crosses between Gossypium hirsutum and G. herbaceum. - IJPB

Evidence of unreduced gamete production from interspecific
crosses between Gossypium hirsutum and G. herbaceum.
Catherine Pannetier1,2, Jean-Luc Hofs1, Emilie Montes,2, Frederique Eber3, Olivier Coriton3, Virginie Huteau3, Bernard Hau1, Anne-Marie Chèvre3.
1: CIRAD Département PERSYST UR 102, Av Agropolis 34398 Montpellier cedex 5 France; !
2: Laboratoire de Biologie Cellulaire INRA Centre de Versailles-Grignon 78026 Versailles cedex France; !
3: INRA, UMR 118 Amélioration des Plantes et Biotechnologies Végétales, BP35327, 35653 Le Rheu cedex, France!
Introduction!
In order to analyse the gene flow between the allotetraploid cultivated cotton (Gossypium hirsutum, AADD, 2n=52) and the wild diploid (G. herbaceum, AA, 2n=26),
the possibility of natural hybridization between these two cotton species has been investigated. In South Africa and particularly in the KwaZulu Natal Province,
where commercialisation of transgenic Bt cotton began in 1998, it is not uncommon to find the wild species (G. herbaceum) growing alongside cultivated cotton
fields. The rate of interspecific hybridization was assessed from reciprocal crosses without emasculation and genomic structure of hybrids was analysed. !
Flow cytometry (method described by Eber et al, 1997 with barley as internal standard), morphology, meiotic behavior and rDNA-FISH analysis were used to
characterise the plants obtained. !
G.hirsutum (A1A1DD) x G.herbaceum (A2A2)!
From 504 seeds, 335 plants were obtained. Neither examination of the
morphological characteristics (plant architecture; leaf shape; colour, size, shape of
flowers…) nor the flow cytometry analysis have shown any to be hybrid plants. !
Table 1: Flow cytometer values
!
* mean on 20 plants (3 analyses per sample)!
** mean on 3 measurements per sample
G.herbaceum (A2A2) x G.hirsutum (A1A1DD) !
From 330 seeds, 148 plants were obtained.
Flow cytometry:
Three hybrids were detected whose two hybrids showed
intermediate value which might correspond to AAD hybrids (H1 and H2). The third
(H3) exhibited a value in flow cytometry slightly higher than G. hirsutum (Table 1).!
Morphological characterization:
Among the 3 hybrids, H1 and H2
exhibited the same phenotype typical of a triploid hybrid (AAD). The third one (H
3) has shown intermediary phenotype between G. herbaceum and the triploid. These
two types of hybrid phenotype (Figs. 1-2) have been also observed for flowers:
corolla size, red spot intensity and size, stamens colour and size (Fig.3). Hybrid 3
had bigger and brighter spots, intermediary between AAD and G. herbaceum which
can support the hypothesis that it carries more genome A from G. herbaceum than the
G. herbaceum parent.!
Fig 1: from left to right: G. herbaceum, G. hirsutum, Hybrids 1 and 2 (AAD),!
Hybrid 3 (AAAD), after 8 weeks of culture.!
Pollen viability evaluated by Alexander and Aceto-carmin staining are presented in
Fig. 3. Hybrid 3 considered to be AAAD has shown a slightly higher viability than
the two triploid hybrids.!
Meiotic behavior: The meiotic behavior (Table 2 and Fig. 4) of the two AAD
hybrids was established from 26 pollen mother cells (PMC); only 35% of the cells
showed the expected behavior with 13 univalents and 13 bivalents. In the third
hybrid, the chromosome number as well as the high frequency of multivalents, 1 per
PMC, are in accordance with the presence of 3 A genomes (Table 2) with a AAAD
structure. !
Fig 3: pollen viability!
Fig 2: from left to right: G. herbaceum, Hybrid 1
(AAD), Hybrid 3 (AAAD), G hirsutum.!
4
2
3
II
12
4
XI
13
XI
VII
X
5
VI
IX
9
IV
VIII
V
X
XII
6
11
10
14
I
1
II
III
3
’’’
’’’
XIII
’’’’
V
VIII
XIV
IV
XV
2
III
13
I
VI
XVI
IX
7
12
8
8
VII
1
9
5
7
Table 2: Meiotic behavior of hybrids (* range)
Fluorescent in situ hybridization:
We confirmed that G.hirsutum
(A1A1DD) and G.herbaceum (A2A2) carry the same number of 45S loci i.e. six.
However, the A genome carried 2 or 6 loci in both species, respectively (Hanson et al.
1996). In agreement with the genomic structure expected, we found 6 loci in A1A2D
hybrids (H1 and H2) and 9 loci in A1A2A2D hybrid (H3) (Fig. 5). !
6
10
B
A
Fig 4: PMC in Metaphase I !
A- From a AAD hybrid: 13 univalents (1-13), 11 bivalents (I-XI) and 1 quadrivalent !
B- From AAAD hybrid: 14 univalents (1-14), 16 bivalents (I-XVI) and 2 trivalents!
11
x
Conclusion!
Interspecific hybrids were detected only when G. herbaceum is used as female with a
rate of 2%. Two hybrids were formed from reduced gametes of the parents but one
was obtained from female unreduced gamete giving to a AAAD structure. This is, to
our knowledge, the first description of the occurrence of a non-reductional meiosis in
the species G. herbaceum. This plant material could provide a useful tool for the study
of the expression of genes duplicated on the A and D cotton genome. The possibility
of obtaining an interspecific hybrid between cultivated and diploid cotton through
fertilization with an unreduced gamete raises the question of its evolution in natural
populations.!
International Conference on Polyploidy, Hybridization and Biodiversity!
May 17-20, 2009 - Saint Malo - FRANCE!
G. hirsutum (A1A1D1D1)
G. herbaceum (A2A2)
H1 and H2 hybrids (39K)
H3 hybrid (52K)
Fig 5: Identification of the genomic structure using rDNA-FISH. Probe: Alexa
488- 45S rDNA (green)!