Evidence of spontaneous triploidy in cassava Manihot esculenta Crantz

Evidence of spontaneous triploidy in cassava Manihot

esculenta Crantz

J. Sardos

1

_{, M. Rodier-Gould}

2

_{, D. Triaire}

2

_{, D. Dambier}

1

_{, R. Malapa}

3

_{, J.-L. Noyer}

2

_{and V. Lebot}

4

_{CIRAD UPR Multiplication Végétative, Montpellier, France.}

_{CIRAD UMR DAP 1098, Montpellier France.}

_{VARTC Santo, Vanuatu.}

_{CIRAD UPR}

Multiplication Végétative, Port-Vila, Vanuatu.

Corresponding author: [email protected]

Spontaneous polyploidy has never been reported in strictly Manihot

esculenta individuals although artificial polyploids are currently created in

breeding programs using colchicine-induced tetraploids (Sreekumari et al.

1999) and interspecific triploids (M. esculenta x M. glaziovii) have been

reported (Hahn et al. 1990). We here describe two M. esculenta

spontaneous triploid clones that were selected, conserved and propagated

by farmers in Vanuatu (Oceania, South Pacific). These two clones, exhibiting

typical triploid morphology, are grown in traditional home-gardens and are

highly appreciated for domestic uses. The first clone, biskit, has already

spread throughout the archipelago and is now cultivated throughout the

country. The second clone, mariango red, was collected in the village of

Lamlu in the island of Tanna but has not spread yet to the other islands

despite its outstanding characteristics. We present hereafter, cytological and

molecular evidence of their triploid level.

Flow cytometry

Chromosome counting

Root tips were treated in 0.04% hydroxyquinoline for 4 h in the dark at room

temperature. Then they were rinsed two times with a fixative solution

(ethanol : glacial acetic acid, 3 : 1 v/v) and stored in 70% ethanol. Then,

chromosome preparations were made according to the method described by

D’Hont et al. (1995). After staining with 4’,6-diamino-2-phenylindole /

Vectashield, chromosomes were visualized and counted under fluorescent

light with a microscope Leica DMRAX 2.

Mariango red

Leaves from Biskit

6

4

5

2

A B C

7

C B C

3

1

4

6

A C

2

3

4

5

For each clone and a 2X control individual, DNA was extracted from 150 mg

of dried leaves following the method described by Risterucci et al. (2000).

They were genotyped with 11 SSR markers developed by Mba et al. (2001)

that were already used to assess the extent of the genetic base of cassava in

the country (Sardos et al. Unpublished).

SSRY 179

NS 376

SSRY 19

A: Biskit ; B: Mariango red ; C: 2X Control individual

Control Individual

2n=2X=36

Mariango red

2n=3X=54

Biskit

2n=3X=54

Biskit and mariango red shared their alleles with the M.

esculenta national sample (77 genotypes) and didn’t present any

specific allele. Moreover, we genotyped a hybrid between M.

esculenta and one of its wild relative, probably M. glaziovii,

collected in Vanuatu. This hybrid exhibited 41% of specific

alleles that were not present in the national germplasm neither in

biskit nor mariango red.

Flow cytometry analysis shows in

both cases that the peak of the

control diploid individual of M.

esculenta is on the channel 50

and that the peaks for the triploid

clones are on the channel 75.

Biskit and mariango red exhibited 3 alleles at two SSR loci each

(

respectively NS 376 – SSRY 19 and SSRY 179 - SSRY 19

).

Leaves from a diploid clone

The chromosome counts in metaphase at mitosis confirmed the

triploid level of biskit and mariango red: we counted 54

chromosomes for both samples and 36 chromosomes for the

diploid control individual of M. esculenta.

Conclusion

This is the first time that spontaneous and intra-specific

triploid clones of cassava are documented at the cytological

and molecular level. High yields (50-60 tonnes/ha in 9 months,

with no inputs) were obtained after the first agronomic

evaluations of these two clones. It is now necessary to

characterize the physico-chemical characteristics of their

roots.

References:

D’Hont, A., Rao, P.S., Feldmann, P. and Grivet, L. 1995. Identification and characterisation of sugercane intergeneric hybrid,

Saccharum offiicinarum x Erianthus arundinaceus, with molecular markers and DNA in situ hybridisation. Theor. Appl.

Genet. 91: 320-6. Hahn, S. K., Bai, K.V. and Asiedu, R. 1990. Tetraploids, triploids, and 2n pollen from diploid interspecific crosses with

cassava. Theor. Appl. Genet. 79: 433-439.

Mba, R. E. C., Stephenson, P., Edwards, K., Melzer, S., Nkumbira, J., Gullberg, U., Apel, K., Gale, M., Tohme, J., and Fregene, M. 2001. Simple sequence repeat (SSR) markers survey of the cassava (Manihot esculenta Crantz) genome: towards an SSR-based molecular genetic map of cassava. Theor. Appl. Genet. 102: 21-31.

Risterucci, A. M., Grivet, L., N’Goran, J. A. K., Pieretti, I., Flament, M. H., and Lanaud, C. 2000. A high density linkage map of Theobroma cacao L. Theor. Appl. Genet. 101: 948-955.

Sardos, J, McKey, D., Duval, M.F., Malapa, R., Noyer, J.L. and Lebot, V. Evolution of Cassava (Manihot esculenta Crantz) after Recent Introduction into a South Pacific Island System: the Contribution of Sex to the Diversification of a Clonally Propagated Crop. Submitted.

Sreekumari, M.T., Jos, J.S. and Nair, S.G. 1999. ‘Sree Harsha’: a superior triploid hybrid in cassava. Euphytica. 106:1-6.

Genotyping

Acknowledgements:

This study would not have been possible without the financial support of the «

Agrobiodiversity of Root Crops Project in Vanuatu » funded by the FFEM (Fonds

Français pour l’Environnement Mondial), CIRAD and the Ministry of Agriculture in

Vanuatu.

We used for this analysis a 2X individual as

a control. Preparation procedure was

conducted following Partec ready-to-use

reagent kits and protocols: 0.5 cm² of young

leaves were chopped in 0.5 ml of buffer of

CysStain® UV Ploidy (5001) and then

filtered at 30 µ. The nuclei suspensions were

incubated 1 mn, 1.5 ml of buffer 5001 were

added and the analysis were conducted with

the UV excitation flow cytometer Partec II.