Copyright Ó 2008 by the Genetics Society of America
DOI: 10.1534/genetics.108.089599
The Origin of a ``Zebra'' Chromosome in Wheat Suggests Nonhomologous
Recombination as a Novel Mechanism for New Chromosome Evolution
and Step Changes in Chromosome Number
Peng Zhang,1
Wanlong Li, Bernd Friebe2
and Bikram S. Gill
Department of Plant Pathology, Wheat Genetic and Genomic Resources Center, Kansas State University, Manhattan, Kansas 66506-5502
Manuscript received March 27, 2008
Accepted for publication April 15, 2008
ABSTRACT
An alloplasmic wheat line, TA5536, with the ``zebra'' chromosome z5A was isolated from an Elymus
trachycaulus/Triticum aestivum backcross derivative. This chromosome was named ``zebra'' because of its
striped genomic in situ hybridization pattern. Its origin was traced to nonhomologous chromosome 5A of
wheat and 1Ht
of Elymus; four chromatin segments were derived from chromosome 1Ht
and five
chromatin segments including the centromere from 5A. In this study, our objective was to determine the
mechanism of origin of chromosome z5A, whether by nonhomologous recombination or by multiple
translocation events. Different crossing schemes were used to recover recombinants containing various
Elymus chromatin segments of the z5A chromosome. In addition, one z5AL telocentric chromosome and
three z5AL isochromosomes were recovered. The dissection of the Elymus segments into different stocks
allowed us to determine the chromosomal origin of the different chromosome fragments on the basis of
the order of the RFLP markers employed and suggested that the zebra chromosome originated from
nonhomologous recombination. We present a model of possible mechanism(s) of chromosome evolution
and step changes in chromosome number applicable to a wide range of organisms.
CLASSICAL phenomena of changes in multiple sets
of basic chromosome number by polyploidy and
step changes in basic chromosome number through
aneuploidy have been documented for over a century.
Stebbins (1950) provided the first synthesis of the
enormous literature on chromosome variation and evolution
in plants. However, such studies were limited to
groups of species that could be hybridized and their
meiotic behavior analyzed. With the dawn of the genomics
era, comparative genomics and fluorescence in
situ hybridization (FISH) are providing new insights
into mechanisms of chromosome evolution, including
changes in size, morphology, and number at the levels
of tribes, families, or higher hierarchies sharing and
spanning tens or even hundreds of million years of coevolutionary
history. Typically, one or more rounds of
whole-genome duplications, followed by diploidization
and step changes in basic chromosome numbers, have
been hypothesized. In grasses, Salse et al. (2008) have
provided a synthesis of the whole-genome duplications
and step changes in basic chromosome numbers, including
chromosomefissions,fusions,and translocations
that have spawned basic chromosome changes of 1x  5,
7, 10, or 12 in maize (1x  10), wheat (1x  7), rice (1x 
12), and sorghum (1x  10), sharing 50 million years of
evolutionary history. Apart from chromosomefission and
fusion, unequal translocations followed by the elimination
of a diminutive chromosome have been hypothesized
in Arabidopsis species, leading to step changes of
decrease in chromosome number from 1x  8 to 1x  5
(Lysak et al. 2006; Schubert 2007).
However, there have been only a few experimental
demonstrations of step changes in basic chromosome
number (reviewed in Schubert 2007). In wheat, there is
a long history of interspecific hybridization and experimental
introgression aimed at transferring useful genes
from alien species into wheat. There is also a large body
of literature on chromosome behavior and structural
aberrations in interspecific hybrids. One particularly
useful aspect of the wheat genetic system is buffering
of the genome due to polyploidy that allows study of
chromosome structure and behavior in isolation and
over a large number of generations. Some key aspects of
these studies were summarized by Gill (1991): (1)
Interspecific hybridization is mutagenic; (2) many chromosomes
from either one of the parents exhibit meiotic
drive and show preferential transmission; and (3) cytoplasmic
sterility resulting from nucleo-cytoplasmic interactions
is common and leads to unilateral introgression
of fertility restoration (Rf) and/or genes specific to the
cytoplasmic donor in backcross derivatives.
1
Present address: Plant Breeding Institute, University of Sydney, Camden,
NSW 2570, Australia.
2
Corresponding author: Department of Plant Pathology, Wheat Genetic
and Genomic Resources Center, 4024 Throckmorton Plant Sciences
Center, Kansas State University, Manhattan, KS 66506-5502.
E-mail: friebe@ksu.edu
Genetics 179: 1169­1177 ( July 2008)
We have studied one particular wide hybrid between
Elymus trachycaulus (Link) Gould ex Shinners (2n  4x 
28, St
St
Ht
Ht
) and wheat for .25 years. The original
hybrid could only be made using E. trachycaulus as female
and common wheat as male (Sharma and Gill 1981).
Upon backcrossing with wheat, ``unfavorable nucleocytoplasmic
interactions were evident that led to seed
shriveling, embryo abortion, failure of seed or cultured
embryos to germinate, seedling death, plant weakness
and sterility in the backcross derivatives'' (Sharma and
Gill 1983a,b). All viable and partially fertile alloplasmic
derivatives carried chromosomes 1Ht
or 1St
or their short
arms as telocentrics. Twelve simple or complex trans-
locationsinvolving1St
or1St
S withother E.trachycaulusor
wheat chromosomes were recovered (Morris et al. 1990;
Jiang et al. 1994). Among them, an alloplasmic wheat
line carrying a zebra chromosome designated as z5A--a
complex translocation involving 1Ht
S and wheat chromosome
5A that replaced wheat chromosome 5A--was
recovered (Jiang and Gill 1993). Chromosome z5A
gave a striped appearance upon genomic in situ hybridization
(GISH) and was made up of four segments from
E. trachycaulus and five segments including the centromere
from wheat. This line was fully fertile and flowered
10 days earlier than the wheat recurrent parent.
In this article, our objective was to determine the
mechanism of origin of chromosome z5A, whether by
nonhomologous recombination or by multiple translocation
events. By separating and genetically isolating
the four E. trachycaulus segments of z5A using centric
breakage and homologous recombination, we obtained
evidence that chromosome z5A likely arose by nonhomologous
recombination, resulting in a mosaic chromosome
comprised of alternate blocks of Triticum
aestivum and E. trachycaulus chromatin segments. A
model is presented and the significance of this phenomenon
is discussed in relation to chromosome evolution
and repatterning forming new chromosomes and also
leading to step changes in basic chromosome number in
diverse organisms.
MATERIALS AND METHODS
Plant material: An alloplasmic wheat line, TA5536, carrying
the zebra chromosome z5A pair substituting for chromosome
5A of wheat in addition to 20 pairs of wheat chromosomes, was
isolated from a derivative of an E. trachycaulus/T. aestivum cv.
``Chinese Spring'' (CS) hybrid (Sharma and Gill 1983a,b;
Morris et al. 1990; Jiang 1993; Jiang and Gill 1993; Jiang
et al. 1994). Chromosome z5A was recovered in progeny that
had a complete 1Ht
chromosome, a 1Ht
S telosome or isochromosome,
or a T1Ht
SÁ5AL translocation chromosome in
addition to a monosomic wheat chromosome 5A. These
observations suggested that the E. trachycaulus chromatin in
z5A was derived from chromosome 1Ht
(Jiang and Gill
1993). Alloplasmic euploid wheat plants in E. trachycaulus
cytoplasm are male sterile with reduced vigor. The short arm of
chromosome 1Ht
has the fertility restoration gene, Rf-Ht
1. The
alloplasmic stock TA5536 is vigorous and fully fertile, indicating
that the 1Ht
segments present in chromosome z5A
have the Rf-Ht
1 gene. The long arm of chromosome 5A has the
major domestication gene Q, which controls the free-threshing
character and square spike morphology (Huskins 1946;
Unrau et al. 1950; Sears 1952, 1954; Mackey 1954; Simons
et al. 2006). TA5536 has a square head and is free threshing,
indicating that the chromosome 5A segment containing the Q
gene is present in z5A. Meiotic metaphase I pairing revealed
that the short arm of z5A paired with the short arm of
chromosome 1Ht
, whereas the distal region of the long arm of
z5A paired with the distal region of 5AL. These data confirmed
that chromosome z5A was derived from chromosomes 1Ht
of
E. trachycaulus and 5A of wheat (Jiang and Gill 1993).
Different crossing schemes were used to separate the four
Elymus chromatin bands in chromosome z5A. For obtaining
recombinants with different Elymus chromatin bands, the CS
double ditelosomic dDt5A stock (20$ 1 5AS$ 1 5AL$) was
crossed as female with TA5536. The F1 was either backcrossed
with CS or selfed. To obtain z5A telosomes, the CS monosomic
5A stock was crossed as a female with TA5536. Because
chromosome 5A is expected to be absent in $75% of the
female gametes, the F1 plants were expected to segregate 25%
with the chromosome constitution 20$ 1 z5A9 1 5A9 (42
chromosomes) with normal square spike morphology (two
doses of Q) and 75% with 20$ 1 z5A9 (41 chromosomes) and
speltoid (spelled-like) spikes (one dose of Q). Plants with
speltoid spikes were selfed and screened for z5A telosomes
using GISH. In addition, ditelosomic addition (DtA) stocks
DtA1Ht
S and DtA1Ht
L were included in Southern hybridization
analysis to enable the assignment of DNA markers to
either the 1Ht
arms or Elymus chromatin bands. All materials
are maintained at the Wheat Genetic and Genomic Resources
Center, Kansas State University, Manhattan, Kansas.
Mitotic and meiotic analyses: Chromosome measurements
were performed on 20 z5A and 1Ht
chromosomes using 3B of
wheat as a standard. Anthers from F1 plants of dDt5A/TA5536
undergoing meiotic metaphase I were fixed in Carnoy's
solution I (100% ethanol:glacial acetic acid  3:1), stained
in 1% acetocarmine, and squashed in one drop of 45% acetic
acid. Meiotic metaphase I pairing was analyzed in pollen
mother cells (PMCs) after GISH.
FISH and GISH analyses: Seeds were germinated in
distilled water on filter paper in a petri dish at room
temperature for 2­3 days until roots were $2-cm long. Roots
were cut, pretreated in ice water for 24 hr, and fixed in
Carnoy's solution I. FISH and GISH were according to Zhang
et al. (2001).
The Aegilops tauschii Coss. plasmid clone pAet6-J9, containing
a 750-bp sequence with high sequence similarity to the gagpol
polyprotein of the Ty3/gypsy retrotransposon cereba, was
cloned into the plasmid pCR4Blunt-TOPO (Invitrogen Life
Technologies, Carlsbad, CA) (Zhang et al. 2004). This clone
contains a centromere-specific repetitive sequence that hybridizes
to the centromeric regions of wheat, rye, barley, and
maize.
Plasmid DNA was isolated using a QIAprep Spin miniprep
kit (QIAGEN, Valencia, CA). Genomic DNA from E. trachycaulus
was isolated using the standard CTAB method. One
microgram of plasmid DNA was labeled with either rhodamine-6-dUTP
using rhodamine-nick translation mix or fluorescein-12-dUTP
(Roche Applied Science, Indianapolis) using
nick translation according to the manufacturer's protocol.
One microgram of total genomic DNA from E. trachycaulus was
labeled with fluorescein-12-dUTP. Probes were purified with
the QIAquick nucleotide removal kit (QIAGEN).
The composition of the FISH hybridization solution was as
described in Zhang et al. (2004). The GISH hybridization
solution ($77% hybridization stringency) contained 50%
1170 P. Zhang et al.
deionized formamide (Fisher Scientific, Pittsburgh), 1.53
SSC, 10% dextran sulfate (Sigma, St. Louis), 0.17 mg/ml of
sheared salmon testes DNA (Sigma), 1.3­2 mg/ml of labeled
probe, and unlabeled total genomic DNA of wheat as a
blocker. The probe:blocker ratio was $1:40­50. The hybridization
procedures and post-hybridization washes were according
to Zhang et al. (2004). Slides were counterstained with
49,6-diamidino-2-phenylindole (Molecular Probes, Eugene,
OR) or propidium iodide (PI) (Molecular Probes) for FISH
when the probe was labeled with rhodamine-6-dUTP or
fluorescein-12-dUTP, respectively, and mounted in Vectashield
(Vector Laboratories, Burlingame, CA). GISH slides
were counterstained with PI. Slides were analyzed with a Zeiss
Axioplan 2 epifluorescence microscope and images were
captured with a SPOT CCD (charge-coupled device) camera
operated with Spot 2.1 software (Diagnostic Instruments,
Sterling Heights, MI) and processed with Photoshop v5.5
software (Adobe Systems, San Jose, CA).
Southern hybridization: Total genomic DNA isolation,
digestion, gel electrophoresis, and Southern blot hybridization
followed Faris et al. (2000). Probes used were selected
from the consensus map of wheat homeologous group 1
chromosomes (Van Deynze et al. 1995).
RESULTS
Characterization of chromosome z5A by GISH,
FISH, and meiotic pairing analyses: Chromosome z5A
has a length of 9.47 6 0.79 mm (67% of 3B length) with
an arm ratio (long/short) of 4.6. Chromosome 1Ht
has a
length of 8.23 6 0.68 mm (60% of 3B length) with an
arm ratio of 1.8. Chromosome 3B is the largest wheat
chromosome with a size of 13.8 mm (Gill et al. 1991).
GISH revealed that 33% of chromosome z5A (3.13 mm)
was derived from E. trachycaulus and 67% (6.34 mm) was
derived from wheat. The calculated size of 1Ht
S (2.99
mm) is similar to that of the 1Ht
S segment present in
chromosome z5A as determined after GISH (3.13 mm).
Similarly, the 5AL segment in z5A is 6.34 mm, which is
just a little shorter than that of the 5AL reported earlier
(7.7 mm, Gill et al. 1991). The measurement data suggested
that almost the complete 5AL and 1Ht
S arms
were retained in chromosome z5A.
GISH analysis confirmed that chromosome z5A consists
of four E. trachycaulus segments designated as E1,
E2, E3, and E4, and five wheat segments, including the
centromere, designated as W1, W2, W3, W4, and W5
(Figure 1A, Figure 2). The W1 segment contains the
centromere derived from chromosome 5A. FISH with
the centromeric clone pAet6-J9 on the disomic addition
line DA1Ht
revealed strong centromeric FISH sites
on all wheat chromosomes, whereas chromosome 1Ht
showed a very weak centromeric FISH signal (Figure
1B). Clone pAet6-J9 on TA5536 revealed three FISH
sites on chromosome z5A (Figure 1E). Sequential
GISH, performed on the same slide after stripping off
the FISH probe, revealed that the major pAet6-J9 FISH
site mapped to the centromeric W1 segment, whereas
the second pAet6-J9 FISH site mapped between the W2
and W3 segments and the third minor pAet6-J9 FISH
site mapped to the distal region of the E4 segment
(Figure 1E). Colocalization of the minor pAet6-J9 FISH
site to the E4 segment suggested that this site was
derived from E. trachycaulus, in agreement with the
much smaller centromeric pAet6-J9 FISH site in chromosome
1Ht
compared to those of wheat chromosomes.
Meiotic metaphase I pairing was analyzed in 197
PMCs of plants with the chromosome constitution 20$
1 z5A9 1 5AS9 1 5AL9. Chromosome z5A was univalent
in 37 PMCs (19%) (Figure 1C) and paired in the distal
region of the long arm with the 5AL telosome as a rod
bivalent in 160 PMCs (81%) (Figure 1D). These data
confirmed that the distal W5 segment in chromosome
z5A was derived from the distal region of 5AL. The
chiasmata were almost exclusively in the distal regions of
z5A and 5AL, with no chiasma formed in proximal
regions.
Identification of z5A recombinants: The F1 hybrids of
the cross dDt5A/TA5536 were either backcrossed with
CS or selfed. One hundred and nineteen BC1 plants and
68 F2 plants were screened for recombinants using
GISH. Almost all detected recombination events (55
of 56) occurred in the W4 segment, resulting in 28
recombinants having the first three E. trachycaulus
segments (E1, E2, and E3) on a complete z5A chromosome,
designated as rec1, and 27 recombinants with
only the E4 segment on a 5AL telosome, designated as
rec3 (Figure 1F). The frequent recovery of the rec1
and rec3 recombinants further suggested that the W4
segment in z5A is homologous and not rearranged
relative to the corresponding region of wheat chromosome
5A. Only one recombinant event occurred in the
W3 segment and resulted in a z5A recombinant chromosome
with only the E1 and E2 segments (rec2, Figure
1F). The recovery of the rec2 recombinant likewise
suggested that the W3 segment in z5A is homologous
and not rearranged relative to the corresponding region
of wheat chromosome 5A.
Separation of the first E. trachycaulus chromatin
segment (E1) from the remaining three segments (E2,
E3, and E4): F1 hybrids from the cross of a monosomic
5A plant with TA5536 with speltoid spikes, and thus
putatively monosomic for z5A, were selfed. One hundred
and ten F2 individuals were screened for telocentric
chromosomes using GISH. One plant with a tz5AL
telosomic chromosome and a normal z5A chromosome
(2n  41 1 t) having a square spike (two doses of the Q
gene), one plant with a z5AL isochromosome (iz5AL)
(2n  40 1 iz5AL) (square spike, two doses of the Q
gene), and two plants with an iz5AL and a normal z5A
chromosome (2n  41 1 iz5AL) (compactoid spike,
three doses of the Q gene) were recovered (Figure 1G).
In addition, plants with three different types of translocation
chromosomes were identified (Figure 1G),
including (1) T1, with the short arm from an unknown
wheat chromosome and the long arm from z5AL; (2)
T2, with three Elymus chromatin bands (one band in
Origin of a ``Zebra'' Chromosome 1171
the short arm and two bands in the long arm); and (3)
T3, with five Elymus chromatin bands. The centromerespecific
probe pAet6-J9 was used to determine the
location of the centromere in T2 by FISH and identified
T2 as a small metacentric chromosome (Figure 1G).
The GISH pattern of T2 suggested that the GISH band
in the short arm was derived from the E4 segment of
z5A, whereas the two Elymus segments in the long arm
corresponded to the E2 and E3 segments of z5A. The
GISH pattern of T3 suggested that the two Elymus
segments in the short arm correspond to the E3 and E4
segments of z5A, whereas the long arm of T3 is identical
to the long arm of z5A. The T3 translocation stock had
speltoid spikes and low fertility, with only 20 seeds
produced on 16 tillers. Although z5A long-arm telosome
and isochromosomes were recovered, no z5A
short-arm telosome was obtained.
Southern hybridization analysis: Chromosome 1Ht
is
homeologous to the group 1 chromosomes of wheat.
Molecular markers spanning the short and long arms of
wheat group 1 chromosomes were used to map the
Elymus segments in chromosome z5A by Southern
hybridization analysis (Figure 2). The recovered recombinants,
derived tz5AL telosome, and iz5AL isochromosomes
allowed the assignment of wheat group
1 short-arm markers to specific Elymus segments
in chromosome z5A. None of the group 1 long-arm
markers hybridized to z5A, indicating that all the
Elymus segments in z5A were derived from the short
arm of chromosome 1Ht
. The presence of centromeric
marker BCD1072 in 1Ht
S and its absence in z5A
indicated that not all the short arm of 1Ht
is present
in chromosome z5A. Jiang and Gill (1993) similarly
reported that the short-arm marker PSR161, previously
mapped to 1Ht
S, was absent in chromosome z5A. Later
Figure 1.--GISH and FISH pattern on mitotic (A, B, E, F,
and G) and meiotic metaphase chromosomes (C and D) of
TA5536 (A, C, D, and E), disomic addition line DA1Ht
(B),
z5A recombinant chromosomes (rec1­rec3) (F), telosomic
chromosome tz5AL, isochromosome iz5AL, and translocation
chromosomes (T1­T3) (G). Total genomic DNA from E. trachycaulus
and the centromeric clone pAet6-J9 were labeled
with fluorescein-12-dUTP (except for the pAet6-J9 FISH pattern
on T2 in G) and visualized by yellow-green fluorescence.
Chromosomes were counterstained with propidium iodide
and fluoresced red. (A) The total genomic DNA from E. trachycaulus
generated a striped GISH pattern on a pair of z5A
chromosomes in TA5536. The four yellow-green chromosome
segments originated from chromosome 1Ht
of E. trachycaulus
and the four red segments, including the centromere, originated
from wheat chromosome 5A. (B) Clone pAet6-J9 hybridized
strongly to all 42 wheat centromeres in line DA1Ht
, but
very weakly to the centromeres of chromosomes 1Ht
(arrowheads).
(C and D) GISH on the meiotic metaphase I cells in
pollen mother cells from anthers of F1 plants of dDt5A/
TA5536 with the chromosome constitution 20$ 1 z5A9 1
5AS9 1 5AL9. Chromosome z5A was univalent (arrowhead)
in C and was paired in the distal region of the long arm with
the 5AL telosome (arrowhead) in the form of a rod bivalent in
D. (E) (Middle) Three FISH sites on chromosome z5A
probed with clone pAet6-J9; (left) The sequential GISH result
on the same chromosome; and (right) a merged image from
the first left and middle images. The chromosome, the GISH
signals, and the FISH signals were pseudocolored blue, green,
and magenta, respectively. The merged image shows that the
top FISH site mapped to the centromeric wheat segment
(W1), the second site mapped to the second wheat segment
(between W2 and W3), and the third site mapped to the distal
region of the last Elymus segment (E4) (arrowhead). (F)
GISH pattern on z5A chromosomes and three recombinant
chromosomes derived from either selfed or backcrossed derivatives
of dDt5A/TA5536. The chromosomes rec1, rec2,
and rec3 had the first three (E1, E2, E3), first two (E1, E2),
and the last Elymus segment(s) (E4), respectively. Rec3 is a
telosomic chromosome. (G) tz5AL and iz5AL show a telosomic
chromosome and an isochromosome, respectively; T1
shows a translocation chromosome with the short arm from
an unknown wheat chromosome and the long arm from
z5AL; T2 (GISH) shows a translocation chromosome with
three Elymus bands (one in the short arm and two in the long
arm). T2 (pAet6-J9) shows a sequential FISH pattern using
clone pAet6-J9 labeled with rhodamine-6-dUTP and visualized
by red fluorescence, identifying T2 as a small metacentric
chromosome, with the GISH band in the short arm derived
from the last Elymus segment of z5A and the two in the long
arm corresponding to the middle two Elymus segments of
z5A. T3 shows a translocation chromosome with five Elymus
bands, two in the short arm corresponding to the last two Elymus
segments of z5A, and the long arm of T3 is identical to the
long arm of z5A. Bars, 10 mm.
1172 P. Zhang et al.
it was shown that marker PSR161 mapped to the functional
centromere of wheat group 1 chromosomes
(Francki et al. 2002), indicating that the 1Ht
S chromatin
missing in z5A is closely associated with the
centromere.
Separation of the four E. trachycaulus segments in
chromosome z5A also allowed the determination of the
marker order in these segments. Southern hybridization
analysis revealed that the marker order in the Elymus
segments E1 and E4 was conserved relative to wheat,
whereas they were reversed in segments E2 and E3
(Figure 2). The reversed marker order in the latter two
segments was probably caused by a paracentric inversion
in the short arm of chromosome 1Ht
. The short-arm
marker BCD200 was mapped to both E3 and E4 E.
trachycaulus segments, suggesting that a duplication
event occurred during the origin of chromosome z5A.
DISCUSSION
Southern hybridization analysis revealed that the four
E. trachycaulus segments present in chromosome z5A
were derived from the short arm of 1Ht
. However, 1Ht
Sspecific
centromeric marker PSR161 and the adjacent
marker BCD1072 were missing in z5A, indicating that
not all of the 1Ht
S arm was included in the formation of
5A. The mapping data further indicated the presence of
a paracentric inversion in 1Ht
S.
Although Southern hybridization analysis did not
allow the assignment of 5AL markers to the wheat
segments in z5A, the structure of these segments can
be inferred from GISH and meiotic pairing analyses.
This study confirmed previous reports by showing that
the distal part of the short arm of chromosome z5A
paired with the distal region of the short arm of 1Ht
and
that the distal region of the long arm of z5A paired with
the distal region of wheat arm 5AL (Jiang and Gill
1993). In the presence of the major meiotic pairing
locus Ph1 in wheat, homology at chromosome ends
triggers the formation of chiasmate metaphase I associations
(Curtis et al. 1991; Jones et al. 2002; Qi et al.
2002). Thus, E1 was derived from the distal region of
1Ht
S, and W5 was derived from the distal region of 5AL.
GISH analysis showed that the functional centromere
was derived from wheat and was located within the W1
segment. The homology of wheat segments W3 and W4
was inferred from the recovered recombinants rec2,
rec1, and rec3. In the presence of Ph1, recombination
occurs normally only between homologous regions in
wheat. The recovery of these recombinants indicated
that the W3 and W4 segments in chromosome z5A are
not structurally rearranged but are homologous to
corresponding regions in chromosome 5AL.
During the formation of the zebra chromosome,
many changes occurred not only in chromosome 1Ht
of Elymus, but also in chromosome 5A of wheat. As seen
Figure 2.--Schematic summary of Southern
hybridization results and the molecular marker
positions on the z5A chromosome. (Top) Schematics
for telosomic chromosomes 1Ht
S and
1Ht
L, z5A, three recombinant chromosomes
(rec1­rec3), and isochromosome iz5AL. A
scaled-up z5A chromosome is shown on the right.
The Elymus short-arm chromatin is in black, the
Elymus long-arm chromatin is in gray, and wheat
chromatin is in white. The Elymus centromere is
shown in blue and the wheat centromere in red.
A schematic of the consensus map of wheat group
1 chromosome is on the left. Only tested markers
are listed. The ``1'' and ``'' indicate ``presence''
and ``absence'' of markers, respectively. The
marker order in Elymus segments E1 and E4 is
conserved relative to wheat, whereas the order
is reversed in segments E2 and E3. None of the
group 1 long-arm markers hybridized to z5A.
Origin of a ``Zebra'' Chromosome 1173
from our sequential FISH and GISH results, the original
centromere on wheat chromosome 5A was split into two
portions in z5A chromosome (Figure 1E, pAet6-J9 on
z5A; see also Figure 3). Although two of the group 5
long-arm markers, including Q (this study and Jiang
and Gill 1993) and H1 (Jiang and Gill 1993), also are
present in the z5A chromosome, some wheat markers in
chromosome 5AL were deleted in z5A.
In addition to the recombinants, telosome, and
isochromosomes, three different types of translocation
chromosomes were recovered. The overall high translocation
frequency (3 of 110) involving the z5A chromosome
indicated that this chromosome is prone to
breakage and translocation.
Jiang and Gill (1993) postulated two possible
origins for the zebra chromosome. One was that it arose
by illegitimate recombination between nonhomologous
chromosome arms 1Ht
S of E. trachycaulus and 5AL of
wheat. In this scenario, the marker order of the four
Elymus and five wheat segments is expected to be linear
from one telomere to the other on chromosome z5A.
The second possibility was that chromosome z5A arose
from multiple breakage and fusion translocation events,
and, in this case, the marker order is expected to be
random. With the exception of Elymus segments E2 and
E3, where the marker orders are reversed relative to
wheat--most likely resulting from a paracentric inversion
in 1Ht
S--the marker orders or homologies of the
remaining Elymus and wheat segments are linear from
one telomere to the other. Such an order would be
highly unlikely if chromosome z5A originated by multiple
translocation events.
On the basis of our data, a possible mode of origin of
chromosome z5A is shown in Figure 3. The alloplasmic
wheat plant was double monosomic for 5A and 1Ht
S and
the two nonhomologous chromosomes may have prealigned
prior to S-phase of meiosis or mitosis in germinal
tissues during transition from the vegetative to reproductive
phase. Chromosome z5A might have originated
from nonhomologous recombination during the DNA
double-strand break (DSB) repair process (Gorbunova
and Levy 1999; Pacher et al. 2007) during the S-phase.
Initially, at least five breaks occurred in chromosome 5A,
resulting in six chromatin blocks. Similarly, four breaks
in the 1Ht
S telosome split it into five chromatin blocks.
Rejoining of the prealigned 5AL and 1Ht
S blocks in
succession resulted in the formation of chromosome
z5A. It was accompanied by the complete loss of 5AS,
including the telomere and the loss of a very small distal
centromeric end of 1Ht
S. The formation of chromosome
z5A was not the result of a reciprocal segment
exchange.
Amazingly, the z5A has the centromere of 5A of
wheat embedded in E1 and E2 segments from 1Ht
S of
E. trachycaulus. Its short arm, including the telomere,
was derived from E. trachycaulus and its long arm is a
chimera of 5AL and 1Ht
S segments with the distal part
and the telomere derived from 5AL. This represents an
aneuploid change in chromosome number from two to
one and the beginning of the evolutionary history of a
new z5A chromosome.
The aneuploid changes in chromosome number and/
or the origin of structurally rearranged chromosomes
may be associated with interspecific hybridization. The
F1 hybrids are usually sterile and one component of
Figure 3.--Proposed origin of chromosome z5A by chromosome
breakage and fusion of the broken chromatin
blocks. (Left) Wheat chromosome 5A, (middle) z5A, and
(right) telosome 1Ht
S, from which the four Elymus segments
present in z5A were derived. The centromeres of wheat and
Elymus are shown in red and blue, respectively. The original
centromere of wheat chromosome 5A was split into two portions
in z5A. The major part is the functional centromere, located
within the W1 segment, and the smaller part is located
in W2. The third and smallest centromere was derived from
part of the centromere of chromosome 1Ht
(E4). Marker
analysis indicated that the entire short arm of 1Ht
was not present
in z5A. GISH and meiotic pairing analyses showed that
the E1 segment was derived from the distal region of 1Ht
S
and W4 was derived from the distal region of 5AL. Because
the marker order in segments E1 and E4 is conserved relative
to wheat, but reversed in segments E2 and E3, either a paracentric
inversion in the short arm preexisted in 1Ht
or fusion
of the broken chromatin blocks occurred, as indicated.
1174 P. Zhang et al.
sterility is due to adverse nucleo-cytoplasmic interactions
(NCI) (Gill 1991; Maan 1991). During the process of
evolutionary introgressive hybridization in subsequent
generations, certain chromosomes that overcome adverse
NCI and restore fertility are exclusively transmitted
to the progeny and are often involved in complex
chromosomal rearrangements (Gill 1991). In alloplasmic
hybrid derivatives between T. aestivum and E. trachycaulus,
all progeny plants in backcrosses to wheat carried
either chromosome 1Ht
or 1St
or their short arms as
telocentrics or in complex rearrangements with wheat
chromosomes (Jiang and Gill 1993). The z5A plant was
recovered in one such progeny. It was fully fertile and
flowered 10 days earlier than the Chinese Spring wheat,
the recurrent parent. In nature, the z5A plant will be a
candidate for a founder population for the evolution of a
new species.
The two genomes in alloplasmic hybrids between T.
aestivum and E. trachycaulus exist in a common nucleus
in an Elymus cytoplasm. Because of the incompatibility
and genomic stress imposed by interspecific hybridization,
certain intergenomic and/or chromosomal structural
rearrangements might have occurred (Gill 1991),
similar to those reported by Naranjo et al. (1987) and
Jiang and Gill (1994). Nonhomologous recombination
might be initiated to repair chromosomal DSBs by
joining sequences with little or no homology. If chromosomes
1Ht
and 5A were in close proximity at the time
of DSB repair, DNA ends or other chromosome breaks
on 1Ht
and 5A might have been joined by nonhomologous
end joining. Alternatively, because there were
free DNA ends from chromosomal DSBs, these free
ends might have enhanced random integration by the
copy-join process of nonhomologous recombination to
link new DNA sequences together (Merrihew et al.
1996). In our case, the sequences from the Elymus 1Ht
chromosome were linked to those from wheat chromosome
5A because these two chromosomes were by
chance physically close at the time.
The molecular and cytogenetic research has documented
extensive chromosome changes, including
inversions, translocations, and fusions/fissions leading
to aneuploid changes and karyotype repatterning in the
Brassica family (reviewed in Schubert 2007). It was
suggested that aneuploid changes as well as extensive
chromosome repatterning--including inversions, unequal
translocations involving nonhomologous chromosomes,
and elimination of minichromosomes--led
to step changes in chromosome number from n  8 in
Arabidopsis lyrata to n  5 in A. thaliana (Lysak et al.
2006). The experimental data on the origin of z5A
allude to additional mechanisms. Results from our study
indicated that the zebra chromosome arose by illegitimate
recombination between nonhomologous chromosomes
of T. aestivum and E. trachycaulus, resulting in a
mosaic chromosome comprised of alternate blocks of T.
aestivum and E. trachycaulus chromatin. Although our
Figure 4.--Hypothetical scheme leading to changes
in chromosome number after interspecific hybridization,
whole-genome duplication, and chromosomal rearrangements.
Species A and B with n  2 chromosomes have differentiated
cytoplasms (indicated by open and shaded outer
circles) and nuclear genomes. Species A has a fertility factor
F1 on chromosome A1 that is essential for restoring fertility to
species-A-specific cytoplasm and species B has another fertility
factor, F2 on chromosome B2, that is essential for gametophytic
or sporophytic viability. Interspecific hybridization between
species A and B followed by whole-genome duplication
created genomic stress that resulted in chromosomal repatterning
and the formation of the novel chromosome Z3 by
nonhomologous recombination, harboring both fertility factors
F1 and F2. Plants with such a chromosomal constitution
have strong selective advantage and will form species D with
n  3 chromosomes.
Origin of a ``Zebra'' Chromosome 1175
findings involved a polyploid species, it is possible that
similar mechanisms might have been involved in diploid
species.
A hypothetical scheme showing possible evolutionary
chromosomal changes during speciation is shown in
Figure 4. We assume species A and species B with n  2
co-evolved from a common ancestor and have differentiated
cytoplasm and nuclear genomes. Species A has
fertility factor F1 on A1 chromosome that is essential for
restoring fertility to species-A-specific cytoplasm. Species
B has another fertility factor, F2 on B2 chromosome, that
is essential for the viability of the gametophyte or the
sporophyte. Next, species A and B come in contact and
undergo hybridization and a whole-genome duplication
to form species C with n  4 chromosomes. The wholegenome
duplication is a polyploidy-associated mechanism
that fixes hybrid vigor. However, hybridization is
also mutagenic and may trigger chromosomal repatterning
events. Genome duplication provides buffering for
the formation of new chromosomes that otherwise may
be deleterious because they involve segmental deletions
and duplications. Most such events will be lethal;
however, those that bring genetic factors F1 and F2 onto
the novel chromosome Z3 will have strong selective
advantage and may be fixed to form species D with n  3
chromosomes. In our experimental hybrids, the z5A
chromosome in the alloplasmic z5A plant had the Rf
gene from E. trachycaulus that restored fertility. The
group 5 long-arm chromosomes in the Triticeae are
known to carry genes essential for the viability of the
gametophyte (Endo and Gill 1996) and alien group 5
chromosomes show exclusive preferential transmission
(Jiang and Gill 1998). This gene is the hypothetical
gene F2 and because the z5A carried these two essential
genes, the hybrid derivatives carrying z5A will be fertile
and this chromosome will be fixed in the population.
We thank Robert A. McIntosh and W. John Raupp for critical
reading of the manuscript, Duane Wilson for excellent assistance, Li
Huang, Lili Qi, Chongmei Dong, Scott Jackson, and Jiming Jiang for
beneficial discussions. This research was supported by the Kansas
Wheat Commission and a special U. S. Department of Agriculture
grant to the Wheat Genetic and Genomic Resources Center. This
article is contribution no. 08-323-J from the Kansas Agricultural
Experimental Station, Kansas State University, Manhattan, Kansas
66506-5502.
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Communicating editor: J. A. Birchler
Origin of a ``Zebra'' Chromosome 1177