全 文 ：Received 17 Apr. 2003 Accepted 29 Aug. 2003
Supported by the Hi-Tech Research and Development (863) Program of China (2003AA207080) and the Key Project of Scientific and
Technological Innovation of The Chinese Academy of Sciences (KSCX2-SW-304-2).
* Author for correspondence. Tel: +86 (0)311 5887272; Fax: +86 (0)311 5815093; E-mail: .
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Acta Botanica Sinica
植 物 学 报 2004, 46 (4): 436-442
Fluorescent In Situ Hybridization Analysis of Rye Chromatin
in the Background of “Xiaoyan No.6”
WANG Zhi-Guo1, AN Tiao-Guo1, LI Jun-Ming1*, Molnar-Lang MARTA 2
JI Jun1, ZHONG Guan-Chang1, MU Su-Mei1
(1. Centre of Agricultural Resources, Institute of Genetics and Developmental Biology,
The Chinese Academy of Sciences, Shijiazhuang 050021, China;
2. Agricultural Research Institute of Hungarian Academy of Sciences, Martonvasar, Hungary)
Abstract : A set of wheat germplasms with rye’s characters was obtained by crossing the Trit icum
aestivum L. cv. “Xiaoyan No.6” and Secale cereale L. cv. “German White”. Eight lines were found carrying
rye chromosomes or segments by genomic in situ hybridization (GISH), including three addition lines, one
substitution line and four translocation lines. Two-color fluorescent in situ hybridization (FISH) was used to
identify the genome composition and individual chromosome of the hybrids using pSc119.2 labled with
fluorogreen and pAs1 labled with fluorored as probes. The partial results showed that BC116-1 is a wheat-
rye 1RS/1BL translocation line; BC152 is a monosomic 1RS/1BL translocation line; BC97-2 is a disomic
substitution line with a pair of 2D chromosomes substituted by 2R; BC122-3 is a 6R monosomic addition
line with one of the long arm of 6B deletion. The application of sequential GISH and two-color FISH in wheat
breeding is discussed.
Key words : rye chromatin; Xiaoyan No.6; fluoroscent in situ hybridization
Int roduct ion of alien genes from relat ive species has
been proven to be a successful way to improve the quality
and yield of common wheat. A large number o f wild rela-
tives of common wheat have been crossed with wheat
(Sharma and Gill, 1983; Jiang et al., 1994). Undoubtedly, it
will be more practical and useful to introduce the beneficial
genes to cu ltivated wheat varieties and overcome their
disadvantages.
As an important source of disease resistance and yield
improvement, rye (Secale cereale L.) has also been widely
used and some wheat-rye substitutions and translocations
have been proven to have high resistance to leaf rust, stem
rust, powdery mildew and insects (Driscoll and Jensen, 1964;
Stewart et al., 1968; Friebe et al., 1989; Heun et al., 1990).
Friebe et al. (1994) found that the rye chromosomes 2R, 6R
presenting in wheat-rye addition and substitution lines are
additional sources of powdery mildew resistance. And the
rye chromosome arm 1RS has been widely used in cu lti-
vated wheat breeding. A lot of wheat varieties carrying 1RS/
1BL have been released all over the world (Rabinorich, 1998;
Villareal et a l., 1998). Koszeg i et al. (2000) reported that
53% of Hungarian-bred wheat varieties registered in Hun-
gary between 1978 and 1999 carried the 1RS/1BL
translocation.
“Xiaoyan No.6” was an elite winter wheat variety ob-
tained by cross ing common wheat with Thinopyrum
ponticum (2n=70). It posses sed the characters o f good
quality and high yield, earliness, stress tolerance and wide
adaptation (Li et al., 1990; Wei et al., 2000; Ma et al., 2000).
By transferring rye chromatin into “Xiaoyan No.6”, it would
be possible to enlarge genetic variation of the cultivated
wheat, improve resistance of this variety to d isease and
produce germplasm enhanced.
Many approaches including chromosome banding, bio-
chemical markers and molecu lar markers (in si tu
hybridization, RAPD, RFLP and PCR markers) were used in
analysis and identification of alien chromatin in hybrid (Ru
et al., 2002; Wei et al., 2001). In situ hybridization appeared
to be a more quick and powerful way. By using one of the
parent’s total genome DNA as a p robe, genomic in si tu
hybridization (GISH) provides a direct and effective method
to determine the origin of genome, chromosome and chro-
mos ome s egment in hybrid. In this way, all alien
chromosomes, segments involving addition, substitution
and translocation could be discriminated. By labeling ge-
nomic DNA with different colors’ fluorescent respectively,
WANG Zhi-Guo et al.: Fluorescent In Situ Hybridization Analysis of Rye Chromatin In the Background of “Xiaoyan No.6” 437
Sanchez-Moran et al. (1999) discriminated the four genomes
(A , B, D, R) o f wheat -rye hybrid in s omat ic cell
simultaneously . By using two or more specific highly re-
petitive DNA sequences labled with different fluorescent
as probes, FISH made it possible to map the repetitive DNA
sequences to s pecific chromosome sites and discriminate
genome constitution. Multicolor FISH was not only a use-
ful method in identifying alien chromatin, but also an alter-
native one in discriminating individual chromosome besides
chromos ome banding (Zhang et a l., 2000). Us ing two re-
peated sequences pSc119.2 and pAs1 as probes, Mukai et
al. (1993) identified 17 of 21 chromosome pairs of hexaploid
wheat, whereas, with two probes pAs1 and pHv38, Pedersen
and Langridge (1997) iden tified the entire chromosome
complement of hexaploid wheat.
In this paper, combining GISH with two-color FISH, we
detected the rye chromatin in wheat-rye hybrids involving
addit ion, subst itution and translocation. The analys is of
genome constitution and individual chromosome was also
carried out.
1 Materials and Methods
1.1 Plant materials
Wheat-rye hybrids were produced by crossing winter
wheat (Triticum aestivum L.) cultivar “Xiaoyan No.6” and
winter rye (Seca le cereale L.) cultivar “German White”.
The wheat-rye hybrids F1 were treated with colchicines. F2
and the subsequent generations were s elf-cros sed and
planted in the field. Each individual plant was selected with
the qualities of seed set, wheat-like plant type and good
resistance to rust and powdery mildew. Fifty-one lines of
F5 were used in this experiment.
1.2 Chromosome preparation
Root t ips of 2-3 cm long were excised and pretreated
in ice water for 22 to 24 h, then fixed in freshly prepared
3:1 mixture solu tion of abso lute ethano l and glacial ace-
tic acid fo r 1-7 d. The roo t cap removed with a razor
blade, and a small p iece of meristem was squas hed in
45% acet ic acid. The s lides were frozen in liquid n itro-
gen and cover slips removed away with razor b lade. The
s lides with moderate number o f cells in s omat ic
meta phas e were s electe d unde r phas e con t ras t
microscope. Subsequen tly , the s lides were dehydrated
with 70%, 90% and 100% ethanol series for 5 min each,
and dried up overnight at room temperatu re and stored
at 4 ℃ fo r fu rther us e.
1.3 GISH: probe labeling, in situ hybridization
Total genomic DNA of rye was sonicated into 500 to 800
bp long and labeled with fluorogreen-11-dUTP (Amersham)
by nick translation. The wheat blocking DNA was mixed
with probe at 35:1. In situ hybridization was carried out as
described by Reader et al. (1994). Prepared slides were pre-
treated with 5 µg/mL RNase for 60 min at 37 ℃ and washed
in 2×SSC. The slides were fixed in 4% paraformaldehyde
for 10 min at room temperature, three times of washing in 2
× SSC. The chromosomal DNA on the slides was dena-
tured in 70% formamide fo r 3 min at 70 ℃. After that, the
slides were dehydrated in cold ethanol series of 70%, 90%
and 100% subsequently. The hybridization mixture, 45 mL
for each slide, contained 10% dextrane sulphate, 0.25% SDS,
2× SSC and 50 ng labeled rye probe. After hybridization
in a humid chamber for 2.5 h at 65 ℃, the slides were washed
in 2×SSC for 5 min twice. The slides were counterstained
with 1 µg/mL DAPI, then covered with Vetashield and stored
at 4 ℃.
1.4 FISH after GISH: probe labeling, in situ hybridiza-
tion
Two highly repeated DNA s equences pSc119.2 and
pAs1 were labeled with Fluorogreen and Fluorored via nick
translation respectively. In 40 mL hybridization mixture per
slide, there were 30 ng labeled probe, 50% formamide, 2×
SSC, 10% dextram sulphate, 0.9 µg salmon sperm DNA and
0.1% SDS.
The slides after GISH were washed in 2×SSC and fixed
in 4% paraformaldehyde for 10 min at room temperature.
The hybridization mixture was boiled for 10 min to denature
the DNA and then was chilled in ice for 5 min. Then the
mixture was added to the slide and the slide was kept at 37
℃ for 6 h. The slides were washed twice respectively in 2
×SSC at 42 ℃ (5 min each), in 25% formamide at 42 ℃ (5
min each), and in 2× SSC at 42 ℃ (fo r 5 min each). The
slides were counterstained with 1 µg/mL DAPI (Amersham),
then covered with Vetashield and stored at 4 ℃.
1.5 Microscopy and photograph
Hybridization signals were detected with Zeiss Axioskop
2 epifluorescence microscope equipped with Filter 10 for
FITC, Filter 15 for Texas Red and a triple band filter (25) set
for FITC, DAPI and Texas Red. Pictures were taken by Spot
CCD camera (Diagnostic Inst ruments Inc., USA). Images
were analyzed and synthes ized with Image-Pro Plus
software.
2 Result and Analysis
2.1 GISH analysis
Using rye genomic DNA as probe and wheat genomic
DNA as blocker, 51 lines derived from “Xiaoyan No.6×
rye” were detected by GISH. Eight lines were found carry-
ing rye chromosomes or s egments . The b righ t-green


Acta Botanica Sinica 植物学报 Vol.46 No.4 2004440
chromosomes, two specific pSc119.2 hybridization bands
were observed, one was at the terminal posit ion and the
other was at the intercalary position. These indicate the
long arms were 1BL (Rayburn and Gill, 1985). No pAs1 hy-
bridizat ion s ignal was detected on the t ranslocated
chromosome. Therefore, the translocation chromosomes
were identified as 1RS/1BL (arrowed). And the BC116-1 line
should be a 1RS/1BL translocation line and its chromo-
some composition was 2n=40W+DT (1RS/1BL) (Fig.5).
2.2.2 Translocation line BC152-1 In the same way, the
single translocated chromosome in the BC152-1 line also
was recognized as a 1RS/1BL (arrowed), and BC152-1 line
was proven to be a 1RS/1BL monosomic translocation line
and its chromosome composition was 2n=41W+MT (1RS/
1BL) (Fig.6).
2.2.3 Substitution line BC97-2 The GISH result showed
that BC97-2 line had forty wheat chromosomes and two rye
chromosomes. The sequential two-color FISH results sug-
gested that two of D-genome chromosomes were replaced
by two rye chromosomes because only 12 wheat chromo-
somes carried the visible pAs1 labeling pattern. The pAs1
hybridization patterns in D-genome chromosomes also in-
dicated two 2D chromosomes were substituted (pAs1 hy-
bridization patterns of 2D were dis played in the Fig.7).
Furthermore, the pSc119.2 hybridization signal on the two
rye chromosomes was also obvious, there were two promi-
nent signals at the terminal position and a signal on the
short arms was stronger. Accord ing to the pSc119.2 hy-
bridization pattern and chromosome morphologic charac-
ters reported by Mukai et al. (1992), the two rye chromo-
somes were primarily thought to be 2R (arrowed). The chro-
mosome composition of BC97-2 was 2n=40W+DS [2R(2D)]
(Fig.7).
2.2.4 Addition line BC122-3 According to the GISH
result, this line had forty-two intact chromosomes (including
a rye chromosome) plus a telomeric chromosome. The two-
color FISH results showed this telomeric chromosome had
a weaker terminal pSc119.2 hybridization signal and con-
veyed Nor region. Its hybridizat ion pattern and morpho-
logic characteristics are quite similar to 6BS. Therefore, it
was identified as a short arm of 6B. The additional rye chro-
mosome had a strong terminal pSc119.2 hybridization sig-
nal and a subterminal pSc119.2 hybridization on the short
arm, as well as two pSc119.2 hybridization signals near its
centromere on the long arm. This additional rye chromo-
some was iden tified as 6R (Mukai et a l., 1992; 1993).
Therefore, the chromosome composition of BC122-3 was
2n=41W+t (6BS)+6R (Fig.8).
3 Discussion
Rye was classified into the tertiary gene pool for wheat
improvement based on the affinity relationship to common
wheat by Jiang et al. (1994) and Dong (2000). Since the first
wheat-rye hybrid was found in 1875, scientists have been
working on transferring rye chromatin into wheat. New de-
sirable genes in differen t rye acces sions are s till being
exploited. Recently, Qi et al. (2000) obtained two wheat-rye
hybrids and two amphiploids with highly resistant genes
to powdery mildew derived from a Chinese local rye variety
“Jinzhouheimai”. Ko et a l. (2002) produced a new wheat
line possessing the wheat-rye 1RS/1BL translocat ion de-
rived from a Korean rye cultivar. All of these proved that S.
cereale has more potential desirable genes to be exploited
and utilized in wheat breeding. In this paper, we considered
not only the transfer of desirable genes of rye, but also the
valuab le background of common wheat. “Xiaoyan No.6”
has been cu lt ivated in China for about twen ty years.
Through t ransferring disease resistance gene o f rye cv.
“German White” into “Xiaoyan No. 6”, we obtained some
lines with good agronomic characters and good dis ease
resistance. For example, substitution line BC97-2 presented
high resistance to the epidemic races o f stripe rust and
powdery mildew, while translocation lines BC116-1 and
BC148-1 have good agronomic characters with resistance
to s tripe rust. These lines are h igh seed-set, cytogeneti-
cally stab le. Therefore, they can be valuable materials for
wheat improvement, and can be directly used in production.
GISH is very effective for detection of alien chromatin in
wheat-rye hybrid. Using genomic DNA of rye as a probe,
the rye complete chromosome or segment in wheat-rye
addition, substitution and translocation lines could be vi-
sualized in Xiaoyan No.6 background. The breakpoints of
the wheat-rye translocation chromosomes also can be rec-
ognized eas ily. After detection of rye chromat in in these
wheat-rye hybrids, identification of individual chromosome
became more crucial. Bes ides chromosome band ing
analys is, in situ hybridization showed its special useful-
ness in individual chromosome identification. Combining
the N- or C-banding with in situ hybridization or genomic
in situ hybridization, Jiang and Gill (1993) reported identifi-
cation of the wheat-rye translocation. In practice, breeders
often first ly cons idered the induction of alien chromatin
and secondly its detailed analysis. In this case, GISH was
often proces sed in the first, but the quality of band ing
after in situ hybridization was often variable and unsatis-
factory (Hutchinson and Seal, 1983). Therefore, sequential
WANG Zhi-Guo et al.: Fluorescent In Situ Hybridization Analysis of Rye Chromatin In the Background of “Xiaoyan No.6” 441
GISH and multi-color FISH technique showed its advantage.
In our study, using s equential GISH and two-colo r FISH,
1RS/1BL wheat-rye translocation lines BC116-1, BC152-1
were verified orderly. Because not all of the hexaploid wheat
chromosomes could be discriminated by probes pSc119.2
and pAs1, its wide application in wheat cytogenetics was
limited. The trans locat ion line BC80-3 with one terminal
GISH signal (result unshown) could not be discriminated
because there was no hybridization signal of probes pSc119.
2 and pAs1 along the translocation chromosome. Even so,
the sequential GISH and FISH technique showed its suffi-
cient resolution to detect the alien chromat in and identify
interested chromosomes in wheat-rye hybrids (Nagy et al.,
2002) and it will serve as a useful way for breeding.
Acknowledgements: The first author is g rateful to the
Chinese-Hungarian Exchange Scholarship Program. Most
of the work was carried out at the Agricultural Institute of
Hungarian Academy of Sciences. We also thank Dr. ZHANG
Xue-Yong for revising the manuscript.
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