全 文 ：Received 6 Jan. 2004 Accepted 16 Apr. 2004
Supported by the National Natural Science Foundation of China (39870423).
* Author for correspondence. Tel: +86 (0)27 87684505; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (6): 724-729
Cytogenetic Identification of a New Hexaploid
Coix aquatica Cyto-type
HAN Yong-Hua1, 2, LI Dong-Yu3, LI Ying-Cai4, XUE Yue-Gui2, HU Zhong-Li1, SONG Yun-Chun1*
(1. Key Laboratory of Ministry of Education for Plant Developmental Biology, Wuhan University, Wuhan 430072, China;
2. College of Life Sciences, Guangxi Normal University, Guilin 541004, China;
3. Guangxi Institute of Animal Sciences, Nanning 530001, China;
4. Institute of Germplasm Resources, Guangxi Academy of Agricultural Sciences, Nanning 530007, China)
Abstract: A new Coix aquatica Roxb. cyto-type, found in the southwest of Guangxi Zhuang Autonomous
Region in China, was identified and analyzed by acetocarmine squashing and fluorescence in situ
hybridization (FISH) technique including genomic in situ hybridization (GISH). This new C. aquatica
cyto-type was different from other C. aquatica cyto-types reported in chromosome structure. It was 2n =
30 in chromosome number, and ten bivalents plus ten univalents were formed in meiotic prophase and
metaphaseⅠ. GISH results indicated that its 20 chromosomes were highly homologous to chromosomes
of allotetraploid species C. lacryma-jobi L. (2n = 20). The 45S and 5S rDNA were respectively located on
the two different chromosomes of the C. aquatica. One of chromosomes carrying 45S rDNA showed its
chromosome shape and signal location in the C. aquatica as the same as that in C. lacryma-jobi. So did one
of chromosomes carrying 5S rDNA. It could be deducted that one of parental candidates for the new C.
aquatica cyto-type may be C. lacryma-jobi. The other parental candidate may be allooctopolyploid C. aquatica
(2n = 40), which is similar to the new cyto-type of C. aquatica in aquatic habit and plant morphology.
Key words: Coix aquatica ; rDNA; fluorescence in situ hybridization (FISH); genomic in situ
hybridization (GISH)
The genus Coix, belonging to Maydeae, is the closest
group to the genus Zea after Tripsacum. Unlike Tripsacum
and Zea, native to America, Coix is of Asia origin (Arora,
1977). It consists of four species with different levels of
ploidy, diploids (2n = 10), tetraploids (2n = 20), hexaploids
(2n = 30) and octoploids (2n = 40) on the basic chromosome
number x = 5 (Clayton, 1981). Tetraploids (2n = 20) predomi-
nate while others are less frequent (Christopher and Singh
Thaya, 1986; Christopher and Benny, 1990). A few cases of
aneuploids have also been reported (Barve and Sapre, 1986).
The C. aquatica species reported include diploid 2n = 10
(Koul, 1965), tetraploid 2n = 20 (Venkateswarlu and Rao,
1976) and octoploid 2n = 40 (Christopher et al., 1989) three
cyto-types. They are all fertile. However, a sterile C.
aquatica was found in the southwest of Guangxi Zhuang
Autonomous Region in China (Lu and Zuo, 1996). It is a
highly stabilized and perennial species and resides in
aquatic environment. It resembles the C. aquatica species
reported in both ecological habit and plant morphology,
but propagate only by vegetative reproduction.
In plants, genomic in situ hybridization (GISH) could
provide important information about the genomic
constitutions and origins of polyploid plant species (Chen
et al., 1998; Tang et al., 2000). And it is also an alternative
tool for the direct characterization of genomic relationships
in intergeneric and interspecific hybrids (Jacobsen et al.,
1995; Snowdon et al., 1997). Therefore, in this study we
adopted GISH techniques to identify genomic constitutions
of the new C. aquatica and its relationship with the culti-
vated species, C. lacryma-jobi. The 45S and 5S rDNA se-
quences were important markers to understand genomic
evolution and taxonomic relationships. The 45S rDNA was
also the most important marker recognizing NOR or satellite
chromosomes (Jiang and Gill, 1994). In order to get more
information for origin and evolution of the sterile C. aquatica,
we also mapped its 45S and 5S rDNA by dual-color FISH.
1 Materials and Methods
1.1 Plant materials and chromosome preparation
Plants of Coix lacryma-jobi L. and the sterile C. aquatica
were kindly supplied by Prof. XUE Yue-Gui in College of
Life Sciences, Guangxi Normal University, and Prof. LI Ying-
Cai in Institute of Germplasm Resources, Guangxi Acad-
emy of Agricultural Sciences, respectively. Actively grow-
ing root-tips and young anthers during meiosis were fixed
and then squashed with aceto carmine. For FISH studies,
HAN Yong-Hua et al.: Cytogenetic Identification of a New Hexaploid Coix aquatica Cyto-type 725
actively growing root-tips treated with saturated
a-bromonaphthalene at 25 ℃ for 3 h and young anthers
were fixed in ethanol-acetic acid (3:1) at 4 ℃ overnight.
After a wash in distilled water, the root tips and anthers
were treated with a mixture of 2% pectinase (Yakult Honsha
Co., Ltd, Tokyo, Japan) and 2% cellulase (Yakult Honsha
Co., Ltd) at 28 ℃ for about 3 h. Finally, they were squashed
on slides and dried over a flame (Song and Gustafson, 1995).
1.2 DNA probes and labelling
Genomic DNA from C. lacryma-jobi and the 45S and 5S
rDNA cloned in the vector pUC18 (Arumuganthan et al.,
1994) were used as probes. The extraction of genomic DNA
was performed using the procedure described by Xu et al.
(1991). The probes were labeled with biotin and/or
digoxigenin, respectively, following the nick translation
protocol detailed in the kit (Sino-American Biotechnology
Company, Luoyang City, China). Dot blots were performed
to detect the labeling efficiency.
1.3 In situ hybridization and fluorescence detection
Hybridization was performed as outlined by Song and
Gustafson (1995), with an additional 5 µg/mL pepsin treat-
ment for 10 min. Biotinylated probes were detected with
streptavidin-Cy3 (Kirkegaard Perry Laboratories), followed
by biotinylated streptavidin (Vector Laboratories) and fi-
nally with streptavidin-Cy3. Digoxigenin-labeled probes
were detected with anti-digoxigenin-FITC (fluorescein
isothiocyanate; Boehringer Mannheim) and amplified by
rabbit anti-sheep-FITC (Vector Laboratories). Each detec-
tion step took place at 37 ℃ for 30 min and slides were
washed with phosphate buffered saline between each step.
After immunological reactions, the slides were counter-
stained with 1 µg/mL DAPI. Chromosomes were examined
with an Olympus BX60 fluorescence microscope equipped
with Sensys 1401E cooled CCD camera. Red, green, and
blue images were captured in black and white with G, B, and
UV exciter filters, respectively. The images were combined
and pseudo-coloured using software V++ (Digital Optics,
Auckland, New Zealand). Images were processed using
Adobe Photoshop software.
2 Results
By using aceto carmine squashing, it was indicated that
somatic chromosome number of the sterile C. aquatica was
2n = 30 (Fig.1A). The chromosomes ranked in relative length
from 4.52 to 2.58 with 25 m-type and five sm-type of
chromosomes. The karyotype was analyzed and it belonged
to the category 2A. Table 1 summarizes the karyotype de-
tails of the species. Two chromosomes (fifth and sixth)
showed secondary constrictions in their short arms. Dur-
ing diakinesis and metaphase Ⅰ ten bivalents and ten uni-
valents were consistently observed (Fig.1B). The univalents
lagged during anaphase Ⅰ (Fig.1C). Therefore, the ga-
metes were sterile and no normal seeds could be produced.
Total genomic DNA from C. lacryma-jobi labeled with
biotin-11-dUTP was hybridized to both mitotic and meiotic
chromosomes of the C. aquatica without blocking. In the
observed mitotic metaphase cells, 20 chromosomes showed
intensive signals, which were as strong as those on chro-
mosomes of C. lacryma-jobi hybridized by its own total
genomic DNA (Fig.2A), and the pericentromeric regions
were the most intensive for each of the chromosomes. The
rest ten chromosomes showed much less strong signals
even though the signals were interspersed in most regions
of them (Fig.2B). GISH of meiotic metaphase Ⅰ cells indi-
cated that the chromosomes with strong signals were syn-
aptic ones, i.e. ten bivalents, while the chromosomes with
less strong signals were ten univalents (Fig.2C). It meant
that in the C. aquatica, only synaptic 20 chromosomes
Fig.1. Kayotype of the Coix aquatica. A. Mitotic metaphase, 2n = 30. B. Meiotic metaphase Ⅰ, showing ten bivalents and ten
univalents. C. Meiotic anaphase Ⅰ, the laggards are the ten univalents while the bivalents have separated to two poles.
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004726
were highly homologous to those of the allotetraploid spe-
cies C. lacryma-jobi. It could be deducted that the synap-
tic chromosomes represented 4x basic chromosome num-
ber (4x), i.e. two different genomes. While the ten univalents
represented 2x basic chromosome number and contained
another two genomes, which were different from the ge-
nomes in the ten bivalents because the univalents neither
synapsed with the bivalents nor with the chromosomes
among themselves.
We have also mapped 5S and 45S rDNA on both mitotic
and meiotic chromosomes by dual-color FISH in the C.
aquatica. In mitotic metaphase cells, the 45S rDNA se-
quences were located on two chromosomes (Fig.2D, green
signals). On one chromosome, the signals were located on
whole satellite and secondary constrition of the short arm
(Fig.2D, arrow). Compared with FISH results of 45S rDNA
in C. lacryma-jobi (Han et al., 2003), it was almost the same
as each other in chromosome shape and distribution of the
signals. On the other chromosome, the signals were only
positioned on the extended secondary constriction instead
of the satellite (Fig.2D, arrowhead). The 5S rDNA sequences
were located on another two different chromosomes (Fig.
2D, red signals). One of chromosomes carrying 5S rDNA
(Fig.2D, arrow) showed its chromosome shape and signal
location in the C. aquatica as the same as that in C.
lacryma-jobi. During meiosis metaphase Ⅰ, FISH results
indicated that signals of the 5S (red) and 45S rDNA (green)
located on the different bivalents and the two members
with signals of the same genes were homologous (Fig.2E).
Two signal spots of 5S or 45S rDNA group paired tightly
together, even a large signal spot of 5S (red) and that (green)
of 45S rDNA could be found on different bivalents during
pachytene stage (Fig.2F).
3 Discussion
Different species in Coix could hybridize naturally pro-
ducing allopolyploids provided that they were growing in
close association (Sapre et al., 1985). The allopolyploids
show strong adaptation and increased vigor. Occurrence
of allopolyploids is an important pathway of plant evolu-
tion and speciation. Based on our results and the basic
chromosome number of 5 for the genus Coix, it may be
thought that this new C. aquatica cyto-type is an
allohexaploid with 6x, which contains four different
genomes, two in diploid condition and two in haploid
condition. We putatively name this allohexaploid AABBCD.
It is evident that the C. aquatica is a population originated
from hybrids. It can be deducted that one of the parental
candidates for the C. aquatica may be C. lacryma-jobi,
because DNA of 20 chromosomes in the C. aquatica is
highly homologous to genomic DNA of C. lacryma-jobi
(putative AABB). The karyotype study also shows that
the chromosomes of the C. aquatica are similar to those of
the C. lacryma-jobi in size and morphology (Lin et al., 1985).
Moreover, one of chromosomes carrying 45S and 5S rDNA
respectively shows its chromosome morphology and sig-
nal location in the C. aquatica as the same as those in C.
lacryma- job i . Another parent i s p robably an
allooctopolyploid C. aquatica, of which the chromosomal
size and the karyotype category are also similar to those of
C. lacryma-jobi (Christopher et al., 1989) and the new C.
aquatica. It has four different basic genomes, which may
contain CC and DD besides AA and BB genomes. Moreover,
it is more similar to the new C. aquatica in aquatic habit
and plant morphology. The allooctopolyploid C. aquatica
has not been found yet in the southwest of Guangxi Zhuang
Municipalities. Whether the new C. aquatica cyto-type
Table 1 Karyotype analysis of the Coix aquatica
Chromosome Relative Arm Centromere
number length ratio position
1 4.52 1.33 m
2 4.52 1.15 m
3 4.20 1.50 m
4 4.04 1.78 sm
5 n 3.88* 1.40* m
6 n 3.88* 1.18* m
7 3.72 1.30 m
8 3.72 1.09 m
9 3.72 1.09 m
10 3.55 1.75 sm
11 3.55 1.00 m
12 3.55 1.00 m
13 3.39 1.33 m
14 3.39 1.33 m
15 3.23 1.86 sm
16 3.23 1.22 m
17 3.07 1.71 sm
18 3.07 1.38 m
19 3.07 1.38 m
20 3.07 1.11 m
21 3.07 1.11 m
22 2.91 1.25 m
23 2.91 1.25 m
24 2.91 1.00 m
25 2.75 1.43 m
26 2.75 1.13 m
27 2.58 2.20 sm
28 2.58 1.29 m
29 2.58 1.00 m
30 2.58 1.00 m
m, metacentric chromosome; n, satellite chromosome; sm, sub-
metacentric chromosome; *, with satellite.
HAN Yong-Hua et al.: Cytogenetic Identification of a New Hexaploid Coix aquatica Cyto-type 727
Fig.2. GISH with total genomic DNA from Coix lacryma-jobi and FISH with rDNA on mitotic and meiotic chromosomes of the C.
aquatica. A. Chromosomes of C. lacryma-jobi hybridized by its own total genomic DNA. B. Mitotic metaphase chromosomes of the C.
aquatica hybridized with total genomic DNA from C. lacryma-jobi, 20 chromosomes show intensive signals and ten chromosomes show
much less strong signals. C. Meiotic metaphase Ⅰ, ten bivalents with strong signals and ten univalents with less strong signals. D.
Mitotic metaphase chromosomes hybridized with the 45S and 5S rDNA. The signals of 45S (green) and 5S (red) rDNA sequences are
respectively located on two different chromosomes. E. Signals of the 5S (red) and 45S rDNA (green) located on the different bivalents.
F. A large signal spot of 5S (red) and that (green) of 45S rDNA on different bivalents during pachytene stage.
originated from Guangxi or not is unsure. C. aquatica is an
original species of the genus Coix and mainly distributes
in the origin regions of Coix plants. Therefore, some re-
searchers looked on C. aquatica as the marker species of
Coix origin regions. Southeast Asia is the origin and evo-
lution center of Coix. China is a migration passage of Coix
plants from the center to the north (Lu and Zuo, 1996). We
speculate the new C. aquatica cyto-type might originate
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004728
from Southeast Asia and spread to Guangxi Zhuang Au-
tonomous Region of China.
Because the new C. aquatica is sterile it could propa-
gate only by vegetative organs instead of seeds. The C.
aquatica is perennial, its roots can grow new buds after
stems wilt. In addition, new buds can also grow from nodes
when stems touch the ground. Plant materials of the C.
aquatica collected from ten different counties or parts in
the southwest of Guangxi Zhuang Autonomous Region
show almost the consistent genetic characteristics (Lu and
Zuo, 1996). Obviously, asexual reproduction keeps the C.
aquatica to be a stable population in chromosome comple-
ments and phenotype although it is a hybrid containing
multiple different basic genomes.
Acknowledgements: We thank Dr. K. Arumuganthan
(University of Nebraska, USA) for kindly providing the 5S
and 45S rDNA probes.
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