Microsporogenesis in tetraploid accessions of Brachiaria nigropedata (Ficalho & Hiern) Stapf (Gramineae)
Karina Sayuri Utsunomiya*, Maria Suely Paglirini* and Cacilda Borges do Valle**
* Department of Cell Biology and Genetics, State University of Maringá, Av. Colombo 5790, 87020-900, Maringá, Paraná,
Brazil.
** Embrapa Beef Cattle, P. O. Box 154, 79002-970 Campo Grande, Mato Grosso do Sul, Brazil.
Address correspondence to: Dr. Maria Suely Pagliarini. State University of Maringá, Department of Cell Biology and Genetics 87020-900 Maringá, Paraná, BRAZIL. E-mail: mspagliarini@uem.br
ABSTRACT: The genus Brachiaria (Trin.) Griseb. has achieved considerable importance to cattle production systems, as a result of the good production and adaptation of a few cultivars to poor and acid soils of the Brazilian savannas. Many of its species and accessions are polyploid and apomictic, which limits direct hybridization. To assist the breeding program, cytogenetic characterization has been undertaken on the accessions of Brachiaria collection at the Embrapa Beef Cattle Research Center. In this study, chromosome number and meiotic behavior are reported for the Brachiaria nigropedata (Ficalho & Hiern) Stapf collection. The 20 available accessions are tetraploid (2n = 4x = 36). Chromosomes paired preferentially as bivalents, but quadrivalents were found in high frequencies in some cells. Meiotic behavior was, in general, irregular, and varied among accessions. Most accessions presented more than 20% of abnormal tetrads. The most common meiotic abnormalities were those related to irregular chromosome segregation due to polyploidy, leading to micronuclei formation in the tetrad stage. A low frequency of other meiotic abnormalities such as the absence of cytokinesis, chromosome stickiness, cell fusion, anaphase bridges, and chromosome transfer among microsporocytes were also recorded in some accessions. Limitations of these accessions for use in hybridization programs are discussed.
Keywords: Brachiaria nigropedata. Chromosome number. Meiosis. Polyploidy. Apomixis. Grasses.
Introduction
Brachiaria (Trin.) Griseb. is a large genus comprising
of about 100 species distributed throughout the tropics,
especially in Africa (Renvoize et al., 1996). The
forage potential of these grasses was first recognized
about 50 years ago, particularly in tropical Australia.
The major impact of the genus, however, was realized
only in the past three decades, when a handful of Brachiaria cultivars, derived directly from natural occurring
germplasm, were widely sown in tropical
America (Miles et al., 1996). Current estimates of the
acreage of the Brachiaria pastures in Brazil range from
50 to 70 million hectares. Brachiaria decumbens Stapf
cv. Basilisk and Brachiaria brizantha (A. Rich.) Stapf
cv. Marandu are the most widely grown varieties in the
acid soils of Brazil.
The rapid expansion of acreage did not occur without
problems, and current available cultivars are now
recognized as having serious limitations: cv. Basilisk
lacks resistance to a serious insect pest - spittlebugs -
and cv. Marandu requires higher soil fertility and good
drainage (Valle et al., 1993).
Two decades ago, an extensive germplasm collection
trip was undertaken by the International Center for
Tropical Agriculture (CIAT Colombia), in East Africa with the support of International Board for Plant Genetic
Resources (IBPGR) (Keller-Grein et al., 1996).
This germplasm was later distributed to several latin
American countries. Studies on the cytology and genetics
of several Brachiaria species have opened up
new opportunities and challenges in the improvement
of this important forage (Miles et al., 1996). To date,
new varieties of Brachiaria are being developed, either
by selecting superior genotypes from natural diversity
or by intra- and interspecific hybridization to
obtain novel genetic combinations. In each case an
adequate germplasm base was essential. An important
part of the CIAT germplasm collection was introduced
into Brazil during 1986 and 1987, and is presently
maintained in plots at the Embrapa Beef Cattle Research
Center. Whereas some species are represented
in the collection by a great number of accessions, other
species, such as B. nigropedata, are represented by a
few accessions only.
Breeding difficulties in the genus Brachiaria are
associated with polyploidy and asexual reproduction.
Natural tetraploid (2n = 4x = 36) populations are widespread,
whereas sexuality is rare in most species and
usually at lower ploidy levels (Valle and Savidan,
1996). Polyploids have generally been classified as
highly to obligate aposporous apomicts, which propagate
clonally by seeds. Apomictic accessions cannot
be improved by using traditional breeding schemes,
but sexual diploid, artificially tetraploidized genotypes
of a few species have been used to facilitate the introgression
of valuable agronomic characteristics from
apomictic species (Valle and Miles, 1994; Miles and
Valle, 1996). Taking into consideration the correlation
between polyploidy and apomixis in the genus, the
knowledge of basic characteristics, such as ploidy level
and chromosome behavior in meiosis, is essential for
a successful breeding program. Brachiaria nigropedata is a species still untested. About 67% of the collection
present at the Roodeplaat Grassland Institute of the
African Research Council (RGI/ARC) belongs to this
species (Keller-Grein et al., 1996). In this case, most
accessions were obtained through direct collection
conducted mainly in Zimbabwe and the diversity gathered
does not represent all the variation present in nature,
according to its collectors (Keller-Grein et al.,
1996). Brachiaria nigropedata grows on soils of granitic origin and is considered a valuable component of
natural grasslands in Southern Africa. It is reported to
have high forage value and tends to disappear under
heavy grazing (Oudtshoorn, 1992). Its agronomical
value is under evaluation at Embrapa Beef Cattle Center.
Cytological characteristics such as chromosome
number and meiotic behavior are reported here for the
accessions available in Campo Grande, Mato Grosso
do Sul, Brazil.
Materials and Methods
Cytogenetic studies were carried out on 20 accessions
of B. nigropedata of the Embrapa Beef Cattle
Center Brachiaria collection grown in Campo Grande
(state of Mato Grosso do Sul, Brazil), which comprises
of approximately 475 accessions of 15 species maintained
in plots. Site characteristics are: (climate type
Aw: tropical humid savanna (average annual precipitation
= 1526 mm; average temperature = 22°C); altitude
520 m; latitude = 20° 28' S; longitude = 55° 40' W); poor Dark Red Latossol (59% sand; 8% silt; 33%
clay; pH = 4.2).
Inflorescences were collected for meiotic studies,
fixed in ethanol: acetic acid (3:1) for 24h and stored
under refrigeration until use. Microsporocytes were prepared
by squashing and then staining with 0.5% propionic
carmine. All meiotic phases were evaluated in five
plants per accession. More than 1400 microsporocytes
were analyzed per accession. Chromosome associations were evaluated in 20 microsporocytes at diakinesis per
accession. Photomicrographs were made using a Kodak
Imagelink - HQ, ISO 25 black and white film.
Results and Discussion
The 20 accessions of B. nigropedata analyzed were
tetraploid (2n = 4x = 36). Previous chromosome
countings for this species revealed the occurrence of
diploid (2n = 2x = 18) (De Wet and Anderson, 1956;
Spies and Du Plessis, 1986) and tetraploid (2n = 4x =
36) (Moffet and Hurcombe, 1949; Hoshino and
Davides, 1988) accessions. Two basic chromosome
numbers, x = 7 and x = 9, have been generally accepted
for the genus Brachiaria, with most species
presenting chromosome numbers multiples of 9
(Basappa et al., 1987; Valle and Savidan, 1996), as
also found for B. nigropedata. The available literature
about the genus Brachiaria indicates a prevalence of
tetraploidy (Sotomayor-Ríos et al., 1968; Bernini and
Marin-Morales, 2001; Mendes-Bonato et al., 2002).
Polyploidy is common among grasses. Stebbins (1956)
estimated that 70% of the Gramineae family are natural
polyploids.
The polyploid condition of B. nigropedata accessions
under analysis was also revealed by the pattern
of chromosome association at diakinesis. Uni-, bi-, triand,
quadrivalents were detected at this stage (Fig. 1a,
b), with prevalence of bivalents (Table 1), as also observed
in tetraploid accessions of B. brizantha (Mendes-
Bonato et al., 2002). However, in some cells, chromosomes
associated mainly as quadrivalents. According
to Stebbins (1947), low frequency of multivalents is an
argument used in advocating segmental allopolyploidy.
In segmental allopolyploids, the genomes are not identical;
they result from hybridization of closely related
diploid species (AA ´ A'A') followed by the doubling of
the chromosome numbers. As a result of preferential
pairing between identical chromosomes, mainly
bivalents are formed and few multivalents are thus
present. Although this hypothesis has been accepted for
many decades, Sybenga (1994, 1996a) pointed out that
this character is not necessarily a reliable indication of
pairing affinity, and thus of homology, because even
true autopolyploids may form quadrivalents with frequencies
substantially lower than the theoretically possible
or expected. Advanced studies of genome constitution
in the genus Brachiaria involving genomic "in
situ" hybridization could help elucidate the origin of
polyploid accessions.
FIGURE 1. Aspects of the irregular microsporogenesis in the tetraploid acessions (2n = 4x = 36) of B. nigropedata. (a) Diakinesis with 18 II, (b) Diakinesis with 2 IV and 14 II (a, b: 1000X), (c) Metaphase I with precocious chromosome migration to the poles, (d) Anaphase I with laggards, (e) Telophase I with micronucleus in the both poles, (f) Metaphase II with micronucleus in the both cells, (g) Metaphase II with the micronuclei eliminated as a microcyte, (h) Anaphase II with laggards, (i) Telophase II with micronuclei in one cell, (j) tetrad with one micronuclei in one microspore, (k) Tetrad with two micronuclei in two microspores, (l) Released microspore with two micronuclei, (m) Pentad, (n) Hexad, and (o, p) Irregular positioning of microspores in the tetrad (c to p: X 400).
TABLE 1.
Accession code, range of chromosome association, and average association/cell.
As the 20 accessions were tetraploid, meiotic behavior was found to be irregular (Table 2). The most common meiotic abnormalities were those related to irregular chromosome segregation, such as precocious chromosome migration to the poles in metaphase I (Fig. 1c), laggards in anaphase I (Fig. 1d) and anaphase II (Fig. 1h), leading to micronucleus formation at telophase I (Fig. 1e) and telophase II (Fig. 1i). The behavior of micronuclei in both telophases was varied. In some cells, after the first division, micronuclei remained as such during the second division (Fig. 1f), whereas in others, these were eliminated in microcytes (Fig. 1g). After telophase II, the micronuclei also showed dissimilar behavior; when containing a single chromosome, they remained as micronuclei inside the microspore (Fig. 1j, k, l) although when constituted by a group of chromosomes, they were isolated as microspores of different sizes, giving rise to polyads (Fig. 1m, n). Tetrads with abnormal shapes were also observed (Fig. 1o, p). Similar meiotic behavior was reported in polyploid accessions of other Brachiaria species (Pritchard, 1967; Sotomayor-Ríos et al., 1968; Valle, 1986; Valle et al., 1987, 1989; Basappa et al., 1987; Mendes-Bonato et al., 2001a; Risso-Pascotto et al., 2003). The percentage of abnormal tetrads among the 20 accessions of B. nigropedata ranged from 14.10 to 65.80%, but in 15 accessions, that is, 75% of the total, the percentage ranged from 20 to 40%. Such frequency of abnormal meiotic products was higher than that found in B. brizantha (Mendes-Bonato et al., 2002), where a considerable number of tetraploid accessions presented less than 15% abnormal tetrads.
TABLE 2.
Accession code, number of cells analyzed, and percentage of abnormal cells in each meiotic phase.
Other meiotic abnormalities were detected in low frequencies in some accessions of B. nigropedata. Absence of cytokinesis after first and/or second meiosis, leading to monad, dyad, and triad formation was found in ten accessions (Fig. 2a to c). In eight of them, the percentage of affected cells was lower than 0.5%, but in two, N 194 and N 195, affected cells reached 2.4 and 2.9%, respectively. This abnormality was also reported in a pentaploid accession of B. brizantha (Risso-Pascotto et al., 2003). Absence of cytokinesis, when accompanied by restitutional nuclei, lead to 2n gamete formation. Restitutional nuclei were described in B. brizantha (Risso-Pascotto et al., 2003), but not found in B. nigropedata. Here all cells affected by absence of cytokinesis maintained isolated nuclei.
FIGURE 2. Aspects of some rare abnormalities. (a to c) Abnormalities related to absence of cytokinesis: telophase II without the first cytokinesis (a), triad originated by absence of the second cytokinesis in one cell (b), and a binucleate microspore (c); (d to f) Meiotic phases showing chromosome stickiness: prophase II (d), telophase II (e), and microspore (f); (g) Bridge in anaphase II; (h) Cell fusion; and (i) Chromosome transfer among microsporocytes (X 400).
Chromosome stickiness (Fig. 2d to f) was detected
in nine accessions, ranging from 0.25 to 5.0%. A severe
case of stickiness was reported affecting meiosis in B.
brizantha (Mendes-Bonato et al., 2001b), B. decumbens (Mendes-Bonato et al., 2001a), and several other accessions
of different Brachiaria species under analysis.
Chromosome bridges (Fig. 2g) affecting less than 0.5%
of cells was found in 12 accessions; they may result
from chromosome stickiness such as found in B.
brizantha (Mendes-Bonato et al., 2001b), and B.
decumbens (Mendes-Bonato et al., 2001a). No more
than 0.5% of cells, in four accessions, was affected by
cell fusion (Fig. 2h). This abnormality has been reported
to occur in B. brizantha (Mendes-Bonato et al., 2001c;
Risso-Pascotto et al., 2003), and also in several accessions
of other Brachiaria species under analysis. An
abnormality, which has never been reported in the genus Brachiaria until now, was observed quite frequently in six accessions analyzed: chromosome transfer among
microsporocytes (Fig. 2i). In accession N 193 up to 9%
of cells displayed it. In general, the phenomenon occurred
between two cells, involving total or partial transference
of the genetic material.
The genus Brachiaria is extensively used and became
the most important tropical pasture in cattle production
systems in the poor and acid soils of the Brazilian
savannas. The two Brachiaria cultivars under wide
use in the country are native to East Africa and natural
apomictic tetraploids. The lack of diversity represents
an obvious risk to the ecosystem as well as to cattle
production. Therefore rational exploitation of the diversity present in the germplasm, especially of species with
good forage value such as B. nigropedata, is a fundamental
alternative. Despite identifying promising accessions,
the breeding program in effect at Embrapa Beef
Cattle Center depends on compatible sexual accessions
to act as female genitor in hybridizations. Tetraploid
apomictic accessions with nearly regular meiosis are
used as male genitor, thus wide cytological screening is
a pre-requisite. Work underway with the Brachiaria germplasm collection at Embrapa has revealed the occurrence
of a single sexual diploid accessions and a few
apomictic tetraploid accessions with considerable low
frequency of meiotic abnormalities in B. brizantha (Mendes-Bonato et al., 2002), a species that provides a
wealth of genetic variation of desired attributes for gene
introgression. Previous studies performed on determination
of mode of reproduction of B. nigropedata (unpublished
data) have revealed that all accessions are
apomictic. Taking into account that the present cytological
studies revealed that all accessions are tetraploid
and display high levels of meiotic abnormalities,
their use in hybridization programs is indeed limited.
Only three accessions (N 209, N 194, and N 191) presented
less than 20% of abnormal tetrads and can be
considered proper genitors in crosses.
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Received on February 21, 2004.
Accepted on April 18, 2005.