Oilseed rape/canola (Brassica napus L.) is among important oil producing plants cultivated extensively in Europe, China, North America and Iran. B. napus is an amphidiploid species with 19 pairs of chromosomes and has been evolved by crossing between B. campestris (2n = 2x = 20) and B. oleracea (2n = 2x = 18). Due to economic importance of oilseed rape, several B. napus cultivars have been introduced in Iran during the last decade and at present, germplasm evaluation as well as hybridization programs are in hand. Some basic cytogenetic information characteristics have already been reported in a few B. napus cultivars available in Iran (Sheidai et al., 2001a, b). The present work considers cytogenetic study of 23 unreported cultivars available in Iran considering the chromosome pairing and segregation, the occurrence and effects of B-chromosomes as well as unreduced gamete formation.
MATERIALS AND METHODS
Plants belonging to 23 B. napus L. cultivars were planted according to a randomized block design in the experimental field of Seed and Seedling Breeding Research Center, Karaj, Iran. For cytogenetical studies, flower buds were collected from 10 randomly selected plants from each cultivar. Fifty to hundred young flower buds were collected randomly during 9-12 A. M and fixed in glacial acetic acid: Ethanol (1:3) for 24 h which were then washed thoroughly and transferred to 85% ethanol until used (Sheidai et al., 2001a). Chromosome pairing and chiasma frequency was determined by using minimum 100 meiocytes showing diakinesis/ metaphase-I stages, while chromosome segregation was studied in minimum 500 anaphase-I and II stages. Pollen stainability as a measure of fertility was determined by staining minimum 1000 pollen grains with 2% acetocarmine: 50% glycerin (1:1) for about ½ h. Round/ complete pollens which were stained were taken as fertile, while incomplete/shrunken pollens with no stain were considered as infertile (Sheidai et al., 2003). Analysis of variance (ANOVA) followed by the least significant difference test (LSD) was performed on cytogenetic characteristics including chromosome pairing, chiasma frequency as well as distribution to indicate any significant difference among the cultivars studied (Sheidai et al., 2001a). Different methods of cluster analysis as well as ordination based on principal components analysis (PCA) was performed to identify the cultivars showing similarities in their meiotic characteristics (Sheidai et al., 2001a). For cluster and principal components analysis, standard values (mean = 0, variance = 1) were used. Squared Euclidean distance was use as a measure of similarity in cluster analysis (Sheidai et al., 2003). In order to detect the occurrence of unreduced (2n) pollen grains along with the normal (reduced = n) pollen grains, about 500 pollens were studied, some of which were sketched by the use of Camera Lucida and analysed by t-test. The statistical analyses used SPSS ver.9 (1998, SPSS Inc.) software.
RESULTS AND DISCUSSION
The B. napus cultivars studied and their meiotic characteristics are
presented in Table 1 and 2 as well as Fig.
1-5. All cultivars possessed n = 19 (2n = 4x = 38) chromosome
number. The highest value of total, terminal and intercalary chiasmata occurred
in Chinese cultivar (36.48, 32.25 and 4.23, respectively), while the lowest
values of total and terminal chiasmata occurred in SWO756 cultivar (21.39 and
20.68, respectively). The lowest value of intercalary chiasma occurred in the
hybrid cultivar R.C-Chinese (4.21).
The highest value of ring bivalents occurred in Modena cultivar (14.94), while
the lowest value occurred in SWO756 cultivar (3.09), which also possessed the
highest value of rod bivalents (14.96). Quadrivalents were formed in most of
the cultivars studied ranging from 0.12 (in Garison and Chinese cultivars) to
0.58 (in the cultivar Cyclone) (Table 1). Interestingly enough
some cultivars possessed hexavalents ranging from 0.03 to 0.06. The residual
homoeologous recombination in the cultivars studied may be the reason for quadrivalent
formation in the cultivars, while hexavalents may have been formed due to subsequent
chromosome translocations. ANOVA test revealed the presence of a significant
difference (p<0.01) for chiasma frequency and distribution as well as bivalent
and quadrivalents among the cultivars studied. Therefore at least two cultivars
differ significantly on their meiotic characteristics. The LSD test showed that
such significant difference is present almost among most of the cultivars, particularly
those cultivars, which are, placed in different clusters/ groups in cluster
analysis and ordination based on PCA (explained in the following paragraphs).
No significant difference was observed for hexavalents and univalents.
a = Metaphase I cell showing 19 bivalents in the cultivar
Modena, b = Anaphase I cell showing stickiness in the cultivar Cylone.
c = Metaphase I cell showing B-chromosomes (arrow) in the cultivar Iris,
d = Metaphase I cell showing B-chromosome (arrow) in the cultivar Modena,
Scale bar = 10 μm
Meiotic characters in rapeseed cultivars studied
TX = Terminal Chiasmata, IX = Intercalary Chiasmata, TOX
= Total Chiasmata, RB = Ring Bivalent, ROB = Rod Bivalent, QU = Quadrivalent,
I = Univalent, IV = Quadrivalent, VI = Hexavalent
Representative meiotic cells and pollen grains in B.
napus cultivars. a and b = Unreduced pollen grain (big pollen) in
the cultivars Artus and Garisson, respectively. c and d = Partial and
complete cytomixis in the cultivar Chinese. e = Meiocyte having double
the chromosome number due to cytomixis in the cultivar Rasmus. f = Meiocyte
showing reduction in chromosome number in the cultivar Chinese. g = Anaphase-II
failure in the cultivar Alexandra. h = Metaphase-I cell showing Desynapsis
(large number of univalents) in the cultivar Hylite-201. I = A pentapolar
cell in the cultivar Elite. j = Abnormal tetrad in the cultivar Cyclone.
k = Unequal segregation in the cultivar Alexandra. Scale bar = 10 μm
The frequency and distribution of chiasma is under genetic control (Quicke,
1993), therefore presence of a significant difference in chiasma frequency and
distribution as well as ring and rod bivalents among the cultivars studied may
indicate partly their genomic differences as these plants were grown under uniform
conditions in the experimental field.
Data with regard to chromosome segregation is provided in Table
2. Chromosome stickiness, laggard chromosomes and micronuclei occurred in
most of the cultivars. The highest percentage of anaphase-I and II tickiness
occurred in the cultivar Talent (14.70 and 7.27, respectively). Paired sample
χ2 test showed a significant difference in the percentage of
chromosome stickiness among the cultivars studied. Genetic as well as environmental
factors have been considered as the reason for chromosome stickiness (De Souza
and Palgliarini, 1996; Baptista-Giacomelli et al., 2000).
Anaphase-I and II laggards were observed in all the cultivars studied (Table
2). The highest value of Anaphase-I and II laggards occurred in Talent and
Ebonite cultivars (14.70 and 10.00, respectively) while the lowest values occurred
in Alexandra (0.39) and Elite and Express cultivars (00.00). χ2
test showed a significant difference in the percentage of laggard chromosomes
among the cultivars studied indicating their genomic differences for such a
Pearson coefficient of correlation determined among meiotic irregularities
themselves and also with pollen fertility revealed a positive significant correlation
(p<0.05) between the percentage of laggard cells in anaphase-I with stickiness
in anaphase-I and II as well as percentage of laggard cells in anaphase-II.
The chromosomes stickiness in anaphase-I showed a positive significant correlation
(p<0.05) with micronucleus formation. However pollen fertility did not show
any correlation with these meiotic irregularities. The cultivars studied showed
a high pollen fertility (> 98%, Table 2), therefore it
seems that meiotic irregularities mentioned do not have a major effect on pollen
Meiotic abnormalities and pollen fertility in rapeseed
A1L = Laggards in Anaphase-I, A2L = Laggards in Anaphase-II,
A1S = Stickiness in Anaphase-I, A2S = Stickiness in Anaphase-II, UD =
Unequal Division, MN = Micronucleus, PF = Pollen Fertility. (All values
It has been suggested that infertility in polyploids is not solely due to
the production of aneuploid gametes formed by improper segregation of chromosomes
during anaphase/telophase stages, the genetic factors may also bring about pollen
sterility as evidenced in different tetraploid strains of rye as well as Avena
sativa cultivars (Baptista-Giacomelli et al., 2000). Therefore reduction
in pollen fertility in B. napus cultivars may also be affected by genetic
factors and not only by meiotic irregularities reported.
Different cluster analyses and ordination of cultivars based on PCA of meiotic
data produced a similar result (Fig. 4-5).
In general four major cluster/groups are formed, separating the cultivars studied.
The first major cluster is comprised of two sub-clusters. The cultivars Opera,
Rafaella, Ebonit, Talent, Elite and RPC702 form the first sub-cluster while
the cultivars Artus, Kristina, Express and Cyclone comprises the second sub-cluster.
Although the cultivars Express and Cyclone are joined the other cultivars of
the second sub-cluster with some distance due to their meiotic differences all
the four cultivars of this sub-cluster formed hexavalents in metaphase-I (Table
1). The second major cluster is also comprised of two sub-clusters. The
cultivars Hylite-201, Garisson, Rasmus, Ryder, Mercure, Alexander, Iris and
Modena form the first sub-cluster while the cultivars R.C X Chinese, R.C X Hylite
and Emblem form the second sub-cluster. The cultivar Chinese alone forms the
third major cluster and cultivar SWO756 alone forms the forth major cluster
and are joined with a great distance to the other cultivars indicating their
cytogenetic differences. It is interesting to mention that two hybrid cultivars
of R.C x Chinese and R.C x Hyalite are obtained from hybridization of the hybrid
cultivar Regen x Cobra with the cultivar Chinese and cultivar Hyalite-201 and
are placed close to each other in one cluster possibly due to genomic effects
of Regen x Cobra. However the parental genotypes of Hyalite-201 and Chinese
are placed in different clusters. As stated earlier, the members of different
clusters differ significantly in their meiotic characteristics from the others.
PCA analysis of meiotic data revealed that the first 3 factors comprise about
82% of total variance. In the first factor, which comprises about 57% of total
variance, meiotic characters like total and terminal chiasmata as well as ring
bivalents and possessed the highest positive correlation (>0.90), while rod
bivalents possessed the highest negative correlation (>-0.90). In the second
factor, which comprises about 14% of total variance, quadrivalents possessed
the highest positive correlation (>0.80) while in the third factor with about
10% of total variance intercalary chiasmata possessed the highest positive correlation
(>0.80). Therefore these are the most variable meiotic characteristics among
the cultivars studied as also revealed by ANOVA test discussed earlier. The
combination of the first and second PCA factors separates mainly the members
of the firs and second clusters from each other, while combination of the first
and third PCA factors separates SWO756 and Chinese cultivars of the third and
forth major clusters (Fig. 5a and b).
The size (μm) of reduced and unreduced pollen grains
in oil rapeseed cultivars (names of the cultivars as in Table
B-chromosomes: Twelve out of 23 cultivars studied showed the occurrence
of 0-4 B-chromosomes (Table 3 and Fig. 1).
These were smaller than the A-chromosomes and did not form any meiotic association
with them, although they could arrange themselves along with the A-chromosomes
on the equatorial plane of the spindle and move to the poles during anaphase.
In some cases they occurred as laggard chromosomes. B-chromosomes are accessory
chromosomes reported in many plant and animal species. B-Chromosomes may affect
the frequency and distribution of chiasmata as well as chromosome association,
either directly or by affecting the genes present on the A-chromosomes that
control meiosis (Camacho et al. 2000). T-test analysis of meiotic characteristics
among the cells possessing B-chromosomes compared to the cells devoid of B-chromosomes
is presented in Table 3. The effect of B-chromosomes varied
in different cultivars studied, for example the presence of Bs did not have
any significant effect on the meiotic characters in four cultivars of Mercure,
Rasmus, Garisson and R.C. X Hyalite while in the cultivars Opera, Iris, Hyalite-201
and Alexandra significantly reduced the number of intercalary chiasmata. The
presence of B-chromosomes significantly increased the number of total chiasmata
in the cultivars Artus, Rafaella and R.C X Chinese and significantly increased
the number of intercalary chiasmata in the cultivars Artus and Ryder. A significant
change in frequency of chiasmata may bring about changes in genetic recombination
of the progenies this is particularly true if a significant increase in the
number of intercalary chiasmata occurs.
T-test analysis of meiotic characteristics among the cells
possessing B-chromosomes (+ B) and cells devoid of B-chromosomes (-B)
in rapeseed cultivars
Meiotic characters as in Table 1, * =
Significant at p = 0.05
PCA ordination of oil seed rape cultivars (cultivars number
as in Fig. 4)
An increase in the number of intercalary chiasmata indicates that genes present
in the middle part of the chromosomes also become involved in genetic recombination,
which may lead to an increase in genetic variation of the next generation. In
general if the cytogenetic differences observed in the cultivars studied is
accompanied by other agronomic differences a better hybridization and selection
program may be planned for B. napus cultivars.
Unreduced gamete formation: The occurrence of unreduced (2n) gametes has been considered important in the evolution of polyploids and also of economic importance in crop plants like potato for obtaining natural tetraploid plants by crossing 2x (producing unreduced gametes) X 4x lines. It was only recently that Sheidai et al. (2003) reported the occurrence of unreduced pollen grains in 6 out of 22 B. napus cultivars studied. Therefore the present study was performed to detect the occurrence of 2n pollen grains and its cytogenetic mechanisms in 23 unreported cultivars available in Iran.
The presence of meiocytes having double the gametic chromosome number as well
as 2n pollen grains was noticed in 17 out of 23 B. napus cultivars studied
(Fig. 3). A numerically unreduced diploid, or 2n gamete is
a meiotic product that bears the sporophytic rather than the gametophytic chromosome
number. Such gametes result from abnormalities during either microsprogenesis
(2n pollen) or megasporogenesis (2n eggs). Unreduced gametes are known to produce
individuals with higher ploidy level through a process known as sexual polyploidization
(Villeux, 1985). Different methods have been used to detect 2n gametes including
morphological, flow cytometery and cytological methods. The most direct method
of screening for 2n pollen involves the examination of the range of size of
pollens produced by an individual, as with increase in DNA content the cell
volume increases which in turn influences the pollen diameter. The presence
of giant pollen grains has been used as an indication of the production of 2n
pollen (Vorsa and Bingham, 1979).
The measurement of the pollen grains in the B. napus cultivars with unreduced meiocytes revealed the presence of a bimodal distribution of pollen grain size. T-test analysis also showed a significant difference (p<0.01) between the pollen grains indicating the possible 2n constitution of the larger pollen grains. The frequency of 2n pollen formation varied from 0.12 in the cultivar Modena to 1.41% in the cultivar Alexandra.
In higher plants, unreduced gametes originate through two types of meiotic nuclear division: First Division Restitution (FDR) and Second Division Restitution (SDR). In case of FDR, usually failure of anaphase-I chromosomes segregation leads to the formation of restitution nucleus while in SDR failure of anaphase-II chromosomes segregation leads to the formation of unreduced gametes. However other cytological abnormalities including tripolar, parallel and fused spindles, abnormal cytokinesis as well as syncyte formation have been considered as the other causes of 2n pollen formation (Vorsa and Bingham, 1979). The present study indicates the occurrence of both FDR and SDR in B. napus cultivars and detailed cytological investigation revealed that the main cytological mechanisms for production of 2n gametes are: 1- anaphase- I and II failure in one of the poles leading to the formation of triads at the end of telophase-II instead of tetrad (one is unreduced), 2-Desynapsis or early chiasma terminalization, 3- multipolar spindles which occurred in most of the cultivars and 4- Cytomixis. Details of cytogenetical mechanisms, leading to the formation of unreduced gametes in B. napus cultivars studied are discussed bellow.
Cytomixis: Among 23 B. napus cultivars studied 13 cultivars (Table
4) showed the occurrence of cytomixis and chromosome migration (Fig.
2). The percentage of cells showing cytomixis varied from 0.79% in the cultivar
Ryder to 15.76% in the cultivar Alexandra. Chromatin/ chromosome migration occurred
in different directions from early prophase to telophase-II. Several metaphase/
diakinesis cells in these species possessed extra or missing chromosomes showing
aneuploid condition, such aneuploid cells may lead to the formation of abnormal
tetrads and infertile pollen grains. However migration of the whole chromosome
complement and production of unreduced (2n) meiocytes occurred in some of the
cultivars (Fig. 1), which may be the reason for production
of unreduced pollen grains there after. Since the whole chromosome migration
occurred before the start of anaphase I, the restitution nucleus (unreduced
cell) is of FDR type. Paired wise X2 test performed for percentage
of cytomixis showed a significant difference among the cultivars studied indicating
their genomic difference.
Migration of chromatin material among the adjacent meiocytes occurs through
cytoplasmic connections originated from the pre-existing system of plasmodesmata
formed within the tissues of the anther. The plasmodesmata become completely
obstructed by the deposition of callose, but in some cases they still persist
during meiosis and increase in size forming conspicuous inter-meiocyte connections
or cytomictic channels that permit the transfer of chromosomes (Falistocco et
al., 1995). Cytomixis is considered to be of less evolutionary importance
but it may lead to production of aneuploid plants or produce unreduced gametes
as reported in several grass species including Dactylis and Aegilops
(Falistocco et al., 1995).
Unreduced pollen grain formation and the related cytogenetic
mechanisms (%) of rapeseed cultivars studied
UNP = Unreduced Pollen grain, CYT = Cytomixis, ST = Stickiness,
DES = Desynapsis, A1F = Anaphase-I failure, A2F = Anaphase-II failure
Anaphase I and II failure: Failure of chromosome movement either during
anaphase I or II occurred in 12 out of 23 cultivars studied. The failure of
chromosome movement occurred in one of the poles of in anaphase cells (Fig.
2), leading to the formation of normal reduced and unreduced daughter cells.
Such unreduced meiocytes may lead to the formation of 2n pollen grains. Since
failure of chromosome segregation occurred in both anaphase-I and II, the restitution
nucleus of both FDR and SDR constitution would be formed. In the cultivars Emblem,
Ebonit, Kristina and Alexandra both anaphase-I and II failure occurred.
Multipolar cells: The occurrence of tripolar or multipolar cells was
observed almost in most of the cultivars studied (Fig. 2).
Formation of tripolar cell leads to the formation of two reduced and one unreduced
pollen grains, while pentapolar cells observed in some of the cultivars may
lead to the formation of abnormal tetrads and infertile pollen grains (Fig.
2). The spindle apparatus is normally bipolar and acts as a single unit,
playing a crucial role in chromosome alignment during metaphase. Any distortion
or breakage in the spindle may result in random sub-grouping of the chromosomes
which function independently (Nirmala and Rao, 1996). In several instances spindle
abnormalities have led to the production of aneuploid gametes for example in
polyploidy hybrids and derivatives of Aegilopsx Triticum hybrids,
amphiploid Triticineae, amphiploids of Solanum hybrids, etc. and also
is considered as a reason for production of unreduced (2n) gametes in Solanum
Desynapsis: Desynapsis is considered as the precocious separation of
bivalents in metaphase of meiosis I leading to the formation of varying degree
of univalents. Partial to complete desynapsis was observed in 4 out of 23 cultivars
studied (Table 4 and Fig. 2). In partial
desynapsis few to some bivalents were separated to form univalents while, in
complete desynapsis all bivalents were separated to form univalents. Such desynaptic
cells with double the chromosome number may form unreduced pollen grains. Desynapsis
occurs either due to the action of recessive ds genes in a homozygous situation
or early chiasma terminalisation which may lead to the formation ofmeiocytes
with double the normal chromosome number.
In several cases such univalents may have difficulty during anaphase-I movement
and become lagged therefore producing aneuploid gametes causing reduction in
pollen fertility of plants. However they may skip the first anaphase and form
restitution nucleus resulting in the formation of unreduced gametes as reported
in Solanum (Villeux, 1985).