Annals of Botany 86: 1135±1142, 2000
doi:10.1006/anbo.2000.1284, available online at http://www.idealibrary.com on
Repetitive DNA, Genome and Species Relationships in Avena and Arrhenatherum
(Poaceae)
A . K AT S I OT I S *{, M. LO U K A S{ and J . S . H E S LO P - H A R R I S O N {
{Laboratory of Genetics, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75,
GR-118 55, Athens Greece and {Karyobiology Group, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
Received: 1 February 2000 Returned for revision: 13 June 2000 Accepted: 21 August 2000 Published electronically: 27 October 2000
Repetitive sequences have been widely used for examining genome and species relationships by in situ and Southern
hybridization. In the present study, double-stranded DNA sequences, from denatured DNA reannealed to Cot ˆ 1,
from Avena strigosa (2n ˆ 2x ˆ 14; A genome; referred to as CotA) and Avena sativa (2n ˆ 6x ˆ 42; ACD genome;
referred to as CotACD) were isolated with a hydroxyapatite column, and were used for in situ hybridization on
hexaploid A. sativa chromosomes. Probe CotACD labelled all chromosomes evenly throughout their length at the
same intensity. Probe CotA labelled the 28 A and D genome chromosomes strongly and the 14 C genome
chromosomes weakly. Three cloned repetitive sequences, pAvKB9 (126 bp), pAvKB26 (223 bp) and pAvKB32
(721 bp) were characterized in the A, B, C and D Avena genomes and the genus Arrhenatherum using molecular and
cytological methods. Clones pAvKB9 and pAvKB26 were absent from the Avena C genome, while both could
identify the presence of the D genome by Southern hybridization. In situ hybridization to diploid and tetraploid
Avena species revealed that the probes showed a dispersed genomic organization and that they are present on both
arms of all chromosomes. These sequences were excluded from areas where tandem repeats, such as rRNA genes
and telomeres, are present. These results indicate the close relationship between A and D genomes and the presence
of common DNA sequences between A and C Avena genomes. All three clones hybridized to Southern blots
containing Arrhenatherum digested genomic DNA, indicating Arrhenatherum's close anity to A, B and D Avena
# 2000 Annals of Botany Company
genomes.
Key words: Cereals, DNA, hydroxyapatite, in situ hybridization, oats, reassociation kinetics, repetitive DNA.
I N T RO D U C T I O N
Repetitive DNAÐsequence motifs from one to thousands
of bases long, repeated hundreds or thousands of timesÐ
constitute the major fraction of most plant genomes. Some
sequences, such as the rRNA genes or telomeres, are highly
conserved between all organisms, but other DNA motifs,
often with no known function, vary extensively from species
to species in absolute amount, sequence and dispersion
pattern. Families of repetitive DNA motifs, related by
similarity in sequence, each have a characteristic genomic or
chromosomal location within a genus, ranging from
tandemly repeated units present at one distinct location,
to dispersed motifs present throughout the genome
(Schmidt and Heslop-Harrison, 1998).
In early experiments investigating the organization and
evolution of the genome, the reassociation kinetics of
denatured DNA in solution was studied to characterize the
proportion and reiteration frequencies of di€erent DNA
classes (Britten et al., 1974). The key parameter in analysis
of reannealing rates is Cot ( ˆ DNA concentration in moles
of nucleotides per litre the incubation time in seconds).
In a typical species, highly repetitive DNA will have a Cot
value of less than 1, and will reanneal very quickly, while
single copy sequences will have a high Cot value. The use of
* For correspondence. Fax ‡30 1 529 4375, e-mail katsioti@auadec.
aua.gr
0305-7364/00/121135+08 $35.00/00
reassociation studies in plants has demonstrated in some
detail the nature of plant DNA (e.g. Smith and Flavell,
1974; Flavell et al., 1977), and the nature of the di€erent
fractions has been described. In the last 10 years, methods
using total genomic DNA as a probe to chromosome
preparations have been developed to identify the origin of
plant genomes, chromosomes and chromosome segments
(Schwarzacher et al., 1989, 1992; in oats: Leggett and
Markhand, 1995; Katsiotis et al., 1996). Genomic DNA
and, increasingly, fractions of reassociated DNA have been
used to block hybridization of repetitive DNA sequences
found in large clones (BACs or YACs) where there is a need
to localize the single-copy sequences found in the clones.
Furthermore, knowledge about the overall distribution and
organization of the di€erent repetitive DNA fractions adds
to our model of the large-scale organization of the plant
genome (Schmidt and Heslop-Harrison, 1998). Hence there
is a need to learn more about the repetitive DNA sequences
and their genomic locations.
The genus Avena L. consists of diploid (2n ˆ 2x ˆ 14
chromosomes, A and C genomes), disomic tetraploid
(2n ˆ 4x ˆ 28 chromosomes, AB and AC genomes), and
disomic hexaploid (2n ˆ 6x ˆ 42 chromosomes, ACD
genome) species. Thus B and D oat genomes are present
only in tetraploid and hexaploid species, respectively. Flavell
et al. (1977) estimated that 83 % of the total DNA of the
cultivated hexaploid A. sativa L. species was repeated
# 2000 Annals of Botany Company
1136
Katsiotis et al.ÐSpecies Relationships in Aveneae
sequences. Using genomic in situ hybridization (GISH), a
method that utilizes mostly these repeating sequences as a
labelled probe in plants, the C genome was clearly
distinguished from the others, but no di€erentiation was
possible between the A, B and D genome chromosomes
(Chen and Armstrong, 1994; Jellen et al., 1994; Leggett and
Markhand, 1995; Katsiotis et al., 1997a). To improve
di€erentiation between Avena genomes, a number of
repetitive sequences from di€erent oat species have been
isolated and characterized (Fabijanski et al., 1990; Gupta
et al., 1992; Solano et al., 1992; Chen and Armstrong, 1995;
Fominaya et al., 1995; Katsiotis et al., 1996, 1997a; Linares
et al., 1998).
In the present work, the objective was to study the
chromosomal organization of highly repetitive DNA
fractions (Cot ˆ 1) from A. strigosa Schreb. (As genome)
and A. sativa (ACD genome) isolated through hydroxyapatite chromatography. Furthermore, we aimed to study
the phylogenetic relationships between the Avena genomes
and the Arrhenatherum genus, by Southern hybridization
using total genomic DNA from Arrhenatherum as a probe
to diploid, tetraploid and hexaploid oat species' DNA and
three repetitive DNA fragments cloned from a partial
Sau3A A. vaviloviana (Malz.) Mordv. (AB genome) library.
The genomic and chromosomal organization of these three
clones within the genus Avena was also studied using
Southern and in situ hybridization.
M AT E R I A L S A N D M E T H O D S
Plant material, DNA isolation, molecular cloning and
Cot DNA
Plant species and accessions used in the present study along
with their ploidy level and genomic designation are
presented in Table 1. Seeds were obtained from the
collection at the John Innes Centre, Norwich, UK, except
for PI412767, which was acquired from the United States
Department of Agriculture, World Small Grain Collection
in Aberdeen, Idaho.
Total genomic DNA was extracted from young leaves
using standard methods. The partial Sau3A A. vaviloviana
library and the identi®cation of clones containing repetitive
DNA fragments has been previously described (Katsiotis
et al., 1996). Three clones, pAvKB9, pAvKB26, and
pAvKB32 were selected for further study.
T A B L E 1. Plant species and accessions used
Species
Accession
Avena strigosa
Avena longiglumis
Avena clauda
Avena murphyi
Avena abyssinica
Avena barbata
Avena vaviloviana
Avena sativa
Arrhenatherum elatius
2080
2687
2201
2190
1171
2146
PI412767
`Image'
2518
Ploidy level
2n ˆ 2x
2n ˆ 2x
2n ˆ 2x
2n ˆ 4x
2n ˆ 4x
2n ˆ 4x
2n ˆ 4x
2n ˆ 6x
2n ˆ 4x
ˆ 14
ˆ 14
ˆ 14
ˆ 28
ˆ 28
ˆ 28
ˆ 28
ˆ 42
ˆ 28
Genome
designation
AsAs
AlAl
CpCp
AACC
AABB
AABB
AABB
AACCDD
±
Fractions of Cot DNA were isolated from diploid
A. strigosa and A. sativa. Samples containing approx.
100 mg of total genomic DNA in 0.03 M sodium phosphate
(NaH2PO4 and Na2HPO4 , pH 6.8) bu€er (PB) were
sonicated to break them to an average length of 400 bp.
The samples were then denatured in boiling water for
10 min, followed by incubation at 608C for DNA reannealing until the desired Cot value was reached. After
incubation, the samples were applied to a hydroxyapatite
(DNA-Grade Bio-Gel HTP; BioRad, Hercules, CA, USA)
column at 608C. The low-salt fraction, single-stranded
(ssDNA) and double-stranded (dsDNA) DNA was eluted
as 1 ml fractions with 0.01 M PB, 0.12 M PB and 0.5 M PB,
respectively. The column was regenerated according to the
manufacturer's recommendations after each sample run.
Absorption of all fractions was measured with a spectrophotometer at 260 nm and total amounts of the low-salt
fraction, ssDNA and dsDNA were determined (measurements were adjusted for hypochromicity). The total volume
of samples containing the dsDNA was decreased with 2butanol to a volume of 100 ml, followed by removal of
phosphates by spun-column-chromatography using Sephadex G-50 (Pharmacia, Uppsala, Sweden) (Sambrook et al.,
1989). After desalting, S1 nuclease (Sigma, St. Louis, MO,
USA; 400 u/ml) digestion at 378C for 1 h was used to
remove the single-stranded portions from the re-annealed
DNA fractions. Single nucleotides and enzymes were
removed by 2 M ammonium acetate precipitation. The
average length of the re-annealed fragments was determined
in a 1 % agarose gel.
Southern hybridization and DNA sequencing
DNA from species used in this study was digested with
HaeIII and TaqI restriction endonucleases, separated on
1 % agarose gels and transferred to Hybond2 N‡
(Amersham, Bucks, UK) membranes. Southern hybridization was performed using the ECL2 (Amersham) Random Prime labelling and detection system (version II) for
pAvKB9, pAvKB26 and pAvKB32, and the ECL2
(Amersham) Direct Nucleic Acid labelling and detection
system for the total Arrhenatherum genomic DNA.
Sequencing for all clones was performed on an automated
sequencer (Pharmacia) using the dideoxy chain-termination
procedure for both strands. The FASTA and GAP
programs of the GCG package were used for homology
searches of the clones.
Labelling of DNA probes and in situ hybridization
Clones pAvKB9, pAvKB26 and pAvKB32 were labelled
using the polymerase chain reaction, with either biotin-11dUTP (Sigma) or digoxigenin-11-dUTP (Roche, Mannheim, Germany). Double-stranded Cot DNA from diploid
A. strigosa and hexaploid A. sativa species were also
labelled with ¯uorochrome-conjugate nucleotides (Fluorored or Fluorogreen, Amersham) using random prime
labelling as described by Schwarzacher et al. (1994).
Chromosome preparations were made as previously
described by Katsiotis et al. (1996). Preparation of slides
Katsiotis et al.ÐSpecies Relationships in Aveneae
for in situ hybridization was carried out according to
Schwarzacher et al. (1994), with minor modi®cations. Slides
were denatured in an Omnislide in situ hybridization
machine (Hybaid, Teddington, UK) at 658C for 5 min,
followed by 558C for 2 min, 508C for 1 min, 428C for
5 min, and 388C for 1 min, and ®nally transferred to a
humid chamber at 378C and left overnight. Post-hybridization treatment of slides included washes in 2 SSC (0.3 M
NaCl, 0.03 M Na citrate) for 3 min at 428C, 20 %
formamide in 0.1 SSC for 10 min at 428C, 2 SSC for
2 5 min at 428C, 2 SSC for 2 5 min at room
temperature, and a ®nal wash in 4 SSC/Tween (0.2 %)
for 5 min at room temperature. Digoxigenin-labelled
probes were detected with anti-digoxigenin-¯uorescein
Fab fragments (Roche) and biotin-labelled probes were
detected with streptavidin-Cy3 conjugate (Sigma). Slides
were counterstained and mounted in antifade solution
(AF1, Citi¯uor, Canterbury, UK). Photographs were taken
on Fujicolor Super HG400 colour print ®lm.
R E S U LT S
Cot DNA in Avena species
Three di€erent molarities of sodium phosphate bu€er, 0.01,
0.12 and 0.5 M, were used to elute the DNA from the
hydroxyapatite column. The three fractions, after butanol
concentration, were run on a 1 % agarose gel and stained
with ethidium bromide. Sodium phosphate bu€er at 0.12
and 0.5 M was used to elute ssDNA and dsDNA,
respectively, from the hydroxyapatite column. No ¯uorescence was visible for the eluted fraction with the low-salt
(0.01 M) bu€er (Fig. 1, lanes 7, 8 and 9), while the fraction
eluted with the 0.12 M bu€er (corresponding to ssDNA)
showed similar sizes to the sonicated DNA fragments (data
not shown). The fraction eluted with the 0.5 M bu€er
(corresponding to dsDNA) showed a smear at higher
molecular weights than the sonicated DNA (Fig. 1, lanes 1,
3 and 5). However, after this fraction was treated with S1
nuclease, the respective sizes of the fragments were similar
to, or smaller than, the sonicated DNA (Fig. 1, lanes 2, 4
and 6). The dsDNA that was eluted from the hydroxyapatite column and had values of Cot ˆ 1, was found, after
spectrophotometer readings, to correspond to 35±40 % of
the total DNA amount. At the same Cot value, 40±55 % of
the initial DNA concentration corresponded to ssDNA.
The remaining percentage, eluted with the low-salt bu€er,
was found to correspond to mono- and oligonucleotides.
This fraction has been previously described by Flavell et al.
(1974) as an `unknown fraction'.
After hybridization with CotACD (detected red) and
CotA (detected green), visualization with a double bandpass ®lter showed the C genome chromosomes labelled red
and A/D labelled yellow, on chromosomes of the cultivated
hexaploid oat species A. sativa (Fig. 2C). The probes
labelled the chromosomes evenly throughout their length,
with no site-speci®c localization. Fraction CotA labelled 28
chromosomes intensely and 14 chromosomes faintly
(Fig. 2B).
M
1
2
3
4
1137
5
6
7
8
9
F I G . 1. Ethidium bromide-stained gel with reannealed DNA fractions
from diploid A. strigosa (lanes 1 and 2), tetraploid A. vaviloviana (lanes
3 and 4) and hexaploid A. sativa (lanes 5 and 6) species. In lanes 2, 4
and 6 the double-stranded DNA was treated with S1 nuclease to digest
the single-stranded portions from the reannealed DNA fractions.
Lanes 7, 8 and 9 contain the fractions eluted with the low salt (0.01 M)
bu€er from the hydroxyapatite column from the diploid, tetraploid and
hexaploid species, respectively. The marker (M) is a 100 bp ladder.
Homologous DNA repetitive sequences between
Arrhenatherum and Avena
To establish the relationship between closely related
genera and Avena, total genomic DNA from the genus
Arrhenatherum was used as a probe for Southern hybridization on DNA from di€erent oat species digested with either
HaeIII or TaqI restriction enzymes. A strong hybridization
signal was observed in lanes containing the Arrhenatherum
DNA, and a weaker hybridization signal with clear bands
(abundant restriction fragments) was observed in lanes
containing DNA from di€erent oat species (Fig. 3).
In AB tetraploids, a characteristic band present at 4.2 kb
was related to retrotransposons (Katsiotis et al., 1997a).
This band is also present in the genomic Southern,
indicating the presence of this class of retrotransposons in
Arrhenatherum DNA as well. The presence of common
sequences in Arrhenatherum and Avena genomes was also
established by Southern hybridization for the three highly
repetitive probes ( pAvKB9, pAvKB26 and pAvKB32)
isolated from Avena. All three probes were found to be
present in the genus Arrhenatherum in lower copy numbers
but with similar banding patterns to Avena A genome
digestions (Figs 4±6). In Fig. 4, a common band at low
molecular weight is present between Avena species containing the A genome and Arrhenatherum digested with HaeIII,
while in TaqI digestions a common band is present at higher
molecular weights (approx. 0.95 kb). In Fig. 5, four bands
are in common between the Avena species containing the A
genome and Arrhenatherum at approx. 0.4 kb, 0.95 kb,
1138
Katsiotis et al.ÐSpecies Relationships in Aveneae
F I G . 2. Localization of dispersed repetitive DNA sequences on root tip metaphase chromosomes of diploid A. strigosa (As genome), tetraploid
A. vaviloviana (AB genome) and hexaploid A. sativa (ACD genome) by ¯uorescence in situ hybridization. A, D, G and J, Chromosomes stained
with DAPI. B, A. sativa chromosomes probed with double-stranded DNA Cot fractions isolated from A. strigosa (As genome). A and D genome
chromosomes are stained green, while 14 C genome chromosomes are stained faintly green (arrows). C, The same metaphase spread as in A and B,
probed with double-stranded DNA Cot fractions isolated from A. sativa (ACD genome), viewed with a double ®lter. A and D genome
chromosomes are yellow stained, while C genome chromosomes appear red. E and F, A. strigosa chromosomes probed with pAvKB9 and
pAvKB26, respectively. H and I, A. vaviloviana chromosomes probed with pAvKP9 and pAvKB26, respectively. K, A. strigosa chromosomes
probed with pAvKB32. Arrows indicate NORs, double arrows indicate centromeric regions and triple arrows subtelomeric regions. Bar ˆ 10mm.
1.6 kb and 1.8 kb in HaeIII digests, while in TaqI digestions
common bands are present at approx. 0.6 kb and 1.5 kb.
Finally, in Fig. 6 at least one band at approx. 1.9 kb is
present in the HaeIII digests common for A genome Avena
species and Arrhenatherum.
Genomic and chromosomal organization of pAvKB9,
pAvKB26 and pAvKB32
The clones pAvKB9, pAvKB26 and pAvKB32 were
126 bp (Fig. 7), 223 bp (Fig. 8) and 721 bp (Fig. 9) long,
with an AT content of 48 %, 51 % and 54 %, respectively.
Six-mer sequences were repeated twice in pAvKB9 (Fig. 7)
and pAvKB26 (Fig. 8), while a 6-mer sequence in pAvKB32
(Fig. 9) was repeated three times and a 9-mer and an 8-mer
were repeated twice each. No signi®cant homologies to
sequences present in the GenBank/EMBL database (release
104) were found for any of the three sequences; in particular,
despite their dispersed localization, no homology to retrotransposons was found.
To study the genomic organization of pAvKB9,
pAvKB26 and pAvKB32, Southern blots of DNA from
oat species digested with either HaeIII or TaqI were
hybridized with each probe. Probes pAvKB9 and
pAvKB26 were found to be essentially absent from the oat
C genome species (Fig. 4, lane 2, and Fig. 5, lane 2), while
Katsiotis et al.ÐSpecies Relationships in Aveneae
Hae III
1
2
3
4
5
1139
Taq I
6
7
8
9
1
2
3
4
5
6
7
8
9
kb
4.2
1.9
0.9
F I G . 3. Southern blot analysis of Arrhenatherum elatius (lane 1), A. strigosa (As genome, lane 2), A. longiglumis (Al genome, lane 3), A. abyssinica
(AB genome, lane 4), A. barbata (AB genome, lane 5), A. vaviloviana (AB genome, lane 6), A. clauda (Cp genome, lane 7), A. murhpyi (AC genome,
lane 8) and A. sativa (ACD genome, lane 9) genomic DNA digests, probed with Arrenatherum elatius genomic DNA.
Hae III
1
2
3
4
Taq I
5
6
1
2
3
4
5
6
Hae III
1
kb
1.9
1.4
0.95
0.56
2
3
4
Taq I
5
6
1
2
3
4
5
6
kb
1.9
1.4
0.95
0.56
F I G . 4. Southern blot analysis of A. strigosa (As genome, lane 1),
A. clauda (Cp genome, lane 2), A. vaviloviana (AB genome, lane 3),
A. murphyi (AC genome, lane 4), A. sativa (ACD genome, lane 5) and
Arrhenatherum elatius (lane 6) genomic DNA digests probed with
pAvKB9. Arrowheads indicate the presence of extra hybridization
bands in ACD genome species.
F I G . 5. Southern blot analysis of A. strigosa (As genome, lane 1),
A. clauda (Cp genome, lane 2), A. vaviloviana (AB genome, lane 3),
A. murphyi (AC genome, lane 4), A. sativa (ACD genome, lane 5) and
Arrhenatherum elatius (lane 6) genomic DNA digests probed with
pAvKB26. Arrowheads indicate the presence of extra hybridization
bands in ACD genome species.
pAvKB32 was present in all species (Fig. 6). No di€erences
in the hybridization pattern were observed between the A,
AB and AC genome species for all probes. A similar
hybridization pattern to the A genome species was observed
for C genome species when pAvKB32 was used as a probe
(Fig. 6). In C genome HaeIII digests, no band was present at
1.5 kb while an extra band was present at approx. 1.1 kb
(Fig. 6, lane 2). Di€erences were also observed between A or
AB and ACD genome species when hybridized with
pAvKB9 and pAvKB26. Probe pAvKB9 showed an extra
band in HaeIII digests at 0.5 kb and in TaqI digests at
1.5 kb compared to A or AB genome species' DNA when
hybridized to ACD genome species' DNA (Fig. 4, lane 5).
Probe pAvKB26 showed an extra band in ACD genome
species HaeIII digests at 0.65 kb and two extra bands in
TaqI digests at 0.8 and 1.3 kb compared with A or AB
genome species (Fig. 5, lane 5).
Fluorescent in situ hybridization on diploid A. strigosa
(As genome) and tetraploid A. vaviloviana (AB genome)
metaphase chromosomes was used to study the physical
1140
Katsiotis et al.ÐSpecies Relationships in Aveneae
Hae III
1
2
3
4
Taq I
5
6
1
2
3
4
5
6
kb
1.9
1.4
0.95
0.56
F I G . 6. Southern blot analysis of A. strigosa (As genome, lane 1),
A. clauda (Cp genome, lane 2), A. vaviloviana (AB genome, lane 3),
A. murphyi (AC genome, lane 4), A. sativa (ACD genome, lane 5) and
Arrhenatherum elatius (lane 6) genomic DNA digests probed with
pAvKB32.
distribution of the three probes. No probe was found to be
localized at a distinct site on any speci®c chromosome. The
probes were found to be present on all chromosomes
scattered throughout their chromosome length, excluded
from areas where tandem repeats were present, such as
rRNA genes and telomeres (Fig. 2E, F, H, I and K).
Furthermore, in some centromeric regions no hybridization
of the probes was observed (Fig. 2E, F, H, I and K).
DISCUSSION
In the present study, three fractions (low-salt fraction, ssDNA and dsDNA) have been separated using
hydroxyapatite chromatography at Cot ˆ 1. The eluted
low-salt fraction gave a spectrophotometer reading but did
not ¯uoresce in gels stained with ethidium bromide. This
low-salt fraction corresponds to mono- or oligonucleotides
produced by the sonication process: single-stranded DNA
fragments less than 40 nucleotides and double-stranded
DNA fragments less than 100 nucleotides were eluted from
the hydroxyapatite column, whereas larger fragments were
retained using the same molarity phosphate bu€er (Wittelsberger and Hansen, 1979).
The dsDNA fraction, corresponding to 35±40 % of the
total oat nuclear DNA amount, includes foldback DNA,
highly repetitive DNA and some middle repetitive DNA
sequences (Flavell et al., 1977). This fraction, prior to S1
nuclease treatment, showed higher molecular weights
compared with the initial sonicated DNA fragments,
because after DNA renaturation the single-stranded overhangs of the reassociated repetitive DNA fragments tend to
reanneal partially with one another and form long chains of
repetitive sequences (Smith et al., 1975).
Using in situ hybridization, dsDNA (Cot ˆ 1) isolated
from the diploid species A. strigosa (As genome) was found
to hybridize largely to the chromosomes of the genome-oforigin (A genome) and to those of the D genome. The
close relationship between the two genomes, A and D,
has been revealed by using genomic in situ hybridization
(Chen and Armstrong, 1994; Jellen et al., 1994; Leggett
and Markhand, 1995). However, a satellite DNA
sequence, As120a, has been isolated which discriminates
A and D genome chromosomes (Linares et al., 1998). C
genome chromosomes were labelled weakly using CotA as a
probe, indicating the presence of common sequences
throughout the Avena genus, some of which corresponded
to retrotransposons that are also found throughout the
family Poaceae (Katsiotis et al., 1997b).
Repetitive sequences common to Arrhenatherum and
Avena were identi®ed in Southern hybridizations using total
DNA from the former as a probe. The close relationship
between the two genera within the tribe Aveneae has long
F I G . 7. The DNA sequence of pAvKB9. Repeated six-mers are underlined, double underlined, dotted underlined, and in bold font. The
nucleotide sequence appears in the EMBL Nucleotide Sequence Database under Accession Number AJ297384.
F I G . 8. The DNA sequence of pAvKB26. Repeated six-mers are underlined and double underlined. The nucleotide sequence appears in the
EMBL Nucleotide Sequence Database under Accession Number AJ297385.
Katsiotis et al.ÐSpecies Relationships in Aveneae
1141
F I G . 9. The DNA sequence of pAvKB32. The six-mer repeat is underlined, the eight-mer repeat is in bold font and the nine-mer repeat is double
underlined. The nucleotide sequence appears in the EMBL Nucleotide Sequence Database under Accession Number AJ297386.
been established (Baum, 1968). Also, all three isolated
probes, pAvKB9, pAvKB26 and pAvKB32 were found to
be present in Arrhenatherum DNA in low copy numbers.
Interestingly, although pAvKB9 and pAvKB26 clones did
not hybridize to C genome oat species, homology to
Arrhenatherum DNA sequences existed under the same
stringency conditions, giving a similar hybridization pattern
to A genome oat species. This result is in agreement with a
previous suggestion (Katsiotis et al., 1996) that Arrhenatherum is more closely related to the Avena A genome than
to the C genome. So far, all repetitive clones isolated from
oat libraries have exhibited homology to sequences present
in Arrhenatherum elatius (Katsiotis et al., 1996 and present
study). We suggest that members of these repetitive DNA
families were present in the common ancestor of the species
studied, but there has since been some divergence in
sequence, seen by the di€erences in restriction patterns
between A/D, B, C and Arrhenatherum genomes (Figs 4±6).
Probes pAvKB9 and pAvKB26 revealed banding di€erences in Southern blots between the A and the ACD
genome species. Since these two probes do not hybridize to
the C genome, RFLPs should be related to the presence of
the D genome and could be useful for identifying the
presence of this genome in oat species. No RFLP di€erences between A genome diploid and AB genome tetraploid
species were observed, as in previous reports with other
probes (Leggett and Markhand, 1995; Katsiotis et al.,
1997a) questioning the validity of AABB designation, and
suggesting an AAA0 A0 genomic designation for the barbata
group tetraploids.
All three probes examined in the present study were
dispersed along the chromosomes, but showed no sequence
homologies to retrotransposons. Thus far, only one
tandemly-repeated sequence has been isolated from Avena
C genome (Solano et al., 1992), while other non-homologous dispersed sequences have been isolated from Avena
A, B, D and C genomes (Fabijanski et al., 1990; Linares
et al., 1992; Solano et al., 1992; Chen and Armstrong, 1995;
Katsiotis et al., 1996, 1997a). Even sequences with a tandem
organization in other genera, such as pSc119.2 (Castilho
and Heslop-Harrison, 1995), were found to have a
dispersed organization in Avena (Katsiotis et al., 1997a).
C-banding studies in diploid (Fominaya et al., 1988a) and
tetraploid (Fominaya et al., 1988b) oat species revealed two
distinct types of staining appearances of oat chromosomes:
(1) a group of chromosomes with euchromatin uniformly
stained and prominent C-bands located at the telomeres (A
and B genomes); and (2) the C genome chromosomes,
characterized by higher chromatin condensation with
several intercalary heterochromatic bands. These results
are in agreement with the presence of a limited number of
tandemly-repeated families (telomeric sequences) in A, B
and D genome chromosomes, since tandem repeats tend to
be localized to C-banded regions. Random cloning of
genomic DNA sequences from the tribe Triticeae has
allowed isolation of multiple, tandemly-repeated sequences,
but a similar strategy in the related Aveneae has found
1142
Katsiotis et al.ÐSpecies Relationships in Aveneae
largely dispersed sequences (except for the C-genome
sequence discussed above). Within the Avena genomes, the
signi®cance of the accumulation of dispersed sequences,
with no known relationship to retroelements, is unknown;
although possible functions for both tandem and dispersed
repeats have been considered (Smyth, 1991; Singer and
Berg, 1991), prevalence and accumulation of one over the
other and potential e€ects on genomes have not been
studied and explained.
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