Multilocus DNA-fingerprinting using oligonucleotide probes (GATA)4 and (GGAT)4 in rainbow trout (Oncorhynchus mykiss)

SHORT COMMUNICATION

Multilocus DNA-fingerprinting using oligonucleotide probes (GATA)₄ and (GGAT)₄ in rainbow trout (Oncorhynchus mykiss)

N. Vergara¹, P. Iturra¹ and R. Aguirre²

¹Departamento de Biología Celular y Genética and ²Centro de Síntesis y Análisis de Biomoléculas, Facultad de Medicina, Universidad de Chile, Casilla 70061-7 Santiago, Chile. Fax: [phone], E-mail: [e-mail] Send correspondence to P.I.

ABSTRACT

Oligonucleotide probes (GATA)₄ and (GGAT)₄ for minisatellite loci produced DNA fingerprinting patterns which are unique in individual rainbow trout. These patterns can be used to monitor the efficiency of manipulation of production characters in gynogenetic individuals. The (GATA)₄ probe produced highly individual patterns that differentiated individuals among gynogenetic offspring, and could be easily scored.

INTRODUCTION

DNA fingerprinting of salmonid fishes was first reported by Lloyd et al. (1989). They found polymorphic patterns in rainbow trout (Oncorhynchus mykiss Walbaum) using a DNA fragment with repeats of the GATA-GACA sequence as a probe. Taggart and Ferguson (1990), using the probes developed by Jeffreys (1985a,b), found very complex multilocus patterns in three species of salmonid fish. In an attempt to obtain more simple and yet specific patterns, locus specific probes for different salmonid fishes have been cloned (O'Reilly and Wright, 1995). Here, we report that the onligonucleotides (GATA)₄ and (GGAT)₄ generate highly informative multilocus DNA fingerprints in rainbow trout. The (GATA)₄ probe produced highly individual patterns, that differentiates individuals among gynogenetic offspring, and can be easily scored.

MATERIAL AND METHODS

Artificial fertilization and experimental gynogenesis (i.e., production of individuals with only maternal genetic information) were performed according to Colihueque et al. (1992). In brief, eggs were activated with UV-irradiated sperm followed by diploid restoration by disruption of the second meiotic division. Genomic DNA was extracted from whole embryos before hatching and from blood in adult specimens of rainbow trout, Scottish strain (Río Blanco-UCV Fish Hatchery, Los Andes, Chile), according to a modified version of the method described by Sambrook et al. (1989).

Ten micrograms of DNA per fish was digested with HaeIII or HinfI, and fragments were separated in 0.6% agarose gel. The 20-cm long gels were dried under vacuum and hybridized with the ³²P-labeled oligonucleotide probe as described by Schäfer et al. (1988). (GATA)₄ and (GGAT)₄ were labeled by the reaction catalyzed by polynucleotide kinase (Sigma, 10 units/ml) using 50 pmol of oligonucleotides, 10 units of enzyme and 336 mCi of g-³²P ATP (ICN, 7000 Ci/mmol). The labeled probe was used without further purification, and one fifth of the probe solution was used per hybridization. The solution was reused for up to three gels. After hybridization and washing, the gel was exposed to X-ray film (Kodak XAR-5). The gels were rehybridized with a second probe after denaturation and neutralization (Epplen and Zischler, 1990).

RESULTS AND DISCUSSION

Probes (GATA)₄ and (GGAT)₄ produced fingerprints of several bands with DNA digested with either HaeIII or HinfI as shown in Figures 1 and 2. The four probe/enzyme combinations showed that the variable and well-resolved fragments in the gel are in the size range defined by 23.1 and 6.6-Kb markers. The patterns generated differed among individuals in number, position and intensity of the bands. The mean band sharing (similarity index (S ± ES); Lynch, 1990) was estimated to be 0.24 ± 0.14 and 0.33 ± 0.11 for (GATA)₄ and (GGAT)₄, respectively.

Figure 1
- Genomic DNA of five rainbow trout (Oncorhynchus mykiss) specimens was digested with HinfI and subsequently hybridized in a vacum-dried gel with multilocus probes (GATA)₄ (A) and (GGAT)₄ (B). Positions of l HindIII marker fragments (Kb) are indicated.

Figure 2
- DNA fingerprinting patterns of five gynogenetic embryos of Oncorhynchus mykiss. A) Lanes 1-5: GATA/HaeIII. Lanes 6-10: GATA/HinfI. B) Lanes 1-5: GGAT/HaeIII. Positions of l HindIII marker fragments (Kb) are indicated.

The HinfI/(GGAT)₄ combination resulted in more bands larger than 6.6 Kb ( = 16.4 ± 2.5) than HaeIII/(GATA)₄ (10.6 ± 2.6 fragments). All of the combinations used produced patterns with band densities that were low enough for comparison, and allowed clear parental assignment when analyzed with their offspring. Crowded band patterns were obtained with probes 33.6 and 33.15 that produced average band numbers of 37.5 ± 3.7 and 30.5 ± 2.2, respectively (Taggart and Ferguson, 1990). DNA fingerprinting with similar band numbers was obtained in salmonids with Ssal-rep probe by Bentzen et al. (1993), but fragment size was smaller than the minisatellites detected in the present study.

In general, the probe (GATA)₄ generated fingerprints with fewer bands which were darker, more variable in position and with less background than those produced by (GGAT)₄ (Figure 1A and B). These highly variable (GATA)₄ patterns also allowed individualization of some members of a gynogenetic sibship as compared with the (GGAT)₄ patterns where differences among gynogenetic individuals could not be established (S = 1) (Figure 2A and B). (GATA)₄/HinfI patterns showed a reduced heterozygosity value (Stephens et al., 1992) of 0.07 in the gynogenetic progenies. Both restriction enzymes produced (GATA)₄ patterns that individualized gynogenetic fishes (Figure 2a) with small differences in S (0.94 ± 0.03 for HaeIII and 0.88 ± 0.08 for HinfI).

Our results show that the synthetic oligonucleotides (GGAT)₄ and (GATA)₄ provide reproducible and scorable fingerprint patterns for individualization of rainbow trout specimens, and that the latter probe is a useful tool to diagnose the efficiency of gynogenetic manipulation in this species. Minisatellites detected in the rainbow trout genome showed stability. Within 64 embryos analyzed in a single offspring, only one anomalous fragment was identified probably due to a mutation in the hypervariable minisatellite. Analysis of 36 gynogenetic embryos with (GATA)₄ showed no exclusive paternal bands in contrast to results from control offspring produced by normal sexual mating (data not shown).

Synthetic oligonucleotides used as multilocus probes allow in-gel hybridization, a fast and reliable technique omitting transfer and prehybridization steps. This study reports a procedure that can be easily applied to monitor the genetic variability in cultivated fish populations and breeding stocks. In contrast to other technologies, the use of (GATA)₄ and (GGAT)₄ did not require cloned probes and produced fingerprinting patterns that were easy to score.

ACKNOWLEDGMENTS

Research supported by FONDEF-PI10. We wish to thank Río Blanco-UCV Fish Hatchery and Dr. L. Jara for laboratory facilities.

RESUMO

Probes de oligonucleotídeos (GATA)₄ e (GGAT)₄ para locos mini-satélites produziram padrões de "fingerprinting" de DNA que são específicos para cada espécimen de truta do arco-íris. Esses padrões podem ser usados para monitorar a eficácia da manipulação de caracteres de produção em indivíduos ginogenéticos. O probe (GATA)₄ produziu padrões altamente individuais que diferenciaram os indivíduos entre proles ginogenéticas e puderam ser facilmente quantificados.

(Received May 27, 1997)

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Publication Dates

• Publication in this collection
  06 Jan 1999
• Date of issue
  June 1998

History

• Received
  27 May 1997