全 文 ：Chromosomes and Karyotypes of Three Seagrass
Species of Hydrocharitaceae
Wenjie YANG, Jingjie DU, Bo HUANG*, Ren’en WANG, Yu ZHANG
College of Ocean, Hainan University, Haikou 570228,China
Supported by the State key Subjecet of Botany at Hainan University (071001); the
Malor Scientific Research Protect of Hainan Province, China(20080137).
*Corresponding author. E-mail: huangbohb1@163.com
Received: March 5, 2012 Accepted: April 20, 2012A
Agricultural Science & Technology, 2012, 13(6): 1175-1178
Copyright訫 2012, Information Institute of HAAS. All rights reserved Agricultural Biotechnology
Abstract [Objective] This study aimed to investigate the chromosomes and kary-
otypes of three seagrass species of hydrocharitaceae-Enhalus acoroides, Thalassia
hemprichii and Halophila minor collected from Li’an Lagoon, Hainan Province, China.
[Method] The root-tips of E. acoroides, T. hemprichii and the axillary buds of H. minor
were selected as the materials in this study. The conventional crushing method was
used to prepare the slice specimens of chromosomes, and the karyotypes of the three
species were analyzed. [Result] The chromosome numbers of E. acoroides, T. hemprichii
and H. minor were 2n=18, 18, 28, respectively. The karyotype formulas were K (2n)=18=
12m+6sm, K(2n)=18=12m+6sm and K(2n)=28=16m+8sm+4st, respectively. According to the
standard of Stebbins, the karyotypes of E. acoroides and T. hemprichii were 2A, and
that of H. minor was 2B. There was no B chromosome and satellite in these three
species. [Conclusion] According to the comparison on the karyotypes of this three
species, E. acoroides and T. hemprichii are similar in karyotypes to each other, indicat-
ing that there is close, inter-relationship between E. acoroides and T. hemprichii.
Key words Enhalus acoroides; Thalassia hemprichii; Halophila minor; Chromosome;
Karyotype
S eagrasses are the mono-cotyledons which are adapt tothe submerged marine life dis-
tributed in the shallow waters of tropi-
cal and temperate seas [1]. Seagras is
not a taxon, but an ecological group,
and there are totally 59 species of 12
genera of seagrass worldwide[2], which
belong to four families: Zosteraceae,
including Zostera, Phyllospadix and
Heterozostera; Posidoniaceae, only
including Posidonia; Cymodoceaceae,
including Cymodocea, Halodule, Sy-
ringodium, Amphibolis and Thalasso-
dendron; Hydrocharitaceae, including
Enhalus, Thalassia and Halophila. Ex-
cept for Heterozostera and Amphibo-
lis, 20 seagrass species of the other
10 genera have been found in China,
in which Zostera and Phyllospadix
mainly distributed in Liaoning, Hebei
and Shandong, and the other genera
are mainly distributed in Guangdong,
Guangxi, Hainan and Fujian[3-4].
Enhalus acoroides (L. F.) Royle is
the only species in Enhalus distributed
in Wenchang, Qionghai and Lingshui
of Hainan Province, China. Thalassia
include two species, in which Thalas-
sia hemprichii (Ehrenberg) Ascherson
is the species found in China, mainly
distributed in the coastal areas of
Guangdong, Guangxi, Hainan, Tai-
wan, Xisha and other places. Halophi-
la includes about 14 species world-
wide, and only 4 species are found in
China, in which the studied Halophila
minor (Zollinger) den Hartog is dis-
tributed in the coastal areas of
Guangdong, Hong Kong, Guangxi,
Hainan and Xisha[1].
There are reports on the chromo-
some number of E. acoroides and
T . hemprichii [ 5 ] that the chromosome
number of E. acoroides is 2n=14, and
that of T. hemprichii is 2n=18, but still
no record on their karyotype especially
on the chromosome of H. minor. In this
study, we researched the chromo-
somes and karyotypes of E. acor-oides,
T. hemp-richii and H. minor, compared
the karotypes of the three seagrass
species, with the aim to provide tech-
nical preconditions and basis for the
study on the system evolution, band-
ing pattern, genome and DNA se-
quence of seagrasses.
Materials and Methods
Materials
Experimental materials were tak-
en from Li’an Lagoon, Hainan Pro-
vince in November 2009. After sam-
pling, the root tips with the length of 1-
2 cm of E. acoroides and T. hemp-
richii, and the axillary buds of Halophila
were put in the pretreatment solution.
Experimental methods
The conventional tabletting was
used to make the tablet for micros-
copy[6]. The root tips with the length of
1 -2 cm of E. acoroides and T. hem-
prichii, and the axillary buds of
Halophila were placed in the 0.1%
colchicine seawater solution at room
temperature for 1.5 -2.0 h. the pre-
treated materials were fixed by the
Kano fixative (ethanol∶acetic acid=3 ∶1)
for 4 - 24 h, then moved to the 70%
ethanol to preserve at 0 -4 ℃ . After
rinsed by the water, the fixed root tips
were put in the 1 mol/L of HCl, dissoci-
ated in the water bath for 8-18 min at
60℃. After rinsed by the purified water
for three times, the materials were
placed on the slides. The meristematic
zones of the root tips and axillary buds
were carefully cut with a blade, stained
by the Kapo fuchsin for 15 -20 min.
conventional tabletting and frozen
tabling method were used to make the
tablets.
More than 50 cells with well split
phases were selected to count the
chromosome number. And then, five
metaphase cells with complete and
DOI:10.16175/j.cnki.1009-4229.2012.06.005
Agricultural Science & Technology
Agricultural Science & Technology Vol.13, No.6, 2012
2012
Table 1 Somatic chromosome number of the three seagrasses
Species
Distribution of diploid chromosome number Total number
of cells 2n mode
Frequency of
2n=18∥%<16 16 17 18 <27 27 28 29
E. acoroides 2 3 2 49 56 18 87.5
T. hemprichii 3 2 4 59 68 18 86.8
H. minor 3 6 67 2 75 28 85.3
clear chromosome morphologies were
selected. After photographed by the
micro digital camera, Adobe Photo-
shop was used to measure the length
and full length of each chromosome
arm, and the karyptype diagrams were
established. The karyotype standard
of Li et al. was adopted for the kary-
otype analysis[7], and the relative length,
arm ratio and type of chromosomes
were named according to the naming
system of Levan et al [ 8 ] . The kary -
otypes were classified according to the
standard of Stebbins [9]. The chromo-
somal asymmetry was determined ac-
cording to the Karyotype asymmetric
coefficient (As K%) of the ratio of full
arm length to the total length of the
whole chromosome groups proposed
by Arano[10].
Results and Analysis
Chromosome number
The 2n modes of the 3 seagrass
species were all over 85%, therefore, it
could be affirmed that the diploid chro-
mosome numbers of E. acoroides, T.
hemprichii and H. minor were 2n=18,
2n=18, 2n=28, respectively.
The somatic chromosome mor-
phology of E. acoroides was shown in
Fig.1a, karyotype of Fig.1b, karyotype
model of Fig.2, and karyotype analysis
parameters were shown in Table 2.
According to the measurements, the
No. 5, 7, 9 pairs of the root tip somatic
chromosomes of this seagrass spe-
cies were the subcentric chromo-
somes (sm), and the other six pairswere
metacentric chromosomes (m). The
chromosome karyotype formula was
2n=18=12 m+6 sm. Its brachial index
(N.F.) was 36, and arm ratio varied
from 1.052 to 2.455. The ratio of the
longest to the shortest chromosome
was 1.81. There were three pairs of
chromosomes with the arm ratio of
greater than 2, accounting for 0.33,
and they were the 2A type in Stebbins.
The relative length variation ranged
from 7.511% to 13.622% , and the
composition of relative length was
8M2+8M1+2S. The variation range of
centromere index was 28.932-48.811.
The Karyotype asymmetric coefficient
(As·K) was 59.72.
The somatic chromosome mor-
phology of T. hemprichii was shown in
Fig. 1c, karyotype of Fig. 1d, karyotype
model of Fig. 3, and karyotype analy-
sis parameters were shown in Table 2.
According to the measurements, the
No. 5, 8, 9 pairs of the root tip somatic
chromosomes of this seagrass species
were the subcentric chromosomes
(sm), and the other six pairs were
metacentric chromosomes (m). The
chromosome karyotype formula was
2n=18=12m+6 sm. Its brachial index
(N.F.) was 36, and arm ratio varied
from 1.036 to 2.066. The ratio of the
longest to the shortest chromosome
was 1.71. There were two pairs of
chromosomes with the arm ratio of
greater than 2, accounting for 0.22,
and they were the 2A type in Stebbins.
The relative length variation ranged
from 8.274% to 14.179% , and the
composition of relative length was 2L+
6M2+8M1+2S. The variation range of
centromere index was 32.611-49.105.
The Karyotype asymmetric coefficient
(As·K) was 62.55.
The somatic chromosome mor-
phology of H. minor was shown in
a-b, the chromosomes and karyotypes of E. acoroides; c-d, the chromosomes and kary-
otypes of T. hemprichii; e-f, the chromosomes and karyotypes of H. minor.
Fig.1 The chromosomes and karyotypes of the three seagrass species
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2012
Table 2 The Karyotypic Parameters of the three species
Species Num-ber Relative length∥% (S+L=T )
Index of
relative
length
Arm ratio
(Long/Short)
Cen-
tromere
index∥%
Type
E. acoroides 1 8.023 5.599 13.622 1.226 1.438 41.104 m
2 7.483 5.951 13.434 1.209 1.262 44.298 m
3 6.725 6.039 12.764 1.149 1.118 47.313 m
4 7.348 5.202 12.550 1.130 1.413 41.448 m
5 7.549 3.178 10.727 0.965 2.408 29.630 sm
6 5.576 4.630 10.206 0.919 1.207 45.356 m
7 7.077 2.881 9.958 0.896 2.455 28.932 sm
8 4.736 4.516 9.252 0.833 1.052 48.811 m
9 5.222 2.289 7.511 0.676 2.358 30.479 sm
T. hemprichii 1 7.217 6.963 14.179 1.276 1.036 49.105 m
2 7.614 6.032 13.646 1.228 1.262 44.203 m
3 6.836 6.007 12.843 1.156 1.138 46.772 m
4 6.497 5.195 11.692 1.052 1.251 44.428 m
5 6.997 3.418 10.415 0.937 2.047 32.819 sm
6 5.347 4.831 10.178 0.916 1.107 47.465 m
7 5.838 3.959 9.797 0.882 1.474 40.415 m
8 6.049 2.927 8.976 0.808 2.066 32.611 sm
9 5.364 2.910 8.274 0.745 1.843 35.174 sm
H. minor 1 7.895 2.370 10.265 1.437 3.330 23.092 st
2 5.963 4.148 10.110 1.415 1.438 41.023 m
3 7.268 2.419 9.687 1.356 3.005 24.967 st
4 6.422 2.505 8.926 1.250 2.564 28.060 sm
5 5.800 2.746 8.546 1.196 2.113 32.127 sm
6 4.918 3.531 8.450 1.183 1.393 41.794 m
7 4.606 3.755 8.361 1.171 1.227 44.906 m
8 3.481 2.725 6.206 0.869 1.277 43.913 m
9 3.379 2.444 5.823 0.815 1.383 41.968 m
10 3.719 1.863 5.582 0.782 1.996 33.379 sm
11 2.751 2.408 5.159 0.722 1.142 46.683 m
12 2.616 2.107 4.723 0.661 1.242 44.605 m
13 2.756 1.331 4.087 0.572 2.070 32.568 sm
14 2.365 1.709 4.074 0.570 1.384 41.942 m
Fig.1e, karyotype of Fig.1f, karyotype
model of Fig.4, and karyotype analysis
parameters were shown in Table 2.
According to the measurements, the
No.1, 3 pairs of the axillary bud somat-
ic chromosomes of this seagrass
species were the acrocentric chromo-
somes (st), No.4, 5, 10, 13 pairs were
the subcentric chromosomes (sm),
and the other eight pairs were meta-
centric chromosomes (m). The chro-
mosome karyotype formula was 2n =
18=16 m+8 sm+4 st. Its brachial index
(N.F.) was 52, and arm ratio varied
from 1.142 to 3.330. The ratio of the
longest to the shortest chromosome
was 2.52. There were five pairs of
chromosomes with the arm ratio of
greater than 2, accounting for 0.36,
and they were the 2B type in Stebbins.
The relative length variation ranged
from 4.074% to 10.265% , and the
composition of relative length was 6L+
8M2+6M1+8 S. The variation range of
centromere index was 23.092-46.683.
The Karyotype asymmetric coefficient
(As·K) was 63.94.
Discussion
According to the observation of
this experiment, the somatic chromo-
somes number of E. acoroides is 18,
while the previously reported number
is 14[5]. There are two reasons for the
inconsistent chromosome number of
the same species[11], namely, hybrid-
ization and environmental factors .
As for E . acoroides, there is only
one reason for the occurrence of such
phenomenon. There is no report on
the intergeneric hybridization in Hy-
drocharitaceae, and the intergeneric
hybridization is not common in the bio-
sphere, therefore, the chromosome
variation of E. acoroides is most prob-
ably caused by the environmental fac-
tors in the waters. However, the sam-
pling environment and specific ex-
perimental methods were not men-
tioned in the previous reports, further
research is needed to determine the
specific environmental factor for the
chromosome number variation, and
the variation pattern of E. acoroides
chromosome. The chromosome num-
ber of T. hem-prichii is 18, consistent
with the previous studies. The somatic
chromosome number of H. minor is
28, but there is no record of this
species. The chromosome number in
existed reports of some plants in
Halophila (H. stipulacea, H. ovalis,
H. ovata) are 18, but there is no
record on the karyotype , therefore ,
specific comparative analysis cannot
be conducted .
There is no B chromosome and
satellite in the somatic cells of the
three seagrass species. The compar-
ative analysis on the karyotypes of the
somatic chromosomes of the three
seagrass species show that the simi-
larity between the chromosome kary-
otype features of E. acoroides and
T. hemprichii is great, showing the
same chromosome number, karyotype
composition and karyotype, and the
ratios of longest chromosome to
shortest chromosome is also very
close; the chromosome karyotype
feature of H. minor is significantly dif-
ferent from that of E. acoroides and
T. hemprichii. The chromosome num-
ber of H. minor is 28, more than that E.
acoroides and T. hemprichii. H. minor
Fig.2 Idiogram of E. acoroides
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Agricultural Science & Technology Vol.13, No.6, 2012
2012
水鳖科三种海草的染色体及其核型的研究
杨文杰，都晶洁，黄 勃 *，王仁恩，张钰 （海南大学海洋学院，海南海口 570228）
摘 要 [目的]对采自海南黎安港的 3种水鳖科海草—海菖蒲（Enhalus acoroides）、泰来藻（Thalassia hemprichii）和小喜盐草（Halophila minor）的
染色体及其核型进行研究。[方法]采用海菖蒲和泰来藻的根尖及小喜盐草的腋芽为材料，采用常规压片法制备染色体玻片标本，并对其进行核型
分析。[结果]海菖蒲和泰来藻的染色体数目为 2n=18，小喜盐草的染色体数目为 2n=28，两者均未发现 B染色体和随体；海菖蒲、泰来藻和小喜盐
草的核型公式分别为 K(2n)=18=12m+6sm，K(2n)=18=12m+6sm，K(2n)=28=16m+8sm+4st；按 Stebbins的核型分类标准，海菖蒲和泰来藻的染色体属
2A类型，小喜盐草为 2B型。[结论]通过对 3种海草核型进行比较发现，海菖蒲与泰来藻的核型差距很小，而它们与小喜盐草的核型差距较大，说
明海菖蒲与泰来藻的亲缘关系更近。
关键词 海菖蒲；泰来藻；小喜盐草；染色体；核型
基金项目 海南大学植物学国家重点学科（071001）资助，海南省重大科研项目（20080137）。
作者简介 杨文杰（1986-），男，湖北荆州人，硕士研究生，研究方向：海洋生物学，E-mail: yangwenjie_@163.com。*通讯作者，教授，博士生导师，从
事海洋生物研究，E-mail: huangbohb1@163.com。
收稿日期 2012-03-05 修回日期 2012-04-20
Responsible editor: Na LI Responsible proofreader: Xiaoyan WU
Fig.4 Idiogram of H. minor
has acrocentric chromosomes, while
E. acoroides and T. hemprichii only
have subcentric chromosomes and
metacentric chromosomes. The long-
est chromosome to shortest chromo-
some ratio of H. minor is greater than
that of E. acoroides and T. hemprichi,
and its karyotype is 2A, different from
the other two seagrass species. The
somatic chromosomes of H. minor are
not as symmetrical as the other two
species. However, according to the
classification of Stebbins, 2A and 2B
are all symmetric karyotypes, the evo-
lution species of primitive plants. In ad-
dition, the experiment also clearly indi-
cates that the genetic relationship be-
tween E. acoroides and T. hemp-
richi is very close, which is consistent
with the results of the system evolution
study on the seagrasses using rbcL
gene sequences[12].
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Fig.3 Idiogram of T. hemprichii
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