Production of hexaploid triticale by a synthetic hexaploid wheat-rye hybrid method

Abstract

Hexaploid triticale, including its primary and secondary forms, is an important forage crop and a promising energy plant. Primary forms are usually developed by crossing Triticum turgidum L. with rye, with secondary forms obtained by crossing primary hexaploid triticale and/or hexaploid wheat with octoploid triticale. In this study, we developed an effective method for production of hexaploid triticale via hybridization of synthetic hexaploid wheat (SHW) with rye. The three employed SHW lines were derived from hybridization of T. turgidum with Aegilops tauschii Cosson, and inherited meiotic restitution genes, which can promote the formation of functional gametes in haploid status, from their T. turgidum parents. Although the resulting tetraploid F1 hybrids with rye (genome ABDR) produced amphiploids (octoploid triticale) and partial amphiploids, the final hybrid products obtained through fertility selection over several generations were hexaploids. These hexaploids were the result of preferential elimination of D-genome chromosomes. In addition to complete hexaploid triticale with 28 intact A/B and 14 intact R chromosomes, we obtained hexaploid triticales with other chromosome constitutions, including monosomic, substitution, and translocation lines. Chromosomes 2D and 5D from the wild species A. tauschii were incorporated into the hexaploid triticales. Out of eight analyzed stable lines derived from three different SHW-L1/rye F1 plants, we observed four lines with small-fragment translocations between wheat and rye chromosomes. Rapid production of hexaploid triticales using this method involves two factors: (1) hybridization between hexaploid wheat with a meiotic restitution gene(s) and rye and (2) selection for good fertility during F3 and subsequent generations.

Full text

Production of hexaploid triticale by a synthetic hexaploid
wheat-rye hybrid method
Ming Hao •Jiangtao Luo •Lianquan Zhang •Zhongwei Yuan •
Youwei Yang •Meng Wu •Wenjie Chen •Youliang Zheng •
Huaigang Zhang •Dengcai Liu
Received: 27 January 2013 / Accepted: 20 April 2013 / Published online: 30 April 2013
ÓSpringer Science+Business Media Dordrecht 2013
Abstract Hexaploid triticale, including its primary
and secondary forms, is an important forage crop and a
promising energy plant. Primary forms are usually
developed by crossing Triticum turgidum L. with rye,
with secondary forms obtained by crossing primary
hexaploid triticale and/or hexaploid wheat with octo-
ploid triticale. In this study, we developed an effective
method for production of hexaploid triticale via
hybridization of synthetic hexaploid wheat (SHW)
with rye. The three employed SHW lines were derived
from hybridization of T. turgidum with Aegilops
tauschii Cosson, and inherited meiotic restitution
genes, which can promote the formation of functional
gametes in haploid status, from their T. turgidum
parents. Although the resulting tetraploid F
1
hybrids
with rye (genome ABDR) produced amphiploids
(octoploid triticale) and partial amphiploids, the final
hybrid products obtained through fertility selection
over several generations were hexaploids. These
hexaploids were the result of preferential elimination
of D-genome chromosomes. In addition to complete
hexaploid triticale with 28 intact A/B and 14 intact R
chromosomes, we obtained hexaploid triticales with
other chromosome constitutions, including monoso-
mic, substitution, and translocation lines. Chromo-
somes 2D and 5D from the wild species A. tauschii
were incorporated into the hexaploid triticales. Out of
eight analyzed stable lines derived from three different
SHW-L1/rye F
1
plants, we observed four lines with
small-fragment translocations between wheat and
rye chromosomes. Rapid production of hexaploid
Ming Hao and Jiangtao Luo contributed equally to this work
M. Hao J. Luo L. Zhang Z. Yuan 
Y. Yang M. Wu Y. Zheng D. Liu
Triticeae Research Institute, Sichuan Agricultural
University at Chengdu, Wenjiang 611130, Sichuan,
People’s Republic of China
Present Address:
Y. Yang
College of Life Science, Hunan University of Arts
and Science, Changde 415000, Hunan,
People’s Republic of China
Present Address:
M. Wu
Department of Botany, Miami University, 316 Pearson
Hall, Oxford, OH 45056, USA
W. Chen H. Zhang D. Liu (&)
Key Laboratory of Adaptation and Evolution of Plateau
Biota, Northwest Institute of Plateau Biology, Chinese
Academy of Sciences, Xining 810001,
People’s Republic of China
e-mail: dcliu7@yahoo.com
123
Euphytica (2013) 193:347–357
DOI 10.1007/s10681-013-0930-2

triticales using this method involves two factors: (1)
hybridization between hexaploid wheat with a meiotic
restitution gene(s) and rye and (2) selection for good
fertility during F
3
and subsequent generations.
Keywords Chromosome elimination Meiotic
restitution Translocation Triticale
Introduction
Triticale (9Triticosecale Wittmack) is the first man-
made crop derived from a cross of wheat (Triticum) and
rye (Secale). It combines the high yield potential and
grain quality of wheat with the growth vigor and
tolerance to non-optimal growing environments pos-
sessed by rye (Varughese et al. 1997; Larter 2009). In
2009, 15.0 million tons of triticale grains were
harvested from 29 countries (FAOSTAT Database
2010). Triticale is mainly used for forage or fodder,
serving as a good source of protein, lysine, B vitamins,
and readily digested starch; it is also used on a smaller
scale as an ingredient in human food and in the brewing
industry (Bird et al. 1999; McKevith 2004; Glatthar
et al. 2005; Mcgoverin et al. 2011; Rakha et al. 2011).
This hardy crop can be grown in marginal areas with
poor soils and unsuitable climates, producing high
yields where other cereal crops, such as wheat, perform
unsatisfactorily (Griffith and Lang 2004). It thus has
potential in fighting world hunger (Hansen 2010).
Triticale has also recently been recognized as a
promising energy crop because of its high biomass
and grain yield (Oettler 2005; Davis-Knight and
Weightman 2008; Bilgili et al. 2009; Bassu et al.
2011; Gowda et al. 2011; Estrada-Campuzano et al.
2012). In 2005, the Canadian Triticale Biorefinery
Initiative (CTBI) was established to develop triticale as
a dedicated industrial biofuel feedstock crop.
The first triticales to be developed were octoploids
(2n=8x=56, AABBDDRR) generated by chromo-
some doubling of hybrids between common wheat
(Triticum aestivum L.) and rye. Following the intro-
duction of successful embryo culture techniques in the
late 1940s, hexaploid triticales (2n=6x=42,
AABBRR) were developed by crossing tetraploid
wheat (T. turgidum L.) with rye. These primary
hexaploid triticales are amphiploids containing all 28
wheat and 14 rye chromosomes. Because of their
greater meiotic stability and fertility, hexaploid
triticales are more successful crop plants than octoploid
triticales (Lukaszewski and Gustafson 1987; Fox et al.
1990; Cheng and Murata 2002). Secondary hexaploids
are primarily derived from crosses of primary hexa-
ploid triticale and/or hexaploid wheat with octoploid
triticale. In addition, hexaploid triticales can spontane-
ously appear in the selfing progenies of octoploid
triticale (Nakata et al. 1984; Dou et al. 2006; Zhou et al.
2012). The majority of triticale grown worldwide today
is secondary hexaploid triticale because of its desirable
agronomic traits (Fox et al. 1990).
Aegilops tauschii Cosson (2n=2x=14, DD) is the
source of the D-genome of common wheat. Although
this wild species has been not used in triticale develop-
ment, its D-genome may accelerate environmental
adaptation (Gororo et al. 2001; Trethowan and Mu-
jeeb-Kazi 2008) and enhance early plant vigor (Ter
Steege et al. 2005). In addition, genetic variability within
the D-genome of A. tauschii is much higher than within
the D-genome of common wheat (Van Ginkel and
Ogbonnaya 2007;Yangetal.2009). The A. tauschii
D-genome can be quickly incorporated into synthetic
hexaploid wheat (SHW) using spontaneous chromo-
some doubling in haploid hybrids of T. turgidum–A.
tauschii via unreduced gametes resulting from meiotic
restitution (Zhang et al. 2010). Meiotic restitution also
occurs in SHW-rye F
1
hybrids and results in the
production of amphiploids or partial amphiploids (Zhang
et al. 2007). While these observations suggest that SHW-
rye hybridization may be useful for triticale develop-
ment, its effectiveness needs to be further evaluated.
In this study, we identified the chromosome con-
stitutions of some highly fertile derivatives of three
SHW-rye hybrid combinations. These derivatives
were usually found to be hexaploid triticales, and
included monosomic, substitution, and translocation
forms. The results of our study demonstrate that using
meiotic restitution in hexaploid wheat-rye hybridiza-
tion provides a simple method for hexaploid triticale
development via the preferential elimination of
D-genome chromosomes in the hybrid derivatives.
Materials and methods
Plant materials
Three synthetic hexaploid wheats, SHW-L1, Syn-
SAU-8, and Syn-SAU-18, were used as female parents
348 Euphytica (2013) 193:347–357
123

in crosses with Chinese rye (Secale cereale L.,
2n=2x=14, RR) landrace Qinling (AS156). SHW-
L1 was derived from a cross of T. turgidum ssp.
turgidum line AS2255 with A. tauschii accession AS60,
and inherited a gene(s) for meiotic restitution from
AS2255 (Zhang et al. 2007). Syn-SAU-8 and Syn-
SAU-18 were produced by spontaneous chromosome
doubling via unreduced gametes resulting from meiotic
restitution in T. turgidum ssp. durum ‘Langdon’ 9A.
tauschii AS2386 and T. turgidum ssp. turgidum
AS2236-1 9A. tauschii AS77 hybrids (Zhang et al.
2010). These hexaploids also inherited the gene(s) for
meiotic restitution from their T. turgidum parents
Langdon and AS2236-1 (Zhang et al. 2010).
Emasculation and pollination were carried out as
described by Liu et al. (1999). No embryo rescue or
hormone treatment was applied for the production of
F
1
seeds. F
2
and F
3
hybrid seeds were obtained by
selfing individual F
1
or F
2
plants, respectively. F
1
–F
3
hybrid seeds were germinated in petri dishes on filter
paper, and the seedlings were transplanted into an
experimental field using within-row spacing of 10 cm,
with 30 cm between rows. F
3
plants having relatively
good seed set of over 50 seeds were selected and then
advanced to the F
4
generation. F
4
plants with high
fertility, having at least 100 seeds, were advanced to
the F
5
generation.
The crossability of the two species in each hybrid
cross was calculated as the percentage of F
1
seeds
obtained relative to the number of florets pollinated for
that cross. The selfed seed set was calculated as the
percentage of total seeds obtained relative to the total
number of spikelets.
Cytological observations
Cytological observation of chromosome numbers in
root-tip cells and chromosome pairing in pollen
mother cells (PMC) in hybrid plants was according
to procedures described previously (Zhang et al.
2007). For meiotic analysis, at least 20 PMCs were
observed for each plant. Univalents (I), bivalents (II),
trivalents (III), and quadrivalents (IV) were counted
and their average numbers calculated.
For genomic in situ hybridization (GISH), tips of
fixed roots were squashed in a drop of 45 % acetic
acid. After freezing with liquid nitrogen, the squashed
slides were uncovered, air-dried, and stored at -20 °C
until use. GISH was performed as described
previously (Hao et al. 2011). The GISH hybridization
mixture included 100 % formamide, 209SSC, probe
DNA, sheared genomic DNA of common wheat
cultivar Chinese Spring (as a blocker), sheared salmon
sperm DNA, and 5 % dextran sulfate.
Clones pAs1 (Nagaki et al. 1995), pSc119.2
(Contento et al. 2005), and pTa71 (Fujisawa et al.
2006) were used as probes for fluorescence in situ
hybridization (FISH). pAs1 is a D-genome-specific
clone. Although weak hybridization signals are
observed on some B and A-genome chromosomes,
the FISH pattern obtained with this clone permits
identification of the D-genome chromosomes (Ray-
burn and Gill 1986; Pedersen and Langridge 1997).
pSc119.2 was used to identify B and R-genome
chromosomes, and pTa71 was used to identify satel-
lites. pAs1 and pSc119.2 were labeled with biotin-16-
dUTP (Roche Diagnostics Gmbh, Mannheim, Ger-
many REF 11745816910) or digoxigenin-11-dUTP
(Roche Diagnostics Gmbh, Mannheim, Germany,
REF 11745816910) according to the manufacturer’s
instructions. pTa71 was labeled with 50 % biotin-16-
dUTP and 50 % digoxigenin-11-dUTP (Roche). Total
genomic DNA of the rye cultivar Qinling was labeled
with digoxigenin-11-dUTP (Roche) or biotin-16-
dUTP (Roche). Unlabeled genomic DNA of the
common wheat cultivar Chinese Spring was used as
blocking DNA. Detection of the biotinylated probe
was accomplished with streptavidin-Cy3 (SIGMA-
ALDRICH CHEMIE Gmbh, steinheim, Germany,
Pcod 1001238444) and digoxigenin using anti-digox-
igenin-fluorescein (Roche Diagnostics Gmbh, Mann-
heim, Germany, REF 11207741910). The preparations
for in situ hybridization were counterstained for
analysis with 40,6-diamidino-2-phenylindole (DAPI)
(Vector Laboratories, Inc., Burlingame, USA) or
propidium iodide (PI) (Vector Laboratories, Inc.,
Burlingame, USA). Chromosome observations were
made and documented using an Olympus BX-51
microscope coupled to a Photometric SenSys Olym-
pus DP70 CCD camera. Raw images were processed
using Photoshop ver. 7.1 (Adobe Systems Incorpo-
rated, San Jose, CA, USA).
Field evaluations
Field trial evaluations were conducted during the
2011–2012 growing season at the Triticeae Research
Institute Experimental Station in Wenjiang, Sichuan
Euphytica (2013) 193:347–357 349
123

Province, China. Individual plants were spaced 10 cm
apart within 2-m-long rows, with row spacings of
30 cm. For new triticale lines derived from SHW-L1/
rye hybrids, experiments consisted of two replicates.
At maturity, plant height, tiller number per plant, spike
length, spikelet number, and 1000-grain weight were
evaluated from 10 plants randomly selected from each
plot. Plant height was calculated as height in centi-
meters measured from the soil surface to the tip of the
spike (awns excluded). The main spikes of the selected
plants were measured for spike length, spikelet
number, and 1000-grain weight. The average value
for each trait was then calculated.
Results
SHW-L1/rye, Syn-SAU-8/rye, and Syn-SAU-18/rye
intergeneric hybrid combinations had high crossabil-
ities, i.e., 32.6 % (140/430), 22.6 % (19/84), and
53.9 % (55/102), respectively. The 19 analyzed F
1
plants (10 from SHW-L1/rye, 2 from Syn-SAU-8/rye,
and 7 from Syn-SAU-18/rye) all grew vigorously and
were partially fertile. They produced a total of 112 F
2
seeds (6 seeds/plant) by selfing. F
2
plants showed a
large range of variation with respect to seed set by
selfing: of 18 plants investigated, 10 did not set F
3
seeds, while the remaining eight produced a total of
245 seeds. F
3
plant seed set also varied greatly,
ranging from 0 to 510 seeds/plant. Vigorous F
3
plants
with good seed-setting ability, derived from eight F
1
plants, were advanced to the next generation and used
for further investigation.
SHW-L1/rye hybrid combinations
One F
5
and seven F
7
lines derived from SHW-L1/rye
F
1
hybrid plants 10, 56, 186, 188, and 190 were
analyzed. The plants grew vigorously and had a high
level of seed set (Table 1). GISH and FISH were
further used to identify their chromosome constitu-
tions. By using PTa71, Psc119.2 and PAs1 as probes,
we can identify the chromosomes of wheat parent
SHW-L1 (Fig. 1). The two F
7
lines 186-1-4-18-8-8-27
(Fig. 2a) and 186-3-5-26-14-14-31 had the same
chromosome constitution (28A/B and 14R chromo-
somes) as the primary hexaploid triticale between T.
turgidum and rye. The F
7
line 190-2-3-42-23-33-39
was 6B-monosomic (Fig. 2b). Interestingly, five of the
Table 1 Chromosomal constitutions and agronomic traits of triticale lines from SHW-L1/rye hybrids
Line Chromosome constitutions
a
No. of
tillers
Plant
height
(cM)
Spikelet
length
(cM)
No. of
spikelets
on main
spike
1000-grain
weight (g)
Seed set
b
(seeds/spikelet)
186-1-4-18-8-8-27 42 (28AB ?14R) 9.3 139.7 14.3 31.3 26.8 1.1 (233.7/222.3)
186-3-5-26-14-14-31 42 (28AB ?14R) 6.6 157 13.3 34.8 27.5 1.4 (274.6/192.6)
186-4-2-31-17-17-33 42 (24AB ?14R ?four T) 8.3 139.4 16.3 33.8 23.4 1.1 (224.3/222)
188-2-6-37-20-20-35 42 (26AB ?two 2D ?12R ?two T) 5.6 148.4 12.7 27 23 1.4 (204.1/144.5)
190-2-3-42-23-33-39 42 or 41 (27AB with one 6B ?14R) 6.1 167 13.6 35.6 28.6 1.5 (279.7/196.1)
10-1-1-57-28-28-43 41 (28AB ?13R) or (28AB ?12R ?one T) 7.8 159 17.4 37.8 27.8 1.6 (387.5/242.4)
10-1-1-57-28-29-45 42 (28AB ?12R ?two T) 5.3 168. 9 15.4 38.7 38.5 1.6 (257.6/171.4)
56-5-1-31-49 42 (28AB ?12R ?two T) 3.8 171.7 14.2 36.4 40.2 1.7 (188.7/108.9)
a
AB A and B chromosomes, Ttranslocation
b
Seed set was calculated as the ratio of the total number of seeds obtained relative to the total number of spikelets per plant
350 Euphytica (2013) 193:347–357
123

eight lines carried translocations. Lines 10-1-1-57-28-
29-45 (Fig. 2c) and 56-5-1-31-49 had a pair of
terminal translocations involving large rye and small
wheat fragments (Fig. 2d) in addition to possessing
28A/B and 12R chromosomes. The translocations in
the two lines were of different origins, as they were
derived from two different F
1
plants. The PMCs of
lines 10-1-1-57-28-29-45 and 56-5-1-31-49 showed
normal meiotic behavior, possessing approximately
21 bivalents, with chromosome pairing configurations
of 0.71 I ?6.18 rod II ?14.47 ring II and 1.00
I?7.47 rod II ?12.78 ring II ?0.13 III ?0.03 IV,
respectively. Line 186-4-2-31-17-17-33 had 24A/B
and 14R chromosomes and two pairs of centric
translocations (Fig. 2e), i.e., 6BS-?RS/L and ?RS/
L-6BL (Fig. 2f). A pair of 2D chromosomes and a pair
of wheat-rye terminal translocations were identified in
line 188-2-6-37-20-20-35 (Fig. 2g). We observed two
cytotypes in F
7
line 10-1-1-57-28-28-43. One con-
sisted of 28A/B and 13R chromosomes; the other
comprised 28A/B and 12R chromosomes and one
translocation chromosome (Table 1; Fig. 2h), indicat-
ing that this line was still cytologically unstable.
Syn-SAU-8/rye hybrid combinations
All material used for cytological observation of Syn-
SAU-8/rye hybrid combinations was derived from F
2
plant 228(1)-1, which produced 51 F
3
seeds. Three F
4
plants were randomly analyzed. One of them, 228(1)-
1-20-3, was a hexaploid triticale with 28A/B and 14R
chromosomes (Table 2). Plant 228(1)-1-32-18 had
26A/B chromosomes and 15 rye chromosomes,
including 3 1R chromosomes and 1 rye chromosome
fragment (Table 2; Fig. 3a). Its F
5
seed 228(1)-1-32-
18-3 possessed all the A/B chromosomes except for
1B and had 16 rye chromosomes including 4 1R
chromosomes (Fig. 3b). Loss of D-genome chromo-
somes was also observed in six other analyzed F
5
seeds, as they showed no FISH signal specific for the
D-genome. Of these six, plants 228(1)-1-32-4-2
(Fig. 3c), 228(1)-1-32-19-2, 228(1)-1-32-25-2, and
228(1)-1-32-26-1 were all hexaploid triticales. Plant
228(1)-1-32-4-1 was an aneuploid hexaploid with a
monosomic 1B (Fig. 3d). Plant 228(1)-1-32-1-2 had
28A/B and 13R chromosomes plus 3 rye chromosome
fragments (Table 2).
Syn-SAU-18/rye hybrid combinations
Two fertile F
1
plants, 233(1) and 233(2), produced a
total of 22 F
2
seeds. The F
2
plants 233(1)-2 and
233(1)-6 produced 21 and 75 F
3
seeds, respectively.
Three F
3
seeds 233(1)-6-2 (Fig. 4a), 233(1)-2-20, and
233(1)-6-12 were analyzed; they all had 14 intact rye
chromosomes (Table 2). The seed 233(1)-2-20 also
had one wheat-rye translocation chromosome and one
rye chromosome fragment (Fig. 4b). The translocation
chromosome formed a pair in the F
4
seed 233(1)-2-20-
9 (Table 2). A pair of 5D chromosomes in F
4
seed
233(1)-2-20-4 (Fig. 4c) were inherited by its F
5
hybrid
233(1)-2-20-4-2, which had 26 A/B and 14 R chromo-
somes plus a pair of 5D chromosomes (Fig. 4d).
Discussion
Formation of functional gametes in F
1
haploid
hybrids of synthetic hexaploid wheat and rye
Because they often produce non-functional reduced
gametes, wheat-rye F
1
haploid hybrids are usually
sterile or have very low fertility. In an earlier study
(Zhang et al. 2007), we observed partially fertile
tetraploid F
1
hybrids (genome ABDR) when synthetic
hexaploid wheat line SHW-L1 was crossed with rye,
and hypothesized that production of functional
Fig. 1 Fluorescent in situ hybridization (FISH) using PAs1
(red), PSc119.2 (green), and PTa71 (yellow) as probes on
mitotic chromosomes of wheat parent SHW-L1
Euphytica (2013) 193:347–357 351
123

gametes was induced by a meiotic restitution
gene(s) from SHW-L1. This observation was further
confirmed in the present study by the generation of
partially fertile Syn-SAU-8/rye and Syn-SAU-18/rye
F
1
hybrids. Both Syn-SAU-8/rye and Syn-SAU-18/rye
have inherited meiotic restitution genes from their T.
turgidum parents (Zhang et al. 2010). We therefore
infer that the production of functional gametes in the
present study was also promoted by meiotic restitution
genes. When 10 SHW-L1/rye F
2
plants were randomly
selected in the earlier study (Zhang et al. 2007), three
were found to be amphiploids (octoploid, 2n=56),
with the remaining plants determined to be partial
amphiploids. This observation indicates that a large
fraction of the F
2
seeds produced were derived from
aneuploid gametes. A high ratio of functional aneu-
ploid gametes to euploid gametes has also been
reported in other haploid hybrids possessing meiotic
restitution genes, including T. turgidum–A. tauschii
(Zhang et al. 2008) and synthetic hexaploid wheat
SHW-L1-A. variabilis with genome ABDUS
l
(Yang
et al. 2010). It is thus obvious that meiotic restitution
can result in aneuploid functional gametes in addition
to euploid gametes.
Production of hexaploid triticale by preferential
elimination of D-genome chromosomes
The original goal of this study was incorporation of
the D-genome of the wild species A. tauschii into
octoploid triticale (2n=8x=56, AABBDDRR)
using synthetic hexaploid wheat, which has a
meiotic restitution gene(s). When hybrid plants with
high fertility were analyzed, however, no octoploid
Fig. 2 Chromosome constitutions of SHW-L1/rye lines. In a,c,
d,e, and g, chromosome constitutions were analyzed using
R-genome (green), pTa71 (yellow), and pAs1 (red) probes, with
unlabeled wheat genome DNA used as a blocker. aLine 186-1-
4-18-8-8-27 had 28A/B and 14R chromosomes. bLine 190-2-3-
42-23-33-39 had 27A/B and 14R chromosomes. It was first
determined to have 27A/B and 14R chromosomes using
R-genome, pTa71, and pAs1 probes. The slide was then re-
analyzed with a pSc119.2 (green) probe, which revealed the
absence of one 6B chromosome (arrow). cLine 10-1-1-57-28-
29-45 had 28A/B and 12R chromosomes and a pair of wheat-rye
translocation chromosomes (arrows). dLine 56-5-1-31-49 had
28A/B and 12R chromosomes and a pair of wheat-rye
chromosomes (arrows). eLine 186-4-2-31-17-17-33 had 24A/
B and 14 rye chromosomes and two pairs of centric wheat-rye
chromosomes (arrows). fThe two pairs of translocations in line
186-4-2-31-17-17-33 were reciprocal translocations between
6B and 2R or 3R based on analyses using pSc119.2 (green),
pTa71 (yellow), and R-genome (red) probes, with unlabeled
chinese spring genome DNA used as a blocker. gLine 188-2-6-
37-20-20-35 exhibited 26A/B and 12R chromosomes, a pair of
wheat-rye translocations (arrows), and a pair of 2D chromo-
somes (arrows). hLine 10-1-1-57-28-28-43 had 28A/B and 12R
chromosomes and one translocation (arrow). It was first
analyzed with R-genome (green), pTa71 (yellow), and pAs1
(red) probes, and then re-analyzed with rye (green), pTa71
(yellow), and pSc119.2 (red) probes. The analysis, however, was
unable to identify which chromosomes were involved in
translocations
352 Euphytica (2013) 193:347–357
123

triticales were found; we instead obtained hexaploid
triticales. Although we cannot exclude the possibility
that some octoploid F
2
plants were formed, the
typically low fertility of any such plants must have
led to their elimination through fertility selection
during production of the next generation (Lukaszew-
ski and Gustafson 1987). Octoploid triticale is
known to be unstable with respect to meiotic and
mitotic processes, resulting in elimination of chro-
mosomes from its progenies (Weimarck 1974;
Lukaszewski and Gustafson 1987; Fu et al. 2010;
Kalinka et al. 2010; Tang et al. 2012). Such
chromosome elimination in octoploid triticale may
result in hexaploid triticale (Nakata et al. 1984;
Sasaki et al. 1985; Dou et al. 2006; Zhou et al.
2012). When we selected for high fertility, most of
the products we obtained from hybrids between
synthetic hexaploid wheat and rye were hexaploids.
High fertility depends on cytologically stable condi-
tions that may be achieved through chromosome
elimination. Two chromosome elimination mecha-
nisms have recently been reported in octoploid
triticale. Kalinka et al. (2010) observed direct
chromosome elimination in a cytomixis-like fashion
from PMCs in newly synthesized octoploid triticales.
Tang et al. (2012) detected unequal chromosome
division in somatic cells and proposed that it may be
a chromosome elimination pathway in octoploid
triticales. In our study, we observed chromosome
fragments without centromeres in some somatic cells
of hybrid derivatives (Table 2). Centromere loss
may thus be another mechanism for chromosome
elimination, as centromere-less chromosomes cannot
be passed along to future generations.
The FISH pattern due to pAs1-clone repeated
sequences permits identification of D-genome chro-
mosomes (Nagaki et al. 1995; Rayburn and Gill 1986;
Pedersen and Langridge 1997; Dou et al. 2006). Our
analysis using pAs1 as a probe indicated that most of
the analyzed plants or lines contained chromosomes
from A, B, and R genomes, with D-genome chromo-
somes being preferentially eliminated from hybrid
progenies between synthetic hexaploid wheat and rye
(Tables 1,2). Nakata et al. (1984), Dou et al. (2006),
and Zhou et al. (2012) observed a similar phenome-
non. When they analyzed hexaploid lines spontane-
ously derived from progenies of some primary
octoploid triticales, they found that while all of them
retained complete sets of A and B-genome chromo-
somes and most of the R-genome set, most of the
D-genome chromosomes were eliminated. These
studies all suggest that with respect to existence in
Table 2 Chromosome
constitutions of partial
Syn-SAU-8/rye and
Syn-SAU-18/rye hybrid
plants
a
?unknown, Wwheat; AB
A and B chromosomes, RF
rye chromosome fragments,
WF wheat chromosome
fragment, w-r wheat-rye
translocation
Plant code No. of chromosomes
(chromosome constitution)
a
Syn-SAU-8/rye
228(1)-1-20-3 F4 42 (28AB ?14 R)
228(1)-1-32-18 F4 41 (26AB absent two 1B ?15R with three 1R) ?1RF
228(1)-1-32-1-2 F5 41 (28AB ?13R) ?3RF
228(1)-1-32-4-1 F5 41 (27AB absent one 1B or 6B ?14R)
228(1)-1-32-4-2 F5 42 (28AB ?14 R)
228(1)-1-32-18-3 F5 42 (26AB absent two 1B ?16R with four 1R)
228(1)-1-32-19-2 F5 42 (28AB ?14 R)
228(1)-1-32-25-2 F5 42 (28AB ?14 R)
228(1)-1-32-26-1 F5 42 (28AB ?14 R)
Syn-SAU-18/rye
233(1)-2-20 F3 ? (14R ??W ?one w-r translocation) ?1RF
233(1)-6-2 F3 44 (30 W ?14R)
233(1)-6-12 F3 42 (28 W ?14R)
233(1)-2-20-9 F4 ? (? ?two 5D ?3WF ?1RF ?two? w-r translocations)
233(1)-2-20-4 F4 ? (? ?one 2D ?two 5D ?one 7D ?14R)
233(1)-2-20-4-2 F5 42 (26AB ?two 5D ?14R)
Euphytica (2013) 193:347–357 353
123

cell nuclei, the D-genome is at a competitive disad-
vantage compared with A, B, and R genomes.
Preferential elimination of the D-genome has also
been reported in other hybrids, such as the amphiploid
between hexaploid wheat and A. kotschyi (genome
AABBDDU
k
U
k
S
k
S
k
) (Tiwari et al. 2010) and trigen-
eric hybrids between hexaploid wheat, rye, and
Psathyrostachys huashanica (genome AABBDRNs)
(Xie et al. 2012). It will consequently be interesting to
further elucidate this phenomenon.
The utility of new hexaploid triticales derived
from hexaploid wheat-rye hybrids
The results of our study indicate that complete and
substituted hexaploid triticales can be produced using
the hexaploid wheat-rye hybrid method. It is believed
that many characteristics of hexaploid triticale can be
improved by the introduction of D-genome chromo-
somes (Lukaszewski and Gustafson 1987; Dou et al.
2006). Previous studies focused on the incorporation
of D-genome chromosomes from common wheat into
hexaploid triticale. The D-genome of common wheat
is derived from that of the diploid wild species A.
tauschii. Direct incorporation of A. tauschii chromo-
somes into current triticale lines is valuable because of
the high diversity (Van Ginkel and Ogbonnaya 2007;
Yang et al. 2009), wide environmental adaptation
(Gororo et al. 2001; Trethowan and Mujeeb-Kazi
2008; Mizuno et al. 2010), and high early plant vigor
(Ter Steege et al. 2005) of this wild species. In this
study, we obtained a F
7
line (188-2-6-37-20-20-35)
Fig. 3 Chromosome constitutions of Syn-SAU-8/rye hybrids.
Chromosome constitutions were analyzed using R-genome,
pTa71, and pAs1 probes, with unlabeled wheat genome
DNA used as a blocker. aPlant 228(1)-1-32-18 had 15
rye chromosomes, including 3 1R chromosomes and 1R
chromosome fragment (red). bPlant 228(1)-1-32-18-3 had
26A/B and 16R chromosomes, with 4 1R chromosomes but
lacking 1B. cPlant 228(1)-1-32-4-2 had 28A/B and 14R
chromosomes. dPlant 228(1)-1-32-4-1 had 27A/B chromo-
somes, with 1 1B and 14R chromosomes
354 Euphytica (2013) 193:347–357
123

that carried a pair of 2D chromosomes from A. tauschii
accession AS60 (Fig. 2g). The photoperiod response
gene Ppd-D1 on 2D is one of the critical genes for
adaptation to growth habitats. Compared with its allele
in common wheat variety Chinese Spring, Ppd-D
t
1in
A. tauschii AS60 shortens heading times (Xiang et al.
2009). We also obtained an F
5
line (233[1]-2-20-4-2)
that carried a 5D chromosome pair from Chinese A.
tauschii accession AS77 (Fig. 4d). The vernalization
response gene Vrn-D1, another important gene for
adaptation to growth habitats, is found on chromo-
some 5D. In addition, chromosome 2D of A. tauschii
includes QTLs for leaf elongation rate and duration,
cell production rate, and cell length, while 5D harbors
QTLs for total leaf mass, total leaf area, and number of
leaves and tillers (Ter Steege et al. 2005). These QTLs
for early seedling growth are important for early
establishment and eventual success of triticale plants.
Our observations also demonstrate that transloca-
tion lines of hexaploid triticale can be effectively
produced using the hexaploid wheat-rye hybrid
method. Translocation lines are important genetic
materials for genetic improvement. Out of the eight
SHW-L1/rye hybrid lines analyzed, five carried
translocations. Because unpaired chromosomes in
hybrids remain as univalents, they have a chance to
misdivide and then fuse (Lukaszewski and Gustafson
1983). The centric translocations in line 186-4-2-31-
17-17-33 (Fig. 2f) were probably produced in this
way, i.e., by centric break-fusion. Terminal translo-
cations involving small chromosome fragments were
observed in four lines (Fig. 2c, d, g, h). Other studies
Fig. 4 Chromosome constitutions of some Syn-SAU-18/rye
hybrids. aand bwere analyzed with R-genome (green) probe
and unlabeled chinese spring genome DNA as a blocker; cand
dwere analyzed with R-genome (redincand green in d), pTa71
(yellow), and pAs1 (red) probes, with unlabeled chinese spring
genome DNA used as a blocker. aPlant 233(1)-6-2 had 30A/B
chromosomes and 14R chromosomes. bPlant 233(1)-2-20 had
one wheat-rye translocation (short arrow) and one rye
chromosome fragment (long arrow). cPlant 233(1)-2-20-4
had a pair of 5D chromosomes, 1 2D chromosome, and 1 7D
chromosome. dPlant 233(1)-2-20-4-2 had 26A/B chromo-
somes, 14 rye chromosomes, and a pair of 5D chromosomes
(arrow)
Euphytica (2013) 193:347–357 355
123

have demonstrated that small fragment translocations
are rare, and only a few small fragment translocations
between wheat and rye chromosomes have previously
been reported (see review by Zhou et al. 2012).
Although terminal translocations can arise from
homoeologous pairing, the level of homoeologous
chromosome pairing in wheat-rye F
1
hybrids is usually
low. The observed translocations were thus more
likely the result of non-centric breakage of chromo-
somes and subsequent fusion (Lukaszewski 1997;
Oleszczuk et al. 2011) or arose from abnormal
chromosome pairing in somatic cells (Fu et al. 2010;
Tang et al. 2012). The exact mechanism of origin
remains to be clarified, however.
‘‘Genome shock’’ resulting from interspecific
hybridization induces genomic changes (McClintock
1984). Interestingly, as reviewed by Ma and Gustafson
(2008), wheat hybrids with rye have higher rates of
genomic changes than wheat hybrids with other
related species. Novel alleles can be induced in
wheat-rye hybrids (Yuan et al. 2011). In this study,
hexaploids, such as 186-1-4-18-8-8-27 and 186-3-5-
26-14-14-31, with identical chromosome constitutions
were derived from the same F
1
hybrid plant (Table 1).
These are desirable materials for studying the evolu-
tionary biology of distant hybridization: because an F
1
plant originates from the union of a female gamete
with an ABD haploid genome and a male gamete with
an R haploid genome, the variations in its derivatives
can be attributed to the action of distant hybridization.
Acknowledgments We thank Prof. Yang Yen, South Dakota
State University, USA, for help with manuscript improvement.
This research was supported by the National Natural Science
Foundation of China (31271723), the 863 Program
(2011AA1001), and the 100-Talent Program of the Chinese
Academy of Sciences.
References
Bassu S, Asseng S, Richards R (2011) Yield benefits of triticale
traits for wheat under current and future climates. Field
Crop Res 124:14–24
Bilgili U, Cifci EA, Hanoglu H, Yagdi K, Acikgoz E (2009)
Yield and quality of triticale forage. J Food Agric Environ
7:556–560
Bird SH, Rowe JB, Choct M, Stachiw S, Tyler P, Thompson RD
(1999) In vitro fermentation of grain and enzymatic
digestion of cereal starch. Rec Adv Anim Nutr 12:53–61
Cheng ZJ, Murata M (2002) Loss of chromosomes 2R and 5RS
in octoploid triticale selected for agronomic traits. Genes
Genet Syst 77:23–29
Contento A, Heslop-Harrison JS, Schwarzacher T (2005)
Diversity of a major repetitive DNA sequence in diploid
and polyploid triticeae. Cytogen Gen Res 109:34–42
Davis-Knight HR, Weightman RM (2008) The potential of
triticale as a low input cereal for bioethanol production.
Report 434. Home-Grown Cereals Authority, Caledonia
House, London
Dou QW, Tanaka H, Nakata N, Tsujimoto H (2006) Molecular
cytogenetic analyses of hexaploid lines spontaneously
appearing in octoploid Triticale. Theor Appl Genet 114:41–47
Estrada-Campuzano G, Slafer GA, Mirallesa DJ (2012) Dif-
ferences in yield, biomass and their components between
triticale and wheat grown under contrasting water and
nitrogen environments. Field Crop Res 128:167–179
FAOSTAT Database (2010) http://faostat.fao.org/site/567/defa
ult.aspx#ancor.
Fox PN, Skovmand B, Thompson BK, Braun HJ, Cormier R
(1990) Yield and adaptation of hexaploid spring triticale.
Euphytica 47:57–64
Fu S, Tang Z, Ren Z (2010) Inter and intra-genomic transfer of
small chromosomal segments in wheat-rye allopolyploids.
J Plant Res 123:97–103
Fujisawa M, Yamagata H, Kamiya K, Nakamura M, Saji S,
Kanamori H, Wu J, Matsumoto T, Sasaki T (2006)
Sequence comparison of distal and proximal ribosomal
DNA arrays in rice (Oryza sativa L.) chromosome 9S and
analysis of their flanking regions. Theor Appl Genet
113:419–428
Glatthar J, Heinisch JJ, Senn T (2005) Unmalted triticale cul-
tivars as brewing adjuncts: effects of enzyme activities and
composition on beer wort quality. J Sci Food Agric
85:647–654
Gororo NN, Flood RG, Eastwood RF, Eagles HA (2001) Pho-
toperiod and vernalization responses in Triticum turgi-
dum 9T. tauschii synthetic hexaploid wheats. Ann Bot
88:947–952
Gowda M, Hahn V, Reif JC, Longin CFH, Alheit K, Maurer HP
(2011) Potential for simultaneous improvement of grain
and biomass yield in Central European winter triticale
germplasm. Field Crop Res 121:153–157
Griffith N, Lang H (2004) Plant triticale, cut hay bills, boost
lamb profits. Sheep Magazine. http://www.sheepmagazine.
com/issues/25/25-5/Nathan_Griffith.html. Accessed 5 Nov
2012
Hansen R (2010) Triticale. http://www.agmrc.org/commodit
ies__products/grains__oilseeds/triticale.cfm. Accessed 5
Nov 2012
Hao M, Luo JT, Yang M, Zhang LQ, Yan ZH, Yuan ZW, Zheng
YL, Zhang HG, Liu DC (2011) Comparison of homoeol-
ogous chromosome pairing between hybrids of wheat
genotypes Chinese Spring ph1b and Kaixian luohanmai
with rye. Genome 54:959–964
Kalinka A, Achrem M, Rogalska SM (2010) Cytomixis-like
chromosomes/chromatin elimination from pollen mother
cells (PMCs) in wheat-rye allopolyploids. Nucleus 53:69–83
Larter EN (2009) Triticale agriculture. The Canadian
Encyclopedia
Liu DC, Yen C, Yang JL, Zheng YL, Lan XJ (1999) The
chromosomal locations of high crossability genes in tet-
raploid wheat Triticum turgidum L. cv. Ailanmai native to
Sichuan. China. Euphytica 108:79–82
356 Euphytica (2013) 193:347–357
123

Lukaszewski AJ (1997) Further manipulation by centric mis-
division of the 1RS.1BL translocation in wheat. Euphytica
94:257–261
Lukaszewski AJ, Gustafson JP (1983) Translocations and
modifications of chromosomes in Triticale 9wheat
hybrids. Theor Appl Genet 64:239–248
Lukaszewski AJ, Gustafson JP (1987) Cytogenetics of triticale.
In: Janick J (ed) Plant breeding reviews, vol 5. AVI Pub-
lishing, New York, pp 41–93
Ma XF, Gustafson JP (2008) Allopolyploidization-accom-
modated genomic sequence changes in triticale. Ann Bot
101:825–832
McClintock B (1984) The significance of responses of the
genome to challenge. Science 226:792–801
Mcgoverin CM, Snyders F, Muller N, Botes W, Fox G, Manley
M (2011) A review of triticale uses and the effect of growth
environment on grain quality. J Sci Food Agric
91:1155–1165
McKevith B (2004) Nutritional aspects of cereals. Nutr Bull
29:111–142
Mizuno N, Yamasaki M, Matsuoka Y, Kawahara T, Takumi S
(2010) Population structure of wild wheat D-genome pro-
genitor Aegilops tauschii Coss: implications for intraspe-
cific lineage diversification and evolution of common
wheat. Mol Ecol 19:999–1013
Nagaki K, Tsujimoto H, Isono K, Sasakuma T (1995) Molecular
characterization of a tandem repeat, Afa family, and its
distribution among Triticeae. Genome 38:479–486
Nakata N, Sasaki M, Mochida M, Kishii Y, Yasumuro Y (1984)
Genome composition of 42-chromosme lines spontane-
ously derived from octoploid triticales. J Fac Agric Tottori
Univ 19:1–7
Oettler G (2005) The fortune of a botanical curiosity-Triticale:
past, present and future. J Agric Sci 143:329–346
Oleszczuk S, Rabiza-Swider J, Zimny J, Lukaszewski AJ (2011)
Aneuploidy among androgenic progeny of hexaploid trit-
icale (X Triticosecale Wittmack). Plant Cell Rep
30:575–586
Pedersen C, Langridge P (1997) Identification of the entire
chromosome complement of bread wheat by two-colour
fish. Genome 40:589–593
Rakha A, A
˚man P, Andersson R (2011) Dietary fiber in triticale
grain: variation in content, composition, and molecular
weight distribution of extractable components. J Cereal Sci
54:324–331
Rayburn AL, Gill BS (1986) Molecular identification of the
D-genome chromosomes of wheat. J Hered 77:253–255
Sasaki M, Yasumuro Y, Nakata N (1985) Development of
chromosome substitution triticale lines and their utility
value as feed. In: Proceedings of the 15th International
Grassland Congress, Kyoto, pp 253–255
Tang Z, Fu S, Yang B, Zhang H, Ren Z (2012) Unequal chro-
mosome division and inter-genomic translocation occurred
in somatic cells of wheat-rye allopolyploid. J Plant Res
125:283–290
Ter Steege MW, den Ouden FM, Lambers H, Stam P, Peeters
AJM (2005) Genetic and physiological architecture of
early vigor in Aegilops tauschii, the D-genome donor of
hexaploid wheat: a quantitative trait loci analysis. Plant
Physiol 139:1078–1094
Tiwari VK, Rawat N, Neelam K, Kumar S, Randhawa GS,
Dhaliwal HS (2010) Random chromosome elimination in
synthetic Triticum-Aegilops amphiploids leads to devel-
opment of a stable partial amphiploid with high grain micro
and macronutrient content and powdery mildew resistance.
Genome 53:1053–1065
Trethowan RM, Mujeeb-Kazi A (2008) The use of novel
germplasm resources to improve the environmental stress
tolerance of hexaploid wheat. Crop Sci 48:1255–1265
Van Ginkel M, Ogbonnaya F (2007) Novel genetic diversity
from synthetic wheats in breeding cultivars for changing
production conditions. Field Crops Res 104:86–94
Varughese G, Pfeiffer WH, Pen
˜a RJ (1997) Triticale: a reap-
praisal. http://www. worldbank.org/html/cgiar/newsletter/
april97/8tritic.html. Accessed 5 Nov 2012
Weimarck A (1974) Elimination of wheat and rye chromosomes
in a strain of octoploid triticale as revealed by giemsa
banding technique. Hereditas 77:281–286
Xiang ZG, Zhang LQ, Ning SZ, Zheng YL, Liu DC (2009)
Evaluation of Aegilops tauschii for heading date and its
gene location in a re-synthesized hexaploid wheat. Agric
Sci China 8:1–7
Xie Q, Kang H, Tao S, Sparkes DL, Fan X, Cui Z, Xu L, Huang
J, Fan X, Sha L, Zhang H (2012) Wheat lines derived from
trigeneric hybrids of wheat-rye-Psathyrostachys huasha-
nica, the potential resources for grain weight improvement.
Aust J Crop Sci 6:1550–1557
Yang W, Liu D, Li J, Zhang L, Wei H, Hu X, Zheng Y, He Z,
Zou Y (2009) Synthetic hexaploid wheat and its utilization
for wheat genetic improvement in China. J Genet
Genomics 36(9):539–546
Yang YW, Zhang LQ, Yen Y, Zheng YL, Liu DC (2010)
Cytological evidence on meiotic restitution in pentaploid
F
1
hybrids between synthetic hexaploid wheat and Aegi-
lops variabilis. Caryologia 63(4):354–358
Yuan ZW, Liu DC, Zhang LQ, Zhang L, Chen WJ, Yan ZH,
Zheng YL, Zhang HG, Yen Y (2011) Mitotic illegitimate
recombination is a mechanism for novel changes in high-
molecular-weight glutenin subunits in wheat-rye hybrids.
PLoS ONE 6:e23511
Zhang LQ, Chen QJ, Yuan ZW, Xiang ZG, Zheng YL, Liu DC
(2008) Production of aneuhaploid and euhaploid sporo-
cytes by meiotic restitution in fertile hybrids between
durum wheat Langdon chromosome substitution lines and
Aegilops tauschii. J Genet Genomics 35:617–623
Zhang LQ, Liu DC, Zheng YL, Yan ZH, Dai SF, Li YF, Jiang Q,
Ye YQ, Yen Y (2010) Frequent occurrence of unreduced
gametes in Triticum turgidum-Aegilops tauschii hybrids.
Euphytica 172:285–294
Zhang LQ, Yen Y, Zheng YL, Liu DC (2007) Meiotic restriction
in emmer wheat is controlled by one or more nuclear genes
that continue to function in derived lines. Sex Plant Reprod
20:159–166
Zhou J, Zhang H, Yang Z, Li G, Hu L, Lei M, Liu C, Zhang Y,
Ren Z (2012) Characterization of a new T2DS.2DL-?R
translocation triticale ZH-1 with multiple resistances to
diseases. Genet Resour Crop Evol 59:1161–1168
Euphytica (2013) 193:347–357 357
123