He edi as
108:
129-131 (1988)
B ie
epo
High-lysine gene seg ega ion dis o ed
in
he ba ley c oss
Ris@
1508
x
C yp
CI
1090:
Pa e ns
o
endospe m p o eins
by
an elec opho e ic me hod
HANNU AHOKAS
Depa men
o
Gene ics, Uni e si y
o
Helsinki, A kadianka u
7,
SF-00100 Helsinki, Finland
(Recei ed Ap il 29, 1987)
The ba ley c oss be ween he Indian line C yp
(CI
1090) and 1508 (Ris~ high-lysine mu an no.
1508
in
c . Bomi) shows, in
F,
spikes, an abe an seg ega-
ion
o
abou 1:63
o
sh unken o plump g ains in-
s ead o he expec ed a io
o
1:3 (AHOKAS 1978,
1979a). A di e en kind o dis o ion was obse ed
in
he c oss o c . Glacie and
1508
(AHOKAS 1979b).
The Glacie supp ession, which seems o be p omis-
ing o b eeding, will be desc ibed la e . P og ess
in
b eeding
o
1508
g ain plumpness and yield has
been epo ed by BANG OLSEN
e
al. (1986). The
p esen a icle deals wi h he case o C yp .
Below, he c osses a e ma ked acco ding o
PURDY e al. (1968), and he gene a ion o seed, ac-
co ding o AHOKAS (1976). When an
F,
spike o
C yp /1508
o
he ecip ocal c oss is pollina ed wi h
1508,
he BC-F,,, g ains seg ega e sh unken and
plump in a a io o
1:1
wi h expe imen al esul s
5850
o he c oss C yp /2*1508
(P>0.30)
and 35:36
o 1508/C yp //1508
(P-I).
Thus he mu an gene
o sh unken pheno ype seg ega es no mally in his
F,,
hough i s pheno ypic e ec is masked in sel ed
F,
spikes, leading o 1:63 a io in he
F(2)
g ains. Ad-
di ionally, when he equency o he mu an gene
(o he p opo ion
o 1508
genome) eaches 75
%
in
he zygo es on a C yp /1508 he e ozygous plan , he
dis o ion is no longe appa en . I was o in e es o
know hen, whe he he geno ypically homozygous
sh unken g ains wi h a plump pheno ype on
F,
spikes ha e a mu an -like p o ein pa e n
in
endo-
spe ms. This ques ion will be answe ed below.
Sample p epa a ion o
SDS-PAGE
om single
g ains
All
emo able husks we e pealed o wi h inge
nails om he g ain. The emb yo was cu away wi h
a scalpel (i s o al emo al may be checked unde
s e eomic oscope). The g ain was i ed in o a ans-
pa en plas ic pipe e- ip (o ange
50
o 200 kl) o
se e as a g ip while d illing. Excess plas ic was cu
away. The endospe m was g ound o a powde
by
d illing wi h an F1 cu ing edge a ached
o
a minia-
u e d ill (Mini Plus
o
equi alen ).
Meal
samples
o
15
o
25
ng
we e weighed
in
a
1.5
ml Eppendo
ube and suspended
in
a modi ied LAEMMLI (1970)
sample bu e con aining
65.8
mM T is-HC1 (pH
7.0), 2.1
YO
SDS,
9.2
'/o
glyce ol and 0.001
%
b omophenol blue. The amoun
o
sample bu e
(~1) used o he suspension was 14.25 imes he mil-
lig ams o he meal. The meal was suspended
in
he
e ening wi h a mic opipe e- ip and by o exing,
and allowed o s and o less han hal -hou . The
ubes we e hen spun o
5
seconds in a mic ocen-
i uge (12500 pm) o a oid ex ensi e swelling du -
ing he subsequen hea ing o 2 min
in
boiling
wa e . The caps we e pie ced be o e boiling. The
boiled ubes we e le o e -nigh a 18-22°C. Any
in e up ion
in
boiling may lead
o
anomalies in he
p o ein pa e n ollowing elec opho esis wi h some
high-molecula weigh p o eins o ally missing. The
esul is simila ,
i
boiling is omi ed. On he ollow-
ing mo ning, 2-me cap oe hanol was added
(PI)
o
0.75 imes he weigh o he meal
in
millig ams, and
he pelle was b oken and pa ially esuspended as
abo e. Be o e loading, he samples we e cla i ied
by spinning in mic ocen i uge a 12000
o
14000
pm o
10
min a oom empe a u e. The dissol ing
p ocess can also be done du ing a couple o hou s
be o e beginning he gel un wi hou any g ea e -
ec on he banding pa e n. The gels used he e we e
essen ially acco ding o LAEMMLI (1970) wi h 9.5
%
ac ylamide in he sepa a ion gel. A e elec-
opho esis, he gels we e s ained wi h B illian blue
R
(Sigma) ollowing a modi ica ion o
FAIRBANKS
e
al.
(1971). The gels we e agi a ed
in
solu ion
1
o
5
h, solu ion 2 o 10 h, and in se e al changes o solu-
ion
4
o cla i y he backg ound. In my hands, he
me hod abo e e eals mo e p o ein bands han he
apid one desc ibed by SMITH and PAYNE (1984) and
He edi as
I08
(1988)
Fig. 1.
SDS-PAGE
dis ibu ion
o
endospe m p o eins om meals wi h he p esen me hod
(a-e,
g-i)
o
wi h he me hod
o
SMITH
and
PAYNE
(1984) on lanes
ii
and
hh.
Solu ion olumes used
o
ex ac ion we e he same
on
a meal-weigh
basis.
60
pl
was loaded pe lane. Lane hal es we e p in ed wi h wo di e en exposu e imes. Lane
a
=
Bomi,
b
=
C yp ,
c
=
C yp /1508
F,,,,
d
=
15081C yp
F,,,,
e
=
1508, =
molecula weigh ma ke s in kilodal ons (kd),
g-h-i
=
F,,,
endo-
spe ms om C yp 11508 c oss wi h majo p o eins be ween 24-45 kd
o
F,,,-,
Bomi- and C yp -like pa e ns, espec i ely.
Lanes
ii
and
hh
=
spli samples
o
meals
o
i
and
h,
co espondingly, wi h less bands isible and wi h di e en mobili ies
han wi h he p esen me hod.
Angles
and do s indica e unique bands
o
mobili y di e ences be ween Bomi and C yp
Some
o
he mobili y di e ences a e exp essed codominan ly in
F,,,’s
o
C yp and
1508.
SMITH
e al.
(1986).
Also
mul iple bands a e e-
ealed whe e he la e displays a single di use band
(Fig. 1). G ound meal
o
ba ley can also be used,
i
whole-g ain p o eins a e o be s udied. The me hod
desc ibed he e ca ied ou on indi idual g ains is a
good es o a ie al pu i y o seeds
o
small g ain
ce eals.
Resul s
and
discussion
Wi h
SDS-PAGE,
Bomi shows wo majo p o ein
bands be ween
24
and
45
kd
(Fig.
1
a),
which a e
also
sppa en when alkyla ed B ho dein p epa a-
ions
o
Bomi a e elec opho esed (DOLL and
AN-
DERSEN
1981; ULLRICH e al. 1986). C yp has a com-
posi e
o
h ee majo bands a he
B
ho dein posi-
ion (Fig.
1
b).
F,,,
endospe ms be ween 1508 and
C yp a e in e media y be ween Bomi and C yp a
hei
24-45
kd bands, whe e 1508 is de oid
o
ol-
uminous bands (Fig. 1
e).
The
F,,,
endospe ms,
being iploid, show some dosage e ec in he eci-
p ocal c osses (Fig. 1
c
and
d).
The F,,, pa e n wi h
1508 as seed pa en (Fig.
1
d)
dis inc ly displays,
also a he B ho dein posi ion, he p esence o mos
Bomi bands no appea ing in
1508
(Fig.
1
e).
This is
in acco dance wi h he lack
o
dele ions in he B ho -
He edi as
108
(1988)
BRIEFREPORT
131
dein locus
o
1508
(FORDE
in
KREIS
e al. 1983) and
wi h he appea ance o
B
ho dein mRNA
in
1508
(THOMPSON
and
BARTELS
1983) as well as wi h some
immuno eac i e end p oduc s o B ho deins in 1508
(ULLRICH
e
al.
1986).
When
F zJ
g ains o he c oss C yp A508 seg ega -
ing abou
1:63
sh unken o plump we e s udied
on
gels, no 1508-like banding pa e n was obse ed
among he 100 plump endospe ms s udied. These
g ains a e expec ed o seg ega e 1:3,
o
p obably
mo e exac ly
(-):(
-
1:3.2 o mu an o
non-mu an pheno ypes
(P<0.001).
Ins ead, he
h ee pa e ns
o
he majo p o eins
(Fig.
1,
g,
h,
i)
we e ound.
All
he a e sh unken F,*, endospe ms
s udied had he 1508-like pa e n. Thus, he
pheno ypically plump, bu geno ypically sh unken
endospe ms a e no ghos -g ains de oid
o
p ope
s o age p o eins.
The
1508
allelic
Ris0
mu an s no. 18 and 19
(JEN-
s N
1979) we e also dis o ed
in
he F zJ g ain seg e-
ga ion
in
c osses wi h C yp . Some o he old Indian
ba ley lines, CI 1085, CI 1086, CI 1087, CI 1088, CI
1089. C1 1091, CI 2229, all om Cawnpo e as is
C yp , and CI
3405
om Que a (p esen ly
in
Pakis-
an) ailed o show dis o ion wi h 1508.
The sh unken g ains
o
BC-F, spikes (see in o-
duc ion) we e he sou ce o a sea ch
o
he pu a-
i e homozygous supp esso s. Supposing ecombi-
na ion o occu be ween sh unken gene and pu a-
i e complemen a y supp esso genes
(AHOKAS
197X), he sh unken BC-F,,, seeds should gi e ise o
plan s seg ega ing sh unken and plump BC-F ,,
g ains. Such plan s showing abou
1:15
seg ega ion
o sh unken o plump we e de ec ed. Thei descen-
den s we e selec ed o s able supp esso s du ing
se e al seasons. Such de i a i es om 1508/C yp //
1508
c oss a e s ill en i onmen ally somewha sensi-
i e, small g ained, six- owed, wi h pedicella e la -
c als (Fig.
2).
Thei endospe m p o ein pa e ns on
SDS
gels a e mos ly simila o hose o 1508, wi h
h ee di e ences conce ning he majo p o eins ( e-
sul s no desc ibed in his pape ). P esen ly, some
backc oss lines based on he supp essi e geno ype
o
c . Glacie a e unde s udy. These Glacie /l508
de i a i es ha e be e g ain and plan cha ac e is-
ics han he C yp de i a i es, and a e expec ed o
be use ul as high-lysine eeding ba ley.
16-1
3x16)
64
64
Acknowledgemen s.
-
I
am obliged o Ms. Leena Naskali, M.
Ag ic. Fo es ., o help a sample p epa a ions. and
o
D . A.
H.
Schulman o eading and e ising he manusc ip . The s udy was
ca ied
ou
unde he auspices o he Academy o Finland ( he Re-
sea ch
Council o Ag icul u e and Fo es y).
Fig.
2.
G ain
sample
o
a
high-lysine
de i a i e
om
he
c oss
lSOX/C yp //1508.
Na u al
size.
Li e a u e
ci ed
AHOKAS,
H.
1976.
A way o ma k he gene a ion
o
he seed.
~
Ba ley
Gcne .
Newsl.
6;
96
AHOKAS.
H.
1978.
Gene ic supp ession o sh unken endospe m
in
Ris@ mu an
1.508.
-Ba ley
News/
21:
62-64
AHOKAS,
H.
1979a.
Fu he esul s on he supp ession
o
sh unk-
en
endospe m in high lysine mu an s.
~
Ba ley
Gene .
Newsl.
Y;
3-7
AHOKAS,
H.
1979b. Dis o ion o FI?] ke nel seg ega ion
iii
he
c osses Ris@ ISOXiGlacic and Rise 1508/High Amylo Glacie .
-Ba ley
News/.
22:
129-131
BANG OLSEN, K., STILLING.
B.
and MUNCK, L.
1986.
B eeding
o yield in high-lysine ba ley.
h i h
In .
Ba ley
Gene .
Symp.,
Okayama,
Abs m ,
p.
222
DOLL.
H.
and ANDERSEN, B.
19x1,
P epa a ion
o
ba ley s o age
p o ein. ho dein. o analy ical sodium dodecyl sul a e-poly-
ac ylamide gel elec opho esis.
-Anal. Biochem.
11.5:
6146
FAIRBANKS,
G
.
STECK,
T.
L. and WALLACH, 0.
.
H.
1971.
Elec iopho e ic analysis
o
he majo polypep ides o he huinan
e y h ocy e memb ane.
-
Biochemis y
10.
2hOk26
17
JENSEN.
J.
1979. Loca ion o a high-lysine gene and DDT- esis -
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oil
ba ley ch omosome
7.
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Euphy icu
28: 47-S6
KREIS. M., SHEWRY,
P.
R., FORDE. B.
G.,
RAHMAN.
S.
and
MIFLIN,
B.
J.
1983.
Molecula analysis
o
a
mu a ion con e ing
he high-lyaine pheno ype
on
he g ain
o
ba ley
(I o d um
w/-
gu e).
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LAEMMLI,
U.
K.
1970. Clea age
o
s uc u al p o eins du ing he
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o
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Nu u e
227:
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685
KING,
W.
0..
KONZAK.
C.
F..
PETFR~
SON,
C.
J.
and ALLAN. R.
E. 1968.
A
p oposed s anda d me h-
od
o
illus a ing pedig ees
o
small g ain a ie ies.
~C' op
Sc .
8:
405406
SMITH. D.
B.
and PAYNE,
P.
I.
1984. A p ocedu e o he ou ine
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o
ba ley and
mal endospe m p o eins.
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J.
Null.
/m .
Ag c.
Bo .
16:
487-
49
8
SMITH,
D.
R.,
LISTER,
P.
R.
and HANSSON,
P.
R.
1986.
Disc i-
mina ion o ba ley a ie ies by elec opho esis o endospe m
p o eins ex ac able in o a mix u e
o
sodium dodecyl sulpha e,
2-me cap oe hanol and dime hyl o mamide.
.-
J.
Ce eal
Sci.
4:
107-1
16
THOMPSON, R.
D.
and BARTELS,
D.
1983. Ho dein messenge
RNA le els in he wild ype and mu an ba lcy endospe m.
-
Plan
Sci. Le .
29:
295-304
ULLRICH,
S.
E.,
RASMUSSEN.
U..
HBYER-HANSEN,
G.
and
BRANDT, A. 1986. Monoclonal an ibodies o ho dein polypep-
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Res.
Commun.
51:
381-399