全 文 ：第28卷 第3期 作　物　学　报 V ol. 28, N o. 3
2002 年5月　 315～ 320页 A CTA A GRONOM ICA S IN ICA pp. 315～ 320　M ay, 2002
RAPD M arkers from M itochondr ia l DNA Can D istinguish M ale Ster ile
and Fertile Cytoplasm s in R ice (O ryza sa tiva L. ) Ξ
HON G D e2L in1, 2, 3 　M asah iko Ich ii2
(1 College of A g riculture, N anj ing A g ricultural U niversity , N anj ing 210095, P. R. China; 2 Faculty of A g riculture, K agaw a U niversity , M iki,
K agaw a, 761- 0795, J apan)
Abstract　U sing to talDNA ofW ild abortion (WA ) type CM S line Zhenshan 97 A and itsm ain tainer Zhenshan
97 B as temp late, a 1600 bp band w as amp lified on ly in Zhenshan 97 A by O PA 12 w h ich w as screened out from
one hundred p rim ers. T he 1600 bp band w as also found in WA type L ongtepu A , Dw arf abo rtion (DA ) type
X ieqingzao A , F 1 and F 2 individual p lan ts of Zhenshan 97A öM inghui 63, but no t found in m ain tainer L ongtepu
B and X ieqingzao B , using to talDNA as temp late and O PA 12 as p rim er. T he results indicate that the 1600 bp
band amp lified by O PA 12 is a specific band of the m ale sterile cytop lasm s. U sing m itochondrial DNA
(m tDNA ) as temp late and amp lified by O PA 12, the 1600 bp band w as found in the 3 CM S lines, but no t
found in the 3 m ain tainers m en tioned above. In addition to the 1600 bp band, two o ther bands (1000 bp and
700 bp) w ere found in DA type X ieqingzao A. T hese results indicated that the 1600 bp band derives from CM S
m tDNA and can be used as a RA PD m arker to distinguish A and B p lan ts at seedling stage, and the bands
o ther than the 1600 bp can be used to distinguish WA and DA cytop lasm s.
Key words　O ry za sativa L. ; M itochondrialDNA ; RA PD m arkers; D istinguish; Cytop lasm s
CLC num ber: Q 943; S511. 032; S339. 5+ 3　　　D ocum en t code: A
线粒体DNA 由来的 RAPD 标记能鉴别水稻雄性不育和可育细胞质
洪德林1, 2, 3 　一井真比古2
(1南京农业大学农学院, 江苏南京 210095; 2 香川大学农学部, 76120795 日本国香川县三木町)
摘　要　以野败型细胞质的水稻雄性不育系珍汕97A 及其保持系珍汕97B 的总DNA 为模板, 从100个引物中筛选到
O PA 12对珍汕97A 能扩增出一条1600 bp 的特异带。用O PA 12扩增野败型细胞质的龙特浦A 及其保持系龙特浦B、矮
败型细胞质的协青早A 及其保持系协青早B、恢复系明恢63、珍汕97A ö明恢63的 F 1和 F 2个体的总DNA , 不育系、F 1和
F 2所有调查个体都有1600 bp 带, 而保持系和恢复系都没有这条带。这个结果表明O PA 12扩增的1600 bp 带是上述不育
细胞质的特异带。提取上述3对不育系和保持系的线粒体DNA 作为模板用O PA 12扩增, 结果是3个不育系都有1600bp
带而3个保持系都没有这条带; 矮败型协青早A 还另有1000bp 和700bp 两条带。这些结果表明1600bp 带源于线粒体
DNA , 可用作苗期鉴别不育系和保持系的RA PD 标记; 1600bp 以外的带可用于鉴别野败细胞质和矮败细胞质。
关键词　水稻; 线粒体DNA ; RA PD 标记; 鉴别; 细胞质
　　H ybrid rice occup ies about 50% of to tal rice
grow ing area in Ch ina[ 1 ]. In o rder to m ax im ize the
use of yield heterosis, purity of hybrid rice seeds
supp lied to farm ers m ust reach 96% or more[ 2 ].
Generally hybrid seed purity iden tification w as
conducted in H ai2nan P rovince from N ovem ber to
A p ril by p lan ting samp le seeds and inspecting them at
heading stage. A cco rding to years′data of fieldΞ Foundation item: Grant2in2aid for U niversity to U niversity Cooperative Research from the M inistry of Education, Science and Culture in
Japan (Grant No. 09045067)
Biographies: Hong De2L in (19572) , A nhui2born, P rofessor, Ph D , Genetics, B reeding, Seed P roduction and Inspection in Hybrid R ice;
M asahiko Ich ii (19452) , Hyogo2born, P rofessor, Ph D , Genetics and B reeding in R ice.
　 3 To w hom correspondence should be addressed.
Received on: 2001205223, A ccep ted on: 2001211218

in spection and research results, it w as found that the
m ajo rity of off2type p lan ts in F 1 generation of ind ica
rice w ere m ain tainer and m ale sterile p lan t[ 3 ]. T hey
com e from the seed m ade by using CM S population
con tam inated w ith m ain tainer seed, because the seed
p roduced in th is case w ill con tain real hybrid,
m ain tainer and CM S seeds. T herefo re, the purity of
CM S seed affects greatly the purity of F 1 seed [ 4 ]。
D iscrim ination of m ain tainer and CM S p lan ts, up to
now , can on ly be conducted at heading stage by
observing an thers and po llens. W e had considered to
in spect seed purity by using sho rt roo t gene[ 5 ] , but it
is no t successful yet [ 6, 7 ]. It w ill be very useful if there
is a m ethod to distinguish m ain tainer and CM S line at
seedling stage. R ecen tly, several papers repo rted the
utilization of RA PD m arkers to iden tify real and false
hybrids, to iden tify differen t hybrids and their
paren ts[ 8～ 16 ]. T heo retically, differences betw een
CM S line and its m ain tainer line ex ist in cytop lasm s.
It is w idely repo rted that CM S in rice is clo sely
related w ith m itochondria[ 17～ 20 ]. In th is paper, w e
repo rted RA PD m arkers that can distinguish
m ain tainer and CM S p lan ts w ith WA type and DA
type cytop lasm s at seedling stage.
1　M a ter ia ls and m ethods
1. 1　Plan t ma ter ia ls
WA type CM S lines Zhenshan 97A , L ongtepu
A , DA type CM S line X ieqingzao A , and their
m ain tainers Zhenshan 97B, L ongtepu B and
X ieqingzao B, resto rer line M inghui 63, F 1 and F 2
p lan ts of Zhenshan 97A öM inghui 63 w ere used in
th is study. Zhenshan 97A has been most w idely used
as fem ale paren t in hybrid rice seed p roduction since
1975. L ongtepu A has good agronom ic characters
and easy to get h igh yield of hybrids. X ieqingzao A
has good rice quality. Bo th L ongtepu A and
X ieqingzao A are also in use comm ercially. M inghui
63 and F 1 of Zhenshan 97A öM inghui 63 are the
resto rer line and hybrid that most w idely used in
Ch ina respectively. Green leaves used fo r to tal DNA
iso lation cam e from two sources. O ne w as harvested
from 142day o ld seedling population cultured in
hydropon ics. T he o ther w as harvested from
individual p lan ts cultured in po ts o r paddy field.
L eaves from individual p lan ts w ere harvested at
various grow th stages befo re heading. E tio lated
shoo ts used fo r m itochondrial DNA iso lation w ere
cultured as fo llow s. F ive hundred to one thousand
seeds of each line tested w ere sterilized and
germ inated using the sam e m ethod described as[ 5 ].
T he germ inated seeds w ere sow n and cultured on a
net float in a p lastic con tainer con tain ing tap w ater in
dark room at 28 ± 2℃ fo r 7 days. T hen the tap
w ater w as changed w ith 1ö2c K im ura′s B so lution
and cultured fo r ano ther 7 days in the sam e dark
room.
1. 2　Tota l D NA isola tion
TotalDNA con tain s nuclear DNA , m tDNA and
ch lo rop last DNA. For each samp le, 0. 5～ 0. 8 gram
of green leaves w as put in to p re2ch illed mortar and
grounded to almost pow der under the condition of
liquid n itrogen′s p resence. A fter the liquid n itrogen
evaporated, the samp le w as put in to 1. 5 mL ′s
Eppendorf tube using a spatula p re2ch illed by liquid
n itrogen. 0. 5 mL of CTAB so lution (1. 5% CTAB,
1. 05 molöL N aC l, 15 mmolöL ED TA , 75 mmolöL
T ris2HC l pH 8. 0) w as added in to the tube. A fter
m ixed w ell, incubated in w ater bath at 65℃ fo r 10
m in. 0. 5 mL of ch lo rofo rm öisoam y1 alcoho l (24÷ 1)
so lution w as added, gen tly m ixed fo r 5 m in by
inversion, cen trifuged at 13000 g fo r 4 m in at room
temperature. T he supernatan t w as co llected and
transferred to a separate Eppendorf tube. 10%
CTAB so lution (10% CTAB, 0. 7 molöL N aC l) p re2
heated at 65℃ w as put in to the supernatan t and
incubated at 65℃ fo r 5 m in. Ch lo rofo rm öisoam yl
alcoho l ( 24 ÷ 1) so lution w as added at 1 ÷ 1 ratio,
cen trifuged at 13000 g fo r 4 m in at room
temperature. T he supernatan t w as saved and
isop ropano l w as added in to it at 1÷1 ratio,
cen trifuged at 13000 g fo r 1 m in at room
temperature. T he p recip itate w as saved, 400 ΛL of 1
molöL N aC l T E so lution ( 1 molöL N aC l,
10 mmolöL T ris2HC l, 1 mmolöL N a2ED TA ) w as
added in to it and incubated in w ater bath at 55℃ fo r
613　　　　　　　　　　　　　　　　　　　　　作　　物 　　学　　报　　　　　　　 　　　　　　　　　　　　28卷

at least 3 hours to m elt the DNA. To purify the
DNA , 400 ΛL of pheno löch lo rofo rm so lution (pheno l
saturated by T E: ch lo rofo rm öisoam yl alcoho l= 1÷ 1)
w as added, cen trifuged at 13000 g fo r 4 m in at room
temperature. T he supernatan t w as saved and the
purification repeated one tim e. 1000 ΛL of abso lute
ethano lw as added and m ixed, cen trifuged at 13000 g
fo r 4 m in at 4. 0℃. T he supernatan t w as discarded,
200 ΛL of 70% ethano l w as added, cen trifuged at
13000 g fo r 4 m in at 4. 0℃. T he pellet w as dried in
desiccato r in asp irating condition fo r 40 m in. 50 ΛL
of 10% T E so lution (10 mmolöL T ris2HC l, 1 mmolö
L N a2ED TA , pH 7. 5) w as added in to the p recip itate
and m ixed w ell. 1. 5 ΛL of 100×RN ase w as added
in to the DNA so lution. T hen incubated at 55℃ fo r
60 m in. DNA quality and concen tration w ere
exam ined by electrophoresis in 1. 5% agarose
( Sigm a, type Ê : M edium EEO ) , and the
concen tration w as quan tified by comparing the
concen trations of differen t amoun t of ΚDNA (10 ngöΛL ). Fo r RA PD analysis, aliquo ts of 10 ngöΛL
DNA in w ater w ere p repared and sto red at - 20℃.
1. 3　M itochondr ia l D NA isola tion
M itochondrial DNA iso lation p rocedure w as
essen tially that of Kadow ak i et al. ( 1986 ) [ 18 ].
F ifteen to th irty gram s of the etio lated shoo ts w ere
harvested, m inced w ith scisso rs and homogen ized
using an ice2ch illed mortar and pestle. T he
homogen ization buffer ( 5 mL ög in itial w eigh t )
con tained 0. 4 molöL sucrose, 50 mmolöL T ris2HC l
pH 7. 5, 5 mmolöL N a2ED TA , 0. 1% (w öv) bovine
serum album in (BSA ) , 5 mM 22m ercap toethano l.
T he homogenate w as filtrated th rough four layers of
gauze and two layers of m iraclo th and cen trifuged fo r
10 m in at 1000 g. T he supernatan t w as co llected and
cen trifuged fo r 10 m in at 17000 g. T he pellets w ere
resuspended (0. 5 mL ög in itial w eigh t) in 50 mmolö
L T ris (pH 7. 5 ) and 0. 3 molöL sucrose. A fter
cen trifuging fo r 10 m in at 1000 g, the supernatan t
w as put in to a clean tube, M gC l2 ( final conc. 10
mmolöL ) and DNA se (10 Λgög in itial w eigh t) w ere
added, m ixed and let stand fo r 1 hour at room
temperature ( 25℃). DNA se and M gC l2 w ere
removed by underlaying the m itochondrial suspension
w ith two vo lum es of 10 mmolöL T ris (pH 7. 2) , 20
mmolöL N a2 ED TA , and 0. 6 molöL sucrose and
cen trifuging fo r 20 m in at 10000 g. Pellets w ere
resuspended and w ashed tw ice in the sam e buffer.
T he final pellet w as resuspended in lysis buffer (0. 1
mL ög in itial w eigh t ) con tain ing 50 mmolöL T ris
(pH 8. 0) , 10 mmolöL N a2 ED TA , 25 g L - 1 sodium
dodecylsulfate and 0. 3 g L - 1 P ro teinase K and
incubated l hour at 37℃. T he lysis so lution w as
dep ro tein ized by adding ammonium acetate ( final
conc. 0. 8 molöL , and equal vo lum es of pheno l and
ch lo rofo rm 2isoam yl alcoho l ( 24 ÷ 1) , m ix ing gen tly
and cen trifuging fo r 5 m in at 7000 g. T he upper
aqueous phase w as saved and the ex traction repeated
two tim es. A bso lute ethano l w as added to the last
upper phase ( final conc. 70% , vöv ) and the
m itochondrial nucleic acid w as p recip itated overn igh t
at - 20℃. Pelletsw ere co llected by cen trifugation at
8000 g fo r 10 m in, w ashed by 70% ethano l and
allow ed to dry in asp irating condition fo r 1 hour at
room temperature. T he p recip itate w as so lubilized in
10 mmolöL T ris (pH 7. 5) and 1 mmolöL N a2 ED TA
to m ake a final vo lum e equal to 0. 01 mL ög in itial
w eigh t. Q uality and concen tration of m tDNA w ere
exam ined using the sam e m ethod as m en tioned
above. T he m tDNA p reparations w ere sto red at
- 20℃.
1. 4　D NA am pl if ica tion
20 ng of to tal DNA or 2 ng of m tDNA w ere
used as temp late in a final vo lum e of 40 ΛL reaction
buffer (10 mmolöL T ris2HC l pH 8. 3, 1. 5 mmolöL
M gC l2, 50 mmolöL KC l, gelatin 0. 001% , 0. 2
mmolöL dN T P, 0. 163 ΛmolöL p rim er (Operon 10
m er) , 0. 5 U Amp li T aq Gold (DNA polym erase).
A n ex tra reaction con tain ing all the componen ts, p lus
w ater in stead of temp late DNA w as included in all
the experim en ts (called negative con tro l). T he DNA
amp lifications w ere perfo rm ed in a GeneAmp PCR
System 2400 therm al cycler ( PER K IN ELM ER ,
N orw alk CT. 06859 U SA ) under the condition: 1
cycle of 12 m in at 95℃, 45 cycles of 1 m in at 94℃,
1 m in at 37℃, 2 m in at 72℃ ( fo r denaturing,
7133期　　　　　　　　　HON G D e2L in et al. : RA PD M arkers from M itochondrialDNA Can⋯⋯　　　　　　　　　

annealing and p rim er ex tension, respectively). T he
last cycle w as fo llow ed by a final incubation fo r 5 m in
at 72℃, and the PCR p roducts w ere sto red at 4℃
befo re analysis. T he DNA amp lification p roducts
w ere analyzed by electrophoresis (using M up id M in i
Gel E lectrophoresis T rough, ADVAN CE Co. L td,
Japan) in 1. 5% agarose gels in 1×TA E buffer (40
mmolöL T ris, 19 mmolöL A cetic A cid, 1 mmolöL
ED TA , pH 8. 0) fo r 50 m in at 50V. T eh gels w ere
stained fo r 20 m in w ith eth idium brom ide ( 3 Λgö
mL ) w h ile shak ing at 50 röm in. DNA w as visualized
on a UV transillum inato r ( 312 nm , M OD EL TC2
312A , TRAN S ILLUM INA TOR, spectron ics
co rpo ration w estbury, N ew York, U S A ) and
pho tographed using Po laro id T ype 667 film.
F ragm en t length w as estim ated by comparison w ith
standard size m arkers ( 200 base pair ladder,
TA KA RA , Code 3410A ).
2　R esults
2. 1　Screen ing of RAPD markers to d istinguish A
and B a t seedl ing stage
O ne hundred of p rim ers ( O PA 012O PE20,
Operon, 10 m er. ) w ere used fo r screen ing m arkers
to distinguish Zhenshan 97 A and Zhenshan 97 B
using to tal DNA ex tracted from 142day2o ld seedlings
( population ). Tw en ty2th ree of the 100 p rim ers
amp lified no bands, 70 p rim ers amp lified bands but
no po lymorph ism betw een Zhenshan 97 A and
Zhenshan 97 B. Seven p rim ers amp lified bands
show ing po lymorph ism , but on ly one p rim er, i. e.
O PA 12 show ed stable po lymorph ism in differen t
samp les ( rep lications). In Zhenshan 97 A , p rim er
O PA 12 can amp lify a 1600 bp band w h ile in
Zhenshan 97 B the 1600 bp band can no t be
amp lified. T hen using to tal DNA ex tracted from
individual p lan t (30 p lan ts fo r Zhenshan 97 A and 30
p lan ts fo r Zhenshan 97 B ) as temp late, amp lification
results w ere the sam e as above, i. e. the 1600 bp
band w as amp lified in Zhenshan 97 A , and w as no t
amp lified in Zhenshan 97 B (F ig. 1). W hen to tal
DNA ex tracted from individual p lan t of L ongtepu A ,
L ongtepu B , X ieqingzao A and X ieqingzao B w as
used as temp late and amp lified by O PA 12, the 1600
bp band w as found in L ongtepu A and X ieqingzao A ,
but w as no t found in L ongtepu B and X ieqingzao B.
It can be seen from the results that the 1600 bp
band amp lified by p rim er O PA 12 can be used as a
RA PD m arker to distinguish WA type A and B , and
DA type A and B , at seedling stage.
2. 2　The 1600 bp band com es from ma le ster ile
cytopla sm
TotalDNA of Zhenshan 97 A , M inghui 63, F 1
and F 2 p lan ts of Zhenshan 97 A öM inghui 63 w as
used as temp late and amp lified byO PA 1 2. A s show n
in F ig. 2, the 1600 bp band w as no t found in
F ig. 1　RA PD p rofile amp lified by p rim er O PA 12, using to tal DNA as temp late 1, 15: DNA size m arker (200 bp ladder) ;
2～ 7: Individual p lants of Zhenshan 97 B; 8～ 13: Individual p lants of Zhenshan 97 A ; 14: N egative contro l
813　　　　　　　　　　　　　　　　　　　　　作　　物 　　学　　报　　　　　　　 　　　　　　　　　　　　28卷

M inghui 63, but appeared in F 1 and all F 2 p lan ts
investigated, indicating that the 1600 bp band
amp lified by O PA 12 com es from m ale sterile
cytop lasm.
2. 3　The 1600 bp band der ives from m itochondr ia l
D NA
U sing m itochondrial DNA of the th ree pairs of
A and B as temp late and amp lified by O PA 12, the
fo llow ing results w ere obtained. In Zhenshan 97 A ,
on ly the 1600 bp band appeared w h ile in Zhenshan
97 B no band w as found. In L ongtepu A , on ly the
1600 bp band w as found, w h ile in L ongtepu B on ly a
1400 bp band w as found. In X ieqingzao A , there
w ere two o ther bands (1000 bp and 700 bp) besides
the 1600 bp band, w h ile in X ieqingzao B there w as
on ly one 1000 bp band ( F ig. 3 ). T hese results
indicate that the 1600 bp band derives from m tDNA
of WA and DA type cytop lasm s, and the bands o ther
than the 1600 bp can be used to distinguish betw een
WA cytop lasm and DA cytop lasm.
F ig. 2　RA PD p rofile amp lified by p rim er O PA 12, using to tal DNA as temp late 1, 17: DNA size m arker (200 bp ladder) ;
2: Zhenshan 97 A ; 3: M inghui 63; 4: F1 p lant of Zhenshan 97 A öM inghui 63; 5～ 16: F2 p lants of Zhenshan 97 A öM inghui 63
F ig. 3　RA PD p rofile amp lified by p rim er O PA 12, using
m tDNA as temp late 1: DNA size m arker (200 bp ladder) ;
2: Zhenshan 97 A ; 3: Zhenshan 97 B; 4: Longtepu A ;
5: Longtepu B; 6: Xieqingzao A ; 7: Xieqingzao B
　　 F rom the results described above, fo llow ing
conclusions can be obtained. 1. T he 1600 bp band
amp lified by p rim er O PA 12 is a characteristic band of
CM S lines w ith WA type and DA type cytop lasm s
and it can be used to distinguish A and B p lan ts at
seedling stage. 2. T he 1600 band derived from
m itochondrial DNA of WA type and DA type m ale
sterile cytop lasm s. 3. U sing m itochondrial DNA as
temp late and amp lified by O PA 12, the bands o ther
than the 1600 bp band can be used to distinguish
betw een WA cytop lasm and DA cytop lasm.
3　D iscussion
In Zhenshan 97 A and Zhenshan 97 B , w e
repeated the DNA amp lification more than 10 tim es
by using to tal DNA ex tracted from green seedlings
and etio lated seedlings cultured at differen t dates,
and operated by 4 persons. A ll results p roves that the
1600 bp band amp lified by p rim er O PA 12 in
Zhenshan 97 A is stable. O n the basis of the these
results, green leaves from individual p lan ts in
L ongtepu A , L ongtepu B , X ieqingzao A and
9133期　　　　　　　　　HON G D e2L in et al. : RA PD M arkers from M itochondrialDNA Can⋯⋯　　　　　　　　　

X ieqingzao B w ere studied and the sam e results w ere
obtained. W hen m tDNA w as used as temp late, the
1600 bp band w as found on ly in the 3 CM S lines and
w as no t found in their m ain tainers. T herefo re the
1600 bp band amp lified by O PA 12 in the m ale sterile
lines could be used to distinguish m ain tainer and
CM S p lan ts w ith WA and DA cytop lasm s at seedling
stage.
T he genom e size of rice m tDNA is about 280
kb [ 19 ] , w h ich is m uch sm aller than that of m uclear
DNA (430 M b) [ 21 ]. W hen to tal DNA w as used as
temp late, it con tain s a very sm all amoun t of
m tDNA , In th is case, it m igh t be difficult to detect
amp lification p roduction if single copy of low
repetitive DNA sequence comp lem en tary to the
p rim er ex isted in m tDNA. W e supposed that th is
m ay be the reason w hy the 1000 bp and 700 bp
bands did no t appeared in X ieqingzao A w hen to tal
DNA w as used as temp late.
Generally 800 p lan ts fo r one samp le w h ich
rep resen ts 2. 5 tons of seed are p lan ted in H ainan
P rovince in w in ter season fo r seed purity in spection.
T he cost is about 10 U S do llars per samp le. To
inspect 800 p lan ts by using the RA PD m arker w ill
cost 800 U S do llars, w h ich is too h igh to accep t at
p resen t. T herefo re it is necessary to develop cheaper
and faster m ethod fo r the purpose, although the
m arker is useful in special cases such as seed disputes
o r seed law suits.
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