全 文 ：第 27 卷 第 6 期 作　物　学　报 V o l. 27, N o. 6
2001 年 11 月 A CTA A GRONOM ICA S IN ICA N ov. , 2001
A New M ethod for the Iden tify ing Pr imary Tr isom ics of R iceΞ
CH EN Hong　Q IN R u i2Zhen
( K ey L abora tory of C rop Genetics and B reed ing , M inistry of A g ricu ltu re, Institu te of C rop B reed ing and Cu ltiva tion,
CA A S , B eij ing 100081, Ch ina)
Abstract　　T he p rim ary trisom ics of rice p lay an im po rtan t ro le in study of genet ics and
b reed ing of rice. R esearchers a lw ays iden t ify p rim ary trisom ics by the karyo type analysis of
pach tene first ly u sed by Shastry (1960). Bu t th is m ethod is no t easy to ob ta in good resu lt
becau se it needs superb sk ill. In th is study, a new w ay to iden t ify p rim ary trisom ics w as
u sed. It m akes u se of the d ifference in the quan t ity of hom o logou s DNA betw een p rim ary
trisom ics and dip lo id rice and dem on stra ted the d ifference w ith m o lecu lar m arker. T he
iden t if ica t ion resu lt by m o lecu lar m arker is the sam e as tha t by karyo type analysis. T h is
m ethod is cred ib le and qu ick, so it w ill be a new and u sefu l m ean s to iden t ify p rim ary
trisom ics.
Key words　　R ice; P rim ary trisom ics; Karyo type analysis of pach; M o lecu lar m arker
一种新的鉴定水稻初级三体的方法
陈　红　　秦瑞珍
(中国农业科学院作物育种栽培研究所, 农业部遗传育种重点实验室, 北京 100081)
提　要　水稻初级三体在水稻遗传育种中起着重要的作用。其归属鉴定通常用 Shastry (1960) 的粗线
期核型分析方法, 但这种方法在实际操作中有一定的难度, 不易得到分散好的粗线期染色体分裂相。
本研究首次尝试利用三体株和正常株同源染色体间DNA 含量的差异, 用分子标记的手段将量的差异
转为自显影带的差异原理去鉴定三体, 发现此鉴定结果与粗线期核型分析结果一致, 而此方法在实验
技术上可靠性高, 快捷, 因此是一种新的有效鉴定初级三体的方法。
关键词　水稻; 初级三体; 粗线期核型分析; 分子标记
In addit ion to m o rpho log ica l m arkers u sed to iden t ify p rim ary trisom ics of rice and
cyto log ica l ob serva t ion of the con st itu t ion of ch rom o som e, rigo rou s cyto log ica l iden t if ica t ion
is a lso needed, w e can u lt im ately confirm the adscrip t ion of addit iona l ch rom o som e, th is is
the basis of u t iliza t ion of p rim ary trisom ics. T he comm on m ethod u sed in terna t iona l to
iden t ify adscrip t ion of p rim ary trisom ics cu rren t ly is kayo type analysis of pach tene [ 1～ 3 ],
w h ich w as first developed by Shastry (1960) [ 4 ]. N um ber ch rom o som e is based on degressiveΞ Foundation item: national 863 p lan (Z16202201203). Received on: Studies on the new w ay to create p rim ary trisom ics
of rice. B iography: Chenhong (1974—) , fem ale, bo rn in Q ingdao city, Shandong p rovince. Received M. S. W e
thank p rofesso r Chuanqing Sun in Ch inese A gricu ltu ral U niversity fo r h is help.
Received on: 2001203201, A ccep ted on: 2001203223

o rder of the length of ch rom o som e in m eio sis pach tene. T he longest ch rom o som e is N o. 1,
the second is N o. 2, ana log ica lly, T he sho rtest ch rom o som e is N o. 12. How ever it is no t
easy to ob ta in w ell d ispersed ch rom o som e u sing th is m ethod. Co rrect iden t if ica t ion can on ly
be ob ta ined by com b in ing m icrograph and handcraft p lo t in kayo type analysis of pach tene.
Exp lo ring new m ethod to iden t ify p rim ary trisom ics is h igh ly needed in o rder to ra ise
efficiency to choo se p rim ary trisom ics. P rim ary trisom ics w ere iden t if ied u sing R FL P
m arkers in th is study. T he p rincip le is tha t there are 25 ch rom o som es in p rim ary trisom ics of
rice. W e suppo se tha t there are th ree ch rom atid in N o. x ch rom o som e (triva len t) , and there
are tw o ch rom atid (b iva len t) in N o. x ch rom o som e in the co rresponding dip lo id. T he DNA
quan t ity in each ch rom atid is p resum ed to be A in N o. x ch rom o som e, then DNA quan t ity in
N o. x ch rom o som e is 3A in p rim ary trisom ics and 2A in dip lo id. T he ra t io betw een them is
3∶2. T he to ta l DNA quan t ity is determ ina te, DNA quan t ity in th ree ch rom atid of p rim ary
trisom ics is m o re than tha t in tw o ch rom atid of con tro lled d ip lo id. R FL P p robes w h ich have
been loca ted in N o. x ch rom asom e w ere u sed to hyb rid ize DNA that has been digested by
enzym e. Since the streng th of the hyb rid band is in d irect ra t io to the quan t ity of DNA
fragm en t. If the rice is p rim ary trisom ics X, then the hyb rid ized band w ill be a lit t le w ider
and darker than tha t in con tro l d ip lo id p lan t. If is no t p rim ary trisom ics X, then the
hyb rid ized band w ill be the sam e as no rm al d ip lo id p lan t. T he experim en t resu lt show ed tha t
the resu lt of iden t ifying the ascrip t ion of p rim ary trisom ics by m o lecu lar m arkers is
con sisten t w ith tha t by kayo type analysis of pach tene. T he resu lt of iden t ifying the
adscrip t ion of p rim ary trisom ics by m o lecu lar m arkers w as repo rted in th is paper.
1　M a ter ia ls and M ethods
1. 1　M a ter ia ls
T risom ic p lan ts u sed in the p resen t study w ere ob ta ined from the p rogen ies of po llen
p lan ts o rig ina ted in tet rap lo id hyb rid rice.
1. 1. 1　T risom ic p lan t 99830072228 ( the p reviou s genera t ion is 912427) , narrow and deep
green leaves, ch rom o som e num ber is 2n= 2x+ 1= 25 ob served in m eiophase of po llen m o ther
cells, the kayo type analysis resu lt of pach tene show ed tha t 912427 w as trip lo28[ 5 ].
1. 1. 2 　 T risom ic p lan t 99830092224, som ew hat narrow and green leaves, ch rom o som e
num ber is 2n = 2x + 1 = 25 ob served in m eiophase of po llen m o ther cells, w h ich have
m o rpho log ica l characters of t rip lo27. Bu t the kayo type of 99830092224 w ere no t ana lyzed in
pach tene.
1. 1. 3　T risom ic p lan t 9980402324 and 9980402225, yellow ish leaves, ch rom o som e num ber is
2n= 2x+ 1= 25 ob served in m eiophase of po llen m o ther cells, the kayo type analysis resu lt of
pach tene show ed tha t 9980402225 w as trip lo25.
1. 2　M ethod
1. 2. 1　Co llect fresh grow ing leaves as m ateria ls
1. 2. 2　A b stract DNA of leaves w ith CTAB (Cetylt riethylamm on ium b rom ide) m ethod
repo rted by Roger and Bendich [ 6 ].
1. 2. 3 　 Sou thern B lo t t ing 　　 Equ ilib ra te a ll DNA sam p les fo r d igest ion to the sam e
concen tra t ion. D igest DNA sam p les w ith rest rict ion enzym e, check the quan t ity of DNA
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sam p les by runn ing and pho tograph ing an agaro se gel. R un gel on 20V and Sou thern b lo t t ing
on to H ybond2N + m em b rane.
1. 2. 4 　 P robe labelling and au to2graphy 　 　 Enhanced chem ilum ilunescence ( ECL )
un rad ioact ivity k it w as u sed in th is experim en t. T he p robes u sed com e from clones in R FL P
m ap repo rted by Sat io [ 7 ] and Ku ra ta [ 8 ] , respect ively.
i　T he p robe so lu t ion concen tra t ion w as dilu ted to 10 ngöu l. P robes w ere bo iled fo r 5
m in, t ran sfer DNA on to ice sw ift ly fo r 5 m in. L abel p robe w ith HR P ( ho rserad ish
perox idase) m arker so lu t ion and then keep it in 37℃w ater fo r 11 m in.
ii　Pu t H ybond2N + m em b rane in to p re2w arm ed (42℃) hyb rid iza t ion so lu t ion, pu t the
hyb rid iza t ion box in to a 42℃ a irshaker w ith gen t le shak ing and p rehyb rid ize fo r m o re than
one hou r.
A dd the denatu red p robe to the so lu t ion and m ix w ell. Pu t the hyb rid iza t ion box back
in to an incubato r and hyb rid ize fo r over 12 hou rs.
iii　W hen hyb rid iza t ion is com p leted, take ou t b lo t w ith a b lun t2end fo rcep s and pu t it
in to a box, then rin se b riefly once w ith 2×SSC.
W ash tw ice w ith adequate am oun t of so lu t ion I ( 6M u rea 0. 5×SSC, 0. 1% SD S )
(p rew arm ed 42℃) fo r 20 m inu tes in a 42℃ shaker w ith gen t le shak ing.
W ash tw ice w ith adequate am oun t of 2×SSC so lu t ion fo r 10 m inu tes in a shaker w ith
gen t le shak ing.
iv　L ay one b lo t in to detect ion so lu t ion, then w rap it. Pu t the w rapped b lo ts in to a film
ho lder. In sert a sheet of m edica l X2ray film betw een the b lo ts and the screen in a dark room.
Expo se the film fo r th ree hou rs .
2　Result and Ana lysis
2. 1　 Iden tif ica tion of tr isom ic plan t 9983007-2-8 by RFL P
99830072228 has the typ ica l m o rpho log ica l character of t rip lo28: tw ist ing, narrow and
deep green leaves ( F ig. 1 ). Ch rom o som e con st itu t ion is 2n = 2x + 1 = 25 ob served in
m eiophase of po llen m o ther cells (F ig. 2). T he kayo type analysis resu lt of pach tene show ed
tha t the p reviou s genera t ion 9124 w as trip lo28 (F ig. 3). F rom trip lo21 to trip lo212, on ly
trip lo27 has sim ila r m o rpho log ica l character w ith trip lo28. T h ree experim en ts w ere done w ith
99830072228.
Experim en t 1: H yb rid izing p robe C20 loca ted in N o. 7 ch rom o som e w ith DNA of
trisom ic p lan t 99830072228 and dip lo id con trast respect ively. T he resu lt show ed tha t hyb rid
bands betw een trisom ic p lan t and dip lo id w ere sam e as each o ther basica lly (F ig. 4).
Experim en t 2: H yb rid izing p robe N pb187 loca ted in N o. 8 ch rom o som e w ith DNA of
trisom ic p lan t 99830072228 and dip lo id con trast respect ively. T here w ere d ifference betw een
the tw o hyb rid bands, the m ain bands in 99830072228 are deep darker and w ider than the
band in con trast, w h ich dem on stra ted tha t 99830072228 is the trip lo28. T h is resu lt w as in
acco rdance w ith tha t by kayo type analysis (F ig. 5).
Experim en t 3: H yb rid izing p robe N pb187 w ith DNA of trisom ic p lan t 99830072228,
99830092224 and dip lo id con trast, respect ively. T he ch rom o som e con st itu t ion is 2n = 2x+ 1
5296 期　CH EN Hong et al. : A N ew M ethod fo r the Iden tifying P rim ary T risom ics of R ice　　　

= 25 in 99830092224 ob served in m eiophase of po llen m o ther cells, w h ich has the typ ica l
m o rpho log ica l character of t rip lo27. R esu lts show ed tha t there w ere d ifference betw een the
bands of 99830072228 and dip lo id con trast . bu t the tw o hyb rid bands betw een 99830092224
and no rm al con trast a re iden t ica l. T hese experim en ts fu rther p roved 99830072228 w as trip lo2
8 (F ig. 6).
F ig. 1　　T he mo rpho logical character of trip lo28 p lan t 99830072228
F ig. 2　　T he ch romo som e constitu tion of
99830072228 in m eiophase of
po llen mo ther cells
F ig. 3　　Karyo type analysis at
pachytene of 912427
　
2. 2　 Iden tif ica tion of tr isom ic plan t 998040-3-4 and 998040-2-5 by RFL P
T he ch rom o som e con st itu t ion of 9980402324 and 9980402225 are 2n = 2x + 1 = 25,
iden t if ied by m o rpho log ica l and cyto log ica l ob serva t ion. T he Kayo type analysis resu lt of
pach tene show ed tha t they w ere a ll t rip lo25. D efine the po ssib le range of t risom ic type
acco rd ing to m o rpho log ica l characters, then determ ine the trisom ic type by hyb rid izing DNA
w ith p robes loca ted in ch rom o som e in the range. T he resu lts a re F ig. 7～ F ig. 10
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F ig. 4　　Southern hybridization w ith
p robe C20 in N o. 7 ch romo som e
lane 1: T risom ic p lan t 99830072228
lane 2: D ip lo id p lan t 99830072121
lane 3: T risom ic p lan t 99830092224 F ig. 5　Southern hybridizationw ith p robe N pb187　　　 F ig. 6　　Southern hybridizationw ith p robe N pb187 inN o. 8 ch romo som e　
F ig. 7　　Southern hybridization w ith p robe
C246 in N o. 5 ch romo som e
lane 1: D ip lo id p lan t 9980402121 (contro l)
lane 2: T risom ic p lan t 9980402324
lane3: D ip lo id p lan t 9980402126 (contro l)
lane4: T risom ic p lan t 9980402225
lane5: M arker
F ig. 8　　Southern hybridization
w ith p robe N bp297 in N o. 5
ch romo som e
　
　
　
　
　　F rom F ig. 7～ 10 w e can know tha t w hen hyb rid ize w ith p robe N pb297 and C246 in N o.
5 ch rom o som e, the hyb rid ized bands in 9980402324 and 9980402225 ( lane 2 and 4) are deep
darker and w ider than tho se bands in con trast ( lane 1 and 3) , w h ich dem on stra ted tha t there
w ere m o re hom o logou s DNA than tha t in con trast p lan ts (F ig. 7 and 8). Bu t w hen w e
7296 期　CH EN Hong et al. : A N ew M ethod fo r the Iden tifying P rim ary T risom ics of R ice　　　

F ig. 9　　Southern hybridization
w ith p robe 395 in N o. 2 ch romo som e
hyb rid ized DNA w ith p robes in o ther ch rom o som e excep t fo r
the N o. 5 ch rom o som e, such as N o. 2 and N o. 11 ch rom o som e,
the hyb rid ized bands w ere the sam e dark and w ide as each
o ther (F ig. 9 and 10). T he tw o differen t t risom ic w ith sam e
m o rpho log ica l characters p lan t w ere u sed in th is experim en t,
and differen t R FL P p robes loca ted in the sam e ch rom o som e
w ere u sed. con sisten t resu lts w ere ob ta ined in these repet it ive
experim en ts, w h ich p roved 9980402324 and 9980402225 w ere
trip lo25. T h is dem on stra te aga in tha t the resu lt of iden t ifying
trisom ic p lan ts by m o lecu lar m arkers is acco rd w ith the resu lt
of kayo type analysis of pach tene.
3　D iscuss ion
It′s w ell know n tha t there are one m o re addit iona l
ch rom o som e in p rim ary trisom ics of rice than no rm al d ip lo id rice. T he ch rom o som e
con st itu t ion is 2n= 2x+ 1= 25. T here are 12 p rim ary trisom ics of rice based on the d ifference
of the addit iona l ch rom o som e. Each p rim ary trisom ics no t on ly has specia l character in
cytogenet ics, bu t a lso has d ifferen t m o rpho log ica l character.
F ig. 10　　Southern hybridization
w ith p robe G1465 in N o. 11
ch romo som e
　　T he basis of u t ilizing p rim ary trisom ics is to determ ine
the adscrip t ion of the addit iona l ch rom o som e. T he comm on
m ethod u sed in terna t iona l to iden t ify the ascrip t ion of
p rim ary trisom ics is kayo type analysis of pach tene[ 1～ 3 ] ,
how ever there are d iff icu lt ies to iden t ify the adscrip t ion of
p rim ary trisom ics by kayo type analysis of pach tene. T here
are th ree reason s: ( 1) Since ch rom o som es have no t been
overly conden sed, sta ined rela t ively sha llow and view
con trast is sm all, M ake again st to m icrography; ( 2 )
Ch rom o som e is long and often in tertw ist together, w h ich
lead to d iff icu lty in d ist ingu ish; ( 3) T he loca t ion of cen trom ere in som e ch rom o som e is
unclear, can no t to ca lcu la te the ra t io of arm. T herefo re bo th the o ld o r tender of the fixed
m ateria l and the changes in d ivision stage w ill have determ ina te effect on kayo type analysis
of pach tene. Som etim es it is d iff icu lt to ob ta in app rop ria te p ictu re of kayo type analysis of
pach tene du ring period of t im e. In addit ion, there a lso ex ist som e b ranch ing by com paring
the resu lt of kayo type analysis of pach tene repo rted by differen t researchers, especia lly in
severa l sho rter ch rom o som e. T he m ain reason lay in the d iff icu lty to co rrect ly detect the
num ber, leng th and seria l num ber of ch rom o som e sa tellite. Cheng zhukuan [ 9 ] repo rted tha t
the phenom enon of reverse appeared in com po sito r in leng th of six ch rom o som e, N o. 2, 3, 9,
10, 11 and 12. A t p resen t w hether in terna t iona l un ifo rm seria l num ber of ch rom o som e is
com p letely acco rd w ith the p rincipa l of degressive ch rom o som e length is st ill need
nego t ia t ion.
F irst ly, T he m ethod of iden t ifying p rim ary trisom ics of rice by m o lecu lar m arkers w as
829　　　　　　　　　　　　　　　　　作　　物 　　学　　报　　　　　　　　 　　　　　　　　27 卷

u sed fo r the first t im e in th is study and the resu lts w ere con sisten t w ith tha t by kayo type
analysis of pach tene, w h ich dem on stra ted tha t th is m ethod is co rrect. M o reover, iden t ifying
p rim ary trisom ics of rice by u sing m o lecu lar m arkers w ill no t p roduce argues above. Becau se
each R FL P p robe u sed in th is m ethod has been loca ted in specia l ch rom o som e, therefo re, the
resu lts a re even m o re exact ly and effect ively.
Secondly, since there is one m o re addit iona l ch rom o som e in p rim ary trisom ics of rice,
there are d ist inct d ifferences in m any characterist ics am ong p rim ary trisom ics and dip lo id,
especia lly in longer ch rom o som e. T hey develop rela t ively slow ly, th is is becau se longer
ch rom o som e w ill cau se m o re im balance in inherit than sho rter ch rom o som e. T herefo re som e
p rim ary trisom ics can be dist ingu ished in seed ing stage ( tw o to th ree leaf stage) such as
p rim ary trip lo21 and trip lo28. T rip lo22, 3, 4, 5, 6, 7 and 9 can be dist ingu ished tw o to th ree
w eek s after t ran sp lan t ing. How ever, T rip lo210 and T rip lo212 w ill be d ist ingu ished un t il
an thesis. T he ascrip t ion of p rim ary trisom ics is iden t if ied by m o lecu lar m arkers in th is
study. T he m ateria ls u sed are leaf w h ich can be taken in each genera t iona l stage, so it
develop space t im e fo r w o rk com pared w ith kayo type analysis of pach tene w h ich m u st f ix and
iden t ify cyto log ica l character in po llen m o ther cellm eio sis stage. In addit ion, severa l p rim ary
trisom ics can be iden t if ied a t one t im e, w ith h igh efficiency, co rrect ly and sho rter t im e are
needed.
T h ird ly, non2rad ioact ive p robes are u sed in th is study. T he resu lts is no t w o rse than
tha t u sed rad ioact ive iso tope labeled p robe. So th is m ethod is safe and effect ive and can
reduce m uch inconven ience tha t cau sed by rad ioact ivity.
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9296 期　CH EN Hong et al. : A N ew M ethod fo r the Iden tifying P rim ary T risom ics of R ice