Compatibility of interspecific Manihot crosses presaged by protein electrophoresis .

Abstract

Cross incompatibility of wild Manihot species with cassava (M. esculenta) can impede their utilization for improving this cultigen. We tested whether compatibility could be determined based on electrophoresis results. Manihot pilosa, M. glaziovii, M. reptans, and M. cearulescens were tested. These species were allowed to hybridize with cassava to determine whether hybridization coincides with the similarity index based on electrophoresis analysis. Gene markers of leaf shape, stem surface, disk color, and fruit shape were used to confirm hybridization. Manihot pilosa and M. glaziovii successfully hybridized with cassava, while the others failed to do so under natural conditions. This result coincided with the similarity index from electrophoresis.

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©FUNPEC-RP www.funpecrp.com.br
Genetics and Molecular Research 9 (1): 107-112 (2010)
Compatibility of interspecific Manihot crosses
presaged by protein electrophoresis
N.M.A. Nassar, N. Bomfim, A. Chaib, L.F.A. Abreu and P.T.C. Gomes
Departamento de Genética e Morfologia,
Universidade de Brasília, Brasília, DF, Brasil
Corresponding author: N.M.A. Nassar
E-mail: nagnassa@rudah.com.br
Genet. Mol. Res. 9 (1): 107-112 (2010)
Received September 24, 2009
Accepted October 19, 2009
Published January 19, 2010
ABSTRACT. Cross incompatibility of wild Manihot species with
cassava (M. esculenta) can impede their utilization for improving
this cultigen. We tested whether compatibility could be determined
based on electrophoresis results. Manihot pilosa, M. glaziovii,
M. reptans, and M. cearulescens were tested. These species were
allowed to hybridize with cassava to determine whether hybridization
coincides with the similarity index based on electrophoresis analysis.
Gene markers of leaf shape, stem surface, disk color, and fruit shape
were used to confirm hybridization. Manihot pilosa and M. glaziovii
successfully hybridized with cassava, while the others failed to do
so under natural conditions. This result coincided with the similarity
index from electrophoresis.
Key words: Interspecific crosses; Marker genes; Manihot;
Wild cassava species

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N.M.A. Nassar et al.
INTRODUCTION
Wild Manihot species are sources of many useful characters for improving cassava
(Nassar, 1978a,b,c,d; 2000, 2006a; Nassar and Sousa, 2007). Transfer of these genes, how-
ever, faces the problem of interspecific barriers, which impede successful crosses.
To facilitate crosses aimed at transferring useful genes from the wild plants to the
cultigen, it is necessary to know how much is the distance between a certain wild species and
the cultivated crop, and consequently how strong or weak are the barriers.
Grattapaglia et al. (1986) analyzed biosystematically the relationship between cassava and
its wild relatives on the basis of protein electrophoresis. They constructed a species similarity matrix
based on band density and number. Some enquiries were raised as to how much this relationship
is reflected in trial crosses and in fertility. In this paper, a trial was designed to hybridize 4 species
representative of wild Manihot groups with the cultigen. They were selected because they represent
botanically the extreme ends of distance to cassava (Rogers and Appan, 1973; Nassar, 2003a,b). Our
idea is that the more hybrid seed obtained the greater the relationship and compatibility are.
MATERIAL AND METHODS
Four wild species in addition to cassava were used in this experiment. These species
are: Manihot pilosa, M. glaziovii, M. reptans, and M. cearulescens. Seeds and or cuttings of
these species were planted in September 2000.
The seeds were treated thermally by alternating temperatures of 16º and 28ºC for periods
of 8/16 h (Nassar, 1983; Nassar, 2006b; Nassar and Hashimoto, 2006) for one week to break dor-
mancy. Mode of planting was in circles where one plant of the wild species was in the center of a
circle of 8 cassava plants. They were allowed to pollinate by insects. Fruits were collected from the
wild species (maternal parent) in the third year, June 2002. Seeds were extracted from fruits, treated
thermally to break dormancy and planted in rows. Raised plants were observed using gene markers
to identify interspecific hybrids. These marker genes are dominant genes of prominent nodes on
stem (which came from cassava, the paternal parent) versus smooth stem, red color of flower disk,
which is dominant to yellow, setaceous bracteole, which is dominant to foliaceous, and winged fruit,
which is dominant to globose one. The plants raised were also examined for growth habit, height,
stem texture, and tuber formation.
RESULTS AND DISCUSSION
Of 200 seeds of M. pilosa, only 39 seedlings emerged of which 4 hybrids were identi-
fied by dominant markers from cassava: noded stem, setaceous bracteoles, ribbed fruit, and
tuberous roots (Table 1). Other characters proved to be indirect evidence of hybridization.
Character

Growth habit
Young stem texture
Bracteoles
Fruits
Flower disk color
Tuber formation
M. pilosa Cassava Hybrid
Tall shrub 4 m in height
Hairy
Setaceous
Globose without ribs
Yellow
None
Small shrub 1.5-2 m
Glabrous
Setaceous
Ovoid, ribbed
Red
Forms tubers
Medium shrub 3 m
Hairy
Setaceous
Ovoid, ribbed
Red
Forms tubers
Table 1. Growth habit and marker genes of Manihot species.

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Genetics and Molecular Research 9 (1): 107-112 (2010)
Compatibility of interspecific Manihot by protein electrophoresis
The 200 seeds collected from M. glaziovii gave rise to 78 seedlings. Of these, three
seedlings showed characteristics of interspecific hybridization. Hybrid plants exhibited domi-
nant phenotypes of cassava, namely ribbed fruit, red color of flower disk, noded stem, and
tuberous roots (Figures 1-4; Table 2).
Character M. glaziovii Cassava Hybrid
Growth habit
Young stem texture
Bracteoles
Fruits
Flower disk color
Steam nodes
Tuber formation
Tree 10 m in height
Glabrous
Setaceous
Globose without ribs
Yellow
Smooth
None
Erect shrub 1.5-2 m
Glabrous
Setaceous
Ovoid, ribbed
Red
Prominent
Forms tubers
Erect shrub 2.5-3 m
Glabrous
Setaceous
Ovoid, ribbed
Red
Prominent
Forms tubers
Table 2. Comparison of morphological characters of Manihot glaziovii, cassava and their hybrid.
Figure 1. Marker gene of fruit shape; winged fruit.
Figure 2. Red flower disk (left), globose fruit (right) and hybrid fruit (middle), and yellow disk.

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N.M.A. Nassar et al.
These results show that glabrous stem, setaceous-folicaceous bracteoles, red-
creamy color of flower disks, variegated-green color of fruit, and ribbed-nonribbed fruit
are simple marker genes that can be used to recognize interspecific hybridization. This
is in accordance with what was found by Nassar in 1989 while working with broadening
the genetic base of Cassava by controlled hybridization.
Species of M. reptans and M. cearulescens did not produce any hybrid within 200
seeds collected from each of them. In their biosystematic analysis of Manihot species us-
ing electrophoresis of soluble protein, Grattapaglia et al. (1986) elaborated a matrix
of similarity index for cassava and other wild Manihot species examined as shown
in Table 3.
Figure 3. Foliaceous (above) and setaceous bracteole (below).
Figure 4. Smooth stem (right), noded stem (left) and hybrid form (middle).

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Compatibility of interspecific Manihot by protein electrophoresis
This index was based on quantifying density and distance of bands. From this matrix, it
is seen that M. pilosa and M. glaziovii (referred to by the letters H and I, and cassava referred to
by the letter A) have the highest similarity indices, i.e., 68% for M. pilosa and 64% for M. glazio-
vii. The similarity indices for species M. reptans and M. cearulescens are far lower. These spe-
cies are referred to by the letters S and U, having a similarity index of 32 and 47%, respectively.
Apparently the weak barriers between cassava and M. glaziovii and M. pilosa could
be broken, while the stronger ones with the other two species could not be easily overcome
(Nassar 2006c,d).
From our observations in this experiment, insect pollination played an important role in
obtaining successful crosses. This is probably due to the fact that insects as vectors carry abun-
dant amounts of cassava pollen grains from one flower to another (Nassar, 2004, 2007a). This
means a great diversification of gametes, which is available when manual crosses are applied.
We can conclude that barriers between cassava and other Manihot species are weak
and recently evolved. A similar deduction has been previously made by Nassar et al. (1995,
1997). It seems that they arose not as a primary isolating event, but secondarily after geo-
graphic isolation. Nassar (1978c, 1984, 1985, 2007b) postulated that cassava itself is an inter-
specific hybrid, which appeared by domestication some 3000 years ago or less.
ACKNOWLEDGMENTS
Research supported by Conselho Nacional de Desenvolvimento Cientifico - CNPq.
The above mentioned living collection was established with the support of the Canadian Inter-
national Development Research Center, Ottawa, to which we are grateful.
Section
species
Section
I II III IV V VI VII VIII IX X -
A B C D E F G H I J K L M N O P Q R S T U V
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
-




















78
-



















54 45 67 64 58
49 38 68 68 68
- 62 51 65 48
- 47 53 65
-
















66
61
51
40
70
67
51
-













64
60
49
45
75
71
54
74
-












58
56
51
40
63
67
51
71
59
-











58
54
54
50
74
70
65
60
64
74
-










58
50
54
54
66
70
65
55
70
55
71
-









50
52
59
47
62
71
52
61
45
52
59
59
-








45
42
31
30
46
42
38
49
45
43
41
38
40
-







43
41
30
29
44
40
39
50
43
41
39
39
35
78
-






43
44
32
30
45
41
38
48
43
42
38
46
50
55
-






54
52
45
39
60
58
50
56
62
52
52
56
50
50
51
36





30
28
33
32
36
36
34
35
41
45
32
33
32
-







32
31
33
33
39
38
36
37
45
44
34
36
35
88
-






54
53
50
50
66
58
50
64
70
36
37
38
37
38
39
-





47
43
44
39
53
56
41
54
69
49
47
51
47
43
37
48
49
49
50

43
50
50
40
59
62
56
78
52
60
48
54
59
50
42
43
41
56
36
38
75 61
-















58
-













58
Table 3. Matrix of similarity between the Manihot species studied.