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RESEARCH ARTICLE
Divergent Development of Hexaploid
Triticale by a Wheat –Rye –Psathyrostachys
huashanica Trigeneric Hybrid Method
Houyang Kang
1☯
, Hao Wang
1☯
, Juan Huang
2
, Yujie Wang
1
, Daiyan Li
1
, Chengdou Diao
1
,
Wei Zhu
1
, Yao Tang
1
, Yi Wang
1
, Xing Fan
1
, Jian Zeng
3
, Lili Xu
1
, Lina Sha
1
, Haiqin Zhang
1
,
Yonghong Zhou
1
*
1Triticeae Research Institute, Sichuan Agricultural University, 211 Huimin Road, Wenjiang, Chengdu,
611130, Sichuan, China, 2Dazhou Institute of Agricultural Science, 188 Jianmin Road, Dazhou, 635000,
Sichuan, China, 3College of Resources, Sichuan Agricultural University, 211 Huimin Road, Wenjiang,
Chengdu, 611130, Sichuan, China
☯These authors contributed equally to this work.
*zhouyh@sicau.edu.cn
Abstract
Hexaploid triticale is an important forage crop and a promising energy plant. Some forms
were previously reported for developing the hexaploid triticale, such as crossing tetraploid
wheat or hexaploid wheat with rye, crossing hexaploid triticale and/or hexaploid wheat with
octoploid triticale, and spontaneously appearing in the selfed progenies of octoploid triticale.
In the present study, we developed an effective method for production of diverse types of
hexaploid triticale via wheat—rye—Psathyrostachys huashanica trigeneric hybrid. Genomic
in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) karyotyping
revealed that D genome chromosomes were completely eliminated and the whole A, B, and
R genome chromosomes were retained in three lines. More interestingly, the composite
genome of the line K14-489-2 consisted of complete A and B genomes and chromosomes
1D, 2R, 3R, 4R, 5R, 6R, and 7R, that of line K14-491-2 was 12 A-genome (1A-6A), 14 B-
genome (1B-7B), 12 R-genome (1R-3R, 5R-7R), and chromosomes 1D and 3D, and that of
the line K14-547-1 had 26A/B and 14R chromosomes, plus one pair of centric 6BL/2DS
translocations. This finding implies that some of D genome chromosomes can be spontane-
ously and stably incorporated into the hexaploid triticale. Additionally, a variety of high-
molecular-weight glutenin subunits (HMW-GS) compositions were detected in the six hexa-
ploid triticale lines, respectively. Besides, compared with its recurrent triticale parent
Zhongsi828, these lines showed high level of resistance to stripe rust (Puccinia striiformis f.
sp. tritici,Pst) pathogens prevalent in China, including V26/Gui 22. These new hexaploid
triticales not only enhanced diversification of triticale but also could be utilized as valuable
germplasm for wheat improvement.
PLOS ONE | DOI:10.1371/journal.pone.0155667 May 16, 2016 1/14
a11111
OPEN ACCESS
Citation: Kang H, Wang H, Huang J, Wang Y, Li D,
Diao C, et al. (2016) Divergent Development of
Hexaploid Triticale by a Wheat –Rye –
Psathyrostachys huashanica Trigeneric Hybrid
Method. PLoS ONE 11(5): e0155667. doi:10.1371/
journal.pone.0155667
Editor: Aimin Zhang, Institute of Genetics and
Developmental Biology, CHINA
Received: January 14, 2016
Accepted: May 1, 2016
Published: May 16, 2016
Copyright: © 2016 Kang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was funded by the National
Natural Science Foundation of China (No. 31501311),
the National High Technology Research and
Development Program of China (863 program, No.
2011AA100103), and the Science and Technology
Bureau and Education Bureau of Sichuan Province.
HYK and LLX received the fundings. The funders had
no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Introduction
The small grain cereal triticale (× Triticosecale Wittmack), a man-made wheat—rye hybrid, is
considered a promising crop due to its high genetic variation for several traits of agronomic
importance. It is intended to combine the high productivity and nutritional qualities of wheat
with the growth vigor and environmental tolerance possessed by rye [1–2]. Triticale is widely
adapted to abiotic stress conditions such as aluminum toxicity, drought, salinity, and acidic or
waterlogged soils, and also to biotic stresses including powdery mildew, leaf rust, stripe rust,
stem rust, Fusarium head blight, scald, and leaf and glume blotch [3–7]. Originally, triticale is
mainly used for forage or fodder in animal feed, serving as a good source of protein, lysine, B
vitamins, and readily digested starch [8–9]. However, ongoing research indicates that triticale
has some potential for use in human food consumption and remarkable improvement has
been made on bread making quality during the last decades [10–11]. In recent times, environ-
mental awareness has aroused interest in the use of triticale within bio-energy and bio-fuel pro-
duction owing to its high biomass and grain yield [12–14]. Moreover, the use of triticale in the
brewing industry has gained much attention [15–16].
Since the first octoploid triticale (2n=8x= 56, AABBDDRR) has been developed by chromo-
some doubling of hybrids between common wheat and rye [17], thousands of primary triticale
lines with a variety of ploidy levels and chromosome constitutions, including octoploid triticale
(× Triticosecale rimpaui Wittm., AABBDDRR), hexaploid triticale (× Triticosecale neoblaringhe-
mii A. Camus, AABBRR), and tetraploid triticale (× Triticosecale semisecale (MacKey) K. Ham-
mer et A. Filat., DDRR) have been successfully produced [18–21]. On account of their superior
meiotic stability and fertility, hexaploid triticale is deemed to be more successful than octoploid
and tetraploid triticale [22–24]. Hexaploid triticale is primarily derived from direct crosses of tet-
raploid wheat with rye. Secondary hexaploid triticale was also synthesized by hybridizing hexa-
ploid triticale and/or hexaploid wheat with an octoploid triticale [25]. Furthermore, many
hexaploid derivatives can spontaneously emerge in the selfed progenies of octoploid triticale, with
the elimination of the wheat D genome chromosomes [26–28]. Recently, Hao et al. [29] reported
that some hexaploid triticale lines with complete 28 intact A/B and 14 R chromosomes and other
chromosome constitutions could be rapidly produced by hybridization of synthetic hexaploid
wheatwithrye.Lietal.[30] developed two hexaploid triticales with great morphologic divergence
derived from common wheat cultivar M8003 × Austrian rye, which contained the whole A, B,
and R genome chromosomes. Kwiatek et al. [31] successfully obtained hexaploid triticale carrying
leaf rust resistance gene Lr32 via crossing triticale with the Aegilops tauschii–rye amphiploid.
We previously reported that the trigeneric germplasms involving Triticum,Psathyrostachys
and Secale were successfully created by crossing wheat–Psathyrostachys huashanica amphi-
ploid (PHW-SA, 2n=8x= 56, AABBDDNsNs) with triticale (Zhongsi828, 2n=6x= 42,
AABBRR) [32]. While these observations suggest that trigeneric hybridization may be useful
for triticale development, its effectiveness needs to be further evaluated [33]. The objectives of
this study were to characterize the chromosome constitution of six hexaploid derivatives of
wheat—rye–P.huashanica trigeneric hybrids expressing high stripe rust resistance and diverse
high-molecular-weight glutenin subunits (HMW-GS) compositions by genomic in situ hybrid-
ization (GISH), fluorescence in situ hybridization (FISH), and biochemical marker.
Materials and Methods
Plant materials
A hexaploid triticale (×Triticosecale Wittmack, 2n=6x= 42, AABBRR) line Zhongsi828, with
the characteristics of large spike and grain, cold tolerance, lodging resistance, and high resistance
Divergent Development of Hexaploid Triticale
PLOS ONE | DOI:10.1371/journal.pone.0155667 May 16, 2016 2/14
Competing Interests: The authors have declared
that no competing interests exist.
Abbreviations: CTAB, Cetyltrimethylammonium
bromide; FISH, Fluorescence in situ hybridization;
GISH, Genome in situ hybridization; HMW-GS, High-
molecular-weight glutenin subunits; LMW-GS, Low-
molecular-weight glutenin subunits; PMC, Pollen
mother cell; Pst,Puccinia striiformis f. sp. tritici; SDS-
PAGE, Sodium dodecyl sulfate polyacrylamide-gel
electrophoresis.
to rust and powdery mildew, was kindly provided by Dr. LQ Zhang, Triticeae Research Institute,
Sichuan Agricultural University, Sichuan, China. A wheat–P.huashanica amphiploid PHW-SA
(2n=8x= 56, AABBDDNsNs) was originally produced in our laboratory [34–35]. PHW-SA
and Zhongsi828 were crossed in 2008 [32]. Then seeds selected from the F
1
plants were bulked
and advanced to the F
6
generation by single seed descent. Six derivative lines K14-488-1, K14-
489-2, K14-491-2, K14-493-1, K14-545-2, and K14-547-1, with phenotypic divergence and high
resistance to stripe rust over two years of observation, were isolated from the F
6
generation.
Wheat line SY95-71 was used as a susceptible check in the tests to determine stripe rust resis-
tance. For GISH analysis, wheat cultivar J-11 (2n=6x= 42, AABBDD) was used as blocking
DNA, and the entire genomic DNA of Chinese rye landrace “Qinling”was used as a probe.
Meiotic pairing analysis
Meiotic pairing analysis followed the procedures described by Kang et al. [34]. Young spikes at
metaphase I (MI) stage were fixed in Carnoy’s fixative (ethanol: chloroform: glacial acetic acid,
6:3:1, v/v/v) for 24 h and stored at 70% ethanol until use. The macerated root tips and anthers
were squashed in 1% acetocarmine. At least 50 pollen mother cells (PMCs) were observed for
each plant.
GISH and FISH analysis
Total genomic DNA of rye landrace “Qinling”was isolated using the cetyltrimethylammonium
bromide (CTAB) method [36]. Rye genomic DNA was labeled with digoxigenin-11-dUTP by a
nick translation mix (Roche, Mannheim, Germany) and used as a probe in GISH. Chromo-
some spreads of materials, probe labeling, and in situ hybridization were carried out as previ-
ously described by Han et al. [37], with slight modifications. A total volume of 30 μL denatured
hybridization solution, containing 2 × SSC, 10% dextran sulphate, 0.2% sodium dodecyl sul-
phate, and 1 ng/μL labeled probe DNA together with the competitor DNA, was loaded per
slide and incubated for 12 h at 37°C. Finally, the chromosomes were counterstained with the
propidium iodide (PI) solution (Vector Laboratories, Inc., Burlingame, USA). The in situ
hybridization images were visualized using an Olympus BX-51 microscope coupled to a Photo-
metric SenSys Olympus DP70 CCD camera.
FISH analysis was subsequently used to identify the chromosome constitution of six deriva-
tive lines, using pSc119.2 and pTa535 as probes. Probe pSc119.2 from rye repetitive sequences
was used to determine the B genome chromosomes of wheat and R-genome chromosomes of
rye [38]. Probe pTa535 from wheat repetitive sequences hybridizes preferentially to A and D
genome chromosomes [39]. The two oligonucleotide probes were synthesized by Shanghai
Invitrogen Biotechnology Co. Ltd. (Shanghai, China). Probe labeling was operated according
to Tang et al. [40]. The FISH procedure was performed as described by Han et al. [37], with
minor modifications. The probe mixture (4 ng/μL of each probe in 2 × SSC and 1 × TE buffer)
and chromosomes were denatured together by heating for 5 min at 80°C. The chromosomes
were finally counterstained with DAPI (4, 6-diamidino-2-phenylindole) solution (Vector Lab-
oratories, Inc., Burlingame, USA). The detection and visualization of FISH patterns were the
same as the aforementioned GISH protocol.
Seed storage protein electrophoresis
HMW-GS and Low-molecular-weight glutenin subunits (LMW-GS) compositions of the six
derivative lines were determined by sodium dodecyl sulfate polyacrylamide-gel electrophoresis
(SDS-PAGE) following the procedure described by Yan et al. [41]. The parents PHW-SA and
Zhongsi828 were used as controls in the SDS-PAGE.
Divergent Development of Hexaploid Triticale
PLOS ONE | DOI:10.1371/journal.pone.0155667 May 16, 2016 3/14
Stripe rust resistance screening
Six derivative lines, the parental species PHW-SA and Zhongsi828, and SY95-71 were evalu-
ated for seedling and adult plant responses to stripe rust at the experimental station of Sichuan
Agricultural University, Chengdu, Sichuan, China. Individual plants of each line were grown
rows at 10-cm space in 30-cm wide beds and 2-m in length. The plots were surrounded by the
susceptible wheat line SY95-71. Artificial inoculation was made by spraying the SY95-71 rows
at the two-leaf stage with a mixture of Pst races CYR-32, CYR-33, V26/Gui22-9, V26/Gui22-
14, Su4, and Su5 suspended in light weight mineral oil. The Pst races were supplied by Dr. QZ
Jia, Plant Protection Institute of Gansu Academy of Agricultural Sciences, Gansu, China. Stripe
rust infection type (IT) was scored based on a scale of 0, 0;, 1, 2, 3, and 4, where 0 = immunity,
0; = necrotic flecks, and 1–4 = increasing sporulation and decreasing necrosis or chlorosis. The
plants scored with IT 2 or lower were considered as resistant while the plants with ITs 3 and 4
were considered as susceptible. The ITs were recorded three times when uniform severity levels
were observed on susceptible check SY95-71 at booting, flowering, and milky stages [42].
Results
Meiotic behavior of hexaploid triticale lines
Six stable derivative lines with great phenotypic divergence were obtained in 2014, namely
K14-488-1, K14-489-2, K14-491-2, K14-493-1, K14-545-2, and K14-547-1. The plants of these
lines grew vigorously and had a high level of seed set. These lines were selected to observe chro-
mosome behaviors at meiotic MI, and data on chromosome pairing frequency was presented
in Table 1. The results showed that all the lines with 2n = 42, the average pairing configuration
was 0.94 univalents, 16.51 ring bivalents, 3.99 rod bivalents, and 0.02 trivalents per PMC. The
mean number of univalents varied from 0.12 to 1.76. The number of bivalents varied from 18
to 21, and averaged 20.50 per cell (Fig 1a–1c). Trivalents were found in only one line, K14-547-
1(Fig 1d). No lagging chromosomes or chromosome bridges were observed at anaphase I and
II. This indicated that the six triticale lines presented normal meiotic behavior, and they were
cytologically stable.
GISH and FISH analysis
The GISH analysis was performed to determine the chromosome constitution of six derivatives
using total genomic DNA of rye as the probe and J-11 total genomic DNA as the block. Lines
K14-488-1, K14-493-1, K14-545-2, and K14-547-1 were found to have 14 rye chromosomes
(Table 2,Fig 2a), and other lines K14-489-2 and K14-491-2 carried 12 rye chromosomes (Fig
2b and 2c). At meiosis, all these lines always contained six or seven pairs of bivalents with
hybridization signals (Fig 2d and 2e), and regular segregation of each bivalent at anaphase I
Table 1. Chromosome pairing at metaphase I in the pollen mother cells of hexaploid triticale lines.
Lines 2nChromosomes pairing
I II (Ring) II (Rod) II (Total) III IV
K14-488-1 42 1.36 (0–4) 16.92 (13–20) 3.40 (1–7) 20.32 (19–21) ––
K14-489-2 42 0.58 (0–4) 17.08 (12–21) 3.63 (0–8) 20.71 (19–21) ––
K14-491-2 42 1.76 (0–6) 14.20 (10–18) 5.92 (2–10) 20.12 (18–21) ––
K14-493-1 42 0.80 (0–4) 17.84 (15–20) 2.76 (0–6) 20.60 (19–21) ––
K14-545-2 42 0.12 (0–2) 16.61 (12–21) 4.33 (0–8) 20.94 (20–21) ––
K14-547-1 42 1.04 (0–6) 16.40 (13–18) 3.93 (1–8) 20.33 (18–21) 0.10 (0–1) –
doi:10.1371/journal.pone.0155667.t001
Divergent Development of Hexaploid Triticale
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Fig 1. Meiotic metaphase I pairing analysis of hexaploid triticale lines. (a) Line K14-488-1, 2n= 42 = 1 II (rod) + 20 II
(ring). (b) Line K14-489-2, 2n= 42 = 2 I + 4 II (rod) + 16 II (ring). (c) Line K14-491-2, 2n= 42 = 4 I + 3 II (rod) + 16 II (ring). (d)
Line K14-547-1, 2n= 42 = 1 I + 1 II (rod) + 18 II (ring) + 1III (arrow).
doi:10.1371/journal.pone.0155667.g001
Table 2. Chromosome constitutions of six hexaploid triticale lines.
Line Pedigree No. of plants tests No. of chromosomes (chromosome constitution)
K14-488-1 PHW-SA/Zhongsi828 F
6
5 42 (28AB + 14 R)
K14-489-2 PHW-SA/Zhongsi828 F
6
5 42 (28AB + two 1D + 12R absent two 1R)
K14-491-2 PHW-SA/Zhongsi828 F
6
5 42 (26AB absent two 7A + two 1D + two 3D + 12R absent two 4R)
K14-493-1 PHW-SA/Zhongsi828 F
6
5 42 (28AB + 14 R)
K14-545-2 PHW-SA/Zhongsi828 F
6
5 42 (28AB + 14 R)
K14-547-1 PHW-SA/Zhongsi828 F
6
5 42 (26AB absent two 6B + two 6BL/2DS translocations+ 14 R)
A, B, D, and R mean A, B, D-genome chromosomes of wheat and R-genome chromosomes or rye, respectively
doi:10.1371/journal.pone.0155667.t002
Divergent Development of Hexaploid Triticale
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Fig 2. GISH identification of hexaploid triticale lines at mitotic metaphase (a–c), and meiotic metaphase
I and anaphase I (d–f). The rye genomic DNA was used as probes for in situ hybridization. Chromosomes in
red and yellow-green are wheat and rye chromosomes, respectively. (a) Line K14-488-1, 2n= 42 = 14R +28W.
(b) Line K14-489-2, 2n= 42 = 12R +30W. (c) Line K14-491-2, 2n= 42 = 12R +30W. (d) Line K14-493-1,
2n= 42 = 14R +28W, rye chromosomes with 1 rod and 6 ring bivalents were labeled. (e) Line K14-489-2,
Divergent Development of Hexaploid Triticale
PLOS ONE | DOI:10.1371/journal.pone.0155667 May 16, 2016 6/14
was also found (Fig 2f). It seems that the behavior of rye chromosomes was normal during
meiosis.
Further FISH analysis showed that these derivatives could be grouped into four types with
respect to their chromosome constitutions. Three lines K14-488-1, K14-493-1, and K14-545-2
displayed similar chromosome constitutions (28A/B and 14R chromosomes) as the primary
hexaploid triticale between T.turgidum and rye (Fig 3a, 3d and 3e). Consequently, all the D
and Ns genome chromosomes were completely eliminated during the derivation of the three
hexaploid triticale lines. Line K14-489-2 consisted of six pairs of rye chromosomes and one
pair of D genome chromosomes in addition to the complete A and B genomes (Fig 3b). Assum-
ing that the FISH pattern of Chinese Spring using pSc119.2 and pTa535 as probes corresponds
to the wheat cultivar used in this work, the D genome chromosomes was identified as 1D,
which presented faint pTa535 signals in the terminal regions of both arms. FISH with pSc119.2
demonstrated that rye chromosomes 1R were absent in this line. More interestingly, line K14-
491-2 comprised twelve rye chromosomes (Fig 2c). Reprobing with pSc119.2 and pTa535
indicated that it carried 26 A and B genome chromosomes and two pairs of D genome chromo-
somes (Fig 3c). One of the D genome chromosomes was identified as 1D, and another chromo-
some seemed to be 3D because it carried strong pTa535 signals at the terminal region of the
short arm, and faint pTa535 signals in terminal region of the long arm. Probing with pSc119.2
and pTa535 revealed that rye chromosomes 4R and wheat chromosomes 7A were not included
in this type (Fig 3c). Additionally, Line K14-547-1 had 14A (1A-7A), 12B (1B-5B, 7B), and 14R
chromosomes, plus one pair of centric 6BL/2DS translocations (Fig 3f). Furthermore, the
PMCs of lines K14-489-2, K14-491-2, and K14-547-1 showed normal meiotic behavior, pos-
sessing approximately 21 bivalents (Table 1).
Storage protein variations detected in hexaploid triticale lines
The HMW-GS compositions of the six hexaploid triticale lines together with their donor
parents were analyzed by SDS-PAGE. As illustrated in Fig 4, six completely different HMW-
GS variations were observed in these triticales. 1Dx2+1Dy12 encoded by 1D chromosome was
detected in two lines K14-489-2 and K14-491-2 owing to the presence of 1D chromosome.
This finding was in agreement with the FISH analyses described above. 1Bx7+1By8 encoded by
1B chromosome, which derived from the parent PHW-SA, were present at all triticale lines. In
addition, not all the HMW-GS of the parent Zhongsi828 were simultaneously expressed. A sin-
gle but inconsistent band was inherited in K14-488-1, K14-491-2, K14-545-2, and K14-547-1,
respectively (Fig 4, indicated by black arrows), and a specific band was only expressed in K14-
488-1 (Fig 4, indicated by red arrow). An exception to this was that in lines K14-493-1 and
K14-489-2, all HMW-GS of the Zhongsi828 was absent. Similarly, polymorphic bands were
also distinguished at these triticales in the LMW-GS region. Above results implied that multi-
ple HMW-GS variations were discriminated in these six hexaploid triticale lines and differen-
tial expression of HMW-GS could result from mutation or expression silencing of this locus.
Stripe rust resistance evaluation
Six hexaploid triticale lines, PHW-SA, Zhongsi828, and SY95-71were evaluated stripe rust
resistance with mixture of Pst races CYR-32, CYR-33, V26/Gui22-9, V26/Gui22-14, Su4, and
Su5 at Chengdu, Sichuan, China. At seedling and adult plant stages, Zhongsi828 and SY95-71
2n= 42 = 12R +30W, rye chromosomes with 4 rod and 2 ring bivalents were labeled. (f) Line K14-491-2, rye
chromosomes showed regular segregation at anaphase I.
doi:10.1371/journal.pone.0155667.g002
Divergent Development of Hexaploid Triticale
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Fig 3. FISH karyotypes of hexaploid triticale lines using pSc119.2 and pTa535 as probes. Probes pSc119.2 and
pTa535 signals were pseudo-colored as green and red in the FISH patterns, respectively. (a) Line K14-488-1 had
complete 28 A/B and 14 R chromosomes. (b) Line K14-489-2 had complete 28A/B and 1D (arrows), 2R, 3R, 4R, 5R,
6R and 7R chromosomes. (c) Line K14-491-2 had 12 A (1A-6A), 14 B (1B-7B), 12 R (1R-3R, 5R-7R), and 1D and 3D
(arrows) chromosomes. (d) Line K14-493-1 had complete 28 A/B and 14 R chromosomes. (e) Line K14-545-2 had
Divergent Development of Hexaploid Triticale
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were susceptible to these races, showing infection types 4, respectively. PHW-SA, K14-488-1,
K14-489-2, K14-491-2, K14-493-1, K14-545-2, and K14-547-1 were highly resistant to the
races, all showing 0 infection type (Table 3).
Discussion
Development of hexaploid triticale by loss of most D genome
chromosomes
The primary objective of this work was production of diverse chromosome constitutions
(AABBDDRR, AABBRR, AABBDDNsNs, or AABBNsNs) and introgression of rye and P.
complete 28 A/B and 14 R chromosomes. (f) Line K14-547-1 had 14 A (1A-7A), 12 B (1B-5B, 7B), 14 R, and a pair of
6BL/2DS translocation chromosomes (arrows).
doi:10.1371/journal.pone.0155667.g003
Table 3. Infection types of hexaploid triticale lines and their parents for stripe rust with a mixture of races at seedling and adult plant stages.
Materials No. of plants observed Infection type Resistance/susceptibility
PHW-SA 15 0 R
Zhongsi828 15 4 S
K14-488-1 20 0 R
K14-489-2 20 0 R
K14-491-2 20 0 R
K14-493-1 20 0 R
K14-545-2 20 0 R
K14-547-1 20 0 R
SY95-71 15 4 S
Wheat line “SY95-71”was used as susceptible control
R resistance, S susceptibility
doi:10.1371/journal.pone.0155667.t003
Fig 4. HMW-GS variations in hexaploid triticale lines and their parents. 1Zhongsi828, 2PHW-SA, 3K14-488-1, 4K14-
491-2, 5K14-493-1, 6K14-545-2, 7K14-547-1, 8K14-489-2. Black arrows indicate position of inherited HMW-GS subunit
of Zhongsi828. Red arrow indicates a specific band was only expressed in K14-488-1.
doi:10.1371/journal.pone.0155667.g004
Divergent Development of Hexaploid Triticale
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huashanica chromatins into common wheat background by crossing a wheat–P.huashanica
amphiploid (PHW-SA, 2n=8x= 56, AABBDDNsNs) with hexaploid triticale (Zhongsi828,
2n=6x= 42, AABBRR). However, after selfed generations, Xie et al. [33] found that the rye
chromosome number of F
3
lines with 2n = 41–44 ranged from 12 to 14, and only 1–2P.hua-
shanica chromosomes were observed in three lines (20%). In F
4
progenies, GISH results illus-
trated that 21 lines of 2n = 42 (91.3%) had 12 or 14 rye chromosomes exhibiting complete
pairing, and no P.huashanica chromosomes were detected. Therefore, these lines were cytolog-
ically stable during meiosis and may therefore be considered as new hexaploid triticale (unpub-
lished data). This present study demonstrated that no octoploid triticale was found, and
instead hexaploid triticale was obtained. In addition, our results revealed that these lines
retained the most chromosomes of the A, B, and R genomes, while most of the D and Ns
genome chromosomes were eliminated. Although we cannot exclude the possibility that some
octoploid triticale was formed in F
2
plants [32], the typically abnormal behavior of rye and
wheat chromosomes of F
3
plants must have led to their elimination during production of the
subsequent generations [43]. It is known that octoploid triticale shows meiotic instability and
high aneuploid frequency [22] because chromosomes from rye and wheat were eliminated
[44–45]. Such chromosome loss in octoploid triticale may result in hexaploid triticale, with the
retention of most of A, B, and R genome chromosomes and the elimination of most of the D
genome chromosomes [26–28,46]. To date, some genetic mechanisms involved in chromo-
some elimination, including cytomixis-like fashion chromatin elimination from PMCs,
unequal chromosome division in somatic cell, centromere loss of chromosome fragments, and
chromosomal variations and asynchronous chromosome-division, were reported in octoploid
triticale [29–30,45,47]. Xie et al. [43] indicated that chromosome elimination in early progenies
of PHW-SA/Zhongsi828 may associate with chromatid lagging, fragmentation and micronu-
cleation, or the immobilization of certain univalents during meiosis instead of mitosis in the
relatively advanced generations. Therefore, this mechanism permitted the generation of vari-
ous hexaploid triticale lines in this study.
D genome chromosomes can be stably incorporated into the hexaploid
triticale
Although the stability of the D genome is more strongly affected by the R genome in the octo-
ploid triticale, comparing to the A and B genomes of common wheat, the more researches
suggested that hexaploid triticale contained D genome chromosomes in lower frequency.
Lukaszewski and Gustafson [22,48] reported very frequent 2D (2R) substitution in secondary
hexaploid triticale produced by crosses between hexaploid triticale and hexaploid wheat. Tams
et al. [49] and Leonova et al. [50] demonstrated that chromosomes 2D and 7D were detected in
some European hexaploid triticale by SSR markers. Using FISH to characterize the chromo-
some constitution of 14 hexaploid lines derived from octoploid triticale, Dou et al. [27] revealed
that all of the lines showed 2D (2R) substitution, and additionally two showed 1D (1R) and one
showed 6D (6R). Since 1D, 2D, 6D, and 7D were found in these hexaploid triticale, Dou et al.
[27] concluded that genes on chromosomes 3D, 4D, and 5D may promote the instability of the
R genome, and the D genome and R genome are incompatible in the common wheat genetic
background. However, the present study indicated that the composite genome of the line K14-
489-2 consisted of complete A and B genomes and chromosomes 1D, 2R, 3R, 4R, 5R, 6R, and
7R, that of line K14-491-2 was 12 A genome (1A-6A), 14 B genome (1B-7B), 12 R genome
(1R-3R, 5R-7R), and chromosomes 1D and 3D, and that of the line K14-547-1 had 26A/B and
14R chromosomes, and one pair of centric 6BL/2DS translocations. Moreover, the PMCs of
lines K14-489-2, K14-491-2 and K14-547-1 showed stable cytology, possessing approximately
Divergent Development of Hexaploid Triticale
PLOS ONE | DOI:10.1371/journal.pone.0155667 May 16, 2016 10 / 14
21 bivalents, with chromosome pairing configurations of 0.58 I + 3.63 II (rod) + 17.08 II (ring),
1.76 I + 5.92 II (rod) + 14.20 II (ring), and 1.04 I + 3.93 II (rod) + 16.40 II (ring) + 0.10 III,
respectively. Recently, chromosomes 2D, 5D, and 7D from the wild species Ae.tauschii were
involved in the hexaploid triticale using a synthetic hexaploid wheat—rye hybrid method [29].
Kwiatek et al. [31] successfully transferred the 3D genome chromatin carrying leaf rust resis-
tance gene Lr32 into hexaploid triticale by crossing triticale with the Ae.tauschii–rye amphi-
ploid. It was formerly reported that 3DS promotes homoeologous chromosome pairing, and
5D carries a promoter on each arm [51–52]. Accordingly, all these findings imply that genes on
chromosomes 4D may promote the instability of the R genome, and some of D genome chro-
mosomes can be spontaneously and stably incorporated into the hexaploid triticale. The stabil-
ity of D genome chromosomes in hexaploid triticale may be affected by common wheat genetic
background, rye genetic composition as well as their interaction.
The potential value of the new hexaploid triticale for wheat improvement
The present results suggest that complete and substituted hexaploid triticale can be produced
using the wheat—rye–P.huashanica trigeneric hybrid method. It is believed that one of the
main breeding strategies for triticale improvement was to introduce D genome chromosomes
into hexaploid triticale [22,27,29,31]. Here, we developed three hexaploid triticale lines that
carried 1D, 2D, and 3D chromosomes, respectively. It is known that chromosome 1D carries
the Glu-D1 locus, which plays a major role in the bread-making quality of bread wheat [53].
The dwarfing gene Rht8 and the photoperiodic insensitivity gene Ppd-D1 are linked on the
short arm of chromosome 2D of bread wheat and play an important role in determining the
geographic adaptation of modern wheat varieties [54]. The homoeologous chromosome pair-
ing gene Ph2 on 3DS is one of the critical genes for maintaining genetic stability in the transfer
of alien genes to wheat [51,55]. Furthermore, a number of important traits are known to be
controlled by loci on these chromosomes, including grain yield and seed weight, seed dor-
mancy, glume blotch resistance, stripe rust resistance, stem rust resistance, and leaf rust resis-
tance [56]. Besides, a variety of HMW-GS variations were detected in these hexaploid triticale
lines comparing with their recurrent triticale parent Zhongsi828. Similar phenomenon
occurred in recent studies of new synthesized hexaploid triticale and hexaploid trititrigias
derived from Triticum durum and Thinopyrum elongatum [21,30]. Differential expression
of HMW-GS could result from gene silencing or mutation of this locus occurred among the
derivatives of wheat—rye–P.huashanica trigeneric hybrids. Although these triticales were
derived from the same pedigree, great morphological divergences were displayed in these lines
(unpublished data). Thus, these lines might be potential materials for further quality and yield
improvement of hexaploid triticale.
The occurrence of new virulent stripe rust races, such as V26/Gui 22, which are effective
against all previously identified Pst races and have been deployed in commercial cultivars to
fight predominant races of the fungus in China, represents a destructive serious threat to
wheat production in the Sichuan Basin and potentially in other regions of China [57]. It is,
therefore, urgent to search for and transfer novel sources of resistance and to use more effec-
tive genes to counterbalance the continuous evolution of rust pathogens in wheat breeding
programs. In our work, six hexaploid triticale lines were selected and identified from
PHW-SA/Zhongsi828 F
6
generation. Compared with the parent Zhongsi828, these lines were
highly resistant to prevalent Chinese Pst races, including V26/Gui 22. Consequently, the new
triticale lines provide novel and valuable bridge resources for improving stripe rust resistance
in wheat.
Divergent Development of Hexaploid Triticale
PLOS ONE | DOI:10.1371/journal.pone.0155667 May 16, 2016 11 / 14
Acknowledgments
We thank Dr. ZX Tang (Sichuan Agricultural University, China) for technical guidance in
FISH analysis. We also thank anonymous reviewers for their very useful comments on the
manuscript.
Author Contributions
Conceived and designed the experiments: HYK YHZ. Performed the experiments: HYK HW
JH YJW. Analyzed the data: HYK DYL CDD WZ YT. Contributed reagents/materials/analysis
tools: YW XF JZ LLX LNS HQZ. Wrote the paper: HYK HW.
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