were carried out with two

probes simultaneously: the ribosomal 45S pTa71 (Gerlach

and Bedbrook 1979) and the genomic DNA from Aegilops

tauschii Coss. (2n = 2x = 14; DD). The chromosomes

were counterstained with DAPI. The satellite chromosomes

were identified based on previously described silver

staining and FISH patterns (Amado et al. 1997; Mukai

et al. 1993).

Extraction and digestion of genomic DNA,

and Southern-blot technique

Genomic DNA was extracted from frozen (-80 �C) young

leaves using the CTAB protocol (Doyle and Doyle 1987).

The DNA samples (10 lg) were digested overnight with

the restriction enzymes MspI and HpaII, separately,

according to the manufacturer instructions (New England

Biolabs, Beverly, MA, USA). The recognition sequence of

both enzymes is 50-CCGG-30. However, they differ in their

sensitivity to cytosine methylation, HpaII fails to cleave

the 50-CCGG-30 sequence when the inner C is methylated

while MspI cleaves the sequence in that situation. Both

enzymes do not cleave the 50-CCGG-30 sequence if the

external cytosine is methylated. The digestion products

were observed after electrophoresis on 1 % agarose gels.

Southern-blots were prepared by transferring the diges-

ted genomic DNA to a nylon N? membrane (Roche) using

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the saline transfer buffer 209 SSC (sodium citrate; sodium

chloride). The pTa71 cloned sequence was digested with

the restriction enzyme EcoRI (New England Biolabs,

Beverly, MA, USA), in order to isolate the rRNA genes

18S-5.8S-25S and the intergenic spacer region (IGS). This

8.9 kb fragment was labelled with the ‘‘DIG-High Prime

DNA Labeling and Detection Starter Kit II’’ (Roche) fol-

lowing the manufacturer’s instructions. The hybridisation

signals were detected by conventional autoradiography

after 40 min of exposure.

The NOR methylation patterns achieved with both

restriction enzymes in F1 and F2 plants were compared

among them and with the respective parent cultivars.

Results and discussion

rRNA gene expression analysis and FISH

Our previous work indicated a differential rRNA genes

expression related to cytosine methylation, because culti-

vars with a maximum of four Ag-NORs showed higher

levels of cytosine methylation at the rDNA than those with

a maximum of six Ag-NORs (Carvalho et al. 2010). In the

present study, the treatment of seeds of parent cultivars

with a maximum of four Ag-NORs with 5-azacytidine

revealed the activation of the repressed Nor-D3 locus in the

chromosome 5D (Table 1; Fig. 1a, b), confirming our

previous results.

These results corroborated that the differential rRNA

genes expression in bread wheat was related to cytosine

methylation, which is particularly affecting the minor Nor-

D3 locus, once the major ones were always consistently

expressed (Table 1). Despite the present data seem to be

similar to the phenomenon of nucleolar dominance, in this

study, the suppressed Nor-D3 is not from one particular

parent instead it belongs to one parental genome of one of

the parents on an intraspecific cross of bread wheat, being

very different from the concept of nucleolar dominance.

The nucleolar dominance phenomenon has been exten-

sively described in triticale and wheat-rye hybrids (Neves

et al. 1995, 1997, 2005; Viegas et al. 2002; among many

others) where the Ag-NORs of wheat induce the suppres-

sion of the 1R NOR from rye. In bread wheat there are

cultivars that naturally present a maximum of six Ag-

NORs even without 5-azacytidine treatment (Guedes-Pinto

et al. 1998; Lima-Brito et al. 1998). If nucleolar dominance

effectively occurred in bread wheat, all cultivars presented

a maximum of four Ag-NORs per metaphase cell and four

nucleoli per interphase nucleus. Thus, the present results

could only be explained based on the occurrence of natural

variation in the rDNA cytosine methylation which affects

the rRNA genes expression, as reported for other plant

species, such as Arabidopsis thaliana (Riddle and Richards

2002, 2005; Woo and Richards 2008). Besides, Lima-Brito

et al. (1998) also reported that the bread wheat cultivars

‘Chinese Spring’ and ‘Barbela’ line 1T presented low

frequencies of metaphase cells with five and six Ag-NORs.

These authors also reported that in bread wheat, which is of

Table 1 Frequency of interphase cells with a variable number of nucleoli and number of metaphase cells with a variable number of Ag-NORs in

15 bread wheat cultivars after treatment with 5-azacytidine and silver nitrate staining

Cultivar name (code) Frequency of interphase cells with the following number of

nucleoli:

No. of metaphase cells with the following

number of Ag-NORs:

1 2 3 4 5 6 Total of cells 4 5 6

‘Anafil Claro’ (5) 0.06 0.17 0.35 0.40 0.007 0.009 1,341 125 2 6

‘Ardito’ (7) 0.05 0.12 0.30 0.50 0.011 0.019 1,073 208 3 5

‘Belem’ (14) 0.08 0.09 0.40 0.41 0.010 0.015 1,647 340 5 4

‘Grecia’ (36) 0.07 0.20 0.33 0.39 0.003 0.007 965 167 2 6

‘Mocho Cabecudo’ (48) 0.05 0.25 0.33 0.35 0.002 0.011 1,205 209 4 8

‘Mocho de Espiga Branca’ (50) 0.09 0.26 0.30 0.32 0.007 0.025 1,068 139 0 6

‘Mocho de Espiga Ruiva’ (51) 0.019 0.22 0.35 0.41 0.006 0.002 1,391 142 0 3

‘Ribeiro b’ (74) 0.05 0.23 0.32 0.40 0.003 0.003 1,977 287 7 10

‘Rieti’ (75) 0.06 0.23 0.33 0.36 0.008 0.01 1,721 100 1 12

‘Saloio a’ (80) 0.13 0.15 0.32 0.37 0.008 0.013 1,479 125 0 2

‘Santareno’ (82) 0.16 0.24 0.25 0.31 0.027 0.017 875 212 2 14

‘Serrano b’ (84) 0.10 0.21 0.28 0.35 0.035 0.024 736 216 5 8

‘Tremes Ruivo’ (91) 0.09 0.23 0.26 0.39 0.015 0.010 733 302 6 7

‘Transmontano a’ (93) 0.06 0.20 0.31 0.42 0.002 0.009 932 176 5 9

‘Transmontano b’ (94) 0.08 0.20 0.35 0.36 0.004 0.008 1,135 256 1 12

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relatively recent evolutionary origin, it is notable that the

rDNA loci of the D- and A- wheat genomes have become

largely inactive and diminished in size when compared

with the diploid ancestors.

After performing direct and reciprocal crosses among

the cultivars with four and six Ag-NORs the F1 hybrids

showed mostly a maximum number of four or six

Ag-NORs per metaphase cell, being equal to that of the

female parent, while other cases showed four Ag-NORs or

six Ag-NORs in both senses of the cross (Table 2).

Among the 60 direct and reciprocal crosses performed: 34

cases presented hybrids with a maximum number of

Ag-NORs equal to that of the female parent (light grey

shadow, Table 2); 24 produced hybrids with a maximum of

six Ag-NORs (not shadowed), and only two cases showed

hybrids with a maximum of four Ag-NORs in both senses of

Fig. 1 a Mitotic metaphase cell from bread wheat cultivar ‘Saloio a’

(passport code: 80) after treatment with 5-azacytidine and silver

nitrate staining presenting six Ag-NORs (arrows) per metaphase; and

b interphase nuclei with a maximum of six nucleoli per nucleus

(upper nucleus); c, e mitotic metaphase cells after silver nitrate

staining showing five (c) and six (e) Ag-NORs (arrows); and d,

f sequential FISH performed with genomic Ae. tauschii and pTa71

probes, which allowed the discrimination of the chromosomes from

the D wheat genome (green) and the detection of the rDNA loci (red),

respectively. The chromosomes from wheat A and B genomes (d,

f) were counterstained with DAPI (blue). (Color figure online)

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Table 2 Frequency of interphase cells with a variable number of nucleoli and maximum number of Ag-NORs observed per metaphase cell after

silver nitrate staining in the F1 hybrids

F1 hybrids Frequency of interphase cells with the following no. of nucleoli: Maximum no. of Ag-NORs

per metaphase cell1 2 3 4 5 6 Total cells

80 9 79a 0.16 0.19 0.24 0.42 0 0 1,459 4

79 9 80b 0.10 0.20 0.27 0.41 0.01 0.078 3,174 6

80 9 81 0.07 0.19 0.21 0.53 0 0 1,456 4

81 9 80 0.11 0.14 0.22 0.49 0.03 0.004 1,605 6

80 9 32 0.10 0.19 0.22 0.49 0 0 1,028 4

32 9 80 0.08 0.15 0.19 0.55 0.02 0.01 1,219 6

80 9 31 0.09 0.25 0.26 0.39 0 0 743 4

31 9 80 0.04 0.10 0.31 0.50 0.005 0.04 1,224 6

80 9 46 0.08 0.15 0.33 0.44 0 0 714 4

46 9 80 0.05 0.11 0.30 0.51 0.02 0.009 1,014 6

80 9 77 0.16 0.20 0.23 0.41 0 0 1,333 4

77 9 80 0.14 0.17 0.27 0.38 0.03 0.012 1,633 6

7 9 101 0.095 0.20 0.34 0.37 0 0 588 4

101 9 7 0.16 0.18 0.28 0.37 0.0074 0.0026 1,074 6

48 9 24 0.096 0.197 0.33 0.37 0 0 1,130 4

24 9 48 0.15 0.19 0.26 0.40 0.008 0.0006 1,805 6

36 9 52 0.09 0.14 0.26 0.50 0 0 777 4

52 9 36 0.11 0.21 0.28 0.39 0.0046 0.0023 860 6

80 9 101 0.09 0.13 0.28 0.50 0 0 817 4

101 9 80 0.10 0.20 0.30 0.39 0.0026 0.0065 769 6

5 9 52 0.05 0.12 0.39 0.44 0 0 918 4

52 9 5 0.07 0.14 0.26 0.53 0.0018 0.0018 548 6

91 9 44 0.08 0.23 0.33 0.37 0 0 874 4

44 9 91 0.08 0.20 0.34 0.38 0.0012 0.0006 1,678 6

91 9 43 0.07 0.26 0.29 0.37 0 0 718 4

43 9 91 0.05 0.13 0.37 0.44 0.0056 0.0079 878 6

51 9 52 0.14 0.22 0.29 0.34 0 0 1,694 4

52 9 51 0.10 0.16 0.32 0.41 0.02 0.004 792 6

50 9 53 0.14 0.21 0.25 0.41 0 0 879 4

53 9 50 0.15 0.18 0.27 0.37 0.03 0.007 1,202 6

36 9 31 0.11 0.18 0.31 0.40 0 0 670 4

31 9 36 0.09 0.22 0.27 0.41 0.008 0.006 355 6

80 9 Barb 0.15 0.22 0.28 0.36 0 0 420 4

Barb 9 80 0.07 0.18 0.19 0.24 0.31 0.017 1,068 6

91 9 47a 0.13 0.25 0.30 0.33 0 0 942 4

47 9 91b 0.095 0.198 0.31 0.395 0 0 397 4

51 9 49 0.13 0.20 0.25 0.41 0.006 0.0012 824 6

49 9 51 0.045 0.09 0.37 0.47 0.017 0.005 2,375 6

93 9 92 0.12 0.76 0.28 0.36 0.02 0.005 770 6

92 9 9 93 0.14 0.19 0.27 0.39 0.015 0.002 960 6

75 9 101 0.12 0.20 0.30 0.38 0.005 0.002 1,916 6

101 9 75 0.19 0.20 0.25 0.35 0.001 0.003 873 6

50 9 46 0.12 0.24 0.26 0.34 0.03 0.005 1,488 6

46 9 50 0.14 0.18 0.28 0.39 0.006 0.002 870 6

51 9 101 0.08 0.22 0.33 0.36 0.006 0.0024 834 6

101 9 51 0.06 0.13 0.32 0.48 0.0095 0.0024 837 6

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the cross (dark grey shadow, Table 2). As reported by

Volkov et al. (2007), the inheritance of the 18S-5.8S-26S

rRNA genes in natural allopolyploids could be: a) biparental

(additive); b) uniparental, as result of the elimination of the

rDNA from one parent; or c) uniparental due to subsequent

structural rDNA rearrangements. Our results showed that in

F1 plants with a maximum of six Ag-NORs the Nor-D3 locus

of one of the parents became active after hybridization while

in those with a maximum of four Ag-NORs it became

repressed. These results could arise from the different

degrees of methylation inherited by the F1 hybrid. As we

reported previously, the parent cultivars with a maximum of

six Ag-NORs showed variable indexes of NOR cytosine

methylation as well as those with four Ag-NORs, but in

general, those with a maximum of four Ag-NORs demon-

strated the highest indexes (Carvalho et al. 2010). Those

variable indexes of NOR methylation were transmitted to

the F1 probably because of the absence of a global methyl-

ation resetting between generations (Kakutani et al. 1996;

Martienssen and Colot 2001; Shiba and Takayama 2007).

The hypothesis of nucleolar dominance at the F1 generation

had to be discarded because we found out different types of

inheritance of the Ag-NORs number. If nucleolar domi-

nance of the major NOR loci over the minor locus Nor-D3

took place in the bread wheat hybrids, one would expect only

one type of inheritance, namely four Ag-NORs in all

hybrids, which was not the case (Table 2). In addition, the

analysis of rRNA genes expression in the F2 performed in a

total of 2,400 plants showed plants with four, five and/or six

Ag-NORs per metaphase cell in each progeny (Table 3),

suggesting a null, a partial and/or a total activity of the minor

locus Nor-D3, respectively (Table 3).

The F2 progenies did not show any specific ratio or

mendelian proportion (Table 2).

The physical location of the Ag-NORs in both F1 and F2

plants was determined by the sequential technique of silver

nitrate staining and FISH performed with the pTa71 and

genomic Aegilops tauschii probes, simultaneously. Fig-

ure 1c, e presents metaphase cells from plants with a

maximum of five and six Ag-NORs per metaphase cell

after silver nitrate staining. The NORs location was con-

firmed by sequential FISH (Fig. 1d, f).

In contrast to the detection of the major NOR loci from the

B wheat genome, the minor locus Nor-D3 was difficult to

detected in the F1 and F2 plants just after the silver staining

technique. The reduced intensity of silver impregnation in

this minor locus could be explained by its reduced number of

rDNA copies and low transcriptional activity comparatively

to the major NOR loci. In addition, the coexistence of

interphase nuclei with five or six nucleoli in the same chro-

mosome spread of F1 or F2 plants with a maximum of five

and/or six Ag-NORs, even in reduced frequencies, allowed

us the confirmation of the rRNA genes expression of the

major and minor NOR loci. By other hand, when we per-

formed merely the silver staining technique in the bread

wheat cultivars treated with 5-azacytidine, this minor locus

stained more intensely (Fig. 1a). We can argue that this

feature could be a consequence of the realization of the pre-

treatments inherent to the FISH protocol made just before the

silver staining, that could affected the silver impregnation in

the minor locus Nor-D3. Nonetheless, the sequential FISH

helped us to determine the minor locus Nor-D3 location.

The unique constant throughout the rRNA gene

expression analyses performed in the F1 and F2 plants was

Table 2 continued

F1 hybrids Frequency of interphase cells with the following no. of nucleoli: Maximum no. of Ag-NORs

per metaphase cell1 2 3 4 5 6 Total cells

48 9 30 0.20 0.22 0.25 0.33 0.0024 0.0023 1,277 6

30 9 48 0.09 0.11 0.25 0.48 0.07 0.003 1,077 6

74 9 79 0.07 0.23 0.32 0.37 0.004 0.002 1,192 6

79 9 74 0.12 0.20 0.32 0.36 0.0035 0.0018 562 6

36 9 Barb 0.20 0.34 0.59 0.70 0.158 0.011 4,053 6

Barb 9 36 0.15 0.38 0.65 0.76 0.049 0.012 1,755 6

7 9 Barb 0.08 0.21 0.32 0.34 0.044 0.0075 3,726 6

Barb 9 7 0.16 0.23 0.30 0.30 0.01 0.0064 3,395 6

84 9 Barb 0.08 0.20 0.33 0.37 0.0032 0.0048 617 6

Barb 9 84 0.09 0.20 0.29 0.39 0.02 0.0072 2,078 6

50 9 Barb 0.11 0.16 0.30 0.35 0.03 0.06 552 6

Barb 9 50 0.14 0.18 0.25 0.42 0.0092 0.006 542 6

48 9 Barb 0.10 0.23 0.29 0.36 0.009 0.002 890 6

Barb 9 48 0.13 0.18 0.31 0.38 0.003 0.001 1,747 6

a Direct cross (4 Ag-NORs 9 6 Ag-NORs); b Reciprocal cross (6 Ag-NORs 9 4 Ag-NORs)

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the consistent expression of the major NOR loci (sug-

gesting their codominance). The codominance of major

NOR loci was previously reported by May and Appels

(1992) and May and Zhiyong (1996).

NOR methylation patterns analyses

In order to verify if the expression of the minor NOR locus

of chromosome 5D was related to the NOR cytosine

methylation patterns, we performed genomic Southern-

blots with the restriction enzymes MspI and HpaII in the F1

and F2 plants, and compared them with the patterns of their

respective parents.

The NOR methylation patterns achieved by HpaII were

diffuse and not discriminative among parents, F1 and F2

plants (results not shown). For that reason they were dis-

carded from the further analysis.

Considering only the MspI patterns, some of the frag-

ments present in the parents were transmitted to the F1

plants. The fragments with high molecular weight from the

wheat cultivar ‘Mocho Cabecudo’ (passport code: 48) were

eliminated from the F1 plants (Fig. 2). In addition, novel

fragments (not present in any parent cultivar) were detected

in the F1 plants, suggesting a rearrangement of the NOR

methylation patterns after the process of hybridization

(Fig. 2). These results were observed on both direct and

reciprocal crosses. Another interesting result it is that

parental cultivars showed different NOR patterns, but the

F1 hybrids, independently of their parents, evidenced an

invariable NOR pattern among them (Fig. 2). It seems that

Table 3 Number of F2 plants with a maximum of four, five and/or six Ag-NORs per metaphase cell observed after silver nitrate staining

F2 plants resultant

from the crosses:

No. plants with the following no. of Ag-NORs per

metaphase cell:

F2 plants resultant

from the crosses:

No. plants with the following no. of Ag-NORs per

metaphase cell:

4 5 6 4 5 6

80 9 79 22 10 8 91 9 44 25 5 10

79 9 80 20 10 10 44 9 91 19 7 14

80 9 81 21 8 11 91 9 43 24 7 9

81 9 80 23 10 7 43 9 91 21 10 9

80 9 32 28 6 6 91 9 47 26 11 3

32 9 80 26 6 8 47 9 91 17 10 13

80 9 31 20 4 16 51 9 49 2 5 33

31 9 80 17 13 10 49 9 51 12 15 13

80 9 46 22 3 15 51 9 52 27 3 10

46 9 80 22 4 14 52 9 51 20 2 18

50 9 46 29 2 9 93 9 92 20 2 18

46 9 50 19 13 8 92 9 93 18 4 18

80 9 77 29 6 5 75 9 101 27 2 11

77 9 80 20 12 8 101 9 75 24 3 13

7 9 101 27 3 10 50 9 53 21 10 9

101 9 7 22 8 10 53 9 50 24 6 10

51 9 101 25 8 7 36 9 31 20 12 8

101 9 51 23 12 5 31 9 36 27 8 5

48 9 30 19 12 9 80 9 Barb 22 10 8

30 9 48 26 7 7 Barb 9 80 6 10 24

74 9 79 27 7 6 36 9 Barb 19 11 10

79 9 74 25 7 8 Barb 9 36 5 25 10

48 9 24 21 3 16 7 9 Barb 13 10 17

24 9 48 27 6 7 Barb 9 7 28 2 10

36 9 52 18 13 9 84 9 Barb 20 9 11

52 9 36 12 9 19 Barb 9 84 0 30 10

80 9 101 26 0 14 50 9 Barb 16 17 7

101 9 80 15 7 18 Barb 9 50 0 31 9

5 9 52 29 2 9 48 9 Barb 27 13 0

52 9 5 23 5 12 Barb 9 48 0 32 8

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the elimination and creation of NOR fragments were

selective in order to achieve stability. The tendency to

stabilize was also noticed with the analysis of the F2

results, since the NOR methylation patterns visualized in

the F1 plants were transmitted and maintained in their

respective F2 plants (Fig. 2). Previous studies developed in

A. thaliana reported that NOR methylation patterns were

transmitted in a stable way from the parents to the hybrids,

being maintained in the progeny as a result of combination

of genetic and epigenetic mechanisms (Riddle and Rich-

ards 2005; Woo and Richards 2008). In the present study,

the first assumption was not fully accomplished once only

some fragments were inherited by the F1 plants but a stable

NOR methylation pattern was shared by all F1 plants. The

stability also noticed at the F2 generation might be a result

of rapid concerted evolution of the rRNA multigene family

and their spacers. The hybridization of different genomes

in the formation of newly allopolyploids or the cross

between two allopolyploid plants, which is the case here,

might induce rapid genomic and chromosome rearrange-

ments which tend to achieve stability. In addition, the

invariable NOR pattern observed at the F1 (Fig. 2) did not

show any correlation with the maximum number of Ag-

NORs scored for each F1 plant (Table 1). Moreover, the

maintenance of that NOR methylation pattern by the F2

plants that previously showed a segregate number of

Ag-NORs also revealed no correlation between these two

features. The maintenance of the NOR methylation patterns

from F1 to F2 might be due to the action of DNA

methyltransferases.

Our previous (Carvalho et al. 2010) and present results

revealed that cytosine methylation was only correlated with

the expression of the rRNA genes on the parent cultivars.

After the process of hybridization, several genomic, chro-

mosome and/or epigenetic events took place on the F1

hybrid nucleus, and the interactions of those mechanisms

could probably explain these complex results. The differ-

ential transcription of the rDNA in interspecific hybrids

and allopolyploids might result from chromosomic/geno-

mic context, factors that act preponderantly at the chro-

matin level, structural characteristics of the rDNA loci like

the intergenic sub-repetitions, unlinked genes regulation

(Volkov et al. 2007), and/or imprinting (Neves et al. 1995).

The high variability found out within the intergenic spacer

(IGS) of the rDNA among the parent bread wheat cultivars

used here (see Carvalho et al. 2011), could be influencing

the expression of the rRNA genes in the F1 hybrids,

because most of the recognition sites of MspI are located

within the IGS (see Carvalho et al. 2010). Some authors

stated that the degree of cytosine methylation in regulatory

sequences of the rRNA genes located in the IGS could

explain the differential expression of those genes (Appels

et al. 1980; Molnar et al. 1989; Sharma et al. 2005). Sar-

dana et al. (1993) correlated the IGS length variability with

the cytosine methylation status in wheat. Sharma et al.

(2005) reported that longer IGS regions had more unme-

thylated CCGG sites than shorter IGS regions, which could

explain the consistent expression of the major NOR loci

and the repression of the minor NOR-D3 locus of the 5D

chromosome that we found out in parent cultivars, and in

some F1 and F2 plants. Nevertheless, besides the methyl-

ation levels the subrepeat sequences and their number of

repetitions that contain the regulatory elements could also

control the rRNA genes transcription (Polanco and Perez

Fig. 2 NOR methylation

patterns of the parent cultivars

‘Egıpcio’ (E, code 24); ‘Mocho

Cabecudo’ (MC, code 48); and

‘Fronteirico’ (F, code 30), of

their F1 hybrids (lanes 1, 2, 5,

6), and respective F2 plants

(lanes 3, 4, 7, 8), produced by

genomic Southern blots

performed with the restriction

enzyme MspI, and hybridized

with the probe pTa71. Lanes 1

F1 48 9 30 (direct cross, 4

Ag-NORs 9 6 Ag-NORs); 2 F1

30 9 48 (reciprocal cross, 6

Ag-NORs 9 4 Ag-NORs); 3 F2

48 9 30; 4 F2 30 9 48; 5 F1

48 9 24 (direct cross); 6 F1

24 9 48 (reciprocal cross); 7 F2

48 9 24; and 8 F2 24 9 48.

M DNA Molecular Weight

Marker II, Digoxigenin-labeled

(Roche)

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De La Vega 1997). After PCR–RFLP performed with TaqI

several IGS length variants was detected among the parent

bread wheat cultivars (Carvalho et al. 2011) and those

patterns were inherited by their F1 hybrids. In a near future

we intend to use just the IGS region as probe in Southern-

blot techniques, in order to evaluate the degree of cytosine

methylation within this rDNA region and to study its

structure and inheritance by the F1 and F2 plants. The

results presented here allowed us to suggest the absence of

correlation between the rRNA genes expression and NOR

methylation patterns for the F1 and F2 generations. None-

theless, the use of alternative approaches such as Chro-

matin Immunoprecipitation (ChIP) would provide new

insights about the methylation pattern of the minor NOR

locus in bread wheat.

Acknowledgments This work was supported by the PTDC/AGR-

GPL/65876/2006 project and the Ph.D. grant SFRH/BD/17348/2004

both financed by the Portuguese Foundation for Science and the

Technology (FCT).

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