Genetic Analysis of Mutations at Loci

Controlling Leaf Form in Cowpea

(Vigna unguiculata [L.] Walp.)
I. Fawole

Mutations affecting leaflet number and shape occurred at high frequencies in some
cowpea crosses. Mutant plants were nonpetiolate and unifoliolate as opposed to
normal plants which were petiolate and trifoliolate. Two types of unifoliolate mu-
tants were distinguishable on the basis of leaf shape which was ovate in one mu-
tant and orbicular in the other. The nonpetiolate and the unifoliolate traits in the
two mutants are each controlled by single recessive genes, but the genes control-
ling the traits in the different mutants were nonallelic. The orbicular leaf shape was
also under the control of a single recessive gene. In the F2 and subsequent gen-
erations of the cross IBS 2497 3 IBS 2625, orbicular-shaped unifoliolate leaf mu-
tants were regularly produced, although the two parents involved in the cross were
both trifoliolate. Linkage tests showed that the genes pt-1 for nonpetiolate trait, un-
2 for unifoliolate leaf, and orb for orbicular leaf shape in one of the mutants were
located on the same chromosome, while the genes pt-3 and un-3 in the other were
also linked on a different chromosome. The results of this study provide further
evidence indicating the involvement of transposable elements in the mutations ob-
served in the cowpea lines used in this study.

From the Department of Crop Protection and Environ-
mental Biology, University of Ibadan, Ibadan, Nigeria.
The author thanks the University of Ibadan for provid-
ing facilities for the research reported in this article.
Address correspondence to I. Fawole 0 Dr. N. Q. Ng,
IITA, 0 L. W. Lambourn & Co., Carolyn House, 26 Ding-
wall Road, Croydon CR9 3EE, England, or e-mail: li-
brary@kdl.ui.edu.ng.
q 2001 The American Genetic Association 92:43–50
In the cowpea (Vigna unguiculata ( L.)
Walp.) the first pair of simple leaves is suc-
ceeded by alternate trifoliolate leaves held
on long, grooved petioles. The leaflets are
ovate to lanceolate, sometimes hastate,
and are subtended by inconspicuous sti-
pels (Summerfield and Roberts 1985). Ra-
wal et al. (1976) reported a nonpetiolate,
unifoliolate cowpea leaf mutant plant in
which the flowers were also malformed.
The unifoliolate trait is controlled by a sin-
gle recessive gene to which the symbol un
was assigned. I ( Fawole 1988) observed
high frequency mutations at a petiole lo-
cus in several cowpea crosses. Data from
inheritance study and allelic tests showed
that the nonpetiolate trait is controlled by
a single recessive gene pt and all the ob-
served mutations occurred at the same lo-
cus. The pt gene is nonallelic to a second
gene designated pt-2 that controls the ex-
pression of the nonpetiolate trait in the
unifoliolate mutant plant described by Ra-
wal et al. (1976). The F1 plants obtained
by crossing the two different nonpetiolate
mutant lines were petiolate and the F2 gen-
eration segregated in the complementary
epistatic ratio of 9 petiolate:7 nonpetiolate
( Fawole 1990).
A major cause of unusually high muta-
tion frequency in living organisms is in-
herent instability in gene expression re-
sulting from the presence of transposable
elements ( Fedoroff 1983; McClintock 1951,
1956; Nevers et al. 1986; Peterson 1986,
1995). Integration of a transposable ele-
ment near or within a gene may inhibit
that gene or modify its action. Excision of
the element restores gene activity and an-
other gene may become inhibited through
the insertion of the transposable element
in that gene. Working with originally
crossed lines and progenies derived from
them, I ( Fawole 1988) showed that the
high frequency of recessive mutations that
occurred at a petiole locus in some cow-
pea crosses resulted from the unusual be-
havior of a cowpea cultivar, Ife BPC, when
used as the female parent. In addition, in
advanced generations of some of the
crosses, mutability seemed to be trans-
ferred to a new locus controlling the ex-
pression of leaflet number, where unifoli-
olate true leaves were produced instead of
the normal trifoliolate leaves. I therefore
suggested that transposable elements
might be involved in the induction of the
high frequency of mutations observed in
the cowpea crosses. In this article I pre-
sent further evidence on the nature of the
factors inducing a high frequency of mu-
tations in the cowpea materials used in

43
 Table 1. Origin and some leaf characteristics of cowpea lines used in the study earlier, while the other, designated IBS
Line Origin/source No. of leaflets Petiole type
2625, was petiolate, trifoliolate but with
orbicular-shaped leaflets ( Figure 1A, left).
Ife BPC Cultivar, I.A.R. & T
a
3 Petiolate The flowers of this mutant were mal-
IBCR-4 Breeding line U.I.b 3 Petiolate
TVu1 Germplasm line, IITAc 3 Petiolate formed, which made natural selfing diffi-
TVu 1509 Germplasm line, IITA 3 Petiolate cult, but a few of the flowers produced
TVu 6198 Germplasm line, IITA 3 Petiolate selfed pods. Progeny of selfed orbicular-
IT81D-1137 Breeding line, IITA 3 Petiolate
IBS 401 Ife BPC 3 TVu 4557 3 Nonpetiolate shaped trifoliolate leaf plants bred true for
G942 Ife BPC 3 TVu 1509F8 3 Nonpetiolate the orbicular shape. Some nonpetiolate,
G1001 Ife BPC 3 TVu 1509F4 3 Nonpetiolate
G1004 Ife BPC 3 TVu 4557 3 Nonpetiolate
trifoliolate leaf plants selected from the F6
IBS 2497 Ife BPC 3 TVu1 3 Nonpetiolate row later segregated for plants with orbic-
IBS 2625 Ife BPC 3 IT81D-1137F6 3 Petiolate ular-shaped unifoliolate leaves.
M-2 Ife BPC 3 TVu 1509F6 1 Nonpetiolate
M-3 IBS 2497 3 IBS 2625 1 Nonpetiolate
Experimental Crosses
a
Institute of Agricultural Research and Training, Ibadan Nigeria. In an experiment originally designed to
b
University of Ibadan, Ibadan, Nigeria. test the allelic relationship between non-
c
International Institute of Tropical Agriculture, Ibadan Nigeria.
petiolate, trifoliolate plants from different
crosses, the following crosses were made:
this study, report the inheritance of some wole 1988). The lines IBS 2497 and IBS G 942-1 3 G1001, G942-1 3 G1004, and
new mutations, and establish the allelic 2625 arose in a similar manner from the G942-2 3 G1004. Unexpectedly the proge-
and linkage relationships between the dif- crosses Ife BPC 3 TVu 1 and Ife BPC 3 nies of the crosses segregated for both
ferent mutant genes. IT81D-1137, respectively. IBS 2497 was ob- normal, trifoliolate, and orbicular-shaped
tained from the F2 generation of the cross unifoliolate leaf plants, all of which were
as a single nonpetiolate plant with a dis- nonpetiolate. The plants were scored for
Materials and Methods the normal and mutant phenotypes 6
tinctive nonbranching, indeterminate
The origin and leaf characteristics of the main stem. The determinate habit was weeks after planting.
cowpea lines used in the inheritance study transferred to the line by crossing to TVu The inheritance of the orbicular-shaped
are listed in Table 1. The morphological 6198, a determinate line. On the other unifoliolate leaf mutant was further inves-
characteristics of the germplasm and hand, IBS 2625 originated in an F6 row of tigated by crossing the mutant line to two
breeding lines are described by Porter et the Ife BPC 3 IT81D-1137 cross planted to normal trifoliolate lines, TVu 6198 and
al. (1974), Fawole (1997), and Fawole and seeds from a normal petiolate, trifoliolate IBCR-4. For these crosses, P1, P2, F1, F2, and
Afolabi (1983). The nature of the original selection. Two mutants types emerged the two backcross generations were
crosses that produced lines G942, G1001, from the row. One was a nonpetiolate tri- grown on the Teaching and Research Farm
and G1004 was described previously ( Fa- foliolate mutant similar to those described of the University of Ibadan in the early
planting season of 1986. Six weeks after
planting, individual plants were scored for
leaflet number, presence or absence of
petiole, leaf shape, and flower character-
istics. At maturity, between 40 and 60 F2
plants were randomly selected and those
that produced sufficient seed were ad-
vanced to the F3 generation in the late
planting season of 1987. Individual plants
in each F3 family were scored as earlier de-
scribed.
IBS 2625, the orbicular-shaped trifolio-
late leaf mutant, was also crossed to two
normal lines, Ife BPC and IBS 2497. The
parents, F1, and F2 generations of the
crosses were grown in the field during the
first planting season (April–July) of 1989.
The crosses were handled in the same
manner as described above. At maturity,
100 normal trifoliolate plants were ran-
domly selected from the F2 population of
each cross and were individually harvest-
ed. These F2 selections were advanced to
the F3 generation in the field during the
first planting season of 1990. At the onset
Figure 1. (A) Orbicular-shaped unifoliolate (right) and trifoliolate ( left) mutant leaves. (B) Orbicular-shaped of flowering, individual plants of each F3
leaves at various stages of division toward trifoliolate state on the same plant. (C) Abnormal flowers of the orbic-
ular leaf mutant. (D) Bent, beaked style of normal flower ( left) and nonbeaked style of mutant flower with stigma family were classified for leaf and flower
covered with hair (right). characteristics.

44 The Journal of Heredity 2001:92(1)


Figure 2. (A) Trifoliolate leaf of normal cowpea plant. (B) Flower buds (top), open flower ( bottom right), and
flower with petals removed to show the reproductive organs ( bottom left).
An F3 row of the cross IBS 2497 3 IBS
2625 segregated for a new nonpetiolate,
unifoliolate mutant phenotype with nor-
mal ovate-shaped leaves. The mutant
plants were transferred into plastic pots
filled with field soil and were taken to a
glasshouse. The flowers of the mutant
plants were also malformed, but pods
were easily obtained from them by hand
pollination, and occasional natural selfing
also occurred. The five mutant plants
were true breeding for the unifoliolate
phenotype. The genetic control of the mu-
tant was studied by crossing it to four nor-
mal lines—TVu 1509, TVu 6198, Ife BPC,
and IBS 2497. The parental lines, F1, and F2
generations were planted on the field dur-
Figure 3. (A) Ovate-shaped unifoliolate mutant leaf. (B) Mutant leaves at various stages of division toward tri-
foliolate state on the same plant. (C) Abnormal flowers of the ovate-shaped leaf mutant. (D) Straight, beaked styles
of mutant flower, some flowers possess two styles.
ing the first planting season of 1992. At
flowering, individual plants were scored
for leaf and flower traits. Between 40 and
80 F2 plants were randomly selected in
each cross at maturity. Those that pro-
duced sufficient seed were advanced to
the F3 in the second planting season of
1992 and were scored for the traits of in-
terest.
The cross IBS 2497 3 IBS 2625 segregat-
ed for orbicular-shaped unifoliolate leaf
plants in the F2 and F3 generations even
though both parents were trifoliolate. The
cross was therefore repeated in 1993 and
1996 to ascertain whether the production
of the unifoliolate mutant was of regular
occurrence in this cross. F2 and F3 gener-
Fawole • Genetic Analysis of Leaf Form Mutants in Cowpea 45
ations of the cross were examined on both
occasions and the plants were classified
on the basis of the leaf traits under con-
sideration.
Allelism Test
In April 1994, seeds of IBS 401, IBS 2497,
M-2, and M-3 were grown in a glasshouse,
and during flowering period were crossed
in all possible combinations. The resulting
F1 seeds were planted in the crop garden
of the Department of Crop Protection and
Environmental Biology, University of Iba-
dan. Individual F1 plants were scored for
leaf and flower traits at the onset of flow-
ering.
Results
Morphology of Normal and Mutant
Plants
Two types of unifoliolate plants were
found in the experimental populations and
these can be distinguished on the basis of
leaf shape, which is orbicular in one type
and ovate in the other. The wild-type cow-
pea plants have stipulate, trifoliolate true
leaves with long petioles ( Figure 2A),
while the true leaves of the mutants are
stipulate, nonpetiolate, and unifoliolate
( Figures 1A [right] and 3A). The orbicular-
shaped mutant leaves are further charac-
terized by the leaf apex, which is retuse
( Figure 1A) in contrast to the acuminate
apex of leaves of wild-type plants. Flowers
of normal plants are typically papiliona-
ceous with a large standard petal, two
free-wing petals, and two fused-keel petals.
The reproductive organs are enclosed
within the keel petals. These consist of 10
diadelphous stamens and a bent, bearded,
and beaked style ( Figure 2B). Plants of the
unifoliolate mutants exhibit abnormal
flower development. Many flower buds
drop off prematurely and when large flow-
ers are produced, all five petals are sepa-
rate from each other, the stamens are free,
and the style is straight ( Figures 1C and
3D). Floral abnormality is more severe in
the orbicular-shaped leaf mutant, espe-
cially with respect to the stigma, which is
nonbeaked, smaller in size than normal,
round, and covered with hair ( Figure 1D,
right). As a result of these abnormalities,
natural selfing could not occur but selfed
pods could be obtained easily by hand
pollination in the mutant with ovate leaf
and with great difficulty in the other mu-
tant. Both mutants produced fertile pollen
which was used successfully in crosses
with normal plants as female parents.
 Table 2. Segregation for orbicular shaped unifoliolate leaf in crosses between trifoliolate cowpea lines completely dominant to the orbicular
Expected
shape. F3 progenies derived from selected
Cross Trifoliolate Unifoliolate Total ratio x2 P F2 plants segregated in the expected 2:1
ratio, while pooled data from segregating
G 942-1 3 G 1001 25 9 34 3:1 0.04 .25–.50
G 942-1 3 G 1004 28 6 34 3:1 0.98 .75–.90 F3 families conformed to the 3:1 ratio.
G 942-2 3 G 1004 48 10 58 3:1 1.86 .10–.25 Thus the F3 data confirmed the monohy-
Ife BPC 3 IT81D-1137 20 7 27 3:1 0.01 ..90 brid inheritance of the orbicular shape.
Total 121 32 153 3:1 1.36 .10–.25
Further evidence that the orbicular leaf
shape is a separate and independent mu-
tation was provided by the results of
Inheritance of Orbicular Shaped the cross IBCR-4 3 M-2 and 35 of the 626 crosses between IBS 2625, a petiolate tri-
Unifoliolate Leaf plants in the cross TVu 6198 3 M-2 had foliolate mutant with orbicular leaf shape
Data on the segregation pattern for leaflet the orbicular leaf shape of the unifoliolate and two normal lines, Ife BPC and IBS
number in crosses between nonpetiolate, mutant. No unifoliolate plants with the 2497. The F1 plants in the two crosses
trifoliolate selections from different sourc- normal ovate shape were recovered in ei- were ovate in shape, while the F2 data
es are shown in Table 2. Progenies of the ther the F2 or the F3 generations of the two gave a good fit to the 3 trifoliolate ovate:1
four crosses segregated for leaflet number crosses. Thus the relatively large number trifoliolate orbicular ratio. The F3 data sup-
in the ratio 3 trifoliolate:1 unifoliolate, in- of orbicular-shaped trifoliolate plants ported the monohybrid inheritance of leaf
dicating a monohybrid inheritance of this could not be totally explained on the basis shape in the two crosses ( Table 5). The
trait. The inheritance of this unifoliolate of genetic recombination and most of symbol orb is assigned to the recessive
mutant was further studied by crossing it them probably resulted from reversion gene which in the homozygous condition
to two normal lines. The results of the from the recessive unifoliolate condition determines orbicular leaf shape.
study, presented in Table 3, show that uni- to the wild-type trifoliolate state. Some of
foliolate leaf is recessive to trifoliolate leaf. the unifoliolate plants also had bifoliolate Inheritance of Unifoliolate Leaf Mutant
The backcross to the orbicular mutant and trifoliolate leaves ( Figure 1B). with Ovate Leaf Shape
parent segregated in the ratio 1 ovate:1 or- The F1 generation of crosses between M-3,
bicular. However, the F2 generation devi- Inheritance of Orbicular Leaf Shape the unifoliolate mutant with ovate leaf
ated significantly from the 3 trifoliolate:1 Leaf shape data from the crosses IBCR-4 3 shape, and normal trifoliolate lines were
unifoliolate ratio in both crosses. Further- M-2 and TVu 6198 3 M-2 were also classi- all phenotypically trifoliolate. In the three
more, fewer F2 lines than expected segre- fied as ovate and orbicular, and tested for crosses studied, the F2 generation segre-
gated in the F3 generation, and the pooled goodness-of-fit to the monohybrid ratio. gated in the ratio 3 trifoliolate:1 unifolio-
F3 data also deviated significantly from the All F1 plants and plants of the backcross late ( Table 6). However, several unifolio-
3:1 ratio. In the F2 and F3 generations of to the ovate parent had the ovate leaf late plants also had bifoliolate and
both crosses, there were marked deficien- shape, while the progenies of the back- trifoliolate leaves in addition to unifolio-
cies in the number of unifoliolate plants cross to the orbicular parent and the F2 late leaves ( Figure 3B). F3 families derived
and an excess in the number of trifoliolate generation segregated in the 1:1 and 3:1 from selected trifoliolate F2 plants gave a
plants. Examination of the trifoliolate phe- ovate to orbicular ratios, respectively ( Ta- good fit to the expected 2 segregating:1
notypic class showed that 88 of the 589 ble 4). These results suggest a single-gene nonsegregating family ratio and the
trifoliolate plants in the F2 generation of control of leaf shape with the ovate shape pooled F3 data did not deviate significantly
from the 3 trifoliolate:1 unifoliolate ratio.
Table 3. Inheritance of unifoliolate leaf in two cowpea crosses
The Nonpetiolate Trait
No. of plants
Expected The inheritance of the nonpetiolate trait in
Cross and generation Trifoliolate Unifoliolate Total ratio x2 P mutant line M-3 was investigated in three
Ife Brown CR 3 M-2 crosses ( Table 7). All F1 plants of crosses
Ife Brown CR 126 126 between normal petiolate lines and the
M-2 24 24 mutant were petiolate, while the F2 gener-
F1 28 28
Ife Brown CR 3 F1 88 — 88 ation segregated in the ratio 3 petiolate:1
M-2 3 F1 74 61 135 1:1 1.25 .25–.50 nonpetiolate. The results thus suggest
F2 589 66 655 3:1 77.80 ,.005
F3 families
that the petiolate trait is determined by a
16 S:18 NSa 34 2:1 5.88 .025–.10 single locus with the petiolate condition
F3 (pooled, 1 df ) 235 38 273 3:1 18.47 ,.005 completely dominant to the nonpetiolate
Heterogeneity (15 df ) 9.66 .75–.90
TVu 6198 3 M-2 trait. The F3 data supported the monohy-
TVu 6198 142 142 brid hypothesis except in the cross M-3 3
M-2 24 24 TVu 1509, where the pooled F3 data devi-
F1 27 27
TVu 6198 3 F1 55 — 55 ated significantly from the 3:1 ratio.
M-2 3 F1 24 19 43 1:1 0.58 .25–.50
F2 626 166 792 3:1 6.90 .010–.05
F3 families
Recurrent Mutations at the Unifoliolate
43 S:39 NSa 82 2:1 7.493 .010–.005 Locus
F3 pooled (1 df ) 757 134 891 3:1 47.43 ,.005 The F2 and F3 generations of the cross IBS
Heterogeneity (42 df ) 35.92 .50–.75
2497 3 IBS 2625 segregated for plants with
a
Ratio of segregating to nonsegregating F3 families. unifoliolate leaf, although both parents in

46 The Journal of Heredity 2001:92(1)

 Table 4. Inheritance of leaf shape in crosses of trifoliolate normal and unifoliolate orbicular in pression of the petiolate trait in these
cowpea
crosses. The symbol pt-3 is assigned to the
No. of plants recessive gene in the mutant parental line,
Expected
Cross and generation Normal Orbicular Total ratio x2 P M-3, which in the homozygous condition
determines the nonpetiolate trait.
Ife Brown CR 3 M-2 In addition to being petiolate, the F1
Ife Brown CR 126 126
M-2 24 24 progeny of the cross between the two uni-
F1 28 28 foliolate lines were also trifoliolate, sug-
Ife Brown CR 3 F1 88 — 88
M-2 3 F1 74 61 135 1:1 1.25 .25–.50
gesting that the genes controlling leaflet
F2 508 147 655 3:1 2.28 .10–.25 number in the two mutants are different
F3 families and complementary to each other. The
23 S:11 NSa 34 2:1 0.15 .50–.75
F3 (pooled, 1 df ) 288 101 389 3:1 0.14 .50–.75 symbol un-3 is assigned to the gene in the
Heterogeneity (22 df ) 22.56 .25–.50 unifoliolate mutant with ovate leaf shape.
TVu 6198 3 M-2
TVu 6198 142 142
M-2 24 24 Linkage Relationships
F1 27 27 The linkage relationships of the different
TVu 6198 3 F1 55 — 55
M-2 3 F1 24 19 43 1:1 0.58 .25–.50
genes were analyzed by using Linkage-1, a
F2 591 201 792 3:1 0.06 .75–.90 computer program for the detection and
F3 families analysis of genetic linkage (Suiter et al.
51 S:31 NSa 82 2:1 0.74 .25–.50
F3 pooled (1 df ) 813 268 1081 3:1 0.04 .75–.90 1983). The results, presented in Table 9,
Heterogeneity (50 df ) 59.57 .10–.25 show that of the five F2 linkage tests be-
tween gene pairs, two suggested indepen-
a
Ratio of segregating to nonsegregating F3 families.
dence while three indicated linkage. The
loci Pt-1 and Un-2 are linked with a map
the cross were trifoliolate. The same cross tion between two independently segregat- distance of 19.29, while Pt-1 is also linked
was repeated in 1993 and 1996 to deter- ing factors. with the locus Orb with a distance of
mine whether the occurrence of unifolio- 30.18. Thus Pt-1, Un-2, and Orb are located
late plants was a chance event or a regular Allelism Test on the same chromosome. The distance
feature of the cross. The results are pre- All of the lines used in the allelism test between Un-2 and Orb is 10.89. The genes
sented in Table 8. In all cases, the F2 and were nonpetiolate and two of them, M-2 Pt-3 and Un-3 are both located on the same
F3 generations segregated for orbicular- and M-3, were also unifoliolate. Progenies chromosome and have a map distance of
shaped unifoliolate plants, but the data of three crosses—IBS 401 3 IBS 2497, IBS 3.77. The genes Pt-3 and Un-3 showed in-
did not give a good fit to the 3 trifoliolate: 401 3 M-2, and IBS 2497 3 M-2—were non- dependence with respect to the gene Bpd,
1 unifoliolate monohybrid ratio. However, petiolate, which indicates that the nonpe- which controls peduncle type.
when the trifoliolate plants with orbicular tiolate condition in the parents involved in
leaf shape were separated from normal tri- the crosses is controlled by allelic genes. Discussion
foliolate plants and treated as a different The three other crosses, IBS 401 3 M-3,
phenotypic class, the data gave a good fit IBS 2497 3 M-3, and M-2 3 M-3, produced Mutations occurred in some selections of
to the dihybrid-dominant epistatic ratio of progenies with petiolate leaves. Thus the the cowpea crosses used in this study
12:3:1, except in the F2 generation scored results suggest that two different genes without any apparent cause other than the
in 1989 ( Table 8), suggesting an interac- with complementary action control the ex- nature of their hybrid origin. When non-
petiolate, trifoliolate plants from different
sources were crossed with each other, uni-
Table 5. Inheritance of leaf shape in crosses of trifoliolate normal and trifoliolate orbicular in cowpea foliolate plants appeared in the progenies
in the ratio 3 trifoliolate:1 unifoliolate, sug-
No. of plants
Expected gesting that the two parents in each cross
Cross and generation Normal Orbicular Total ratio x2 P were heterozygous for the unifoliolate
Ife Brown BPC IBS 2625 trait. However, when the unifoliolate mu-
Ife BPC 118 118 tant was crossed to normal trifoliolate
IBS 2625 46 46
F1 36 36
plants, the F2 and F3 generations deviated
F2 418 130 548 3:1 0.48 .25–.50 significantly from the expected monohy-
F3 families brid ratio. An examination of the plants in
22 S:18 NSa 40 2:1 2.45 .10–.25
F3 (pooled, 1 df ) 509 159 668 3:1 0.58 .25–.50 the two phenotypic classes showed an ex-
Heterogeneity (21 df ) 25.85 .10–.25 cess of trifoliolate plants and a marked de-
IBS 2497 3 IBS 2625 ficiency of unifoliolate plants. Further-
IBS 2497 108 108
C 2625 46 46 more, the trifoliolate plants that were in
F1 54 54 excess were those that had the mutant or-
F2 671 197 868 3:1 2.46 .10–.25
F3 families
bicular leaf shape. The orbicular leaf
36 S:14 NSa 50 2:1 0.64 .25–.50 shape results from a separate mutation
F3 pooled (1 df ) 688 217 905 3:1 0.56 .25–.50 from that affecting leaflet number and is
Heterogeneity (35 df ) 23.41 .75–.90
controlled by a single recessive gene. The
a
Ratio of segregating to nonsegregating F3 families. joint segregation data for the two traits,

Fawole • Genetic Analysis of Leaf Form Mutants in Cowpea 47

 Table 6. Inheritance of normal shaped unifoliolate leaf in four cowpea crosses were classified as unifoliolate also had
No. of plants
some leaves that were trifoliolate, bifolio-
Expected late, and even at various stages of incom-
Cross and generation Normal Unifoliolate Total ratio x2 P
plete division toward trifoliolate.
M-3 3 TVU 1509 In the orbicular-shaped unifoliolate mu-
M-3 98 98 tant, three closely linked genes have been
TVU 1509 134 134
F1 48 48 identified and genetically characterized.
F2 780 257 1037 3:1 0.03 .75–.90 These are the genes that control the uni-
F3 families
50 S:28 NSa 78 2:1 0.23 .50–.75
foliolate trait, the nonpetiolate leaf, and
F3 (pooled, 1 df ) 1364 462 1826 3:1 0.07 .75–.90 the orbicular leaf shape. Rawal et al.
Heterogeneity (49 df ) 50.70 .25–.50 (1976) described a unifoliolate mutant in
M-3 3 TVU 6198
M-3 98 a cowpea line and assigned the symbol un
TVu 6198 3 F1 104 to the recessive gene controlling the trait.
F1 28 The symbol un-2 is proposed for the gene
F2 834 250 1084 3:1 2.17 .10–.25
F3 families that controls the unifoliolate trait in the
33 S:11 NSa 44 2:1 1.38 .10–.25 mutant with orbicular leaf shape. On the
F3 pooled (1 df ) 583 208 791 3:1 0.64 .25–.50
Heterogeneity (32 df ) 37.63 .10–.25
other hand, two linked but different genes
M-3 3 Ife BPC control the unifoliolate and the nonpetiol-
M-3 98 98 ate traits in the mutant with the ovate leaf
Ife BPC 144 144
F1 35 35 shape. In a previous report, Fawole (1988)
F2 728 256 984 3:1 0.54 .25–.50 assigned the symbol pt to the petiole lo-
F3 families cus. Subsequently the gene was redesig-
26 S:20 NSa 46 2:1 2.13 .10–.25
F3 pooled (3 df ) 598 203 801 3:1 0.03 .75–.90 nated pt-1 ( Fawole 1990) to distinguish it
Heterogeneity (25 df ) 23.99 .50–.75 from the gene controlling the nonpetiolate
trait in a mutant plant described by Rawal
a
Ratio of segregating to nonsegregating F3 families.
et al. (1976). The pt-1 gene is nonallelic to
the gene controlling the nonpetiolate trait
however, indicated that they are closely ber of these plants could not be explained in M-3, the unifoliolate mutant with ovate
linked. If recombination occurred between solely on the basis of recombination, leaf shape used in this study, and the sym-
the two loci, both orbicular-shaped trifo- therefore reversion from the recessive uni- bol pt-3 is therefore suggested for the pet-
liolate and ovate-shaped unifoliolate re- foliolate to the dominant trifoliolate form iole gene in M-3.
combinant plants are expected in the F2 was probably responsible for the occur- The appearance of a new nonpetiolate
generation. On the contrary, only orbicu- rence of most of these plants. Support for unifoliolate mutant in the F3 generation of
lar-shaped trifoliolate recombinant plants the reversion hypothesis is furnished by the cross IBS 2497 3 IBS 2625 suggests the
were obtained. The relatively large num- the fact that several of the plants that transfer of mutability to a new locus. Un-
like the orbicular-shaped unifoliolate mu-
tant, the inheritance data of this new uni-
Table 7. Inheritance of nonpetiolate leaf in crosses of petiolate 3 nonpetiolate unifoliolate in cowpea
foliolate leaf mutant showed no deviation
No. of plants from the monohybrid ratio. However,
Expected
Cross and generation Petiolate Nonpetiolate Total ratio x2 P some of the unifoliolate plants in the F2
and backcross generations produced
M-3 3 TVU 1509
M-3 98 98
leaves that were bioliolate, trifoliolate, or
TVU 1509 134 134 at various stages of incomplete division
F1 48 48 toward trifoliolate in addition to unifolio-
F2 765 272 1037 3:1 0.84 .25–.50
F3 families late leaves. In both unifoliolate mutants,
56 S:20 NSa 76 2:1 1.68 .10–.25 M-2 and M-3, the genes controlling the
F3 (pooled, 1 df ) 1484 570 2054 3:1 8.14 ..005 nonpetiolate trait are separable by recom-
Heterogeneity (55 df ) 126.05 ..005
M-3 3 TVU 6198 bination from those controlling the unifo-
M-3 98 98 liolate leaf, although the ovate-shaped uni-
TVu 6198 104 104
F1 28 28
foliolate mutant indicated closer linkage
F2 828 256 1084 3:1 1.11 .25–.50 with the nonpetiolate trait than the other
F3 families mutant. Similarly the orbicular leaf shape
33 S:9 NSa 42 2:1 2.68 .10–.25
F3 pooled (1 df ) 581 219 800 3:1 2.28 .05–.10 gene is separable from the nonpetiolate
Heterogeneity (32 df ) 49.39 .025–.05 trait. However, no recombination was ob-
M-3 3 Ife BPC served between the genes controlling leaf
M-3 98 98
Ife BPC 144 144 shape and leaflet number. This is probably
F1 35 35 due to very close linkage of these genes.
F2 738 246 984 3:1 0.00 .995
F3 families
Instances of single gene mutations re-
28 S:17 NSa 45 2:1 1.60 .10–.25 sulting in unifoliolate true leaves have
F3 pooled (1 df ) 662 190 852 3:1 3.46 .05–.10 been reported in some leguminous spe-
Heterogeneity (27 df ) 36.34 .10–.25
cies. Lamprecht (1935) described a highly
a
Ratio of segregating to nonsegregating F3 families. sterile unifoliolate mutant in Phaseolus vul-

48 The Journal of Heredity 2001:92(1)

 Table 8. Segregation for unifoliolate leaf plants in the F 2 and F 3 generations of a cross between two
trifoliolate cowpea lines
No. of
Generation and trifoliolate Trifoliolate Unifoliolate
year of crossa plants orbicular
F2 (1989) 671 164
F3 (1989)a 250 63
F2 (1993) 165 45
F3 (1993)a 179 44
F2 (1996) 560 141
F3 (1996)a 222 47
a
Only selected petiolate trifoliolate F2 plants segregating in the F3 generations were considered.
garis which is controlled by a single reces-
sive gene, while Garrido et al. (1991) re-
ported another unifoliolate mutant in the
same species, encoded by a single domi-
nant gene with some deleteriousness in
unifoliolate homozygotes. In Pisum sati-
vum, Hofer et al. (1997) observed that the
unifoliolate gene has a pleiotropic effect
on flower development and therefore con-
cluded that leaves and flowers share reg-
ulatory processes in their morphogenesis
that may be useful in the study of plant
development. The mutations that are re-
ported in this study affect closely linked
genes that control leaf form traits in cow-
pea. However, abnormal flower traits of
both unifoliolate mutants are inherited to-
gether with unifoliolate leaf, as if flower
malformation results from the pleiotropic
effect of the unifoliolate genes. Results
from other crosses, however, suggest that
the genes controlling flower form are dis-
tinct from but tightly linked to those de-
termining leaflet number ( Fawole I, un-
published data).
The origin and nature of the nonpetiol-
ate unifoliolate mutations are similar to
those reported previously in certain cow-
pea crosses ( Fawole 1988, 1997). In those
crosses the cultivar Ife BPC, when used as
the female parent, facilitated the occur-
rence of high frequency mutations at cer-
tain loci. Further evidence of this unusual
behavior is furnished by the advanced
generation progeny of the cross Ife BPC 3
IT81D–1137. In addition, it appears that
some lines derived from such crosses can
inherit the ‘‘mutator’’ ability in a more po-
tent manner than Ife BPC. For example,
orbicular Total Tested ratio x2 P
33 868 12:3:1 8.947 .010–.025
13 326 12:3:1 2.875 .10–.25
10 220 12:3:1 1.364 .50–.75
23 246 12:3:1 4.044 .10–.25
31 734 12:3:1 5.070 .05–.10
19 288 12:3:1 1.130 .25–.50
the orbicular-shaped unifoliolate mutation
can be induced at will by making the IBS
2497 (female parent) 3 IBS 2625 (male par-
ent) cross and searching for the mutant in
the F2 and F3 generations. Transposable el-
ement activity had been implicated in the
occurrence of the unusually high mutation
frequencies observed in segregating pop-
ulations of crosses involving Ife BPC as the
female parent ( Fawole 1988, 1997).
The behavior of the nonpetiolate, uni-
foliolate mutations described in this arti-
cle also exhibit some features attributable
to the action of transposable elements.
First, the insertion of a transposable ele-
ment at a locus may inhibit the expression
of that gene and confer on it the property
of high mutability. The excision of the el-
ement from the locus restores the activity
of the gene and the reintegration of the
element at another locus may result in in-
stability at the new locus ( Fedoroff 1983;
McClintock 1956; Nevers et al. 1986; Peter-
son 1968). The nonpetiolate unifoliolate
and the orbicular leaf shape mutants prob-
ably resulted from such insertion and ex-
cision events.
Second, mutations mediated by the action
of transposable elements often exhibit un-
stable expression and revert to a phenotyp-
ically wild-type condition in somatic and/or
germinal tissue. Frequent reversions to wild
type in germinal tissue may lead to distort-
ed Mendelian ratios in crosses involving a
mutable allele (Nevers et al. 1986). Data on
the inheritance of the orbicular-shaped uni-
foliolate mutant showed that when stable
unifoliolate plants were crossed to normal
lines, there was significant deviation from
the expected monohybrid ratio. The devia-
tion was caused by an excess of wild-type
trifoliolate individuals that could not be
explained on the basis of genetic recom-
bination and are probably due to rever-
sion of the recessive unifoliolate gene to
the wild-type trifoliolate condition. Evi-
dence of reversion was also provided by
several unifoliolate plants that showed
various degrees of reversion from the uni-
foliolate to the trifoliolate state.
Third, crossing is often necessary to
initiate mutability. For example, in the
nonautonomous two-element system of
maize, the gene influenced by a trans-
posable element remains stable until a
regulatory element is introduced into
the system by crossing. Only then does
a transposable element induce mutation
which may be to a recognizable change
in phenotype ( Dooner 1983; McClintock
1951, 1953, 1956; Peterson 1986, 1995). In
the cowpea lines used in this study, mu-
tability normally occurred only when Ife
BPC and IBS 2497 were used as female par-
ents in crosses to certain selected lines.
This suggests a two-component system in
which factors in the genome of Ife BPC
and IBS 2497 responded to signals from
the lines used as male parents.
The involvement of two independently
segregating factors is indicated in the IBS
2497 3 IBS 2625 cross, which not only re-
peatedly produced a trait not present in
either of the two parents but also consis-
tently showed interaction between factors
that are located on the chromosomes of
the two parental lines. These factors seg-
regated in the dominant epistatic ratio of
12:3:1 in the F2 and F3 generations.
The phenotypic changes reported in
this study represent only a portion of the
total variation that has been generated in
crosses between the mutable and normal
lines. New mutations have been identified
at loci controlling stem growth habit ( Etta
1995), flower form and anthocyanin pig-
mentation on pods and flowers ( Fawole
1998; Oluwatosin 1997), and foliage color
( Fawole 1997). Some of these new mu-
tants have been studied while others are
yet to be genetically characterized.

Table 9. F2 progeny distribution and calculated recombination relationships of some alleles in cowpea
References
No. of plants
Recombination Map distance Dooner HK, 1983. Coordinate genetic regulation of fla-
Alleles a b c d Total x2 linkage (% 6 SE) 6 SE vanoid biosynthetic enzyme in maize. Mol Gen Genet
67:345–355.
Pt-2pt-2Un-2un-2 1011 27 203 206 1447 495.50 18.39 6 2.52 19.29 6 2.52 Etta CE. 1995. Genetic analysis of growth habits and
Pt-2pt-2 Orborb 902 146 197 202 1447 213.03 26.98 6 2.41 30.18 6 2.41 their interrelationships with grain yield in cowpea (Vig-
Pt-3pt-3 Un-3un-3 2279 52 62 711 3104 2522.46 3.76 6 1.79 3.77 6 1.79 na unguiculata ( L.) Walp.) (PhD dissertation). Ibadan,
Pt-3pt-3 Bpbpd 579 163 171 71 984 5.47 44.58 6 2.53 71.48 6 2.53 Nigeria: University of Ibadan.
Un-3un-3 Bpdbpd 569 158 181 76 984 6.44 44.31 6 245 70.20 6 2.54
Fawole I and Afolabi NO, 1983. Genetic control of a

Fawole • Genetic Analysis of Leaf Form Mutants in Cowpea 49

branching peduncle mutant of cowpea, Vigna unguicu-
lata ( L.) Walp. J Agric Sci Camb 100:473–475.
Fawole I, 1988. A nonpetiolate leaf mutant in cowpea,
Vigna unguiculata ( L.) Walp. J Hered 79:484–487.
Fawole I, 1990. Inheritance of petiole development and
unifoliolate leaf in cowpea, Vigna unguiculata ( L.) Walp.
Nigerian J Sci 24:1–3.
Fawole I, 1997. Inheritance of crinkled leaf in cowpea,
Vigna unguiculata ( L.) Walp. Nigerian J Sci 31:35–40.
Fawole I, 1998. Two unstable dominant genes in cow-
pea, Vigna unguiculata ( L.) Walp. Eighteenth Interna-
tional Congress of Genetics, Beijing, China, August 10–
15. Chinese Academy of Sciences and the Genetics So-
ciety of China; 29.
Fedoroff N, 1983. Controlling elements in maize: a sub-
title. In: Mobile genetic elements (Shapiro J, ed). New
York: Academic Press; 1–63.
Garrido B, Nodari R, Debouck DG, and Gepts P, 1991.
Uni-2-A dominant mutation affecting leaf development
in Phaseolus vulgaris. J Hered 82:181–183.
Hofer J, Turner L, Hellens R, Ambrose M, Matthews P,
50 The Journal of Heredity 2001:92(1)
Michael A, and Ellis N, 1997. UNIFOLIATA regulates leaf
and flower morphogenesis in pea. Curr Biol 7:581–587.
Lamprecht H, 1935. Zur Genetik Von Phaseolus vulgaris.
XI. Eine Mutante mit einfachen Blattern und ihre Ver-
erbungsweise. Hereditas 20:238–250.
McClintock B, 1951. Mutable loci in maize. Carnegie In-
stitute Washington Year Book 50:174–181.
McClintock B, 1953. Induction of instability at selected
loci in maize. Genetics 38:579–599.
McClintock B, 1956. Controlling elements and the gene.
Cold Spring Harbor Symp Quant Biol 21:197–216.
Nevers PN, Shepherd S, and Saedler H, 1986. Plant
transposable elements. Adv Bot Res 12:103–203.
Oluwatosin OB, 1997. Inheritance and instability of
genes controlling anthocyanin pigmentation, seed coat
colour and leaf form in cowpea, Vigna unguiculata ( L.)
Walp. (PhD dissertation). Ibadan, Nigeria: University of
Ibadan.
Peterson PA, 1968. The origin of an unstable locus in
maize. Genetics 59:391–398.
Peterson PA, 1986. Mobile elements in maize. Plant
Breed Rev 4:81–112.
Peterson PA, 1995. The development of transposon bi-
ology: from variegation to molecular confirmation.
Maydica 40:117–124.
Porter WM, Rawal KM, Rachie KO, Singh SR, Wien HC,
and Williams RJ, 1974. The cowpea germplasm catalog
no. 1. Ibadan. Nigeria: International Institute of Tropical
Agriculture.
Rawal KM, Porter WM, Franckowiak JD, Fawole I, and
Rachie KO, 1976. Unifoliolate leaf: a mutant in cowpeas.
J Hered 67:193–194.
Suiter AK, Wendel FJ, and Case JS, 1983. Linkage-1: a
Pascal computer program for the detection and analy-
sis of genetic linkage. J Hered 74:203–204.
Summerfield RJ and Roberts EH, 1985. Vigna unguicu-
lata: a subtitle. In: Handbook of flowering, vol. 1 ( Hal-
vey AH, ed). Boca Raton, FL: CRC Press; 171–184.
Received March 20, 2000
Accepted August 31, 2000
Corresponding Editor: Susan Gabay-Laughnan