Cytological characteristics of reciprocal interspecific F hybrids be ...

CARYOLOGIA Vol. 64, no. 4: 377-387, 2011
Cytological characteristics of reciprocal interspecific F 1
hybrids between
the wild species Antirrhinum litigiosum Pau and Antirrhinum
pulverulentum Lazaro
Nikolova 1 Vesselina and Isabel Mateu-Andrés 2
1
Maritsa Vegetable Crops Research Institute; 32, Brezovsko shose Str. 4003 Plovdiv, Bulgaria; phone: + 359 32
991 227; fax: + 359 32 650 177; e-mail: veseto@hotmail.com
2
Instituto Cavanilles de Biodiversidad y Biología Evolutiva y Departamento de Botánica, Universidad de Valencia,
C/ Dr. Moliner 50, E-46100- Burjassot, Valencia; e-mail: isabel.mateu@uv.es
Abstract — The obtained and cytological investigated A. litigiosum x A. pulverulentum and A. pulverulentum
x A. litigiosum F 1
plants are diploids 2n = 2x = 16. Variously shaped bivalents with one or more chiasma formation,
positioned in different chromosome regions were observed. Hybrids presented reduction of the chiasma
frequency and number of ring type of bivalents, in comparison to the parent species. The close connection of
the chromosome condensed chromatin regions with the nucleolus or with the nucleoli was established, as the
assembly continued from the leptoten – zygotene to the dyplotene – early diakinesis stages. This suggested either
transcriptional activity of ribosomal genes before these stages or existence of some activity of the “silenced”
genes. The irregularities in chromosome behaviour during microsporogenesis in the F 1
hybrids, especially the
lower homology expression (66.4 and 61.1% of the pollen mother cells with regular chromosome pairing and
formation of 8II), caused from the phylogenic distance between A. litigiosum and A. pulverulentum genomes on
one hand and chromosome rearrangements (presence of multivalent and univalents, late bivalent disjunction,
bridge formation) on the other had a major effect on the chromosome unbalanced gamete formation and on the
reduction of the hybrid pollen fertility - 71.7 and respectively, 67.5%.
Key words: A. litigiosum, A. pulverulentum, chiasma, chromosome pairing, chromosome rearrangement, incomplete
synapsis, karyotype distance.
*Corresponding author:
INTRODUCTION
Interspecific hybridization is considered to
be a major source of genetic recombination subject
to plant evolution (Arnold 1997: in Aparicio
and Albaladejo 2003).
The classic genomic analysis involves the
evaluation of the meiotic chromosome behavior
in interspecific hybrids in the metaphase one,
with the purpose of establishing phylogenetic relationship
in group of different species (Dewey
1982; Seberg and Petersen 1988).
Investigating the hybrid complex of Phlomis
composita, Aparicio and Albaladejo (2003) concluded
that the translocations, inversions and
deletions, characterised parental taxa, flowing
freely in the hybrid and caused meiotic abnormalities
during microsporogenesis, as multivalent
or univalent formation, late bivalent disjunction
with bridge and chromatin bridges and
fragments. The authors observed relatively high
pollen fertility.
Manisha et al. (2007) reported sufficient high
degree of chromosome pairing in the F 1
hybrid
Gossypium arboreum x G. thurberi, but unequal
separation of chromosomes and chromatids during
anaphase stages, which led to the formation of
unequal sporads and low pollen fertility – 64.2%.
The defining event in meiosis is prophase I,
during which, homologous chromosomes locate

378
vesselina and mateu-andrés
each other, physically connect, and exchange genetic
information (recombination of the genetic
material). The degree of recombination can be
determined by observing the chiasma formation.
According to Darlington (1931), localisation
of the chiasmata occurred in particular regions
along a bivalent. The genetic control of the chiasma
creation has been supposed by Darlington
(1937), Rees (1955), Rees and Thompson
(1956), Roseveir and Rees (1962) and Rees and
Jones (1977). John and Lewis (1965) concluded
that the chiasmata decrease or are completely
absent in the heterochromatin chromosome regions.
The genus Antirrhinum L. (2n = 2x = 16) has
some 25 species, all of them perennial diploid
plants, distributed in the Mediterranean area
(Sutton 1988). The Iberian Peninsula is their
centre of diversification, as 23 species are natural
from this area.
As a model system, the ornamental species
A. majus L. has been genetically and molecularly
well investigated (Schmidt and Kudla 1966;
Stubbe 1966; Lai et al. 2002; Schwarz-Sommer
et al. 2003). Some cytological (Sparrow et al.
1942; Stein 1942; Berger et al. 1951; Stubbe
1996) and molecular cytogenetic investigations
(Zhang et al. 2004; Xue et al. 2009) have been
performed to reveal the origin and the nature of
A. majus. There is very limited information in
the literature about chromosome morphology
and meiotic chromosome behaviour in the wild
Antirrhinum species and in their remote hybrids.
The performed cytological study is the second
step in our research programme. The purpose
of this programme was to investigate the
chromosome pairing at the early meiotic stages,
the type of the bivalent configurations and some
irregularities of the chromosome behaviour during
microsporogenesis in three wild Antirrhinum
species-A. litigiosum Pau, A. subbaeticum
Güemes, Sánchez and Mateu and A. pulverulentum
Lazaro and in their artificially obtained F 1
hybrids. The data can be useful to gain information
about karyotipic distance and relationships
between the studied Antirrhinum species.
Fig. 1 — Microsporogenesis in A. litigiosum x A. pulverulentum F1 plants-Diakinesis: (a) normal, (b, c, d) with 2,4 and
6I, (e, f, g) with different shaped IV; MI: h-normal, (i, j) with 2-4I, (k, l) with IV formation, (m, n) MI with abnormalities;
AI: (o) normal, (p, q, r, s) with lagging chromosomes and bridge formation; (t) MII; AII: u-normal, (v, w) with
irregularities; (y) tetrads, dyads; z- pollen.

cytological characteristics of hybrids between antirrhinum litigiosum and a. pulverulentum 379
MATERIALS AND METHODS
Microsporogenesis and some bivalent characteristics
were investigated in pollen mother
cells (PMCs) of two reciprocal interspecific F 1
hybrids – A. litigiosum x A. pulverulentum and
A. pulverulentum x A. litigiosum – in the laboratory
of the Department of Botany - Valencia
University, Spain in 2010. The hybrids were obtained
by hand pollination during 2009, as the
F 1
plants from the combination between four A.
litigiosum plants (No1, 1/3, 1/4 and No 3) and
pollinator A. pulverulentum No2/1 and from
reciprocal cross A. pulverulentum 2/1 x A. litigiosum
1 were grown in greenhouse conditions
during 2009/2010. Ten F 1
plants: No1, 2 (P 1
A.
litigiosum 1); No3, 4, 5 (P 1
A. litigiosum 1/3); No
6, 7, 8, 9 (P 1
A. litigiosum 1/4) and No 10 (P 1
A.
litigiosum 3) and five reciprocal F 1
hybrids were
studied cytologically. For the evaluation of the
meiotic chromosome behaviour, flower buds of
different size were fixed in 3:1 ethanol:glacial
acetic acid fixative during the blossoming period.
The slides were obtained by temporary
squash preparations and stained with 4% acetocarmine
solution.
The bivalent characteristics were described
at pachytene, diplotene and early diakinesis stages.
The chromosome associations and frequencies
of meiotic configurations were recorded
at diakinesis and metaphase one, as about 100
pollen mother cells (PMCs) were scored from
every studied plant. Some disturbances of the
chromosome behavior were noted at metaphase,
anaphase and telophase stages.
Pollen fertility was estimated by acetocarmine
staining, as the unstained pollen grains
were recognized as sterile.
RESULTS
Chromosome associations and bivalent configurations
- The obtained and cytologically investigated
A. litigiosum x A. pulverulentum and
A. pulverulentum x A. litigiosum F 1
plants are
diploids 2n = 2x = 16, as their progenitors. In the
performed research a simultaneous type of the
microsporogenesis was found in the studied hybrid
PMCs.
The level of synchrony of the initial meiotic
stages (leptotene, pachytene and diplotene) and
at the diakinesis, methaphase (one and second
- MI and MII), anaphase (one and second - AI
and AII) and tetrad formation were higher then
the one, described in the wild progenitors in
2009. Most frequently, subsequent phases of the
microgametogenesis were quite synchronic processes.
Formation of two nucleoli in the meiocyte
nucleus often occurred in the investigated hybrid
plants.
Table 1 — Chromosome associations at diakinesis and metaphase one in pollen mother cells (PMCs) of A. litigiosum
x A. pulverulentum F1 plants.
Chromosome
associations
Plant
No 1
Plant
No2
Plant
No 3
Plant
No 4
Polen mother cells - %
8II 63.4 70.0 66.7 58.9 56.0 75.2 65.0 72.4 69.3 67.8 66.4
8II + fragment 0.4 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.2
7II + 2I 19.3 17.9 25.2 27.2 28.3 18.3 22.8 18.6 22.9 20.1 21.8
6II + 4I 10.7 5.3 4.1 6.7 8.2 3.9 7.8 6.5 6.5 7.5 6.9
6II + 1IV 1.6 3.7 2.7 5.0 1.3 2.0 1.0 1.0 0.0 1.7 2.0
5II + 6I 2.5 0.0 0.0 0.6 4.4 0.7 1.5 1.0 0.0 2.9 1.4
5II + 2I + 1IV 1.2 1,6 1.4 1.1 0.6 0.0 0.5 0.0 1.3 0.0 0.8
4II + 8I 0.8 0.0 0.0 0.0 1.3 0.0 0.0 0.5 0.0 0.0 0.3
4II + 4I + 1IV 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1
3II + 10I 0.0 0.5 0.0 0.0 0.0 0.0 0.5 0.0 0.0 0.0 0.1
2II+5I+1III+1IV 0.0 0.0 0.0 0.6 0,0 0.0 0.0 0.0 0.0 0.0 0.1
Investigated PMCs 243 190 147 180 159 153 206 199 153 174 1804
Pollen fertility 63.6 70.7 83.4 79.1 72.8 70.4 63.0 72.5 70.9 70.5 71.7
Plant
No 5
Plant
No 6
Plant
No 7
Plant
No 8
Plant
No 9
Plant
No10
Average

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vesselina and mateu-andrés
The data from Tables 1, 2 and 3 demonstrates
various chromosome associations and the average
frequency of chromosome configurations at
diakinesis and metaphase one in PMCs of the
studied F 1
progeny.
The homologous chromosomes paired and
formed 8 bivalents (II) in 56.0 to 75.2% (average
value - 66.4%) of the PMCs in the F 1
A. litigiosum
x A. pulverulentum plants (Table 1) (Figs.
1a, h). The normal chromosome conjugation
varied among the investigated ten F 1
hybrids
(VC 34.7%). The data from Table 1 showed that
the disruption of chromosome pairing is clearly
expressed in A. litigiosum 1/3 x A. pulverulentum
2/1 plants. In the reciprocal cross the 8II
were described in 58.3 to 65.8% of the cells (VC
Table 2 — Chromosome associations at diakinesis and metaphase one in pollen mother cells (PMCs) of A. pulverulentum
x A. litigiosum F 1
plants.
Chromosome
associations
Pollen mother cells - %
Plant No1 Plant No2 Plant No3 Plant No4 Plant No5 Average
8II 56.0 62.1 58.3 65.8 63.3 61.1
7II + 2I 24.1 23.3 20.5 21.1 21.8 22.1
6II + 4I 11.2 9.5 12.1 4.4 8.2 9.1
6II + 1IV 0.0 2.6 1.5 3.5 1.4 1.8
6II + 1III + 1I 0.0 0.0 0.0 0.0 0.7 0.1
5II + 6I 4.3 1.7 6.8 2.6 2.7 3.7
5II + 2I +1IV 0.9 0.0 0.0 0.9 0.6 0.5
4II + 8I 2.6 0.9 0.8 0.0 0.0 0.8
3II + 10I 0.9 0.0 0.0 0.0 1.2 0.5
2II + 12I 0.0 0.0 0.0 0.9 0.0 0.1
16I 0.0 0.0 0.0 0.9 0.0 0.1
Investigated PMCs 116 116 132 114 147 625
Pollen fertlity 76.8 62.6 64.8 66.9 66.3 67.5
Fig. 2 — Microsporogenesis in A. pulverulentum x A. litigiosum F1 plants-Diakinesis: (a) normal, (b, c, d) with 2,4and
6I (e, f) with multivalents; MI: (g) normal, (h) with 2I, (i) with IV formation; AI: (j) normal, (k, l, m)with lagging chromosome
and bridge formation; AII: (n, o, p) with irregularities; (q, r) tetrads, dyad and polyad; s- pollen.

cytological characteristics of hybrids between antirrhinum litigiosum and a. pulverulentum 381
15.4%) (Table2) (Figs. 2a, g). The average frequency
of bivalent configurations per PMC was
similar in the two hybrids - 7.51 and respectively
7.36II per cell (Table 3).
In the interspecific hybrids, the type of the
observed bivalent configurations varied significantly
at pachytene stage. Formation of monochiasmatic
bivalents, with single chiasma, always
localized in the distal non-centromeric regions
of the A. litigiosum and A. pulverulentum chromosomes,
was detected in all investigated plants
(Fig. 3a). The shortest A. litigiosum chromosome
(Fig. 4e, Fig. 5d) (Nikolova and Mateu-Andrés
2010) was included in the monochiasmatic bivalent
frequently. Two, three or more chiasmata
formation (Figs. 3 b, c, d, e) were registered in
the remaining bivalents of the hybrid PMCs, as
the chiasmata were positioned in different chromosome
regions.
At pachitene stage it was observed a condensed
chromatin cluster situated in different
chromosome regions (generally in the distal regions)
(Fig. 4a, Fig. 5a) or two and more clusters
(Fig. 4 c, Fig. 5b), as the observed clusters very
often connected closely with the nucleolus or
with the secondary nucleolus (Fig. 4d, Fig. 5c).
Close assembly of the chromosome condensed
chromatin regions with the nucleoli continued
from the leptotene –zygotene to the diplotene –
early diakinesis stages.
The data, obtained from about 50% of the
studied at diakinesis and MI cells, regarding the
bivalent type, the total chiasmata and the mean
chiasma per bivalent is shown in Table 4. The
higher value of ring bivalents (3.36), total chiasmata
(11.03) and mean chiasma number per
bivalent (1.44) was found in A. litigiosum x A.
pulverulentum PMCs, while the lower value of
these characteristics was established in the A.
pulverulentum x A. litigiosum cells (2.94, 10.58
and respectively 1.38).
Abnormalities of chromosome pairing - In case
of incomplete synapsis or of early chiasma terminalisation,
between 2 and 10 univalents (I) were
formed at diakinesis and MI (average frequency
0.85I per cell) in 23.0 to 42.2% (VC35.3%) of
PMCs in A. litigiosum x A. pulverulentum F 1
plants (Figs. 1 b, c, d, i, j). Higher number of
Fig. 3 — Different shaped bivalents in PMCs of A.litigiosum x A.pulverulentum F1 plants with: (a) one chiasma; (b)
two chiasmata; (c, d) more than two chiasmata; (e) different shaped bivalents associated with nucleolus.

382
vesselina and mateu-andrés
univalents (from 2 to 16I) (1.19 per PMC) and
higher percentage of the cells (ranged between
30 and 43.1% - VC 23.8%) with univalents were
established in the reciprocal cross (Figs. 2 b, c, d,
h). In general, two chromosomes did not pair and
the association 7II+2I was the most frequently
observed in studied PMCs of the both hybrids
(Table 1 and Table 2). Our data showed that in
the F 1
progeny, the incomplete synapsis was expressed
stronger in comparison with the parent
components (30.0% and 16.4% of the PMCs
with 2 to10I in A. litigiosum and respectively
in A. pulverulentum cells). Formation of a differently
shaped quadrivalent configuration (IV)
(Figs.1e, f, g, k, l; Figs. 2e, i) or a trivalent (III)
+ univalent (Fig. 2f) were established in 2.4 and
respectively 3.0% of the cells in the F 1
plant from
the two crosses. An additional small fragment to
the 8II in A. litigiosum x A. pulverulentum PMCs
was described at diakinesis. The fragmentation
of small region of the chromosomes and two additional
fragments were noticed in some A. litigiosum
PMCs in 2009. Early orientation of up to
4I toward the spindle poles (Fig. 1m, n) and 1II
outside of the metaphases plate was among the
other disturbances during the above mentioned
stages, as the percentage of PMCs with this type
of irregularities ranged from 2.8 to 9.9% in A.
litigiosum x A. pulverulentum F 1
plants and from
4.2 to 16.6% in the reciprocal hybrid.
Chromosome behaviour at anaphase stages -
Independently of the significant number of the
Table 3 — Average frequency of chromosome configurations at diakinesis and methaphase one per pollen mother cell
(PMC) of A. litigiosum x A. pulverulentum and A. pulverulentum x A. litigiosum F 1
plants.
Hybrids II I III IV fragments
A. litigiosum x A. pulverulentum 7.51 0.85 0.005 0.03 0.002
A. pulverulentum x A. litigiosum 7.36 1.19 0.001 0.02 0.0
Table 3 — Average frequency of chromosome configurations at diakinesis and methaphase one per pollen mother cell
(PMC) of A. litigiosum x A. pulverulentum and A. pulverulentum x A. litigiosum F 1
plants.
Hybrids II I III IV fragments
A. litigiosum x A. pulverulentum 7.51 0.85 0.005 0.03 0.002
A. pulverulentum x A. litigiosum 7.36 1.19 0.001 0.02 0.0
Fig. 4 — Assembly of the nucleoli with chromatin condensed chromosome regions in A. litigiosum x A. pulverulentum
F1 PMCs: (a) one cluster connection; (b) including in connection of the shortest A. litigiosum chromosome; (c) two
and more cluster connection; (d) assembly of the secondary nucleoli with chromatin condensed regions; (e) the shortest
A. litigiosum chromosome.

cytological characteristics of hybrids between antirrhinum litigiosum and a. pulverulentum 383
cells with univalent formation and early orientation
of up to 4 univalents toward the spindle
poles, the high percentages of the PMCs (average
90.1 and 84.8% in the reciprocal hybrids)
showed equal chromosome distribution at AI
stage and 8-8 chromosome number in the spindle
poles (Fig. 1o; Fig. 2j). Some abnormalities
(late bivalent disjunction, up to 4 lagging chromosomes,
unbalanced number of chromosomes
in the spindle poles, bridge formation - one or
two in A. litigiosum x A. pulverulentum F 1
plant
and one bridge in the reciprocal cross, early division
of the univalents to the chromatids) were
defined in the remaining cells. (Figs. 1p, q, r,
s; Figs. 2k, l, m). The meiotic chromosome behaviour
at second metaphase and anaphase was
analogical to that one observed in the MI and
AI. Regular chromosome distribution toward
the spindle poles at AII was registered in 87 to
99% of the PMCs in A. litigiosum x A. pulverulentum
F 1
plants (Fig. 1u) and in 85 to 95% in
the cells of the reciprocal cross. More frequently
in the one spindle, up to 2 and respectively to
4 lagging chromosomes, bridge formation, chromosomes
outside of the division spindle and aneuploid
chromosome number in the poles of the
PMCs in the studied hybrids was possible to detect
(Figs. 1t, v, w; Figs. 2 n, o, p). In some cells,
appearance of two bridges in the two spindles,
three-pole orientations of the chromosomes and
unsynchronized division were established.
Microspore formation and pollen fertility - In
all investigated A. litigiosum x A. pulverulentum
and A. pulverulentum x A. litigiosum F 1
plants,
the microsporogenesis finished with cytokinesis
after second telophase (TII) and tetrad forma-
Table 4 — Some characteristics of the bivalents in A. litigiosum x A. pulverulentum and A. pulverulentum x A. litigiosum
F 1
plants.
Hybrids
| A. litigiosum
| x
| A. pulverulentum
A. pulverulentum
x
A. litigiosum
Studied PMCs
number
Ring
bivalents
mean number
Rod
bivalents
mean number
Chiasmata
per PMC
mean number
Chiasmata
per bivalent
mean number
Univalents
mean number
Quadrivalent
mean number
778 3.36 4.40 11.03 1.44 0.61 0.008
326 2.94 4.73 10.58 1.38 0.79 0.00
Fig. 5 — Assembly of the nucleoli with chromatin condensed chromosome regions in A. pulverulentum x A. litigiosum
F1 PMCs: (a) one cluster connection; (b) two and more cluster connection; (c) assembly of the secondary nucleolus
with chromatin condensed regions; (d) the shortest A. litigiosum chromosome.

384
vesselina and mateu-andrés
tion in about 98.8-100% and respectively in 93.0
to 100% of the cells. As in the progenitors, according
to the position of the division spindles
at MII, two types of tetrads-isobilateral or tetrahedral,
in different proportion (Fig. 1y; Fig.
2q) were established together in the PMCs of
the studied hybrids. Meiotic abnormalities originated
dyad, triad and polyad formation (Fig. 1z;
Fig. 2 r) in some PMCs.
The data from Table 1 showed that the disruption
of the regular chromosome pairing
probably resulted in the reduction of the pollen
fertility (Table 1) (Fig. 1x; Fig. 2s). The relation
between the percentage of the PMCs with regular
chromosome pairing and the pollen fertility
is clearly demonstrated in the investigated hybrid
plants, as only in the progeny of the cross
between A. litigiosum 1/3 and A. pulverulentum
2/1 and in plant No1 from the reciprocal cross
this relation did not exists.
The pollen grains of the studied hybrids were
tricolporate with small generative cell and did
not morphologically differ from those the ones
described in the two wild Antirrhinum progenitors.
The tapetal cells were generally binucleate at
early prophase and, very frequently, there were
more than two nucleoli. At meiotic pachytene
some tapetal cells possessed three or four nuclei.
After endomitotic cycles, tetraploid and octoploid
chromosome number in the poles of the
tapetal cells was observed.
DISCUSSION
The cytologically investigated A. litigiosum
x A. pulverulentum and A. pulverulentum x
A. litigiosum F 1
plants had a diploid genome
2n = 2x = 16, as their progenitors and a simultaneous
type of the microsporogenesis was pre-
Fig. 6 — Ribosome formation in A. pulverulentum x A.
litigiosum F1 PMCs (a) in distal regions, (b) along of the
chromosome arm.
sented in all of them.
The type of the observed bivalent configurations
in PMCs of A. litigiosum x A. pulverulentum
and A. pulverulentum x A. litigiosum F 1
plants varied significantly at pachytene stage and
was more varying then that one described in the
wild parent components (Nikolova and Mateu-
Andrés 2010). Formation of monochiasmatic
bivalent, with single chiasma always localized
in the distal non-centromeric regions of the A.
litigiosum and A. pulverulentum chromosomes
was described in all investigated plants. The
observed in 2009 shortest A. litigiosum chromosome
which had condensed heterochromatin
concentrated in the telomeric regions, was very
often involved in this pair. The bivalents with
two chiasmata formation positioned in different
chromosome regions, either only terminally or
terminally and proximally, were registered. Some
bivalents were asymmetric, having one chiasma
in the terminal part of the two chromosomes and
the second one between distal and centromeric
regions respectively. The presence of a butterflytype
bivalent, as that one described in the progenitors
(Nikolova and Mateu-Andrés 2010)
and bivalents with various complicated shape
with three or more chiasma formation, were observed
in the investigated hybrid PMCs.
The chiasmata frequency differs between
the genotypes and between the female and male
reproductive cells, as chiasmata formation and
distribution is controlled genetically (Darlington
1937; Rees 1955; Rees and Thompson 1956;
Roseveir and Rees 1962; Rees and Jones 1977),
or caused by mutation, or influenced by the environmental
conditions.
The data, regarding the bivalent type, the
total chiasmata and the mean chiasma number
per bivalent showed higher value of ring bivalents
(3.36), total chiasmata (11.03) and mean
chiasma number per bivalent (1.44) in A. litigiosum
x A. pulverulentum PMCs, while the lower
value of these characteristics was established in
the A. pulverulentum x A. litigiosum cells (2.94,
10.58 and respectively 1.38). Our previous studies
(Nikolova and Mateu-Andrés 2010) showed
that the PMC of the wild species A. litigiosum
and A. pulverulentum had higher values of ring
bivalents (4.17 and 5.78), total chiasmata (12.11
and 14.04) and mean chiasma number per bivalent
(1.51 and 1.76 respectively), compared to the
ones established in their hybrids. The increase of
rod type of bivalents in hybrid cells (4.40 and
4.73 in PMCs of the reciprocal crosses - 3.67
and 2.22 respectively in the parent components)

cytological characteristics of hybrids between antirrhinum litigiosum and a. pulverulentum 385
is probably due to reduction of chiasma formation.
Genes controlling chiasma formation and
position in the progenitors may have changed
function in the hybrids and led to reduction of
the chiasma frequency. Such behaviour has been
reported in cotton and rapeseed cultivars and in
their hybrids by Sheida et al. (1998) and Sheida
et al. (2003).
The nucleolus controls functions necessary
for the synthesis of ribosomes and positioned
at the site of the chromosomal nucleolar organizer
regions (NORs). In the hybrid genome the
NORs possess either active or transcriptional inactive
rDNA genes. According to Flavell et al.
(1988) and Thompson et al. (1988), the inactive
rDNA genes are packaged into a transcriptionally
inactive chromatin structure. Lawrence and
Pikaard (2004) concluded that chromatin modification
is an important component of the regulatory
network that controls the effective dosage
of active rDNA genes.
At pachytene stage we have seen a condensed
chromatin cluster, situated in different chromosome
regions (generally in the distal regions) or
two and more clusters, as the clusters mostly
connected very closely with nucleolus or with
two nucleoli in the studied hybrid PMCs. The
shortest A. litigiosum chromosome, with condensed
heterochromatin concentrated in the
telomeric regions, was associated with nucleoli
very frequently (Fig.4b). The closely assembly of
the clusters with the nucleoli visually continued
from the leptoten – zygotene to the dyplotene
– early diakinesis stages. Mapping of the major
heterochromatin domains in the Antirrhinun
majus genome, Zhang et al. (2005) founded that
in general, heterochromatin is mainly located in
the pericentric regions, as in chromosome 3, 4
and 6 major heterochromatin domain are located
et the ends of their short arms.
According to Montijn et al. (1998), in early
mitotic prophase of plants of two Petunia hybrida
varieties, the condensed two chromatin
clusters of chromosome 3 are not associated
with the nucleolus and their ribosomal genes are
transcriptionally inactive.
The close assembling of the chromosome
condensed chromatin regions with the nucleoli
in the observed hybrid cells and the continuance
of the period (from the leptotene – zygotene to
the diplotene – early diakinesis stages), in which
the connection of these regions and nucleoli
were presented, suggest either transcriptional
activity of ribosomal genes before these stages or
existence of some activity of the silenced genes.
The creation of ribosomes near to the distal regions
of some chromosomes (Fig. 6a) or along
of the chromosomal arms (Fig. 6a) at pachytene
stage was detected. In the classic cytological
study the estimation of the chromosome pairing
and formation of bivalent configurations, preferably
at diakinesis, (when this is not possible in
prophase) is used as an indicator of the relation
degree among the different species.
In the performed meiotic study, the normal
chromosome conjugation and formation of 8II
at diakinesis and MI were recognized in average
66.4 and 61.1% of the PMCs in A. litigiosum x
A. pulverulentum and respectively A. pulverulentum
x A. litigiosum F 1
plants. The percentage of
the PMCs with regular chromosome pairing was
smaller and differed from that one established in
the progenitors (Nikolova and Mateu-Andrés
2010) – 71.6% in A. litigiosum and 82.1% in
A. pulverulentum plants. One of the reasons of
the lower homology expression is probably the
karyotipe differences between these wild species
or the genetic system, controlling the regular
chromosome pairing, act differently in the hybrid
genomes. Increasing the percentage of the
cells with meiotic abnormalities such: multivalent
formation (2.4 and respectively 3.0% of the
hybrid PMCs – 1.5% of the wild species cells);
presence of higher number of univalents (in 31.5
and 36.4% of PMCs in the F 1
plants – in 30 and
16.5% of the progenitor cells); bridge formation
(in 3.9 and 5.1% of the hybrid PMCs and 1.3 and
2.0% – in parent components) and bivalent late
disjunction, all of this indicated for escalating of
the heterozygous chromosome rearrangements.
The formation of a quadrivalent configuration
probably was caused either by heterozygous
translocation, concerning very small regions of
non-homologous chromosomes, or by presence
of some homology between small terminal segments
(X shaped quadrivalent configuration
- Fig. 1g). According to Attia and Roblelen
(1986), exhibition of multivalent creation reflects
the pairing between homeologous chromosomes.
Bridge formation suggested the occurrence
of chromosomal inversion and/or translocation.
According to Aparicio and Albaladejo (2003)
the late bivalent disjunction related with bridge
formation in hybrid complex of Phlomis composita.
Sheida et al. (2003) linked chromosome
stickiness and bridge formation in Brassica napus
L. hybrids.
The spindle abnormalities have led to the
production of observed at AI and AII aneu-

386
vesselina and mateu-andrés
ploid type of chromosome distribution toward
the spindle poles in up to 4.5% of the hybrid
PMCs. Nirmala and Rao (1996) proposed different
reasons for the occurrence of spindle
abnormalities – duality of nucleus in foreign cytoplasm,
environmental influence and disharmonious
gene interaction. Another reason for the
increased number of aborted aneuploid pollen
grains probably is that the lagging chromosomes
are not in time to be incorporated into the telophase
nuclei.
The above mentioned irregularities in chromosome
behavior during microsporogenesis in
the observed A. litigiosum x A. pulverulentum
and A. pulverulentum x A. litigiosum F 1
plants,
especially on one hand lower homology expression,
caused from the karyotipe differences
between these wild species and on the other,
chromosome rearrangements, had a major effect
on the formation of the chromosome and gene
unbalanced gamete and on the reduction of the
pollen fertility - 71.7 and respectively 67.5%.
The wild species A. litigiosum and A. pulverulentum
showed a high value of this characteristic
- pollen fertility 96.0 and respectively 98.4%
(Nikolova and Mateu-Andrés 2010).
The performed cytological investigation
shows some influence of mother genotype on
the meiotic chromosome behavior. The mikrosporogenesis
runs with less irregularity in the A.
litigiosum x A. pulverulentum meiocytes, then
the one described in the reciprocal cross, as the
mother progenitor A. litigiosum No1/3 definite
clearly exhibited disruption of chromosome
pairing in comparison of the combination with
P 1
components A. litigiosum No 1, 1/4 and No
3. The chromosome conjugation varied strongly
among the investigated ten F 1
A. litigiosum x A.
pulverulentum plants, as A. pulverulentum x A.
litigiosum genotypes were more constant in this
characteristic.
If we accept that the chromosome pairing
and formation of bivalent configurations is used
as an indicator of the degree of relation among
the different species, we have to conclude that
in genus Antirrhinum there is clearer expressed
karyotype distance between wild species A. litigiosum
and A. pulverulentum in comparison
with the distances between the wild A. litigiosum
- A. subbaeticum and A. pulverulentum - A.
subbaeticum species. The regular pairing of the
homologous chromosomes and formation of 8II
in PMCs was observed: in 84.1 and 80.4% from
the cells of the reciprocal crosses between progenitor’s
A. litigiosum and A. subbaeticum (pollen
fertility in F 1
- 79.0%); in 88.8 and 85.0%
of the PMCs in the F 1
plants A. pulverulentum
x A. subbaeticum and respectively A. subbaeticum
x A. pulverulentum (pollen fertility 97.7 and
95.0%) (unpublished data).
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Received November 18 th 2010; accepted January 12 th 2011