全 文 ：　遗 传 学 报　Acta Genetica Sinica , December 2003 , 30 (12):1147～ 1152 ISSN 0379-4172
收稿日期:2003-03-07;修回日期:2003-05-21
基金项目:国家自然科学基金项目资助(编号:30070376)[ Supported by the National Natural Science Foundation of China(No.30070376)]
作者简介:韩永华(1971-),吉林双阳人 ,博士 ,专业方向:植物分子细胞遗传学。 E-mail:hanyhzhou@sohu.com
①　通讯作者。 E-mail:songyc@whu.edu.cn;Tel:027-87684505
Quantitative Chromosome Map of Coix lacryma-jobi L.
by Imaging Methods
HAN Yong-Hua1 , 2 , QI Cui-Ying1 , LIU Li-Hua1 , SONG Yun-Chun1 , ①
(1.TheKey Laboratory of MOE for Plant Developmental Biology , Wuhan University , Wuhan　430072 , China;
2.College of Life Sciences , Guangxi Normal University , Guilin　541004 , China)
Abstract:Prometaphase chromosomes of Coix lacryma-jobi L.were quantitatively analyzed based on their distribution patterns of
DAPI signals.The DAPI signals showed prominent uneven distribution along the chromosomes.Based on the DAPI signal patterns ,
a quantitative chromosome map was constructed for the first time in C.lacryma-jobi.The quantitative chromosome map will offer
the foundation for genome analysis of C.lacryma-jobi.
Key words:Coix lacryma-jobi L.;prometaphase chromosomes;image analysis;quantitative chromosome map
通过图像分析方法作出的薏苡定量染色体图
韩永华1 ,2 , 亓翠英1 , 刘立华1 , 宋运淳1 , ①
(1.武汉大学植物发育生物学教育部重点实验室 , 武汉　430072;
2.广西师范大学生命科学学院 , 桂林　541004)
摘　要:薏苡染色体数目为 2n=20 , 由于中期染色体的长度差异不大 , 相邻染色体难以区分 , 而薏苡前中期的
染色体相对较长 , 经 DAPI染料染色后染色体上显示明显不同的深染区和浅染区 , 深染区和浅染区分别相当于染
色体上染色质纤维较浓缩和较伸展的区域。前中期染色体上染色深浅不一的染色区可以作为识别薏苡特定染色
体的重要标志 , 也可用于辨别同源染色体。用MetaMorph软件定量分析了薏苡每条前中期染色体上(从短臂到长
臂)DAPI 信号强度的变化 ,结合染色体的长度和臂比作为辅助参数 , 构建了薏苡前中期染色体的定量染色体图。
该定量染色体图不仅描述了每条染色体不同区域浓缩程度的不同 , 而且也反应出不同浓缩区域所占染色体长度的
比例 。因此该定量染色体图是实际的前中期染色体的一种直观模式 , 可作为识别薏苡基因组中每条染色体的有力
依据。
关键词:薏苡;前中期染色体;图像分析;定量染色体图
中图分类号:Q813.4　　　文献标识码:A　　　文章编号:0379-4172(2003)12-1147-06
　　The genus Coix belongs to the tribe Maydeae and
consists of the four species
[ 1] .The chromosome numbers of
this genus reported so far range from 2n=10 to 40 and
tetraploids(2n=20)predominate while others are less
frequent
[ 2] .The species C.lacryma-jobi withmany variet-
ies is a cereal of potentially great economic importance
and cultivated in several warmer parts of the world for var-
ied uses like food , fodder , medicine and ornament[ 3] .The
mitotic metaphase chromosomes of the C.lacryma-jobi
species(2n=20)studied cytologically have proved to be
hardly distinguishable due to their morphological similarity
and the lack of distinct chromosomal landmarks
[ 4] .
It is known that the use of prometaphase cells is ef-
fective for the identification of individual plant chromo-
somes because characteristics caused by the uneven con-
densation of chromatin fibers along chromosomes are more
prominent at this stage
[ 5] .So far , using the condensation
pattern(CP)of the prometaphase chromosomes , reliable
quantitative chromosome maps were constructed in rice
[ 6] ,
oilseed rape
[ 7] , three diploid Brassica species[ 8] , wild
sugarcane
[ 9] , Nicotiana tabacum[ 10] and Arabidosis
thaliana
[ 11] , in all of which complete chromosome identifi-
cation was difficult at mid-metaphase.The condensation
pattern is now considered to be a key parameter to charac-
terize small plant chromosomes and to facilitate the con-
struction of a chromosome map.
In the present research , we analyzed quantitatively
prometaphase chromosomes of C.lacryma-jobi using
MetaMorph software.A quantitative chromosome map of
C.lacryma-jobi was constructed based on the numerical
data of the DAPI signals.
1　Materials and Methods
1.1　Plant material and chromosome preparation
For chromosome preparation , Coix lacryma-jobi L.
var.frumentacea Makino ,which is a cultivated variety of
C.lacryma-jobi ,was used(supplied by professor LI Jian-
Sheng , Department of Agronomy , Huazhong Agricultural
University).The root tips cut from actively growing plants
were treated with saturatedα-bromonaphthalene under the
temperature 25℃ for 3 h and fixed immediately in etha-
nol-acetic acid (3∶1)at 4℃ for over night.After fully
washed with distilled water , the root tips were treated with
a mixture of 2% pectinase (SERVA)and 2% cellulase
(SERVA)at 28℃ for around 3 h.Finally , the treated root
tips were squashed on slides and dried over flame
[ 12] .
1.2　Image analysis
Chromosome preparations were stained with 4 6-dia-
midino-2-phenylindole (DAPI) and observed with an
Olympus BX60 fluorescence microscope equipped with
Sensys 1401E cooled CCD camera.Good chromosomes
samples at metaphase and prometaphase stages were cap-
tured in black and white with UV filter.Karyotypes were
automatically generated using Genus
TM
software from Ap-
plied Imaging Corporation.Five good prometaphase chro-
mosome plates without overlapping and with complete
chromosome numbers were analysed by MetaMorph soft-
ware using DAPI signals intensities as the key character.
Then DAPI intensities along chromosomes were converted
to numerical data for the development of quantitative chro-
mosome map.
2　Results
2.1　Chromosomes features stained with DAPI
In Fig.1 , A and B showed mitotic metaphase and
prometaphase chromosome spreads stained with DAPI from
C.lacryma-jobi.Karyotypes of metaphase and promet-
aphase chromosomes were automatically generated using
Genus
TM
software from Applied Imaging Corporation based
on chromosomal morphological characteristic and size
(Fig.1 ,C and D).The chromosome pairs were ordered in
decreasing size from the largest to the smallest.The cen-
tromeric position was clearly observed with DAPI staining.
Metaphase chromosomes of C.lacryma-jobi were evenly
stained(Fig.1 ,A and C).It was difficult to identify each
chromosome due to similarity in size and arm ratio of
chromosomes.In constrast to the metaphase chromosomes ,
prometaphase chromosomes showed obvious differences in
the distributional patterns of DAPI signals among the
chromosomes , which were depicted by intensely (light)
and faintly (dark)stained regions(Fig.1 ,B and D).The
intensely (light)and faintly (dark)stained regions by
DAPI corresponded to condensed and dispersed chromatin
fibers along chromosomes , respectively (Fig.1 , E).Based
on the distributional patterns of conspicuous DAPI signals
along chromosomes , prometaphase chromosomes of C.
lacryma-jobi could be subdivided into two classes:one of
them included chromosomes 4 ,5 ,6 ,8 and 9 with only in-
tense DAPI signals around centromere regions , the other
with intense DAPI signals in one or two chromosomal ends
besides pericentromeric regions including chromosomes 1 ,
1148 遗　传　学　报 30 卷　
2 , 3 , 7 and 10.The employment of prometaphase spreads
made it possible to identify chromosomes 1 , 2 , 3 , 4 , 6 , 7
and 10 by only visual inspection based on differences in
length and DAPI signal distributional pattern among chro-
mosomes.However , it was difficult to discriminate chromo-
somes 5 , 8 and 9 by only visual inspection due to their
similarity in morphology and distribution of DAPI signals.
Fig.1　C.lacryma-jobi chromosomes at mitotic metaphase and prometaphase stained with DAPI
A:Metaphase chromosomes stained evenly by DAPI;B:Prometaphase chromosomes stained unevenly by DAPI;
C:Karyotype of metaphase chromosomes;D:Karyotype of prometaphase chromosomes;E:Distinctive condensation
pattern of prometaphase chromosomes revealed by DAPI staining.
1149　12 期 韩永华等:通过图像分析方法作出的薏苡定量染色体图
2.2　Measument of DAPI intensities and constrution
of quantitative chromosome map
　　Distributional patterns of DAPI signal intensity along
each chromosome were quantified by MetaMorph software.
Fig.2 showed intensity profiles of DAPI signal along each
of the 10 chromosomes from Fig.1 , D.The vertical and
horizontal axes represented gray level of DAPI signal in-
tensity and the number of pixels along the chromosomes ,
respectively.
Fig.2　Intensity profiles of DAPI signal along each of the 10 chromosomes from Fig.1 ,D
The vertical and horizontal axes represented gray level of DAPI signal intensity
and the number of pixels along the chromosomes , respectively.
　　The 128 pixel on average as threshold was the
boundary between intensely and faintly stained regions in
the profiles according to Ito et al
[ 11] .Image analysis was
run on five different prometaphase plates in order to get a
1150 遗　传　学　报 30 卷　
mean value of arm relative length and DAPI pattern for
each chromosome in C.lacryma-jobi genome.Based on
the DAPI intensities along the chromosomes as the main
feature ,with chromosomal relative length and arm ratio as
supplementary parameters , the quantitative chromosome
map was constructed (Fig.3).The intensely stained re-
gions with the higher gray values were depicted as the
black regions and faintly stained regions with the gray val-
ues below 128 were depicted as the white regions in the
quantitative chromosome map.
Fig.3　Quantitative chromosome map constructed
on the basis of image analysis
Data black and white areas represent the intensely and faint ly
stained regions by DAPI (threshold=128), respectively.
Although chromosomes 5 , 8 and 9 showed similarity
in morphology and distribution of DAPI signals , they could
be discriminated by chromosomal length and arm ratio
shown in Table 1.
Table 1　Relative length and arm ratio of mitotic
prometaphase chromosomes of C.lacryma-jobi
Chromosome
Relative length
(Mean±S.D.)
Arm ratio
(Mean±S.D.)
1 15.07±0.32 1.03±0.14
2n 14.35±0.13＊ 2.60±0.09
3 12.69±0.07 2.30±0.02
4 11.15±0.21 2.82±0.11
5 10.08±0.48 1.39±0.14
6 8.54±0.15 2.22±0.15
7 8.19±0.02 1.17±0.05
8 6.76±0.37 1.02±0.23
9 6.64±0.03 1.48±0.06
10 6.52±0.05 1.46±0.04
　S.D.:standard deviation;n:satellite chromosome;＊:without satellite.
3　Discussion
Cytological studies of C.lacryma-jobi have been
carried out on metaphase chromosomes
[ 13 , 14] .In contrast ,
the use of prometaphase spreads in this study ,made the
features of each chromosome more visible.The key ele-
ment of the present research that made identification and
characterization of individual C.lacryma-jobi chromo-
somes possible was the distinctive condensation pattern
(CP)of prometaphase chromosomes revealed by DAPI
staining.The CP is revealed as a stable and reliable image
parameter , representing essential morphological character-
istics of plant chromosomes
[ 15] .
The idiogram constructed is the first quantitative
chromosome map for C.lacryma-jobi chromosomes.Every
fractional length of each part of the chromosomes was
quantitatively determined.Therefore , the chromosome map
depicts not only the most distinctive chromosome features ,
but also numerical values of fractional length of chromo-
some arms and distribution of differential condensation re-
gions.The idiogram could serve as valuable basic informa-
tion for visual identification of individual C.lacryma-jobi
chromosomes since it so closely represents the patterns of
individual chromosomes.The quantitative chromosome map
based on CP is therefore considered to be not only useful
for cytological studies but also for genomics and molecular
biology on C.lacryma-jobi.It is possible to mapping the
DNA sequences visually on the C.lacryma-jobi chromo-
somes by FISH as already shown in Brassica
[ 7]
and
rice
[ 16] .
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