Endosperm protein and isozyme patterns of Unes derived from the ovoss [Tritioum turgidum (AABB) x Aegilops ventrioosa (DDMVMV)] x T_. aes tivum (AABBDD) were studied. Five bioohemioal oharaoters pvesent in Ae_. ventrioosa (DDM°M°), Ae_. oomosa (MU) and Ae_. uniaris tata (MUMU) , but absent from Ae. squarrosa (DD)t T_. aes tivum (AABBDD) and T_. turgidum (AABB) were investigated. Three of these were identified in several Unes3 indioating gene transfer from the M° genome of Ae. ventrioosa into hexaploid wheat. The wide distribution of one of the markers (CM-4) suggests its previous transfer to the D genome of Ae. ventrioosa

Delibes Castro, Angeles

García Olmedo, Francisco

English

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BIOCHEMICAL EVIDENCE OF GENE TRANSFER FROM THE Mv 
GENOME OF AEGILOPS VENTRICOSA TO HEXAPLOID WHEAT 
ANGELES DELIBES AND F. GARCÍA-OLMEDO 
Department of Biochemistry 
E. T. S. Ingenieros Agrónomos 
Madrid-3, Spain 
SUMMARY 
Endosperm protein and isozyme patterns of Unes derived from the 
ovoss [Tritioum turgidum (AABB) x Aegi lops ventrioosa (DDMVMV)] x 
T_. aes tivum (AABBDDJ were studied. Five biochemical characters pvesent 
in Ae_. ventrioosa (DDM°M°), Ae_. oomosa (MM) and Ae_. uniaris tata (MUMU) , 
but absent from Ae_. squarrosa (DD), T_. aes tivum (AABBDD) and T_. 
turgidum (AABB) were investigated. Three of these were identified in 
several Unes3 indioating gene transfer from the M° genome of Ae. 
ventrioosa into hexaploid wheat. The wide distribution of one of the 
markers (CM-4) suggests its previous transfer to the D genome of Ae. 
ventrioosa. 
INTRODUCTION 
The introduction of extraspecific or extrageneric characters into 
cultivated plants has been long recognized of practical importance and 
of considerable genetic interest (SEARS, 1956; RILEY and KIMBER, 1966). 
The transfer of resistance to leaf-rust from Aegilops umbellulata 
(SEARS, 1956) and to eyespot (Ceroosporella herpotriohoides) from 
Ae. ventrioosa (KIMBER, 1967) into wheat are well-known examples of 
such genetic manipulation. 
A T. turgidum x Ae. ventrioosa amphiploid was obtained by KIMBER 
(1967) and then crossed with T. aestivum. Plants forming 21 bivalents 
at meiosis and resistant to eyespot were recovered in backcrosses to 
T. aestivum. Homologous transfer of a gene or genes from the D genome 
of Ae. ventrioosa to wheat was rightly assumed by Kimber. 
A somewhat different procedure for homologous transfer was 
proposed earlier by SIMONET (1952). It involved obtaining the hybrid 
(tetraploid wheat x Ae. ventrioosa) x T. aestivum and screening the 
progeny for plants with Ae. ventrioosa characters and 21 meiotic 
bivalents. 
We report here biochemical evidence indicating non-homologous 
gene transfer from the Mv genome of Ae. ventrioosa to hexaploid wheat 
by crossing (T. turgidum x Ae. ventrioosa) x T. aestivum. 
MATERIALS AND METHODS 
Seventy F20 unes (H-93-1 through 70) derived from 10 seeds obtained in a 
cross (T. turgidum var. H-l-1 x Ae. Ventrioosa) x T. aestivum var. H-10-15 were 
the g:tft of M. Alonso Peña, who also kindly supplied us with the parental material 
and samples of Ae. squarrosa, Ae. oomosa and Ae. uniaristata. 
Somatic chromosome numbers of the H-93 l ines were counted in the root t ips 
from germinating seeds af ter s ta ining by the Feulgen procedure. 
Single seeds were crushed between two metal plates with a hammer and defatted 
with diethyl e ther . Protein extract ions were usually carried out with five volumes 
of one of the following solvents: Chloroform; methanol (CM) ( 2 : 1 , v /v) ; 3M Urea 
(U); or 70% Ethanol (Et) . Starch gel electrophoresis of the ext rac ts was performed 
in 0.05M aluminum lac ta te buffer, pH 3 .1 , at 10 volts/cm for 3 hours to show 
albumins, globulins and proteins of similar mobility and for 7 hours to show 
gliadins and a th ins . Gels run for the shorter time were stained with nigrosine 
(0.05% in acet ic acidrwater, 1:1 v/v) for 12 hours. Gliadins and athins were 
stained with nigrosine (0.5% in acet ic acid:water, 2 : 1 , v/v) for 20 minutes. 
Destaining was achieved with 70% ethanol. Protein pat terns for each l ine were 
ascertained in at leas t three r epe t i t i ons , and the parental material was included 
in a l l the gels . 
Electrophoresis and s taining of a lkal ine phosphatase isozymes was carried out 
essen t ia l ly as described by BREWER (1970), except that 7.5% acrylamide was used 
instead of s tarch. 
Peroxidase isozymes were electrophoresed and stained according to MacDONALD 
and SMITH (1972). 
Sterol es ters pat terns were screened as previously described (GARCÍA-OLMEDO, 
1968). 
RESULTS AND DISCUSSION 
Composi te d i a g r a m s of p r o t e i n p a t t e r n s o b s e r v e d f o r t h e d i f f e r e n t 
e x t r a c t s a r e shown i n F i g u r e 1 . The s h o r t e r e l e c t r o p h o r e t i c run of 
t h e 3M u r e a e x t r a c t shows t h e p r o t e i n s g e n e r a l l y c o n s i d e r e d as 
b e l o n g i n g t o t h e a l b u m i n s + g l o b u l i n s f r a c t i o n ( F i g . l a ) . The c h l o r -
oform: m e t h a n o l e x t r a c t shows , unde r t h e same c o n d i t i o n s of e l e c t r o -
p h o r e s i s and s t a i n i n g ^ 2 t o 4 bands of u n c e r t a i n c l a s s i f i c a t i o n 
(ARAGONCILLO and GARCÍA-OLMEDO, u n p u b l i s h e d ) . Bands CMl and CM2 of 
t h i s e x t r a c t have beerj shown t o be c o n t r o l l e d by chromosomes 7D and 
7B, r e s p e c t i v e l y (GARCÍA-OLMEDO and CARBONERO, 19 70) . Bands of s i m i l a r 
m o b i l i t y t o t h e CM's and s l o w e r a r e p r e s e n t i n t h e 70% e t h a n o l e x t r a c t . 
C l a s s i c a l g l i a d i n s ( F i g . Ib) a r e i n c l u d e d i n t h e 3 t y p e s of e x t r a c t s , 
b u t w i t h 70% e t h a n o l a c l e a r e r p a t t e r n i s o b t a i n e d f o r t h e s l o w e r 
moving a t h i n s . 
Compos i te a l k a l i n e p h o s p h a t a s e and p e r o x i d a s e i sozymograms a r e 
p r e s e n t e d i n F i g u r e 2 . 
The p a l m i t a t e - l i n o l e a t e 3 - s i t o s t e r o l e s t e r s p h e n o t y p e (PL) i s 
c o n t r o l l e d by a s i n g l e m e n d e l i a n f a c t o r l o c a t e d i n t h e D genome of 
T. aestivum (GARCÍA-OLMEDO, 1 9 6 8 ) . 
A l l H-93 l i n e s have 42 chromosomes w i t h t h e e x c e p t i o n of l i n e 
H-93-1 which showed 56 . 
The d i s t r i b u t i o n of t h e s e l e c t e d b i o c h e m i c a l m a r k e r s i n H-93 
l i n e s as w e l l as i n t h e i r p a r e n t a l and r e l a t e d s p e c i e s i s summarized 
i n T a b l e s 1 and 2 . 
Marker s of AB o r D genomes a r e p r e s e n t i n a h i g h p r o p o r t i o n of 
t h e l i n e s a s e x p e c t e d (Tab le 1 ) . Those p r e s e n t i n t h e homologous 
genomes of two p a r e n t a l s p e c i e s s h o u l d show i n a l l l i n e s . Th i s i s t h e 
c a s e f o r PL and CM-2, b u t n o t f o r CM-1 and G l - 2 . The D genomes of 
«i 
CHÍ:: 2 
3 
Atk 
1 • 
2 * 
Et 
Figure. 1. Composite diagraros of protein electrophoretic patterns with 
(a) 3M Urea (U); ChloroformrMethanol (CM), and 70% Ethanol 
(Et) extracts; 3 hours at 10 volts/cm; nigrosine 0.05%, 
12 hours. (b) CM and Et extracts; 7 hours at 10 volts/cm; 
nigrosine 0.5%, 20 minutes. 
Apk# 
4 
Aph 
+ 
o *-
Px 
Figure 2. Composite alkaline phosphatase (Aph) and peroxidase (Px) isozymograms. 
Table 1. Appearance of biochemical markers from AB and D genomes in H-93 lines. 
(ABD) T. aestivum var. H-10-15; (AB) T. turgidum var. H-l-1; (DMV) 
Ae. ventriaosa; (D) Ae. squarrosa; (M) Ae. cornos a; (M11) Ae. uniaristata. 
~ H-93 lines 
Marker ABD AB D DMV M Mu Assignment with marker (%) 
PL1 
U-2 
CM-1 
CM-2 
Et 
Gl-1 
Gl-2 
Gl-3 
+ 
+ 
+ 
+ 
+ 
-
+ 
+ 
Ath-1 & 2 + 
+ 
-
+ 
-
-
-
-
-
_ 
+ 
-
+ 
-
-
-
-
-
_ 
D, M 
ABD T. aestivum 
D, M 
AB 
D T. aestivum 
AB T. turgidum 
D 
ABD T. aestivum 
ABD T. aestivum 
100 
59 
96 
100 
71 
32 
75 
40 
72 
3-sitosterol palmitate-linoleate phenotype 
Table 2. Apperance of biochemical markers from M genome in species and H-93 lines, 
Marker ABD AB D DMV M MU ABDMV* Marker in H-93 lines No. 
Aph - + + + + 3 3 
Px - - -
U-l - + + + + 8, 35 
U-3 
CM-4 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 2, 5-12, 14, 17, 19, 22, 
24, 26-7, 31, 35, 50-3, 
56, 63-4 
* H-93-1, 56 chromosomes, presumed to be the amphiploid (T. turgidum x 
Ae. ventriaosa) (AABBDDMVMV). 
T. aestivum and Ae. ventvioosa a r e i n v o l v e d i n bo th e x c e p t i o n s and 
t h e i r i n c o m p l e t e homology (SEARS, 1968; SIMONET, 1952; SIDDIQUI and 
JONES, 1967) could supply a r e a s o n a b l e e x p l a n a t i o n of t h e r e s u l t s . 
B iochemica l c h a r a c t e r s p r e s e n t i n Ae. ventvioosa (DDM M ) , 
Ae. oomosa (MM) and Ae. uniaristata (MUMU) and a b s e n t f rom T. aestivum 
(AABBDD), Ae squavvosa (DD) and T. tuvgidum (AABB) w e r e s e l e c t e d a s 
Mv genome m a r k e r s . The d i s t r i b u t i o n of f i v e s u c h m a r k e r s i s s u m m a r i z e d 
i n T a b l e 2 . A much more r e s t r i c t e d d i s t r i b u t i o n of t h e s e c h a r a c t e r s 
i n t h e H-93 U n e s i s t o b e e x p e c t e d , and i n d e e d t h i s i s t h e c a s e . 
On ly t h r e e of t h e m a r k e r s , Aph, U - l and CM-4, w e r e d e t e c t e d b o t h i n 
l i n e H - 9 3 - 1 (56 ch romosomes ) and i n s e v e r a l o f t h e 69 l i n e s w i t h 42 
c h r o m o s o m e s . The o t h e r two m a r k e r s , Px and U-3 w e r e n o t i d e n t i f i e d 
e i t h e r i n H - 9 3 - 1 o r i n t h e o t h e r l i n e s . 
The w i d e r d i s t r i b u t i o n of CM-4 ( i n 25 o u t o f 69 l i n e s ) , s i m i l a r 
t o t h a t o f m a r k e r s p r e s e n t i n t h e A, B , o r D genomes o f o n l y o n e of 
t h e p a r e n t a l s p e c i e s , s u g g e s t s t h a t a p r e v i o u s t r a n s f e r o f t h i s 
c h a r a c t e r f rom t h e Mv t o t h e D genome o f Ae ventvioosa h a d t a k e n p l a c e 
b e f o r e t h e c r o s s was p e r f o r m e d . 
Our r e s u l t s seem t o i n d i c a t e t h a t g e n e t r a n s f e r f rom t h e M genome 
o f Ae. ventvioosa i n t o h e x a p l o i d w h e a t c a n b e a c h i e v e d w i t h o u t t h e u s e 
o f i r r a d i a t i o n o r 5 B - b r e a k i n g s y s t e m s . F u r t h e r c y t o g e n e t i c and 
b i o c h e m i c a l e v i d e n c e t o c o n f i r m t h i s p o s s i b i l i t y i s b e i n g s o u g h t a t 
p r e s e n t . 
ACKNOWLEDGEMENT 
We dedica te t h i s communication to M. Alonso Peña upon r e t i r emen t . 
LITERATURE CITED 
BREWER, G. J . 1970. In t roduc t ion to isozyme techniques . Academlc P r e s s , 
New York. 186 pp. 
GARCÍA-OLMEDO, F. 1968. Genetics of syn thes i s of 6 - s i t o s t e r y l e s t e r s in wheat 
and r e l a t e d s p e c i e s . Nature (London) 220;1144-1145. 
GARCÍA-OLMEDO, F . , and P. CARBONERO 1970. Homeologous p r o t e i n s syn thes i s 
con t ro l l ed by homeologous chromosomes. Phytochem. 9^1495-1497. 
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