全 文 ：收稿日期:2008-05-09 作者简介:王坤波(1956-),男 ,研究员;＊通讯作者:wkbcri@cricaas.com.cn , wkbcri@hotmail.com
基金项目:国家自然科学基金(NSFC , 30170501), 国家高技术计划(“ 863” , 2003AA207051)
基于 45S rDNA 和雷蒙德氏棉 gDNA 为探针的草棉 FISH 核型研究
王坤波1＊ , 宋国立1 , 王春英1 , 刘三宏1 , 刘 方1 , 李懋学2 , 黎绍惠1 , 张香娣1 , 王玉红1
(1.农业部棉花遗传改良重点实验室/中国农业科学院棉花研究所 , 河南 安阳 455000;
2.北京大学生命科学学院 , 北京 100871)
摘要:草棉基于荧光原位杂交(FISH)的核型公式为 2n =2x =26 =16m +10sm (6 sat),
短臂和长臂的相对长度分别为 1.43 ～ 4.14和 3.34 ～ 5.18 ,染色体长度比(最长与最短染色体
的比值)是 1.63。染色体组有 6个随体 ,都定位在最后 3条染色体的短臂上 ,其中位于第 12
和第 13号染色体的随体在 DAPI和罗丹明镜像中明显可见 ,但位于第 11号染色体的随体在
DAPI 镜像中观察不到 。检测到 6个(3对)NOR信号 ,与随体同位 ,1对位于染色体端粒 ,2对
紧接着丝粒 。雷蒙德氏棉基因组 DNA(gDNA)作探针时 ,在体细胞染色体上检测到 GISH-
NOR ,其数量 、位置和大小与 45S 探针的 NOR 相同 , 说明 FISH 核型比以前常规核型(非
FISH 核型)更精确 。结合本试验室其它 FISH 资料 ,推断 A 基因组棉种在作为供体形成异源
四倍体棉种以来 ,一些串连重复序列如 rDNA 可能发生了很大变化 ,包括扩增 、易位或缺失
等。对于 D基因组特有的 GISH-NOR的一个可能解释 ,就是 D基因组棉种的 rDNA 拷贝数
远远多于 A 基因组棉种。NOR或者 GISH-NOR位点等方面的进一步研究 ,有助于探讨 rD-
NA 基因进化和功能 ,并作为一种标记应用于棉属构建染色体序号定位的物理图谱。
关键词:基于荧光原位杂交的核型;草棉;GISH-NOR;rDAN进化
中图分类号:Q343 文献标识码:A
文章编号:1002-7807(2008)04-0264-10
FISH-based Karyotype of Gossypium herbaceum Generated with 45S rDNA
and gDNA of Gossypium raimondii as Probes
WANG Kun-bo1＊ , SONG Guo-li1 , WANG Chun-y ing 1 , LIU San-hong 1 , LIU Fang1 , LI M ao-xue2 ,
LI Shao-hui1 , ZHANG Xiang-di1 , WANG Yu-hong1
(1.Key Laboratory of Cot ton Genet ic Improvement / Cotton Research Insti tute , Chinese Academy o f
A gricul tural Sciences , Ministry o f Agricul ture , Anyang , Henan 455000 , China; 2.Li f e S cience
Col lege , Peking Universi ty , Bei j ing 100871 , China)
Abstract:T he formula for FISH-based ka ry otype o f Gossypium herbaceum was as 2n =2x =26 =16
m +10 sm (6 sat), w ith the range in relat ive leng th o f short and long arms from 1.43 to 4.14 , 3.34
to 5.18 , respect ively .The ratio betw een the largest chromosomes and the smallest one w as 1.63.Six
satel li te lo ci we re mapped on short arms of the last three chromosomes.The satellites on chromo-
somes 12 and 13 w ere clearly visable in both DAPI and rhodamine/DAPI images , how eve r the satel li te
on chromosome 11 w as not detected in DA PI images.Six , in three pai rs , o f NORs w ere obse rved adja-
cent to the satel li te si tes , w ith one pai r on telomere and tw o pairs near centromeres.When gDNA
from G.raimond ii was used as probe , GISH-NORs w ere sco red in mito tic chromosomes of G.herba-
ceum w ith the same numbers , lo cations and sizes as 45S rDNA NORs.It could be therefo re concluded
that FISH-based karyo type analy ses w ere mo re detai led than previous kary otype (non-FISH).Based
on this study in conjunct ion w ith our other FISH resul ts , there might be g reat amplifications or peri-
centric inve rsions o f rDNA in modern A genome species af ter it s cont ribution to allo tet raploid origina-
棉 花 学 报 Co tton Science 2008 , 20(4):264 ～ 273
tions , o r deamplif icat ions/delet ions o f tandem repeats like rDNA in extant allo tet raploids fo llow ing
their polyploidization.An explanation to D genome specif ic GISH-NORs is that rDNA contents in D
genome species may be much more than those in A genome species.The NO Rs o r GISH-NORs herein
may faci li tate future locus-specif ic studies on rRNA gene evolution and function , and also may be use-
ful in developing physical map specif ic to chromosome o rder in Gossy pium.
Key words:FISH-based kary otype;Gossypium herbaceum ;GISH-NOR;rDAN evolution
CLC number:Q343 Document code:A
Article ID:1002-7807(2008)04-0264-10
Molecular researches on Gossy pium devel-
oped very fast including comparisons of co tton
chlo roplast genome to other plants[ 1] .FISH
(f luo re scent in situ hybridization)is an integ rat-
ed tool in cy tog enetic and molecular research ,
which has become mo re pow erful in modern bio l-
ogy sciences[ 2-3] .Karyotypes pro vides fundamen-
tal informat ion to phy sical mapping both on cy-
to logical and molecular levels , as w el l as to dis-
cussions about o rganism speciat ion , evolut ion ,
specific relationship , and even classifications.
Modern ka ry otype researches on co t ton (Gos-
sypium)init iated from 1970s by Edw ards[ 4-6] and
Edw ards et al
[ 7] .Chen Rui-yang et al[ 8] repo rted
a co t ton ka ry otype wi th original pho tograph and
made the data mo re accuracy.Molecular da ta
wi th new techniques in cot ton have provided
mo re useful info rmation to study co t ton origina-
tion , evolution , and genetic diver sity[ 9-1 3] .Spe-
cial at tention to molecular techniques in cy to ge-
netics like in situ hybridization (ISH)may be
mo re helpful and powerful to resolve research is-
sues. Plant genomic in si tu hybridization
(GISH)has been w idely used to advance studies
on po lyploid speciation , genomic constitutions ,
and specif ic relat ionships int ra- o r inter-ge-
nomes[ 14-15] .Ji et al[ 16] co rrected previous t ranslo-
cation linkages of cot ton (G.hirsutum) by
show ing meio tic f luo rescent in si tu hybridization
(FISH)of the tet raploid translo cation stocks.
Wang Kun-bo et al[ 17] pointed out , w ith a FISH-
based karyo type of G.barbadense , that at least
tw o D-sub-genome chromosomes w ere larger
than some A-sub-genome chromosomes , which
could not be dist inguished if only based on kary-
o type (i.e.non-hybridized signal) and which
disag reed w ith the typical hypothesis that all
chromosomes of A-sub-genome in tet raploid cot-
to n w ould be larger than tho se of D-sub-ge-
nome[ 18-20] .More recent ly w e detected six loci of
nucleo lar o rg anizer reg ions(NOR)in G.hirsu-
tum GISH
[ 21] .NOR is the proper ty of 45S rD-
NA , but probes could be generated f rom genom-
ic DNA (gDNA)of Gossypium species.With
tw ice FISHs to the same mito tic cell of G.her-
baceum or G.hirsutum , the number , posi tion
and size for NORs generated f rom 45S rDNA
and gDNA were identified as similar or identical.
The NOR probed by gDNA was therefo re de-
fined as GISH-NOR.Six GISH-NORs in G.
hirsutum were mapped w ith tw o on A- and four
on D-sub-genome chromosomes , respectively.
There w ere also GISH-NORs in mitotic image s
of G.raimondi i w ith its ow n gDNA as probe.
There are four cultiv ated Gossypium specie s
in the w orld.G.herbaceum (A 1A 1 o r A 1 in
brief)is one of the tw o earliest cultiva ted dip-
loids (2n=2x =26), called as Old World Cot-
to n , and now is less planted only in India and
Pakistan.However cot ton scientists are still in-
terested in it because of it s g ermplasm impo r-
tance relat ively easy to improve varieties of G.
hirsutum , a allo tet raploid (2n = 4x = 52 ,
A 1A 1D1D1)and the w o rld leading co t ton crop
now aday s , or i ts special ro le in understanding
speciation history of allotet raploids in Gossypi-
um possibly as the closest dono r to A-sub-ge-
nome chromosome of them[ 10 , 20 , 22] .On G.herba-
ceum kary otype , several papers provided data
wi th pho to graphs[ 23-25] and consistent ly mapped
tw o pairs o f satellites on last pair s of chromo-
some (11 , 12 or 13).The object o f this study
w as to obtain more detail informa tion f rom
FISH-based kary otype of G.herbaceum , mainly
2654 期 沈新莲等:棉属野生种克劳茨基棉(Gossy pium k lotzschianum)基因组向陆地棉的渐渗
fo cusing on physical mapping of satel li te , NOR ,
and even GISH-NOR.
1 Materials and Methods
1.1 Plant material , pretreatment and metaphase
preparation
Hongxin Caomian , a variety of G.herbace-
um developed in China , was used in this study.
Its ro ot tips w ere clipped into 3 ～ 6 cm leng ths
and soaked for 15 h at 4℃in saturated p-Dichlo-
robenzene solution , then f ixed overnight at room
temperature in 5-3-2 Carnoy improved by Wang
Kun-bo[ 26] .Chromosome spreads w ere prepared
as described by Wang Kun-bo[ 26] and Wang
Chun-ying e t al[ 27] .Slides w ere f rozen over liq-
uid ni trog en befo re use.A line o f G.raimondii
(D5D5 o r D5 in brief)was used as probe DNA.
All plant accessions fo r targe t chromosomes and
probe gDNAs w ere obtained from the National
Wild Co t ton Planta tion in Hainan Island , China.
1.2 Probe DNA isolation and labeling
Plasmid DNA of JDH 2-15A (a 8.2 kb XhoI
f ragment of 45S rDNA which contains almost a
full Arabidopsis rDNA repeat , which w as kindly
pro vided by Dai Si-lan and Wu Rui , Cornell Uni-
ve rsity , USA)was iso lated by alkaline ly sis as
described by Wang Chun-ying et al[ 27] .Total ge-
nomic DNA (gDNA)was iso lated f rom imma-
ture leaves of G.raimond ii using the technique
described by Song Guo-li et al[ 28] .Whole plas-
mid and total genomic DNAs w ere labeled w i th
digo xigenin-11-dUTP based on the DIG-High-
Prime Sy stem (Roche Company , German).
1.3 In situ hybridization
The procedure w as a modification of that of
Wang Chun-ying et al[ 27] .Slides were immersed
in 100 mg ·L-1 RNase in 2×SSC at 37℃ for 1 h
fo llow ed by w ashing in 2×SSC for 3×5 min(1
×SSC is 0.15 mol · L-1 NaCl , 0.015 mo l ·L-1
sodium ci t rate), then immersed in 0.01% Pep-
sin(0.01 mol·L-1 HCl)fo r 40 min at 37℃ and
rinsed for 2 ×5 min in 1 ×PBS.Afterw ard the
slides w ere immersed in 1% paraformaldehyde
(1×PBS dilute)fo r 10 min at 37℃ fo llow ed by
w ashing in 2×SSC fo r 3×5 min;then w ere de-
natured at 70℃ in 70% fo rmamide for 3 min and
dehydrated in 70%, 85%, 95%, and 100%eth-
anol fo r 2 min each at -20℃.The hybridization
mix ture solution , containing 100 ng labeled
probe DNA (per slide), 2 μg salmon spe rm
DNA (unlabelled , as block DNA), 50%de-ion-
ized fo rmamide , 10% dex t ran sulfate , 2×SSC
and 0.1% SDS , was denatured at 95℃ for 10
min , chilled on ice fo r at least 10 min , and then
applied to the air dried slide in a 20 μL volume.
The slides were covered wi th a 20 mm×40 mm
plastic membranes and put in a humidity cham-
ber in an 80℃oven fo r 10 min and allow ed to in-
cubate overnight at 37℃.
Fo llowing overnight incubat ion at 37℃,
plastic membranes we re removed and slides w ere
rinsed fo r 3×5 min at 37℃ in 2 ×SSC , slide s
we re rinsed a f inal t ime fo r 5 min in 4×SSC con-
taining 0.2% Tw een-20 at 37℃.The slide s
we re then blocked fo r 30 min at 37℃ by 5%
BSA in 4 ×SSC containing 0.2% Tween-20.
Hybridization si tes w ere detected wi th anti-dig-
oxigenin conjugated by rhodamine.The antibody
binding react ion w as carried out at 37℃ for a-
bout 1 h in 5%BSA in 4×SSC containing 0.2%
Tw een-20.The slides w ere rinsed fo r 3×5 min
at 37℃ in 5% BSA in 4×SSC containing 0.2%
Tw een-20.Slides w ere stained in 4′, 6-diamidi-
no-2-pheny lindo le (DAPI), and mounted wi th
ant i-fade so lution (Vector Labo rato ries Inc.,
Burlingame , CA , USA).
1.4 Microscopy
The hybridization signals w ere observed u-
sing a f luorescence microscope (Leica MRA2).
Images we re captured by a charge-coupled device
(CCD)sy stem (Zeiss)and brought togethe r to
make the plate using Adobe Pho to shop 7.0 sof t-
ware.
1.5 Karyotype calculations
All images w ere used to detect hybridized
signals (mainly nucleolar o rganizer reg ion ,
NOR , probed w ith 45S rDNA , o r GISH-NOR
probed w ith gDNA o f G.raimond ii)but only
metaphase images w ith better mo rpholog y of
chromosomes , especially w ith clear centromere
and be tter spreads of chromosomes w ere used in
calculations fo r kary otype.The procedure fo r
this calculation was described by Wang Kun-bo
et al[ 17 , 26] .
266 棉 花 学 报 20 卷
2 Results
To tal 39 FISH images of G.herbaceum
were reco rded w ith 22 probed from 45S rDNA
and 17 probed from gDNA of G.raimond ii.
Within 16 me taphase images , 10 and 6 probed
from rDN A and gDNA , re spectively , were ap-
plied into karyo type calcula tions.The typical
FISH images for kary otype analy sis w ere docu-
mented in Fig s.1a , 1b , 2a , and 2b.The re sults
of relativ e leng th for satellite chromosome ,
shor t arm , long arm and whole chromosome ,
and arm ra tio and type fo r each chromosome
were given in Table 1.The to tal o r average da ta
of these paramete rs , rat io of the larg est to smal-
lest chromosomes , nume ral orders of satellites
(sat), and kary otype fo rmula fo r w hole karyo-
ty pe w ere show n in Table 2.There w ere six (o r
three pai rs o f) satellites lo cating on the last
three homologous chromosomes wi th an average
relative leng th of 1.17.The sho rt and long arms
fo r G.herbaceum varied in relativ e leng th f rom
1.43(Chr.13)to 4.14 (Chr.1), 3.34 (Chr.
10)to 5.18(Chr.1), with averages of 2.97 and
4.45 , respectively.Arm ratios varied f rom 1.16
(No.10)to 2.98 (No.13)w ith an average of
1.61.Acco rding to the arm ratio s , chromo-
somes in G.herbaceum were divided as 16 m and
10 sm.The largest and smallest chromosomes in
relative leng th w ere 9.32 and 5.71 (in w hich
satel li tes we re not included)with an average of
7.42 and the rat io betw een them was 1.63.The
karyotype fo rmula for the species was as following:
2n =2x =26 =16 m +10 sm (6 sat).
Table 1 Relative length, arm ratio and chromosome type for each chromosome of G.herbaceum
Chromosome
Satellite
chromosome
Shor t arm Long arm
Who le
chromosome＊
Arm
ratio
Chromosome
Type
1 4.14 3.64-4.44 5.18 5.03-5.33 9.32 1.25 m
2 3.75 3.48-4.04 5.03 4.66-5.64 8.77 1.34 m
3 3.74 3.26-4.06 4.69 4.29-5.11 8.43 1.25 m
4 2.72 2.48-3.14 5.35 5.29-5.48 8.07 1.97 sm
5 3.53 3.13-3.77 4.58 4.31-4.83 8.12 1.30 m
6 3.40 2.95-3.72 4.22 3.72-4.35 7.63 1.24 m
7 2.63 2.56-2.71 4.87 4.64-5.21 7.50 1.85 sm
8 3.36 3.30-3.46 3.95 3.79-4.06 7.31 1.17 m
9 2.99 2.35-3.45 3.81 3.40-4.27 6.80 1.27 m
10 2.87 2.77-2.96 3.34 3.18-3.60 6.20 1.16 m
11 0.91 0.82-1.63 2.07 1.62-2.94 4.30 3.35-4.94 6.37 2.08 sm
12 1.10 0.89-1.78 2.00 1.65-2.72 4.25 3.32-5.13 6.26 2.12 sm
13 1.51 1.43-1.57 1.43 1.36-1.52 4.27 4.09-4.65 5.71 2.98 sm
No te:＊The leng ths o f satellites we re no t included in who le chromosome leng ths.
Table 2 Major characteristics of FISH-based karyotype of G.herbaceum
Re lativ e leng th
Satellite Sho rt arm Long arm Whole
A rm ratio LR NOSC Karyo type fo rmula
To tal 3.51 38.65 57.84 96.49 1.63 11 , 12 , 13 2n =26 =16m +10sm(6 sat)
Average 1.17 2.97 4.45 7.42 1.61
No te:Satellites we re no t included in leng ths of w hole chromosomes;LR:Leng th ratio of the lar gest to smallest chromo-
some;NOSC:Numeral o rder o f sa tellite chromosome.
On 45S rDNA probed FISH , we obtained
to tal 22 images , including 18 metaphase and 4
interphase cells , respectively .Six rDNA loci
were observed consistent ly in 15 o f the 18 meta-
phases , accounting fo r 83.33%(Fig.1a).In the
other 3 metaphases , signal numbers w ere four o r
five (Table 3).Within the 15 6-rDNA-locus
metaphases , hybridized signals in each cell , 13
exhibited six major and o ther 2 exhibited four
major plus tw o intermediate (The signal desig-
nation w as referenced from Hanson et al[ 29] and
Ji e t al
[ 19] ).Four interphase cells of G.herbace-
um chromosomes hybridized w ith 45S rDNA
we re examined to cluster the signals mainly
wi thin nucleoli(Fig.4a), demonst rat ing the co r-
respondence of 45S rDNA within nucleolus(nu-
cleolar o rganize r regions , NORs) in nucleus.
Four visible satellites (white t riang les )were
2674 期 王坤波等:基于 45S rDNA 和雷蒙德氏棉 gDNA为探针的草棉 FIS H 核型研究
Figs.1a and 1b. FISH images of rhodamine/DAPI(1a)and DAPI(1b)from metaphase chromosomes of G.herbaceum hy-
bridized with 45S rDNA as probe , with six red hybridized signals(1a)and white triangles pointing 4 visi-
ble satellites(1b).
Figs.2a and 2b FISH images of rhodamine/ DAPI(1a)and DAPI(1b)from metaphase chromosomes of G.herbaceum hy-
bridized with G.raimondii gDNA as probe, with six red hybridized signals(2a)and white triangles point-
ing 4 visible satellites(2b).
Fig.3 Rhodamine/ DAPI image of the anaphase chromosomes of G.herbaceum hybridized with gDNA of G.raimondii as
probe, showing 12 red GISH-NORs.
Figs.4a , 4b and 4c Rhodamine/ DAPI images of the interphase chromosomes of G.herbaceum hybridized with 45S rDNA(4a)and gDNA of G.raimondii as probes(4b , 4c), showing red hybridized signals clustered in nucle-
olus(4a , 4b and 4c)and 6 red distinguishable GISH-NORs(4b).
Fig.5 A standard FISH-based karyogram of G.herbaceum, showing physical mapping to whole chromosomes of the species
and positions of NORs or GISH-NORs with red color.
All FISH images were blocked by Salmon perm DNA.Bars = 10 μm.
268 棉 花 学 报 20 卷
presented in 45S rDNA probed DAPI FISHs
(Fig .1b)and cong ruent to the NO R loci(Fig.
1a), obse rved in all 15 metaphase rDNA FISHs ,
which suggests that there may be the same loca-
tions of satel li tes as NORs on the chromosomes.
Simi larly to sa tellite locations , three pairs of 45S
rDNA NORs in G.herbaceum were mapped on
shor t arms o f chromosomes 11 , 12 and 13 wi th
one pair and tw o pairs loca ted on telomere(Chr.
11)and near centromeres (Chrs.12 and 13),
respectively (Figs.1a , 2a and 5).Signal sizes of
the 45S rDNA NORs w ere uni fo rm betw een the
loci in the same images and also the loci in differ-
ent cells.
Table 3 NORs(GISH-NORs)detected in mitotic FISHs of G.herbaceum blocked with salmon sperm DNA
Probe DNA
Number of cells with NOR
Metaphase
6major
5majo r+1
intermediate
4majo r+2
intermediate
less than 6
NORs
Interphase
Signa ls
cluste red in
nucleo lus
6 NORs
gathered in
nucleolus
Anaphase(12 majo r) To tal
45S rDNA 13 2 3 4 22
D5 gDNA 8 2 1 1 3 1 1 17
To ta l 21 2 3 4 7 1 1 39
On G.raimondii (D5)gDNA probed FISHs ,
six (three pai rs of)hybridized signals were clear-
ly visible in mitotic FISHs of G.herbaceum ,
with the locations cor responding also to the sat-
ellites(Fig s.2a , 2b), and we re therefore defined
as GISH-NOR , which w as an interesting phe-
nomenon and not previously reported in
plants[ 21] .Within the 12 FISH images probed
wi th the gDNA , 11 show ed six GISH-NORs and
1 had less than six GISH-NORs(Table 3).Fig.
3 is an anaphase spread and represents division o f
sister chromatids w ith 6 GISH-NOR loci signals
in each set.Fig s.4b and 4c present the FISH im-
age s that show clustering of the signals w i thin
nucleo li. Besides , the simul taneity betw een
GISH-NORs and NO Rs also existed in the sizes
and dist ribut ions on arms and homolog s.Fig .5
w as a standard kary og ram based on conjunct data
f rom bo th the rDNA and the gDNA probed
FISHs , with no ticing to NOR or GISH-NOR po-
sitio ns(hybridized signals , i.e.red parts)of one
pair on the terminal and tw o pair s near the cen-
t romere regions of the sho rt arms.
3 Discussions
Four satellites of G.herbaceum that w ere
very large , even longer in relative leng th than
the chromosomal short arms , were loca ted on
the last tw o chromosomes wi th bo th DA PI ima-
ges(Figs.1b , 2b)and rhodamine/DAPI image s
(Figs.1a and 2a).This result ag rees wi th previ-
ous kary otypes o f the species[ 23-25] (non-FISH
karyo type.).Tw o (one pair)satellites , mapped
in this FISH-based kary otype on chromosome
11 , we re detected in rhodamine/DAPI image s
(Fig s.1a , 2a)but not in DAPI images(Figs.1b ,
2b).These w ere no t ident ified by the former au-
thors , which demonstrates tha t FISH techno logy
sensi tivity w as able to one distinguished satel li te
(s) more ef ficient ly .The refore , FISH-based
karyo type analy ses proved more accurate
[ 2-3] .
NORs are in theo ry located betw een satellite s
and sho rt arms of chromosomes , and w e could
not observe the satelli tes on chromosome 11 if
NORs(GISH-NORs)were not detected , which
explains the reason that early karyo type paper s
consistently mapped tw o pairs of satel li tes in G.
herbaceum.
Hanson et al
[ 29]
presented six 18S-26S rDNA
FISH loci in tw o A genome species , G.herbace-
um and G.arboreum (2n =2x =26 , A 2A 2),
w ithout mapping phy sically.Therefo re , i t seems
reasonable to infer the same behavior s for 45S
and 18S-26S rDNA repeats w hile being used as
probes in FISH studies in plant.There we re fif-
2694 期 王坤波等:基于 45S rDNA 和雷蒙德氏棉 gDNA为探针的草棉 FIS H 核型研究
teen pairs o f 18S-26S rDNA loci in G.hirsutum
(A 2A 2D2D2), seven and eight pairs in respective
sub-genome A(AD)1 A(AD)1 ve rsus D(AD)1
D(AD)1 [ 16 , 29-31] .Among those loci , three pai rs
were majo r in size w ith one pair and tw o pairs lo-
cating in A- and D-sub-genomes , respective-
ly[ 16] .Four pairs of the rDNA loci w ere visual-
ized in tw o D genome species , G.raimondi i and
G.thurberi(2n=2x=26 , D1D1 .)[ 29] , which w ere
considered as the clo sest to the D-sub-genome
progenitor of the tet raploid.There w ere four
pairs for each o f the both sub-genomes mo re than
their respective diploid genome species (A or D)
and argued a number of po ssible explanations for
these resul ts , why all 18S-26S rDNA loci of G.
hirsutum were not accounted for in it s putative
diploid pro genito rs.But only three pairs o f
NORs(o r satellites)probed wi th 45S rDNA or
gDNA we re detected in all disomic species w e ex-
amined , including G.hirsutum , G.barba-
dense
[ 17 , 21] (A 2A 2D2D2), and G.mustel inum[ 32]
(A 4A 4D4D4).T he NOR sizes (almost the same
as majo r designat ion fo r six loci)and chromo-
some lo cations on the sub-genomes (tw o in A-
and four in D-sub-genomes) were nearly the
same in tw o cult ivated allotetraploids , G.hirsu-
tum and G.barbadense.However the established
data for the NO Rs w i th the same numbers , the
same chromosome posi tions(telomeres o r slight-
ly subtelomeres of sho rt arms and no t near to
centromeres)and diversity of sizes in the three
disomic allotet raploids allow ed a suggest ions ,
that perhaps no mo re sites may be lef t for NO Rs
or at least for 45S rDNA repeats in the disomic
species w e have examined , and aroused ques-
tions , including w hy NOR numbers in tetraploid
co ttons w ere the same as one o f the putative dip-
loid progeni to rs (G.herbaceum)and where the
centromere-near lo ci f rom G.herbaceum had
gone o r how the rDNA repeats f rom the A ge-
nome progeni to r play ed thei r ro les w hile o r af ter
the disomic speciation in Gossyp ium .Li Da-yong
and Zhang Xue-yong repo rted 18S-5.8S-26S rD-
NA FISH studies on a decaploid specie s , Thi-
nopy rum pont icum , and its three possible diploid
donor species[ 33] .Ten pairs o f rDNA signals
w ere located on the terminal of sho rt arms in the
decaploid , which w as the sum o f the three dip-
loids.But one pair o f the rDNA loci , sco red on
inter sti tial regions of sho rt arms in all progeni to r
species , was not obse rv ed in the decaploid ,
which leaded thei r suggestions of rDNA repeat
lo sing subsequent to the polyploid format ion.
They also pointed out that it w as almost impossi-
ble to def ine any sub-genome in the polyploid
completely identical to that in it s putative candi-
date genomes.Feldman et al.studied RFLP pat-
terns in diploid and allopo lyploid w heat using 16
low-copy , non-coding probes and concluded that
polyploidy-induce sequence eliminat ion w as a di-
rected , non-random process[ 34] .Liu et al.[ 35] mo-
ni to red RFLP fragment profiles in synthetic
tet raploids , hexaploids , octaploids , and deca-
ploids in Tri ticum and Aeg ilops using the same
probes as Feldman et al.and obtained nearly the
same results.Similarly , many studies on cot ton ,
such as ITSs wi thin rDN A repea ts by Wendel et
al.[ 9] , repetit ive DNA or dispersed repeat DNA
by Zhao et al[ 36-37] , showed the interlo cus con-
cer ted evolution , polyploid-induced genomic
change or interg enomic invasion.From our FISH
resul ts , it is most likely that there might be
many po ssibly po tential mechanisms , such as
g reat amplif icat ions o r pericent ric inver sions in
modern A genome species af ter it s contribution
to the tet raploid originations , and as deamplifica-
t ions o r deletions of tandem repeats like rDNA in
ex tant allo tet raploids following their
polyploidization[ 10 , 22] .
The number and po sition o f GISH-NORs in
G.herbaceum when probed by G.raimondii gD-
NA were the same as those of 45S rDNA NOR
but , in another study[ 21] , there w as no t any
GISH-NOR signal in mitot ic FISH of G.herba-
270 棉 花 学 报 20 卷
ceum va r.af ricanum (A1-aA 1-a , a wild form of G.
herbaceum)probed by its ow n gDNA.We re-
peated the expe riments to dif ferent phase cells
and go t the same observations (no t show n),
which indicated that the re may no t be the prob-
lem of the probe o r of the w ell hybridized signals
to other chromosome regions.Zhao et al[ 37]
show ed A 1-a mitotic chromosomes we re covered
well by a large number of dispersed repeti tive se-
quences while using as FISH probes.A n expla-
nation to the D genome GISH-NOR may be rD-
NA content in genome D more than in genome
A.Wendel et al[ 9] repor ted rDNA evolution
model in five allopo lyploid specie s and their po s-
sible diploid pro genitor s including A genome spe-
cie s w ith the sequence data f rom the internal
t ranscribed space r regions (I TS1 and ITS2).
They argued that rDNA arrays we re homogene-
ous in all allopolyploids and diploids and , based
on the concerted evolut ion conclusion , rDNA s o f
G.hirsutum were homogenized into D genome
repeat type , o r to say that D genome w as mo re
clo sely related to G.hirsutum in rDNA model
than A genome.Therefo re , comparing to A ge-
nome species , gDNA s from D genome species
may have advantages to hybridize to w hole rDNA
sequences including ITS1 and ITS2 of Gossypium
species w henever w ere used as probes or blo cks
in co t ton GISH studies.So the gDNAs from D
genome species may easily probe GISH-NORs.
Co t ton genetic linkage maps w ere well done
wi th RFLP , STS o r RSAP as molecular mark-
ers[ 38-40] .Ji e t al[ 16] const ructed translo cation
linkages of G.hirsutum in conjunction o f cy toge-
netics wi th mo lecular marke rs by show ing meio t-
ic FISH.However there w as not any phy sical
map specific to cot ton chromosomes (mito sis).
Here in this study the 45S rDNA NORs or
GISH-NORs w ere mapped on short arms of the last
three pairs of mitotic chromosomes of G.herbace-
um , indicating that they may be applied future as a
useful marker in developing physical map.
Acknowledgements:
Various aspects of this w ork w ere supported
by the National N atural Science Foundat ion of
China (G rant No.30170501)and the H igh Tech
Research &Development P lan (863 Plan , G rant
No.2003AA207051).We w ish to thank Dr.
Cheng Zhu-kuan fo r comments on the manu-
script.
References:
[ 1] IBRAHIM R I H , Azuma J , Sakamo to M.Compplete
nucleo tide sequence of the co tton (Gossypium barba-
dense L.)chlo roplast genome w ith a compara tive a-
naly sis of sequences among 9 dico t plants[ J] .Genes
Genet Sy st , 2006 , 81:311-321.
[ 2] JIANG Ji-ming , Gill B S.Cur rent status and the fu-
ture o f fluo rescence in situ hybridiza tion (F ISH) in
plant g enome resea rch[ J] .Genome , 2006 , 49(9):
1057-1068.
[ 3] JIANG Ji-ming , Gill B S.Nonisotopic in situ hybrid-
ization and plant genome mapping:the first 10 yea rs
[ J] .Genome , 1994 , 37:717-725.
[ 4] EDWARDS G A.The kary otype of Gossy pium herba-
ceum L.[ J] .Caryolog ia , 1977 , 30:369-374.
[ 5] EDWARDS G A.Genomes o f the Australia wild spe-
cies of co tton I.Gossypium sturtianum , the standard
for the C genome[ J] .Can J Genet Cyto l , 1979 , 21:
363-366.
[ 6] EDWARDS G A.The ka ryo type of Gossy pium spe-
cie s[ C] // Proceeding s of Beltw ide Co tton P roduc tion
and Research Conference.Nashville:Nationd Co tton
Counlil o f America , 1979:77-79.
[ 7] EDWARDS G A , Mirza M A.Genomes of the Aus-
tralia wild species o f cotton II.The designation o f a
new G genome fo r Gossypium bick ii[ J] .Can J Genet
Cy tol , 1979 , 21:367-372.
[ 8] 陈瑞阳.陆地棉(Gossypium hirsutum)染色体组型的
研究[ J] .植物学报 , 1982 , 24(1)94-97.
CHEN Rui-yang.S tudies on co tten (Gossypium hir-
sutum)[ J] .Acta Bo t Sinica , 1982 , 24(1):94-97.
[ 9] WENDEL J F , Schnabel A , Seelanan T.Bidirectional
interlocus conce rted evolution following allopolyplo id
speciation in cotton(Gossypium)[ J] .P roc Na tl Acad
2714 期 王坤波等:基于 45S rDNA 和雷蒙德氏棉 gDNA为探针的草棉 FIS H 核型研究
Sci USA , 1995 , 92:280-284.
[ 10] WENDEL J F , C ronn R C.Po lyploidy and the Evo-
lutionary History of Co tton[ J] .Adavances in Ag-
r onomy , 2003 , 78:139-186.
[ 11] SMALL R L , Wende l J F.The mito chondrial g e-
nome of allo tetraplo id co tton (Gossy pium L.)[ J] .J
Heredity , 1999 , 90:251-253.
[ 12] CRONN R C , Small R L , Haselbo rn T , e t al.Rapid
diver sification of the co tton genus(Gossypium:Mal-
vaceae) revealed by analysis o f six teen nuclear and
chlor oplast genes[ J] .Am J Bot , 2002 , 89:707-725.
[ 13] ADAMS K L , Cronn R C , Percifield R , e t al.Genes
duplicated by po lyploidy show unequal contributions
to the transcriptome and o rgan-specific recipro cal si-
lencing[ J] .P roc Na tl Acad Sci USA , 2003 , 100:
4649-4654.
[ 14] TANG S , Li X , Jia X , et al.Genomic in situ hy-
bridization(GISH) analy se s o f Thinopy rum inter-
medium , its par tial amphiploid Zhong 5 , and dis-
ease-resistant de riv atives in w heat[ J] .Theor Appl
Genet , 2000 , 100:344-352.
[ 15] SNOWDON R J , Kö hler W , Friedt W , et al.Ge-
nomic in situ hybridization in Brassica amphidi p-
loids and interspecific hybrids[ J] .Theor Appl Gen-
et , 1997 , 95:1320-1324.
[ 16] JI Y , Dona to M D , Crane C F , e t al.New ribo somal
RNA gene locations in Gossypium hirsutum mapped
by meio tic F ISH[ J] .Chromosoma , 1999 , 108:200-
207.
[ 17] 王坤波 , 王文奎 , 王春英 , 等.海岛棉原位杂交及核
型比较[ J] .遗传学报 , 2001 , 28(1):69-75.
WANG Kun-bo , Wang Wen-kui , Wang Chun-ying ,
e t al.Studies of FISH and kary otype of Gossypium
barbadense[ J] .Acta Genet Sin , 2001 , 28(1):69-75.
[ 18] BEAS LEY J O.The o rig in of American tet raploid
Gossypi um species[ J] .Am Na t , 1940 , 74:285-286.
[ 19] BEASLEY J O.Meio tic chromosome behavio r in
specie s , species hybrids , haploids and induced po ly-
ploids of Gossypium[ J] .Genetics , 1942 , 27:25-54.
[ 20] ENDRIZZ I J E , Turco tte E L , Kohel R J.Genetics ,
cy tolog y , and evolution of Gossypium[ J] .Adv Gen-
et , 1985 , 23:271-375.
[ 21] 刘三宏 ,王坤波 ,宋国立 , 等.棉花 GISH-NOR 的初
步探讨[ J] .科学通报 , 2005 , 50(5):425-429.
LIU San-hong , Wang Kun-bo , Song Guo-li , et al.
Primary investiga tion on GIS H-NOR in co tton[ J] .
China Science Bulletin , 2005 , 50(5):425-429.
[ 22] WENDEL J F.Genome evolution in po lyploids[ J] .
Plant Mo lecular Bio lo gy , 2000 , 42:225-249.
[ 23] 聂汝芝 ,李懋学.三种野生和四个栽培棉种的核型研
究[ J] .植物学报 , 1985 , 27(2):113-121.
NIE Ru-zhi , Li M ao-xue.S tudies on kar yo types o f
three wild and four cultiva ted Gossyp ium species[ J] .
Ac ta Bot Sin , 1985 , 27(7):113-121.
[ 24] 宋 平 ,季道藩 ,许复华.三倍体载培棉科草棉和亚
洲棉的核型比较[ J] .作物学报 , 1991 , 17(2):102-
106.
SONG P ing , Ji Dao-fan , Xu Fu-hua.Comparativ e
re searches on kar yo types o f Gossypium herbaceum
and Gossypium arboretum [ J] .Acta Crop Sinica ,
1991 , 17(2):102-106.
[ 25] 王坤波 , 张香娣 , 王春英 , 等.棉属 A 染色体组的核
型研究[ J] .遗传 , 1995 , 17(4):32-34.
WANG Kun-bo , Zhang Xiang-di , Wang Chun-ying ,
et al.Karyo type s fr om A genome o f Gossyp ium[ J] .
Heredity , 1995 , 17(4):32-34.
[ 26] WANG Kun-bo.Identification of ka ryo type o f Gos-
sypium Bickii proch [ C] // Proceeding s of Wor ld
Co tton Research Conference-1.Melburne , Austr al-
ia:CSIRO , 1994:299-302.
[ 27] 王春英 , 王坤波 , 王文奎 , 等.棉花 gDNA 体细胞染
色体 F ISH 技术[ J] .棉花学报 , 1999 , 11(2):79-83.
WANG Chun-ying , Wang Kun-bo , Wang Wen-kui ,
et al.P ro to co l o f cotton F ISH of somatic chromo-
somes w ith gDNA as probes[ J] .Cotton Science ,
1999 , 11(2):79-83.
[ 28] 宋国立 , 崔荣霞 , 王坤波 , 等.改良 CTAB 法快速提
取棉花 DNA[ J] .棉花学报 , 1998 , 10(5):273-275.
SONG Guo-li , Cui Rong-x ia , Wang Kun-bo , et al.
An improved CTAB pro tocol fo r rapid ex tr ac tion o f
cotton genomic[ J] .Co tton Sci , 1998 , 10(5):273-
275.
[ 29] HANSON R E , Islam-Faridi M N , Perciv al E A , et
al.Distribution o f 5s and 18s-28s rDNA loci in a
tet raploid co tton (Gossy pium hirsutum L.)and pu-
tativ e diploid ancestor s [ J] .Chromosoma , 1996 ,
105:55-61.
[ 30] PRICE H J , Stelly D M , McKnight T D , e t al.Mo-
lecula r cy to genetic mapping o f a nucleo lar o rg anize r
region in co tton[ J] .J He redity , 1990 , 81:365-370.
[ 31] CRANE C F , Price H J , S telly D M , et a l.Identifi-
cation o f a homeo logous chromosome pair by in situ
272 棉 花 学 报 20 卷
DNA hybridization to ribosomal RNA loci in meiotoc
chr omosomes of cotton (Gossypium hirsutum)[ J] .
Genome , 1993 , 36:1022-1051.
[ 32] WU Qiong , Song Guo-li , Wang Kun-bo , e t al.U-
niqueness of the Gossypium mustelinum genome re-
vealed by GIS H and 45S rDNA FISH[ J] .Genome ,
(in pre ss).
[ 33] LI Da-yong , Zhang Xue-yong.Physical localization
o f the 18S-5.8S-26S rDNA and sequence ana ly sis of
ITS regions in Thinopy rum ponticum (Poaceae:
T riticeae):Im plication fo r concerted evolution[ J] .
Anna ls of Botany , 2002 , 90:445-452.
[ 34] FELDMAN M , Liu B , Segal G , et al.Rapid elimi-
nation of low-copy DNA sequence in po lyploid
w heat:a possible mechanism for differentiation of
homoeolog ous chromosomes [ J] .Genetics , 1997 ,
147:1381-1387.
[ 35] LIU B , Vega J M , Segal G , et al.Rapid genomic
changes in newly synthesized amphiploids of Tritic-
um and Argilops.I.Changes in low-copy non-coding
DNA sequences[ J] .Genome , 1998 , 41:272-277.
[ 36] ZHAO X P , Si Y , Hanson R E , et a l.Disper sed r e-
pe titive DNA has spread to new genomes since poly-
ploid fo rmation in co tton[ J] .Genome Res , 1998 , 8:
479-492.
[ 37] ZHAO X P , Wing R A , Paterson A H .Cloning and
characteriza tion of the majority of repetitive DNA in
co tton (Gossypium L.)[ J] .Genome , 1995 , 38:
1177-1188.
[ 38] REIN ISCH A J , Dong J , Brubake r C L , e t al.A de-
tailed RFLP map o f cotton , Gossypium hirsutum ×
Gossypium barbadense:Chromosome o rg aniza tion
and evo lution in a disomic polyplo id genome[ J] .Ge-
netics , 1994 , 138:829-847.
[ 39] L IN Zhong-xu , Zhang Xian-long , Nie Y i-chun , et
al.Construction of genetic linkage map with S RAP
in co tton[ J] .Chin Sci Bull , 2003 , 48(19):1676-
1679.
[ 40] RONG J , Abbey C , Bow ers J E , e t al.A 3347-locus
genetic recombina tion map of sequence-tag ged site
rev eals features of g enome o rg aniza tion , tr ansmis-
sion and evo lution o f co tton (Gossypium)[ J] .Ge-
ne tics , 2004 , 166:389-417. ○
2734 期 王坤波等:基于 45S rDNA 和雷蒙德氏棉 gDNA为探针的草棉 FIS H 核型研究