Protocol I - Preparation of metaphasic chromosomes [Pratical guide of protocols: methods of verification of the ploidy]

Karyotyping is a direct method for the verification and identification of triploid fish. The techniques for chromosome preparation are based on obtaining sources of dividing cells to produce high quality metaphase spreads with good chromosome definition. These procedures use specific chemicals, one of the most used reagents is colchicine, to inhibit the formation of the mitotic house and thus, to block dividing cells at metaphase. The objective of this Protocol is to demonstrate that chromosome preparation is an accurate method to corroborate triploidy condition. Determination of ploidy is based on counting the number of chromosomes in colchicine-arrested metaphase spreads from the newly-hatched sea bass larvae with different ploidy levels. The method developed in the present Protocol has been designed for obtaining chromosome plates from newly-hatched sea bass larvae according to the Protocol previously described.

Le caryotypage est une méthode directe pour vérifier et identifier la triploïdie chez les poissons. Il s'agit d'une méthode basée sur le comptage des chromosomes lors de la métaphase à partir de préparations cellulaires. L'utilisation d'agents antimitotiques tels que la colchicine permettent d'inhiber la formation de la mitose. En bloquant la division mitotique au stade métaphase, les chromosomes peuvent être identifiés individuellement et visuellement. La méthode développée dans le présent protocole a été conçue pour obtenir des plaques de chromosomes de larves de bar nouvellement éclos, ce qui permet l'identification positive de différents niveaux de ploïdie chez cette espèce.

Mots-clés    

BAR, CARYOTYPE, CHROMOSOME, COLCHICINE, LARVE DE POISSON, PLOIDIE, PROTOCOLE, TRIPLOIDIE

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Protocol I - Preparation of metaphasic chromosomes [Pratical guide of protocols: methods of verification of the ploidy]. In : Felip A. (ed.), Carrillo M. (ed.), Herráez M.P. (ed.), Zanuy S. (ed.), Basurco B. (ed.). Advances in fish reproduction and their application to broodstock management: a pratical manual for sea bass. Zaragoza : CIHEAM / CSIC-IATS, 2009. p. 61-65. (Options Méditerranéennes : Série B. Etudes et Recherches; n. 63). http://om.ciheam.org/om/pdf/b63/00800915.pdf