BokuaualJoumal ofrhc Linnean Sociep (1999), 129: 239-247. With 9 figures
Article ID: bojl. 1998.0214, available online at http://www.idealibrary.com on
I DE
bL
Sex differences in meiosis between Kcia faba L.
and its close wild relatives
K. K. KOUL', S. N. RAINA, A. PARIDA' AND M. S. BISHT
Laboratoly o f Cellular and Molecular Cytogenetics, Department of Botary, Universig o f Delhi,
DeMi-110007, India
Received May 1998; acceptedfor publication September 1998
Comparison of chiasma frequency was made in male and female meiocytes of the widely
cultivated &a fabu L. and its close wild allies in the Ir,,narbonensk species complex. Contrary
to most of the earlier reports of higher chiasma frequency in female sex cells, a higher
chiasma frequency was observed in male sex cells. This pattern is restricted to the species
representing the Narbonensis complex, since Kcciafaba shows identical chiasma frequency in
male and female sex cells. The genetic significance of these findings is that chiasma formation
in the sex cells of the putative allies is governed and regulated by similar controlling systems
of genes, but that of K fuba indicates a different kind of system.
0 1999 The Linnean Society of London
ADDITIONAL KEY WORDS:-chiasma frequency - Fabaceae - male vs female meiosis.
CONTENTS
Introduction . . . .
Material and methods
Results . . . . .
Karyotypes . .
Meiosis. . . .
Discussion . . . .
Acknowledgements .
References . . . .
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239
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INTRODUCTION
In comparison to the abundant data available on various aspects of male meiosis
in plants, there have been few investigations of female chromosome behaviour,
which represents the second component of two-track heredity (Brock, 1954; Fogwdl,
1958; Ved Brat, 1966; Darlington, 1971; Vosa, 1972; Bennett et al., 1973; Noda,
'
Corresponding author. Present address: Department of Botany, Hindu College, University Enclave,
Delhi-110007, India
Present address: M.S. Swaminathan Research Foundation, Taramani, Madras-600113, India
0024-4074/99/030239+09 $30.00/0
239
0 1999 The Linnean Society of London
240
K. K. KOUL ETAL.
1975; Koul, Gohil & Kaul, 1980, 1981; Koul, 1983; Koul & Gohil, 1989; Koul &
Raina, 1996; Nagpal & Raina, 1995). The main reason for this is the technical
problem related to the strikingly low number of delicately placed embryo-sac mother
cells (EMCs) relative to the thousands of pollen mother cells (PMCs) available for
study in most anthers, and also the extremely low chance that EMCs, once located
in the mass of nucellar cells, would be at the required stage. Nevertheless, the limited
data available from comparative studies of male and female meiosis have revealed
variation in chiasma position, distribution and frequency in the two types of sex
cells of some plant species (Koul, 1983; Koul et al., 1995; Koul & Raina, 1996).
If differences in chiasma frequency, which affect the recombination index, exist
in economically important species such as Eciafaba, the fourth most important of
the food legume crops (Kay, 1979), they could have profound implications for the
controlled handling of genetic variation in breeding programmes within species, and
between cultivated species and their wild relatives. In the genus Ecia no species has
been found which can be identified clearly as the wild progenitor of Vfaba. Zohary
& Hopf (1973) suggested that the V narbonensis species complex ( V narbonensis, K
serrutijilia, I? johannis, V galilaea, V hyaenisyamus, V kalakhensis, V eristalioides) share a
common ancestry with Kfaba (Mated, Callimassia & Bennett, 1991) while others
(Davis& Plitmann, 1970; Schafer, 1973; Kupicha, 1976; Chapman, 1984; Roupakias,
1986) considered K narbonensis and V galilaea to be the closest known wild relatives
of Kfaba. Several workers have reported genetic resistance in many species of the
complex to important pathogens and pests of Kfaba (Birch, Tithecott & Bisby,
1985). This has given an impetus for research on the transfer of such useful traits
from the wild relatives to the cultivated V faba. During the present series of
investigations the meiotic behaviour of chromosomes in the male and female sex
cells of Vfabu and its wild relatives have been studied with a view to establish their
relationship.
MATERIAL AND METHODS
Eczizfaba (2n= 2x= 12), the broad bean, known for its rich protein content, and
its six close wild relatives, i.e. Ecia narbonensis, Vjohannis, V galilaea, V serratijdia, V
hyaenisyamus and V kalukhensis, representing the narbonensis species complex, were
analysed for comparative studies of male and female meiosis. Seeds of the various
accessions were obtained from the International Centre for AgTicultural Research
in the Dry Areas (ICARDA), Aleppo, Syria. The voucher specimens are lodged in
the Department of Botany, Delhi University, Delhi, India. Young buds were collected
from field-grown plants and fixed in 1:3 acetic acid: ethanol. The anthers and
ovaries were removed from the fixed buds and hydrolysed together in 5N HCl for
45 min at room temperature (c. 27°C). Care was taken to hydrolyse and study both
anthers and ovaries from the same individual to minimize any chiasma variation
brought about by ecological or inter-plant genetical differences. After hydrolysis the
material was washed in water and then stained in 0.45% Feulgen (Sigma) for 1 h.
For male meiotic studies the stained anthers were squashed on slides in 1 % acetocarmine whereas for female meiotic studies the ovules were first isolated from the
ovaries using a dissection microscope and then gently squashed in 1OO/ aceto-carmine
under a cover slip.
MEIOSIS IN VICIA SPECIES
241
RESULTS
Karyotypes
In all six species in the narbonensis complex the somatic cells possess 14 mediansubmedian chromosomes (arm ratio> 1:1 <3: 1). Vfaba stands out both in chromosome
number (2n = 2x = 12) and morphology. Barring the first pair of very large submetacentric chromosomes, all the chromosomes are subtelocentrics (arm ratio >3:
1<0: 1).
Meiosis
The chiasma data obtained from PMCs and EMCs in the seven taxa studied are
summarized in Table 1. For the accurate study of chiasma frequency attention was
concentrated on those cells where bivalents showed minimal overlapping. Moreover,
to avoid any error while taking the observations on chiasma frequency, similar
stages of meiosis, whether diplotene, diakinesis and/ or metaphase, were considered
both in PMCs and EMCs of each species.
Narbonensis species complex
Meiosis in all six species was regular with seven bivalents clearly discernible in
all PMCs studied at diplotene/ diakinesis and/ or metaphase I (Figs 1, 3, 6). The
bivalents were tightly paired and showed a high chiasma frequency which ranged
from 15.6± 1.28 per pmc in V.johannis to 28.7 ±0.85 in V. Jv!aeniscyamus.
Meiosis in EMCs of the species in the complex, barring V. kalakhensis and V.
johannis, also showed perfect bivalent formation at diplotene/diakinesis (Figs 2, 4,
5). However, unlike the observations made on PMCs, the bivalents in EMCs were
not so tightly paired and showed a significant decrease in the overall mean chiasma:
frequency per EMC in every species (Table 1). They ranged from 10.0±0.83 in V.
kalakhensis to 22.71 ± 0.87 in V. hyaeniscyamus. In V. kalakhensis and V. johannis up to
four univalents were recorded in 30 and 40% respectively of the cells studied at
metaphase I.
Viciafaba
Meiosis in V. Jaba, unlike that of the six species in the narbonensis complex, did not
show any variation in chiasma frequency between the two sex cells. In both the
PMC and EMC it was regular with six bivalents regularly formed (Figs 7, 8, 9).
The mean chiasma frequencies per EMC and PMC were identical at 18.46±0.49
and 18.72 ±0.44 respectively. Interestingly, the bivalent shape in all the cells studied
at metaphase I did not vary much and the largest bivalent invariably showed five
or six chiasmata in all the cells studied, whether EMC or PMC.
To investigate whether the differences in mean chiasmata frequency in the two
sex cells of the Vicia narbonensis species complex were significant, statistical tests were
employed assuming that (1) the two variables (i.e. chiasma frequency in EMC and
PMC) follow a normal or similar pattern of distribution and/ or (2) the distribution
pattern of the two variables with respect to the parent population was unknown.
The statistical tests employed included Fisher's variance ratio test, students t-test,
2362
3993
4162
4159
2946
l? seratifolk Jacq.
l? narbonmric L.
Kgalilaea Plitm. & Zoh.
K hyoenisyamus Mout
K kalakhensis Khatta. Max & Bis.
K johannis Tamam
4010
1185
1186
1190
V ifaba L.
V i narbonmric species complex
ACC.No.
Species
14
14
14
14
14
14
12
2n
Wild
Wild
Wild
Wild
Wild
Wild
Cultivated
Status
male 50
female 42
male 50
female 40
male 50
female 42
male 50
female 38
male 50
female 39
male 50
female 43
male 50
female 45
No. of cells
analysed
Metaphase I
Diplotene
Diakinesis
Diakinesis
Diakinesis
Metaphase I
Metaphase I
Stage analysed
15.6f 1.28
10. I6k0.84
27.02 k0.96
21.47f1.48
20.56 k0.98
14.66 kO.88
20.88 f 1.08
16.47 kO.78
28.77 kO.85
22.71 k0.87
15.5kO.9
10.0f0.83
18.72f0.44
18.46f0.49
Chiasmata/cell k SD
TASLE
1. Mean number of chiasmata observed in pmc and emc of Ir,,species
,
0
5
h
20.54
35.48
-
21.06
-
21.12
-
28.69
~
-
r
34.87
?
?
-
1.38
-
Oh reduction
of xta in female
e
N
N
MEIOSIS IN VICL4 SPECIES
243
Figures 1-9. Male and female meiosis in V!cia species. Fi~?;. l. PMC at diakinesis in V narhomll.\1~1 (II=
7). Fig. 2. EMC at diakinesis in V narbonensis. Fig. 3. PMC at diakinesis in V smatffolia (11 = 7). Fi~?;. 4.
EMC at diakinesis in V smatijolia. Fig. 5. El\1C at diplotene in V l!)'aenis1yamus (n = 7). Fig. 6. PMC at
diplotene in V l!)'ami.1ryamus. Figs 7, 8. EMCs at metaphase in Vfaba (11 = 6). Fig. 9. PMC at metaphase
in V faba. Scale bar= 10 11m.
K. K. KOUL ETAL..
244
TABLE
2. Statistical values obtained to test the significance for the difference of mean and variance in
various species of Edu
Species
F-ratio
idf)
Hcia faba
1.24
(93)
Narbonmk complex
CIjohannk
2.30
(90)
I .70
c( serralifoka
t-values
Large sample test Z
Mann-Whitney U-b’dcoxon trst
Ho: P I =p2
HI: p l k p 2
U-value
IZI value
2.58
2.6
2.62
840.0
+2.5033
23.29
23.65
24.50
0
+8.315
29.53
30.00
30.83
0
+8.237
29.56
30.10
3 1.05
0
+8.336
20.87
21.20
29.79
0
+8. I 0 2
32.05
32.67
32.37
0
t-8.187
30.04
30.55
30.72
0
+ 8.398
(88)
K narbond
l!g a l i h a
I? hyaenkcyamus
l! kalakhd
1.21
(90)
1.91
(86)
I .06
(87)
1.16
(91)
normal test and Mann-Whitney U-Wilcoxon test, the last being used to test the
mean or equality of such populations where the distribution of two variables is
unknown. The various values obtained are given in Table 2, which shows that the
F-ratios calculated in three species ( k i a galilaea, Kjohannis and K smat$olia) exceeds
the tabulated F-ratio at a 5% level of significance, suggesting that the variance
within the two populations of male and female sex cells is highly significant. However,
in the remaining four species (Table 2) the two populations did not show any
significant variation from the mean value.
The t-test, normal test (Z) and the Mann-Whitney U-Wilcoxon test reveal the
mean values of the two sex cells to be significantly different from each other at the
1YO and 5% levels of significance. This suggests that the two variables did not follow
similar distribution patterns and/or did not represent the same population. In Ecia
faba, on the other hand, the calculated values indicated the chiasmata frequencies
in the populations of two sex cells to be identical.
DISCUSSION
The present observations are interesting in many ways. Unlike the common
observation of higher chiasma frequency on the female side (Pastor & Callan, 1952;
Fogwill, 1958; Ved Brat, 1966; Vosa, 1972; Koul & Raina, 1996), the taxa studied
here showed the reverse trend, with higher chiasma frequency in the PMCs. Such
a situation has been reported in some five cases, i.e. Allium cepa, A. n&m, A. kachrooi,
Brassica olcyrrhina and Phlox drummondii (Gohil & Kaul, 1980; Koul et al., 1995; Koul
& Raina, 1996). Similarly, identical chiasma frequency in the two sexes has also
been found only in a few cases, which include Secale cereale, Hordeum vukare, Calamagrostis
stoliczkai and Lilium species (Brock, 1954; Bennett et al., 1973; Davies &Jones, 1974;
Koul & Gohil, 1989; Koul & Raina, 1996).
In the context of chiasma control, the most important finding from this study is
MEIOSIS IN VICU SPECIES
245
the constant occurrence of lower chiasma frequency on the female side in all the
species within the narbonensis complex, in contrast to the identical chiasma frequency
observed in the two sex cells in Kfaba. The occurrence of higher chiasma frequency
on the male side is more likely to have an important function as far as its contribution
to genic recombination in the species of the complex is concerned. It seems likely
that effective recombination in these species is brought about chiefly by male meiotic
bivalents whereas the female side provides for the retention of certain linkage groups.
Based on cytogenetic, biosystematic and chemotaxonomical evidence (Schafer, 1973;
Ladizinsky, 1975; Raina & Rees, 1983; Raina, 1990; Maxted et al., 1991) there is
no doubt that the genomes of the species within the complex are genetically very
close to each other. The indications are that differentiation between the species
might be mainly a result of segmental interchanges (Ladizinsky, 1975; Hanelt &
Mettin, 1991).Their relatedness is further reflected in the identical meiotic behaviour
in the EMC and PMC. In other words, since homologous synapsis and chiasma
formation are known to be under genetic control, similar to several other events of
meiosis (Baker et al., 1976; Koduru & Rao, 1981), it appears from the present study
that the genetic mechanisms controlling chiasma formation in the meiocytes are
alike in the species of the complex. This is evident from the similar chiasma
frequency/ distribution pattern in the corresponding two sexes. Sex difference in
meiosis in having random distribution of chiasmata in one sex and non-random
distribution in the other (Watson & Callan, 1963; Ved Brat, 1966; Vosa, 1972;
Gohil & Kaul, 1980), higher chiasmata frequency in one of the two sex cells (Fogwill,
1958; Watson & Callan, 1963; Ved Brat, 1966; Vosa, 1972; Gohil & Kaul, 1980;
Koul et al., 1995; Koul & Raina, 1996) and/or achiasmate versus chiasmate meiosis
in the two sexes (Noda, 1975) within species is not uncommon. In this context it is
important to consider whether chiasma formation in the two sexes is governed and
controlled by a single or independent controlling system(s) of genes. The extremely
limited information (inferred by genetic tests) available on this aspect gives evidence
for both (Davies &Jones, 1974; Koul, 1983). Davies &Jones (1974), while discussing
the genetic systems controlling meiosis in two sexes in distinct inbred lines of rye,
concluded that the display of an extreme form of sex difference with respect to
chiasma frequency/distribution and/or complete achiasmy in one sex suggests
separate genetic controls governing meiosis, while taxa with an identical chiasma
condition in the two sexes have a single joint control.
If the views of Davies &Jones (1974) are followed, one could say that in Kfuba,
with similar meiosis and/or chiasma formation in the two sexes, joint or identical
genetic control is operational, but in the taxa in narbonensis species complex two
separate controls appear to work. However, such conclusions, based merely on the
nature of meiosis, may prove wrong. This is so because simple comparison of meiosis
in two sex cells does not necessarily imply the presence or absence of different
genetic controls, as similar chiasma conditions might result from two independent
control systems acting convergently to produce the same effect. Similarly, the sex
difference at meiosis could be the differential response of a joint control to the
differing conditions in two sex cells. Therefore, it becomes rather difficult to underline
the actual genetic system/control working in the two sex cells in the present material.
Nevertheless, whatever control mechanism might exist, the present study has
established beyond doubt the differential behaviour of chromosomes in Kfaba versus
the species of the narbonensis complex. The unavoidable conclusion that can be
drawn from this account is that Kfabu is genetically distinct in its nuclear genome
246
K. K.KOUL E'TAL.
from the presumed allies. This is, in fact, in agreement with the conclusions reached
by several investigators (Schafer, 1973; Ladizinsky, 1975; Roupakias, 1986; Raina,
1990; Maxted et al., 1991; Raina & Ogihara, 1994, 1995). Comparable evidence
on meiotic behaviour in the two sexes in a range of species is clearly needed
to determine the extent of this correlation between wild and cultivated species.
Furthermore, a wide range of genetically different types (e.g. meiotic mutants, inbred
lines) needs to be studied to assess the influence of the genotype in relation to
chiasma control in two sex cells.
ACKNOWLEDGEMENTS
KKK thanks Dr Sudhir Kapoor and Mr Kamal Nain Kapoor of the Statistics
Department, Hindu College, Delhi University, Delhi for their generous help in
statistical analysis.
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