Plant Molecular Biology 53: 745–757, 2003.
© 2004 Kluwer Academic Publishers. Printed in the Netherlands.
Identiﬁcation of differentially expressed cDNA sequences in ovaries of
sexual and apomictic plants of Brachiaria brizantha
Júlio C.M. Rodrigues, Gláucia B. Cabral, Diva M.A. Dusi, Luciane V. de Mello, Daniel J.
Rigden and Vera T.C. Carneiro
Embrapa Genetic Resources and Biotechnology, CP: 02372, CEP: 70770-900 Brasilia-DF, Brazil (
correspondence; e-mail email@example.com)
Received 8 July 2003; accepted in revised form 17 November 2003
Key words: apomixis; differential display; megagametogenesis; megasporogenesis; sequence analysis
The isolation of genes associated with apomixis would improve understanding of the molecular mechanism of this
mode of reproduction in plants as well as open the possibility of transfer of apomixis to sexual plants, enabling
cloning of crops through seeds. Brachiaria brizantha is a highly apomictic grass species with 274 tetraploid
apomicts accessions and only one diploid sexual. In this study we have compared gene expression in ovaries at
megasporogenesis and megagametogenesis of sexual and apomictic accessions of B. brizantha by differential dis-
play (DD-PCR), with 60 primer combinations. Speciﬁcity of 65 cloned fragments, checked by reverse northern blot
analysis, showed that 11 clones were differentially expressed, 6 in apomictic ovaries, 2 in sexual and 3 in apomictic
and sexual, but at different stages. Of the 6 sequences isolated that were preferentially expressed in the apomictic
accession: one sequence was from ovaries at megasporogenesis stage; three were from megagametogenesis stage;
two were from both stages. Of the two sequences isolated from the sexual accessions, one showed expression in
ovaries at megagametogenesis, while the other sequence was shown to be speciﬁc to both stages. Three sequences
were from megasporogenesis stage in apomicts but were also detected at megagametogenesis in sexual plants.
Sequence analysis showed that 5 of the 11 clones had no apparent homologues in the protein database. Some of the
clones identiﬁed as apomictic-speciﬁc shared homology with known genes enabling their functional annotation.
The relationships of these functions to the generation of the apomictic trait are discussed.
Sexual reproduction ensures recombination and ge-
netic diversity. However, in some angiosperms sexual-
ity can be bypassed and the seeds give rise to individu-
als identical to the mother plant, without the traits of
the male parent. This is due to an asexual mode of re-
production named apomixis reported in more than 300
species (Hanna and Bashaw, 1987; Carman, 1997).
In gametophytic apomixis, embryo sacs are derived
either from nucellar cells (apospory) or from a disrup-
ted meiosis of the megaspore mother cell (diplospory).
In both cases, embryos develop parthenogenetically
(Nogler, 1984). In these plants, while the male gam-
etophytes, pollen grains, are normally reduced, the
female gametophytes, embryo sacs, are not.
Nowadays, with the possibility of creating new
cultivars by genetic engineering, there is an increas-
ing interest in the identiﬁcation of the gene, or genes,
responsible for this mode of reproduction. Seed pro-
duction for many crops could be engineered through
apomixis, allowing the ﬁxation and perpetuation of
hybrids and the mass production and maintenance of
elite genotypes. Moreover, breaking apomixis would
allow recombination and the production of new vari-
eties, increasing the gene pool of the species that
reproduce mainly by apomixis, such as forage grasses.
The forage grasses Brachiaria provide an oppor-
tunity to use molecular biology approaches in order
to understand how the embryo is formed in apomictic
plants. The genus comprises around 100 species