He edim
109:
9-3-97
(1988)
Ka yo ypes and meio ic beha io
o
ch omosomes in wo male s e ile
s ains
o
B assica
campes is
L.
YRJO
VIINIKKA’ and MATTI SOVER02
‘
Depa men
o
Biology, Uni e si y
o
Tu ku,
Finland
’
Plan B eeding Ins i u e, Jokioinen, Finland
VIINIKKA.
Y.
and
SOVERO.
M.
1988.
Ka yo ypesand meio ic beha io o ch omosomesin womales e ile
s ains
o
B assica
campes is
L.
-
Hewdi as
109:
93-97.
Lund, Sweden.
ISSN
00184661.
Recei ed No em-
he o,
lVX7
Male s e ili y was examined
in
backc oss amilies
o
he hyb id Diph uxis mu alis
x
B.
campes is (mu -
cy oplasm). Seg ega ion
o
s e ile and e ile plan s did no i o any
o
he Mendelian a ios. Since an ex a
ch omosome
IS
known o occu in s e ile plan s ha ing
a
simila combina ion o mu -cy oplasm and
B.
napus
nucleus, he mi o ic and meio ic ch omosomes we e analysed. Simila ly, he ch omosomes we e examined
in ano he male s e ili y sys em, in backc oss amilies
o
B.
nupus
x
B.
campes is @ol-cy oplasm). The no -
mal ch omosome numbe
(2n=20)
was de ec ed in all he s e ile
as
well
as
he e ile plan s. A e imp o e-
men s
o
he s aining echnique, all he ch omosomes we e iden i ied in some cells. Ten bi alen s we e
o med
in
meiosis and he seg ega ion
o
ch omosomes appea ed o be no mal. The isibili y
o
sa elli es was
examined a diakinesis. Va ia ion among plan s was obse ed bu no co ela ion wi h male s e ili y was
ound
Y j j
Yiinikku,
Depa men!
o
Biology,
Uni e si y
o
Tu ku,
SF-20500
Tu ku,
Finlund
A
sys em ha acili a es c oss pollina ion
is
a neces-
si y o hyb id seed p oduc ion. Cy oplasmic male
s e ili y (CMS) has p o ed o be he mos sui able
me hod in he ield scale. Hence, di e en CMS sys-
ems based
on
na i e
o
alien cy oplasms ha e been
es ed in cul i a ed
B assica
species (e.g., ERICKSON
e al.
1986;
KEMnLEe al.
1986).
One
o
hese is
mu -
cy oplasm o igina ing om he ela ed wild species
Dipio axis
mu alis. HINATA and KCINNO (1979) e-
po ed a CMS sys em wi h mu -cy oplasm and nu-
clea genome om
B. campes is
(2n=20).
On
he
o he hand, when
FAN
e al.
(1985)
c ossed
D.
mu alis
as emale pa en and
B.
nupus
(2n=38),
hey obse ed in he six h backc oss amilies o
B.
napus
ha all he male s e ile plan s had an ex a
ch omosome, while he e ile plan s had he
no -
mal ch omosome complemen
o
B.
napus.
They
concluded ha he p esencc o he ex a ch omo-
some ob iously de i ed om
D.
mu alis
is he sole
cause
o
male s e ili y in hese amilies. In he p e-
sen s udy? we ha e examined backc oss amilies
bea ing mu -cy oplasm and nuclea genome om
B. campes is.
The p opo ion
o
male s e ile plan s
a ied om
0
o
100
YO
in di e en amilies, and
no
Mendelian a ios could be con i med.
In
ano he
male s e ili y sys em,pol-cy oplasm ( om
B.
napus
c . ‘Polima’) wi h
R.
ccwzpes is
genome, he seg e-
ga ion
o
s e ile and e ile plan s was mo e egula ,
one
o
wo dominan genes es o ing he e ili y
(SOVERO unpubl.). Ka yo ypes and meiosis
o
s e ile
and e ile plan s ha e been analysed in bo h sys-
ems.
Ano he aim
o
he p esen wo k was o imp o e
he me hods used o ka yo yping he soma ic
ch omosomes
o
B assica
species. The ch omo-
somes a e small, bu size di e ences ha e been
ob-
se ed al eady by
CAICHESIDE
(1934),
ALAM
(1936)
and RicHHARiA
(1937).
The mos de ailed desc ip-
ions
o
he ch omosomes a e he pachy ene maps
o
RORHELEN (1960) and VENKATESWARLU and KAMALA
(1971). The basic ch omosome numbe in
B assica
species is hough
o
be six
on
he basis o he second-
a ily pai ed g oups
o
bi alen s in meiosis (CATCHE-
SIIE
1937). Hence,
B.
campes is
should be ega ded
as a pa ially polyploid species. Acco ding o
ROBBE-
LEN
(1960)
six basic ch omosome ypes could
be
ec-
ognized. The o mula AABCDDEFFF indica es
ha ch omosome ype A is e asomic (sa elli ed
ch omsomes
I
and
2),
B disomic (ch omosome
3),
C
disomic ( he iny ch omosome
4),
D e asomic
(ch omosomes
S
and
6),
E
disomic (ch omosome
7)
and
F
hexasomic (ch omosomes
8,9
and
10).
In
he p esen con ibu ion, he name
B.
campes-
is
L.
is used as in mos wo ks conce ning he b eed-
94
Y
VIINIKKA
AND
M
SOVERO
He edi as
109 (1988)
ing
o
his species, al hough acco ding o
OOST
(1985)
he mo e co ec name is
B.
apa
L.
on he
basis
o
he nomencla u e ules.
Ma e ial and me hods
The male s e ile s ains wi h mu -cy oplasm o igi-
na ed om a c oss be ween Diplo axis mu alis and
an Indian o ia ype cul i a ‘TL-15’
o
B.
campes-
is. Six h backc oss amilies o
‘TL-15’
we e used.
The plan s wi h pol-cy oplasm we e, simila ly, he
six h backc oss amilies
o
he hyb id be ween
B.
napus and
B.
campes is c . ‘Tobin’. In addi ion,
sp ing u nip ape c . ‘An e’ was used o es ing he
cy ological me hods.
Seeds we e ge mina ed on mois il e pape in
Pe i dishes. Mi oses could be examined a e wo
o
h ee days, bu usually he seedlings we e ans e -
ed in o pape po s illed wi h ga den soil and cul i-
a ed o a week o ob ain enough ma e ial om in-
di idual plan s. The same plan s we e used o he
examina ions o meiosis.
Roo ips we e p e ea ed acco ding o NEWELLe
al.
(1984),
i s in iced wa e o
3
h
(W’C),
he e-
a e in 2mM 8-hyd oxyquinoline sulu ion o
4
h a
18”C,
ixed in absolu e e hanol-glacial ace ic acid
(3:l)
o 24 h, and s o ed in 70
Yo
e hanol a
4°C.
Va ious o he p e ea men s ha e been es ed.
A e he ollowing p e ea men , longe and classi-
iable la e p ophase ch omosomes we e obse ed:
2mM 8-hyd oxyquinoline o
3
h a
18°C
ollowed
by ea men in enzyme solu ion
(1
Yo
pec inase,
2
YO
cellulase) o
90
min. Ma e ial was ixed and
s o ed as abo e. Young lowe s we e ixed in 6:3:1
solu ion
o
absolu e e hanol, chlo o o m and ace ic
acid, and s o ed in he ixa i e in e ige a o un il
used.
All he p epa a ions we e s ained acco ding o
he Feulgen-Giemsa double s aining me hod
(PURO
and NOKKALA
1977).
The ma e ial was ans e ed
om
70
YO
e hanol (o e nigh ) h ough
50
YO
(30
min) and 30
Yo
(5
min) e hanol in o wa e , ollowed
by a ea men in
1N
HCI
o 20 min a oom emp-
e a u e and hyd olysis in
1N
HCI a
60
“C o
8
min,
s ained wi h dilu e Schi ‘s eagen (200 mi boiling
wa e pou ed on lg basic uchsin, il e ed and added
0.8 g
K,S,O,
and 8 ml 1N HCI) o
45
min, and
insed in wa e . The ma e ial was dissec ed in
45
YO
ace ic acid. Co e slip was placed on he ma e ial,
p essed gen ly and emo ed a e d y ice ea men .
Slides we e insed in absolu e e hanol o
5
min, in
glacial ace ic acid o 25 sec, ai -d ied and s o ed
Table
1.
Numbe o ch omowme in
oo
ip cells in di e en
s ains
o
6.
campes i
S ain No.o No.o 2n
plan s cells
c . ‘Tobin’
S
9
20
c .
‘TL-15’
6
8
20
BC6o
4
16 20
B.
napus
x
‘Tobin’
BC6o
9
33
20
D.
mu alis
x
‘TL-
IS’
o e nigh
o
longe a 40°C. Then, he slides we e
insed in SB ensen’s phospha e bu e pH
6.8
o
5
min, s ained wi h 4
‘YO
Giemsa in he same bu e o
15
min, insed
in
wa e , ai -d ied, and moun ed in
En ellan.
Resul s
Soma ic
ch omosomes
Ka yo ypes we e examined om oo ip cells
o
c .
‘Tobin’, o ia ype c .
‘TL-15’,
and he wo
backc oss amilies showing male s e ili y.
In
all
o
hem, he ch omosome numbe 2n=20 was ob-
se ed
(Table
1).
Ka yo ype analysis in B assica
species is complica ed, besides he small size
o
he
ch omosomes,
by
a ying exp ession
o
he nucleo-
lus-o ganizing egions
in
ch omosomes
1
and
2,
and
also by soma ic associa ion
o
ch omosomes. As
shown in Fig. 2 he euch oma ic pa s
o
di e en
ch omosomes a e closely associa ed a hei
elome ic egions
in
p ophase cells. Classi ica ion
o
he ch omosomes is e y di icul a his s age, bu
associa ions ha e been obse ed be ween simila
and e y dissimila ch omosomes. Usually, he as-
socia ions a e absen in ully condensed ch omo-
somes, bu occasionally wo me aphase ch omo-
somes
lie
in close p oximi y (Fig.
3).
A e a combined p e ea men wi h iced wa e
and 8-hyd oxyquinoline (Fig.
l),
cells we e ob-
se ed whe e all he ch omosomes could be clas-
si ied on he basis
o
he pachy ene map o ROBBE-
LEN
(1960). Ch omosome
1
has a la ge and da ke
sa elli e han ch omosome 2. Howe e , he non-
sa elli ed homologues a e usually di icul o dis in-
guish om he es
o
he ch omosomes. Ch omo-
some
3
is subme acen ic and he la ges
o
he
whole complemen in
R.
cnmpes is.
On
he con-
a y, ch omosome
4
is he smalles one, al hough
some imes he size di e ence be ween ha and he
nex smalles ch omosomes
10
and
9
is inadequa e
He edi as
109 (1 988)
MALE
STERILE
BRASSICA CAMPESTRIS
95
Fig.
1-4.
Soma ic ch omosomes
o
B.
campes is.
Fig.
1.
Ka yo ype o BC 6 o
B.
napus
X
B.
campes is
c . ‘Tobin’ a e
cold and 8-hyd oxyquinoline ea men .
Fig.
2. P ophase ch omosomes o c . ‘Tobin’ showing associa ions o simila
(4-4)
and dissimila ch omosomes
(3-9).
Fig.
3. Me aphase ch omosomes
o
BC 6
o
Diplo axis mu alis
X
B.
campes is
c . ‘TL-15’ a e cold and 8-hq ea men .
1
=
sa elli ed ch omosome.
Two
ch omosomes lie close
o
each o he
(94).
Fig.
4.
La e p ophase ch omosomes
o
c . ‘An e’ a e 8-hq and pec inase-cellulase ea men . (Ba s equal
5
pm).
o
classi ica ions. Ch omosome
5
is
o
medium size
and easily iden i iable as an ex eme ac ocen ic.
On ch omosomes
6
and
7
he cen ic he e och oma-
in is conspicuously asymme ical, he long a m
bea ing he la ge block. Ch omosome
7
is a li le
la ge and mo e me acen ic han ch omosome
6.
O
he es
o
he
ch omosomes, numbe
8
is
me acen ic, and
o
he ac ocen ic ones ch omo-
some
9 is
la ge han
10.
Acco ding o
ROBEELEN
(1960)
he cen ic he e och oma in is asymme i-
96
Y
VIINIKKAANDM
SOVERO
He edi as
109
(1988)
Fig.
5-8.
Diakinesis
o
E.
canlpes is.
Ten bi alen s
wi h
a
sa elli e isible
(Fig.
5
and
6)
and in isible
(Fig.
7)
o
BC
6
o
B.
napus
X
B.
campes is,
and
BC
h
o
D.
mu alis
X
E.
campes is
(Fig.
8).
(Ba equals
Spn).
Tuhk
2.
Visibili y
ol he
sa elli e
a
diakincsi in
a
BC
6
amily o
A.
nupus
x
c .
‘Tobin’
No.
o
A
sa elli c isible/
plan s To al
no.
o
cells
0%
Fe ile
plan s
4
4/246
1.6
Pa lys e ile
10
1/86
1.2
plan s
..
-.
_.__
cally loca ed in he wo las men ioned ch omo-
somes, he long a m o ch omosome
Y
ha ing he
la ge block while on ch omosome 10 i is loca ed in
he sho a m. This di e ence could no be con-
i med wi h he p esen echnique.
Since classi iable ch omosoincs we e only in-
equen ly obse ed a e he p e ea men de-
sc ibed abo e, se e al o he ea men s ha e been
es ed. The mos p omising me hod seems o be a
pec inase-cellulase diges ion be o e ixa ion com-
bined wi h X-hyd oxyquinoline (Fig.
4).
The la e
p ophase ch omosomes appea now mo e swollen
wi h clea cons ic ions and sha p bounda ies
o
he e och oma ic egions. Howc e , he esul s
we e a he a iable, and imp o emen s
o
he
me hod a e s ill needed.
No
cons an ch omosomal di e ences be ween
he s ains
could
bc
ound, pe haps because
o
he
small numbe
o
cells wi h classi iable ch omo-
somes.
Meiosis
Usually, en bi alcn s we e obse ed in all he
plan s examined. 0ccasion;illy wo
o
h ee bi a-
len s we e e y closc o each o he . Howe e , i is
e y di icul o examine he eal posi ions o he bi-
alen s in squashed ma e ial, al hough he p epa a-
ions we e made wi h e y gen le p essing. Hence
no
compa isons wi h he “seconda y pai ing” de-
sc ibed by
C,xmiwi)
(1037)
ha e been made.
Seg ega ion a anaphasc
I1
was no mal in nea ly all
he cells o bo h male s e ile s ains examined.
In
one nea ly o ally malc s e ile plan wi h
pol-
cy oplasm
(HC’h
o
l.
nc pus
x
‘Tobin’) hc sa elli e
was s ill isible a diakinesis
in
a la gc numbe
o
cells, when
i
WBS
in isible in all cells a he same
s age in a c ilc sis c
plan .
In
o de
o
examine
i
He edi as 109 (1988)
MALE STERILE
BRASSICA CAMPESTRIS
97
he exp ession o he nucleolus-o ganizing egion is
di e en in male s e ile and e ile plan s, he isi-
bili y o he sa elli e was examined in ano he amily
o
he same c oss.
As
indica ed in Table
2
and Fig.
5-8,
he sa elli e was isible only in a ew cells, and
no co ela ion wi h he male s e ili y could be es ab-
lished. The isibili y o he sa elli e seems o be a h-
e a iable, depending on he s ain and he s aining
me hod.
Discussion
No
ex a ch omosomes we e disco e ed in he male
s e ile plan s bea ing cy oplasm om
D.
mu alis
and nuclea genome om B. cumpes is. In a simila
combina ion be ween
D.
mu alis cy oplasm and B.
nupus genome
(FAN
e al. 1985), male s e ili y was
always combined wi h he p esence
o
an ex a
ch omosome. Hence, he sys ems p oducing male
s e ili y wi h mu -cy oplasm in hese wo B ussica
species seem o di e , e.g., in he es o e genes o
he genomes examined. Meiosis appea ed o be no -
mal, as i was in he o he male s e ile s ain ha ing
pol-cy oplasm.
The soma ic ch omosomes we e iden i ied on he
basis o he pachy ene map o ROBBELEN (1960).
Howe e , he e we e ce ain di e ences in some
ch omosomes. The sa elli ed ch omosomes
1
and
2
appea ed mo e me acen ic han in pachy ene
ch omosomes, and ch omosome
5
mo e ac ocen-
ic. The e is no way o con i m
o
disp o e he
hypo hesis o six ch omosome ypes
(CATCHESIDE
1937;
ROBBELEN
1960) on he basis o he soma ic
ch omosomes. While ch omosomes
1
and
2
(sup-
posed e asomic ype
A)
appea ed o be mo -
phologically simila , ch omosomes
5
and 6 ( e -
asomic ype
D)
we e e y dissimila , and ch omo-
some
8
di e s clea ly om 9 and 10 (hexasomic ype
F).
ARMSTRONG
and
KELLER
(1981) ound, in hap-
loids o B. campes is, he expec ed maximum o
meio ic ch omosome pai ing, wo bi alen s and a
i alen
in
he same cell, bu he size o ch omo-
somes in ol ed in he i alen does no i o he
F
ch omosomes. The inal de e mina ion o he pa -
ial homology
o
he ch omosomes can be made
only wi h he aid
o
mo e e icien molecula me h-
ods.
Li e a u e ci ed
ALAM,
Z.
1936. Cy ological s udies
o
some Indian olei e ous
C uci e ae.
111.
-Ann. Bo . 50:
85-102
ARMSTRONG, K. C. and KELLER,
W.
A. 1981. Ch omosome
pai ing in haploids
o
B assica campes is.
-
Theo . Appl.
Gene . 59
49-52
CATCHESIDE, D.
G.
1934. The ch omosomal ela ionships in he
swede and u nip g oups
o
B assica. -Ann. Bo .
48:
601433
CATCHESIDE,
D.
G.
1937. Seconda y pai ing in
B assica ole acea.
-
Cy ologia Fujii
Jub.
Vol.:
366378
ERICKSON,
L.,
GRANT,
1.
and BEVERSDORF,
W.
1986. cy oplas-
mic male s e ili y in apeseed
(B assica napus
L.)
1.
Res ic ion
pa e ns
o
chlo oplas and mi ochond ial DNA.
-
Theo . Appl.
Gene . 72:
145-150
FAN,
Z.,
TAI, W. and STEFANSON, B.
R.
1985. Male s e ili y in
B assica nupus
L.
associa ed wi h an ex a ch omosome.
-Can.
J.
Gene . Cy ol. 27
467471
HINATA, K. and KONNO, N. 1979. S udies on a male s e ile s ain
ha ing he
B assica campes is
nucleus and he
Diplo axis
mu alis
cy oplasm.
I.
On
he b eeding p ocedu e and some cha -
ac e is ics
o
he male s e ile s ain.
-Jap.
J.
B eed. 29:
305-31
1
KEMBLE, R. J., CARLSON,
J.
E., ERICKSON,
L.
R., SERNYK,
J.
L.
and THOMPSON,
D.
J.
1986. The
B ussica
mi ochond ial
DNA plasmid and la ge RNAs a e no exclusi ely associa ed
wi h cy oplasmic male s e ili y.
-
Mol.
Gen.
Gene .
205:
183-
185
NEWELL, C. A., RHOADS,
M.
L.
and BIDNEY, D.
L.
1984.
Cy ogene ic analysis
o
plan s egene a ed om issue explan s
and mesophyll p o oplas s
o
win e ape,
B ussica napus
L.
-
Can.
J.
Gene . Cy ol. 26:
752-761
OOST,
E.
H. 1985. A p oposal
o
an in aspeci ic classi ica ion
o
B assica apa
L.
-
In
In aspeci ic Classi ica ion
o
Wild
and
Cul i a ed
Plan s
(Ed.
B.
T.
STYLES),
Ox o d Uni . P ess, Ox-
o d,
p.
309-315
PURO, J. and NOKKALA,
S.
1977. Meio ic seg ega ion
o
ch omo-
somes
in
D osophila melanogus e
oocy es.
A
cy ological ap-
p oach.
-
Ch omosoma 63:
273-286
RICHHARIA, R.
H.
1937. Cy ological in es iga ions o 10-ch omo-
some species
o
B assicu
and hei
F,
hyb ids.
-J.
Gene . 34:
45-
55
ROBBELEN,
G.
1960. Bei age
zu
Analyse des
B assica-Genoms.
-
Ch omosomu
11
:
205-228
VENKATESWARLU,
J.
and KAMALA,
T.
1971. Pachy ene
ch omosome complemen s and genome analysis in
B assica.
-
J.
Ind. Ba . Soc. 50A:
442449