Annals of Botany 112: 527– 534, 2013
doi:10.1093/aob/mct121, available online at www.aob.oxfordjournals.org
B chromosomes of rye are highly conserved and accompanied the development
of early agriculture
André Marques1,2, Ali M. Banaei-Moghaddam1, Sonja Klemme1, Frank R. Blattner1, Katsumasa Niwa3,
Marcelo Guerra2 and Andreas Houben1,*
1
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany, 2Laboratory of
Plant Cytogenetics and Evolution, Department of Botany, UFPE, Brazil and 3Laboratory of Plant Breeding, Faculty of Agriculture,
Tokyo University of Agriculture, Japan
* For correspondence. E-mail [email protected]
Received: 11 March 2013 Revision requested: 3 April 2013 Accepted: 16 April 2013 Published electronically: 5 June 2013
† Background and Aims Supernumerary B chromosomes (Bs) represent a specific type of selfish genetic element. As
Bs are dispensable for normal growth, it is expected to observe B polymorphisms among populations. To address
whether Bs maintained in geographically distinct populations of cultivated and weedy rye are polymorphic, the distribution patterns and the transcriptional activity of different B-located repeats were analysed.
† Methods Bs of cultivated and weedy rye from seven origins were analysed by fluorescence in situ hybridization
(FISH) with probes specific for the pericentromeric and interstitial regions as well as the B-specific non-disjunction
control region. The DNA replication, chromatin composition and transcription behaviour of the non-disjunction
regions were determined. To address whether the B-marker repeats E3900 and D1100 have diverged genotypes of
different origin at the sequence level, the genomic sequences of both repeats were compared between cultivated
rye and weedy rye from five different origins.
† Key Results B chromosomes in cultivated and weedy rye have maintained a similar molecular structure at the level
of subspecies. The high degree of conservation of the non-disjunction control region regarding its transcription activity, histone composition and replication underlines the functional importance of this chromosome region for the
maintenance of Bs. The conserved chromosome structure suggests a monophyletic origin of the rye B. As Bs were
found in different countries, it is likely that Bs were frequently present in the seed material used in early agriculture.
† Conclusions The surprisingly conserved chromosome structure suggests that although the rye Bs experienced rapid
evolution including multiple rearrangements at the early evolutionary stages, this process has slowed significantly
and may have even ceased during its recent evolution.
Key words: chromosomal evolution, chromosomal polymorphisms, non-disjunction control, Secale cereale, rye,
parasitic chromosome, genome evolution.
IN T RO DU C T IO N
Supernumerary B chromosomes (Bs) are not required for the
normal development of organisms and are assumed to represent
a specific type of selfish genetic element. As a result, B chromosomes follow their own species-specific evolutionary pathways.
The most widely accepted view is that they are derived from the A
chromosome complement. Despite the high number of species
with B chromosomes, their de novo formation is probably a
rare event, because the occurrence of similar B chromosome variants within related species suggests that they arose from a single
origin (Jones and Houben, 2003; Houben et al., 2013).
As they are dispensable for normal growth, Bs were considered
non-functional and without any essential genes. It is expected to
observe B polymorphisms among populations. Indeed, beside
B-typical numerical polymorphisms, there are several cases of
B structural polymorphisms in plants, e.g. in Brachycome
dichromosomatica (Houben et al., 1999), in Scilla autumnalis
(Guillen and Rejon, 1984; Parker et al., 1991), in Allium schoenoprasum (Bougourd and Parker, 1979), in Aegilops mutica
(Mochizuki, 1960) and in animals such as the grasshopper
Eyprepocnemis plorans (Bakkali et al., 1999; Cabrero et al.,
1999; Bakkali and Camacho, 2004).
One of the best models for a parasitic chromosome is the B
chromosome of rye (Jones and Puertas, 1993). Bs are found in
both cultivated rye (Secale cereale subsp. cereale) and weedy
rye (Secale cereale subsp. segetale) from different countries
(Akita and Sakamoto, 1982; Jones and Puertas, 1993; Niwa and
Sakamoto, 1995, 1996). Based on similar morphology and
meiotic pairing of Bs derived from weedy and cultivated rye
lines of different origins in F1 hybrids, a monophyletic origin
for the rye B is likely (Niwa and Sakamoto, 1995). Employing
next-generation sequencing, we determined the DNA composition of the Bs and the respective A chromosomes of cultivated
rye (Martis et al., 2012). The origin of rye Bs was estimated to
be about 1.1–1.3 million years ago, thus overlapping in time
with the onset of the genus Secale (1.7 million years ago).
Based on the obtained information, we proposed that Bs originated
as a by-product of A chromosome reorganization events during
early evolution of rye. Multiple chromosomal rearrangements
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528
Marques et al. — Highly conserved B chromosomes in rye
involving chromosomes 3RS and 7R, accumulation of repeats and
genic fragments derived from other A chromosomal regions and
insertions of organellar DNA shaped the B (Martis et al., 2012).
The maintenance of Bs in natural populations is possible by
their transmission at higher rates than Mendelian frequencies.
The mechanism of accumulation of the rye B by post-meiotic
non-disjunction requires a factor located at the end of its long
arm and the pericentromere (Müntzing, 1948; Håkanson,
1959; Endo et al., 2008; Banaei-Moghaddam et al., 2012).
Many B-specific repeat families reside in the long arm terminal
non-disjunction control region: E3900 (Blunden et al., 1993),
D1100 (Sandery et al., 1990), and Sc9c11, Sc9c15, Sc9c130,
Sc21c9, Sc21c67 and Sc26c38 (Klemme et al., 2013). The
distal heterochromatin is marked with the euchromatin-specific
histone modification mark trimethylated histone H3 lysine 4
(H3K4me3) (Carchilan et al., 2007). In addition, expression analysis revealed that D1100 and E3900 are transcriptionally active
in anthers. Since both repeats form non-coding RNA, it was proposed that these transcripts themselves are involved in the nondisjunction process of the rye B.
Structural rearrangements of the Bs occur at about 2 % per generation in experimental crosses (Jimenez et al., 1995), giving rise
mainly to B isochromosomes and truncated Bs (Jones and Puertas,
1993). Although such rye B variants occur as non-permanent
products, we have found a rye line showing a high frequency of
B structural alterations (Marques et al., 2012). In addition, early
analysis of pachytene Bs suggested some minor variations in
chromosome structure (Lima-de-Faria, 1963). With the recent
identification of a number of rye B-specific sequences (Martis
et al., 2012; Klemme et al., 2013), a thorough comparative analysis of Bs of different origin became feasible.
As for the classification of cultivated rye and its weedy relatives, Roshevitz (1947) described the three species, S. cereale,
S. afghanicum and S. segetale. Hammer et al. (1987) described
the four subspecies, S. cereale subsp. cereale, subsp. segetale,
subsp. ancestrale and subsp. dighoricum. Frederiksen and
Petersen (1998) recognized only the two subspecies cereale
and ancestrale. Cytogenetic analysis showed the close relationship among subspecies cereale, segetale, ancestrale and dighoricum (Khush, 1963). Recent molecular biological analysis using
rDNA internal transcribed spacer (ITS) sequences (De Bustos
and Jouve, 2002), amplified fragment length polymorphism
(AFLP) (Chikmawati et al., 2005) and microsatellites (Shang
et al., 2006; Ren et al., 2011) also revealed that cultivated and
weedy rye are very closely related. In addition, cultivated rye is
a representative of a secondary crop, i.e. it was domesticated
from weedy relatives (Hawkes, 1983; Vavilov, 1987). In this
context, it is worthwhile elucidating the molecular structure of
Bs in cultivated and weedy rye. As a result, there is the possibility
to deduce the relationship of cultivated and weedy rye by use
of Bs as an indicator.
To address whether Bs maintained in geographically distinct
populations of rye are polymorphic, we analysed the chromosomal distribution patterns and the transcriptional activity of different B-located repeats in Bs derived from cultivated rye
originally collected in Turkey, Iran, Korea, Japan and China
and weedy rye collected from Pakistan and Afghanistan (Niwa
and Sakamoto, 1995, 1996). In particular, we were interested
in determining whether the chromosomal region required for
the control of B non-disjunction is conserved.
M AT E R I A L S A N D M E T H O D S
Plant material
Cultivated rye (Secale cereale L. subsp. cereale) collected from
Turkey, Iran, Korea, Japan (Niwa and Sakamoto, 1995) and
China (no. 37, in Niwa and Sakamoto, 1996), and weedy rye collected from Pakistan (subsp. segetale, no. 34, in Niwa and
Sakamoto, 1996) and Afghanistan (subsp. afghanicum, Niwa
and Sakamoto, 1995), together with seeds of rye reference material of the Japanese line 7415 were genotyped for the presence
of B and kept growing in a greenhouse for tissue collection and
propagation. Subspecies segetale is widely distributed in
eastern Turkey, Transcaucasia, northern Iran, Afghanistan and
Pakistan. Subspecies afghanicum is endemic to Afghanistan
(Roshevitz, 1947; Tsvelev, 1976). Both taxa belong to subsp.
ancestrale in the treatment of Frederiksen and Petersen (1998).
Fluorescence in situ hybridization (FISH) and immunolabelling
Preparation of mitotic chromosome spreads and in situ hybridization were performed as described by Ma et al. (2010). The following probes were used for comparative FISH mapping, the
repeats D1100 (Sandery et al., 1990), E3900 (Blunden et al.,
1993), ScCl11, Sc26c38, Sc36c82, Sc55c1, Sc63c34 and
Sc9c130, Revolver and Sabrina, bacterial artificial chromosomes
(BACs) encoding chloroplast DNA (BAC clone ChHB 100G01)
and mitochondrial DNA (BAC clone MnHB 0205G01) (Martis
et al., 2012; Klemme et al., 2013), the (CAA)10 microsatellite
(Marques et al., 2012) and Arabidopsis-type telomere repeats
(Ma et al., 2010). Chromosome preparation for immunostaining
and localization of anti-histone H3K4me3 (Upstate, Cat. no.
07-473, diluted 1:200) was carried out according to Marques
et al. (2011). Microscopic images were recorded using an
Olympus BX61 microscope equipped with an ORCA-ER CCD
camera and a deconvolution system. Images were analysed
using the SIS software (Olympus).
DNA replication analysis
Roots of young seedlings were treated with 15 mM EdU
(5-ethynyl-2′ -deoxyuridine) for 2 h, and afterwards they were
washed thoroughly with water and subjected to a 2.5 h regeneration time in water. The roots were fixed in ethanol:acetic acid
(3:1) and the slides were prepared as described above. For replication detection, slides were first briefly incubated with 3 %
bovine serum albumin (BSA) in phosphate-buffered saline
(PBS). Afterwards click-it chemistry (Click-it EdU-Kit from
Invitrogen Alexa-Flour 594) was applied according to the protocol supplied by the manufacturer. The slides were washed with
PBS for 2 – 5 min, followed by FISH as described above.
RNA extraction, cDNA synthesis and reverse transcription –PCR
(RT – PCR)
Total RNA was isolated from anthers (microscopically staged
between meiosis and development of mature pollen) using the
Trizol method (Chomczynski and Sacchi, 1987). Genomic
DNA contamination was removed with Ambion TURBO
DNase (www.invitrogen.com). The absence of genomic DNA
contamination was confirmed by PCR with specific primers
Marques et al. — Highly conserved B chromosomes in rye
for Bilby repeats in DNase-treated RNA (Supplementary Data
Table S1). cDNA was synthesized from 1.5 mg of total RNA
(First Strand cDNA Synthesis Kit, Fermentas). The RT – PCR
was performed as described by Carchilan et al. (2007).
The RT – PCR mix contained 75 ng of cDNA, 1 mM of
each primer (regions 1RT, 2RT, 3RT, 4RT, 5RT and 6RT,
Supplementary Data Table S1), buffer, deoxynucleotide triphosphate and 1 U of Taq polymerase. Thirty-five amplification
cycles (45 s at 95 8C, 1 min at 64 8C, and 1 min at 72 8C) for the
amplification of E3900 and D1100 and 25 cycles for transcripts
of the housekeeping genes glyceraldehyde phosphate dehydrogenase (GAPDH) and elongation factor 1a (EF1a) were
conducted, respectively.
Phylogenetic analysis of E3900 and D1100 elements
D1100 and two different regions of E3900 (primer combinations of F2/R2N and F4/R4, Supplementary Data Table S1)
were amplified using genomic DNA of cultivated and weedy
rye with and without Bs. PCR amplicons were gel purified
and cloned using StrataClone PCR Cloning Kits (http://
www.home.agilent.com) and sequenced. Quality control and
trimming of sequences were performed with Sequencher 4.7.
After aligning the sequences with Clustal W and slight manual
adjustments, phylogenetic trees were calculated with the
Neighbor–Joining algorithm in MegAlign (DNASTAR,
Lasergene 8) based on pairwise Kimura-2-parameter distances.
R E S U LT S
The same type of B exists in rye populations of different countries
To compare the overall structure of the B of cultivated rye (subsp.
cereale) and weedy rye (subsp. segetale and subsp. afghanicum)
from different origins (Turkey, Iran, Korea, Japan, China,
A
Japan
Cultivated rye
E
Japan
Sabrina
Cultivated rye
F
China
B
Pakistan and Afghanistan), probes specific for (1) the pericentromeric region (comprising the sequence ScCl11 and mitochondrial DNA), (2) the non-disjunction control region of the long
arm (comprising D1100, E3900, Sc26c38 and Sc9c130
repeats) and (3) the interstitial region (composed of distinct
blocks of repeated sequences Sc36c82, Sc55c1 and Sc63c34,
and chloroplast as well as mitochondrial DNA) were used for
FISH (Supplementary Data Fig. S1).
Regardless of the origin, all Bs of the three subspecies
showed the same distribution of hybridization signals of ScCl11,
Sc36c82 and D1100 in the pericentromeric, interstitial and
terminal regions, respectively (Fig. 1A–D, Supplementary Data
Fig. S2a–c). In contrast, the A chromosomes of different origins
and subspecies revealed distribution polymorphisms for ScCl11
and Sc36c82 (Fig 1A–D, Supplementary Data Figs S2a–c and
S3a, b). The high-copy retroelements Revolver and Sabrina
revealed a genotype-independent distribution pattern (Fig. 1E–H,
Supplementary Data Figs S2h and S3e–f). Hybridization with
Revolver resulted in an enhanced labelling of Bs, while the
retroelement Sabrina is almost absent at Bs.
In situ hybridization with labelled organellar DNA and a
(CAA)10-type microsatellite allowed the identification of two
structural variants of Bs. Bs of cultivated rye from Japan,
South Korea, China and Iran showed the major site of chloroplast
DNA-specific signals at the proximal region of the long arm
distally located to the pericentromeric mitochondrial DNA
site, and an additional small signal in the interstitial region
(Supplementary Data Fig. S2i – m). Two (CAA)10 clusters were
detected at the short arm of the Bs (Fig. 2A). In contrast, the Bs
of cultivated rye from Turkey and weedy rye from Afghanistan
and Pakistan exhibited the major site of chloroplast DNA sites
at the short arm. This B variant showed the major part of the mitochondrial DNA pericentromeric site located on the short arm
(Fig. 2B – E). (CAA)10 signals were revealed at the most terminal
site and the proximal region of the B short arm (Fig. 2B). Minor
C
Pakistan
ScCI11
Sc36c82
Weedy rye
Cultivated rye D1100
Afghanistan
G
H
Revolver
529
Sabrina
Weedy rye
D
Afghanistan
Weedy rye
Revolver
F I G . 1. The probes ScCl11, Sc36c82 and D1100 are constant on the B chromosome, while the former two vary on the A chromosomes. (A– D) FISH was performed
with ScCl11 (red), Sc36c82 (green) and D1100 (yellow) on cultivated rye from (A) Japan and (B) China, as well as weedy rye from (C) Pakistan and (D) Afghanistan.
(E– H) The retroelements Sabrina (yellow) and Revolver (magenta) show similar distributions in (E, F) cultivated rye from Japan and (G, H) weedy rye from
Afghanistan. Arrowheads point to B chromosomes. Scale bar in (H) ¼ 20 mm.
530
Marques et al. — Highly conserved B chromosomes in rye
7415
A
Pakistan
B
C
Afghanistan
C’
Turkey
D
Telomere
Chloroplast
Weedy rye
Cultivated rye
E
F
Standard B
B variant
Weedy rye
Cultivated rye
B variant
Pakistan,
Afghanistan
Turkey
Standard B type
Japan, South Korea,
China, Iran
Pericentric inversion
Mitochondrial DNA
Chloroplast DNA
(CAA)10
Telomere
Mitochondrial DNA
Chloroplast DNA
(CAA)10
E3900
D1100
Non-disjunction
control region
E3900
D1100
Amplification/reduction of E3900
F I G . 2. The rye B shows structural variation in Bs from different locations. (A) Bs from the standard variety from Japan (line 7415) of cultivated rye with chloroplast
signal (red) on the long arm and microsatellite (CAA)10 (light blue) on the short arm (arrows) as well as D1100 (violet) as a B-specific control. (B) Bs of weedy rye from
Pakistan with chloroplast signal (red) on the short arm and microsatellite (CAA)10 (light blue) on both the short and long arm (arrows), as well as D1100 (violet) as a
B-specific control. (C) Bs of weedy rye from Afghanistan with chloroplast signal (red) on the short arm and mitochondrial signal (green) as well as E3900 (yellow) as a
B-specific control. Magnified areas show chloroplast signal on A chromosomes. (C’) Further enlarged B with chloroplast (red) and telomere (green) signals. (D) Bs of
cultivated rye from Turkey with chloroplast signal (red) and mitochondrial signal (green), as well as E3900 (yellow) as a B-specific control. (E) Prophase preparation of
Bs from standard rye (line 7415) and of weedy rye from Afghanistan. Chloroplast signal (red) and mitochondrial DNA (green) are on the long arm for standard Bs and on
the short arm for the variant B. (CAA)10 (light blue) is on the short arm for the standard B and on the long arm for the variant B. (F) Schemata of the structural changes
which occurred during the evolution of the rye B. Arrows indicated the direction of the event. Arrowheads point to Bs. Dashed lines indicate the centromere position.
Scale bars in (D, E) ¼ 20 mm.
signals of chloroplast and mitochondrial DNA were also found on
A chromosomes, although with much less intensity (Fig. 2C,
insets). No interstitial telomeric site was found in this B variant
(Fig. 2C’). The differences between both sub-types of Bs were
better observed in prometaphase chromosomes (Fig. 2E). An inversion of the pericentromeric region was probably responsible
for the origin of the observed B polymorphism (Fig. 2F). Thus,
apart of this inversion, the linear chromosomal distribution of
the analysed repetitive sequences on the rye Bs is still conserved,
regardless of the geographic origin of the populations.
The properties of the non-disjunction control region of the rye
Bs are conserved
To determine whether the non-disjunction control region is
conserved in Bs of both subspecies from different origins, we
compared the DNA composition, distribution of H3K4me3,
the transcriptional activity of D1100 and E3900 elements, and
the pattern of late DNA replication.
All repeats (D1100, E3900, Sc26c38 and Sc9c130) located at
the non-disjunction control region were found in all rye Bs from
different origins (Figs 1 and 2, Supplementary Data Figs S2 and
S3). Only weedy rye B from Afghanistan showed a reduced
E3900-specific signal (Figs 2C, arrow, and 3). To address
whether the B-marker repeats E3900 and D1100 have diverged
genotypes of different origin at the sequence level, the genomic
sequences of both repeats were compared between cultivated rye
and weedy rye from five different origins (Supplementary Data
Fig. S4 and Table S2). Permissive PCR conditions (40 cycles
and a high concentration of template) also allowed the amplification of minor A chromosome-located sequences from 0B plants.
For both repeats, differences were found within and between different accessions with and without Bs, although E3900-like
sequences were more conserved (approx. 97 % sequence similarity) than D1100-like repeats (approx. 88–93 % sequence similarity). For both repeat types, no obvious geographically sorted
sequence clusters were detected in phylogenetic analyses of
their sequences (Supplementary Data Fig. S4). Hence, both
repeats did not diverge into clear accession-specific clusters
after their specific amplification within Bs.
The FISH-based detection of copy number difference between
the Bs of weedy rye prompted us to test whether the reduced copy
Marques et al. — Highly conserved B chromosomes in rye
number of E3900 repeats correlates with a reduced level of
histone H3K4 trimethylation at the sub-terminal end of the B
from Afghanistan. After immunostaining and subsequent
FISH of weedy rye chromosomes, co-localization was found
between hypertrimethylated H3K4 and the E3900-positive
region (Fig. 3). Afghanistan-derived Bs displayed at the terminal
region of the long arm a reduced level of H3K4 trimethylation
(Fig. 3B) compared with Bs from Pakistan(Fig. 3A). In contrast,
the terminal regions of all A chromosomes were negative for
anti-H3K4me3.
To assay whether the observed reduction in the number of
E3900 repeats affects the expression of E3900, we performed a
comparative transcription analysis across all genotypes. E3900
transcripts were amplified from six sub-regions of E3900 (1RT–
6RT, Fig. 4A). As reported by Carchilan et al. (2007)
B-containing plants revealed transcription for all sub-regions of
E3900, although regions 4RT and 6RT showed the highest level
of transcription (Fig. 4B). The expression of all E3900 regions
in cultivated rye from China (+2B) and Turkey (+2B) is higher
than that from from Iran (+2B) and weedy rye from
Afghanistan (+2B) and Pakistan (+2B). Although the copy
number of E3900 in weedy rye B from Afghanistan is reduced
(Figs 2C and 3), no significant expression difference was found
in 2B plants from Afghanistan compared with 2B plants from
Pakistan (Fig. 4B). Control RT–PCR with primers specific for
GAPDH and EF1a showed a comparable yield in all 0B and
+B genotypes. Thus, the differences observed for E3900 were
not due to an uneven amount of cDNA in the reaction. To
compare further the B non-disjunction control region of cultivated
and weedy rye, we analysed the distribution of late DNA replication by EdU incorporation. It is known that this region is undergoing DNA replication last in cultivated rye (Klemme et al., 2013).
After detection of EdU-labelled DNA, FISH was conducted with
labelled Sc26c38 and E3900. As shown in Fig. 5, the nondisjunction control region of the B is undergoing replication last.
A
Pakistan
A’
E3900
E3900
B
H3K4me3
Afghanistan
DAPI/H3K4me3
B’
E3900
E3900
H3K4me3
DAPI/H3K4me3
F I G . 3. Immunostaining against trimethylated histone H3 lysine 4 (H3K4me3)
(red) and sequential FISH with E3900 in weedy rye from (A, A’) Pakistan and (B,
B’) Afghanistan. Inserts in (A’) and (B’) highlight B chromosomes hybridized
with the E3900 probe (yellow). Arrowheads point to B chromosomes. Scale
bar in (A) ¼ 20 mm.
531
Of the tested elements, Sc26c38 co-localized with the
EdU-stained late replicating region, while E3900 did not.
DISCUSSION
Our study revealed that Bs in cultivated and weedy rye have
maintained a similar molecular structure at the level of subspecies. This result confirms the monophyletic origin of the B of
S. secale subspecies proposed by Niwa and Sakamoto (1995).
The high-copy composition of the rye B seems even more conserved than that of the A chromosomes as different hybridization
patterns were found for ScCl11, Sc36c82 and Sc55c1.
Polymorphisms of the sub-telomeric A chromosome regions
were noted after comparison of different accessions of
S. cereale (Zhou et al., 2010).
The high degree of structural and sequence conservation of rye
B among all populations was unexpected, since the apparent nonessential nature of Bs should favour accumulation of mutations
and potential structural polymorphisms. Indeed, a higher rate
of mutations of single nucleotide polymorphisms has been
found in genic sequences of the cultivated rye B compared
with the homologous sequences of A chromosomes, reflecting
a reduced selective pressure for these sequences on the Bs
(Martis et al., 2012). In addition, in many other species, B structural variants derived from a single standard type have been
reported (for a review, see Jones, 1995). Consistent with the
general rapid evolution of pericentromeres (Hall et al., 2006),
the Bs in weedy rye are characterized by pericentric inversion.
Judging from the peripheral samples of weedy rye (from
Pakistan) used in this study, we can suggest that the inversion
took place during the dispersal of weedy rye from
eastern Turkey to Afghanistan and Pakistan. It would be worthwhile to examine Bs of subsp. segetale from eastern Turkey,
Transcaucasia and north-western Iran to define the geographic
region where this mutation probably originiated.
The apparent difference of satellite DNA distribution between
A and B chromosomes might be explained by a population –
genetic interpretation, as initially suggested by Tsujimoto and
Niwa (1994). If amplification of a satellite sequence happens
in an individual without Bs, the amplified sequence will not
become distributed over the Bs. Consequently, sequences that
are amplified in B non-carriers will accumulate only within the
A chromosomes. Amplification events may also occur in individuals with Bs. In this case, the amplified sequence will become
distributed over both chromosome types. After several generations, the amplified repeat in A chromosomes would be diluted
in the large pool of the A chromosomes. However, amplified satellite sequences in Bs would not be weakened so much as in A
chromosomes because the number of carriers of Bs is often
much lower that of the non-carriers.
Repeat composition, transcription activity, histone composition and replication behaviour of the region responsible for the
control of B non-disjunction during gametogenesis are conserved in all genotypes analysed. Compared with all other Bs,
only the B of weedy rye from Afghanistan showed a reduction
in E3900 copy number and the level of histone H3K4 trimethylation. However, it did not alter the late replication behaviour of the
B and the expression of E3900 repeats in anthers. The high
degree of conservation of the non-disjunction control region
underlines the functional importance of this region for B
532
Marques et al. — Highly conserved B chromosomes in rye
A
D1100
E3900
RT
3RT
1RT
4RT
2RT
B
6RT
5RT
Weedy rye
Cultivated rye
gDNA
Anther-oligo dT cDNA
Reference rye (7415)
Iran
0B 2B 0B 2B 0B 2B
0B
China
Turkey
Pakistan Afghanistan H2O
2B 4B 2B 0B 2B 4B 0B 2B
1334 bp
E3900/1RT
E3900/2RT
1108 bp
E3900/3RT
240 bp
499 bp
E3900/4RT
437 bp
E3900/5RT
321 bp
E3900/6RT
800 bp
D1100/RT
GAPDH
EFa-1
C
Cultivated rye
Weedy rye
gDNA
Reference rye Iran
0B
2B
0B
China
2B
0B
Turkey
2B 0B
2B
Pakistan Afghanistan H2O
0B
2B
0B
2B
D1100
800 bp
E3900(F1R6)
3687 bp
F I G . 4. Transcriptional activity of E3900 and D1100 elements located on the non-disjunction control region of the rye B. (A) Primers used for amplification of different regions of the E3900 and D1100 elements as previously described (Carchilan et al., 2007). (B) The transcription of both sequences was estimated using cDNA
from RNA of young anthers. The initial amount of cDNA of each genotype was confirmed with primers for the housekeeping genes GAPDH and EF1a, used as control.
(C) The presence of B chromosome is shown by PCR with gDNA of 0B and 2B plants.
Merged
Edu
A
Afghanistan
B
Pakistan
Sc26c82
E3900
F I G . 5. Late DNA replication of the B of weedy rye from (A) Afghanistan and (B) Pakistan as evidenced by EdU incorporation and sequential FISH with B-specific
repeats. EdU staining of late replicating DNA (red) and FISH with Sc26c38 (green) and E3900 (yellow) probes revealed preferential co-localization of EdU with the
heterochromatic block of Sc26c38. Scale bar in (A) ¼ 20 mm.
Marques et al. — Highly conserved B chromosomes in rye
maintenance. Further, the elevated degree of sequence similarity
of E3900-like repeats across all analysed accessions hints at a
positive selection pressure. Whether E3900-derived non-coding
RNA is interlinked with control of non-disjuction remains to be
demonstrated (Carchilan et al., 2007).
The region showing the latest replicated DNA in +B plants is
exclusively correlated with the repetitive sequence Sc26c38 in
both cultivated and weedy rye (Klemme et al., 2013; this
study). This region does not undergo decondensation at interphase and shows no transcriptional activity, indicating that the
heterochromatic portion of the non-disjunction control region
revealed by C-banding (Carchilan et al., 2007) is mainly composed of Sc26c38 repeats. Because DNA repair systems are
less efficient in heterochromatic regions with late replication
than early replicated euchromatic regions, the former tend to accumulate a higher rate of rearrangements (Herrick, 2011).
However, the block of Sc26c38 sequences did not show any detectable polymorphism among the Bs of the different analysed
populations.
The surprisingly conserved structure prompted us to conclude
that although the rye Bs experienced fast evolution including
multiple rearrangements at the early evolutionary stages
(Martis et al., 2012), this process has slowed significantly and
may have even ceased during its recent evolution. On the other
hand, rye is a young cultivated plant, which originated as a
weed in wheat fields. The earliest remains of wild rye were
found in 11 000- to 12 000-year-old and Epi-Palaeolithic layers
in the Euphrates valley in northern Syria (Hillman et al., 2001;
Willcox et al., 2009). Later, evidence for S. cereale grown as
crop in its own right were found in approx. 6600 BC
(Hillmann, 1978; Fairbairn et al., 2002) records from Turkey.
From East Turkey, the crop spread into other countries, including
the countries used for our comparison of Bs. Thus, the separation
of the different genotypes used for our analyses evolved rather recently in evolutionary terms. As Bs were found in different countries, it is likely that Bs were frequently present in the seed
material used in early agriculture. Hence, the B of rye ‘witnessed’ the early development of agriculture.
S U P P L E M E N TARY D ATA
Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following. Figure S1: distribution
of B-located high-copy sequences along the standard B chromosome of rye. Figure S2: B chromosomes of weedy and cultivated
rye from various locations after FISH. Figure S3: weedy rye B
chromosomes from Afghanistan and Pakistan after FISH.
Figure S4: phylogenetic analyses of D1100 and E3900
sequences. Table S1: primer sequences used in phylogenetic
analysis and RT – PCR. Table S2: comparison of consensus
sequences of D1100 and E3900 from different rye accessions
with published D1100 and E3900 sequences.
ACK N OW L E DG E M E N T S
We thank the National Council for Scientific and Technological
Development (CNPq), the Foundation for Science and
Technology of the State of Pernambuco (FACEPE), the DFG
Germany (HO 1779/14-1) and the Leibniz Institute of Plant
533
Genetics and Crop Plant Research (IPK) for financial support.
We are grateful to K. Meier for her excellent technical assistance.
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