Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-07-03
2004-04-13
Poon, Peter M. (Department: 3643)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C800S021000, C119S215000
Reexamination Certificate
active
06720150
ABSTRACT:
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A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the United States Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
The invention relates to selection of fish for optimum growth. More particularly the invention relates to methods of selecting fish, in particular tilapia, for optimum growth in water of differing salinity. Most particularly the invention relates to using a polymorphism in the tilapia prolactin gene to select and then breed fish, based on genotype, for optimum growth in water of differing salinity.
BACKGROUND OF THE INVENTION
Tilapia are among the world's most important aquacultural finfishes and include any of numerous, mostly freshwater, fish species (genus Tilapia, family Cichlidae) native to Africa. Tilapias resemble North American sunfishes and one species can grow to up to 20 lbs (9 kg). Tilapia species are popular as aquacultural fishes because they are easy to raise and harvest for food. They grow rapidly, resist disease, and eat readily abundant algae and zooplankton. As a result, tilapia have been used in warm-water aquaculture systems since the early Egyptian civilization and have been introduced into many freshwater habitats. In addition, some tilapia also grow well in saltwater, and various strains differ in their tolerance to saltwater. For example, in certain areas of the world it is preferable to grow tilapia in bays or brackish waters, but not all individuals grow well in these conditions. In fact, tilapiine species differ greatly in salt tolerance and growth response in different salinities. Thus, it has been, and continues to be somewhat difficult to select or predict which individuals will grow best at which salinity.
A multitude of studies have addressed means to grow bigger tilapia faster. As with any food animal, producers look for ways to select and grow larger animals in less time. Thus the use of growth hormones has been investigated to various extent with most types of food animals, including tilapia. In addition, there have been experiments with water temperature, salinity and hybridization. However, there is no present method, other than trial and error, to identify tilapia strains for growth in different salinities.
With respect to hormones, a great deal is known about the role of peptide hormones in fish osmoregulation. Prolactin is a member of the GH/Prl gene family whose “freshwater adapting” role is to increase plasma osmolality by reducing gill Na
+
K
+
ATP-ase activity. The tilapiine pituitary produces two forms of prolactin, encoded by different genes, prl 1 and prl 2, which differ in both molecular weight (24 and 20 kD respectively) and number of amino acid residues (188 and 177 respectively). These two forms are roughly 70% identical at the amino acid level and appear to have differential osmoregulatory and somatotropic actions. Past experiments suggest that as fishes move into more saline environments, both prolactin mRNA's as well as serum levels of prolactin decrease, and this effect is more dramatic for prolactin 1.
The tilapia prl 1 promoter shares non-canonical elements with that of the rat. Both promoters have two (CA)
n
microsatellites (short sequences of repeated nucleotides) interspersed among putative binding sites for the pituitary-specific transcription factor Pit-1 (See FIG.
1
). Naylor and Clark (Naylor L. H. and Clarke E. M. Nucleic Acids Res 18: 159501601, 1990) demonstrated in rat that these repeat sequences formed left-handed Z-DNA in vitro and repressed prl 1 expression. At present, microsatellites and non-coding microsatellite length variation among individuals of a species are generally thought to be neutral and lack functional consequences, except perhaps being related to certain genetic diseases, and have generally not been investigated at length. However, despite the textbook interpretation that non-coding simple sequence repeats evolve in a neutral fashion, recent reports have indicated otherwise. In fact, the abundance and position of dinucleotide microsatellites in eukaryotic genomes, coupled with a high rate of mutation, suggest a potential and pervasive role for microsatellites in gene regulation.
Therefore it would be desirable to have a simple method, based on a variety of genotype, to identify and select animals (for example, tilapia) for optimum growth in differing salinity conditions. Such a method would eliminate the need to experiment with the fishes' growing environment/aquaculture conditions, as well as eliminate the need to supplement the fish with growth stimulants or other artificial means.
SUMMARY OF THE INVENTION
The invention most generally relates to providing a reliable means for selecting and breeding fish for optimum growth in different salinities. In developing the invention, Applicants simply used a natural system to evaluate the association between dinucleotide microsatellite variation, quantitative differences in gene expression and the physiological response to contrasting environments. Applicants' work, in fact, provides the first in vivo evidence that differences in microsatellite length among individuals may indeed affect gene expression and that variance in expression has concomitant physiological consequences.
A most basic embodiment of the invention, therefore, preferably uses differences among individual fish in the length of a simple sequence repeat stretch of DNA (a microsatellite) in the prolactin 1 promoter to select individuals for optimum growth in different salinities. The invention includes methods of selecting individual fish for optimum growth in conditions of various salinity. Fish are selected for breeding and raising in a specific salinity based on their prolactin genotype.
Applicants discovered that a simple sequence repeat polymorphism (microsatellite) in the tilapia prolactin 1 (prl 1) promoter is associated with differences in prl 1 expression and growth in “salt-challenged” or “salt-effected” fishes. This discovery suggests that dinucleotide microsatellites may represent an under-appreciated source of genetic variation for regulatory evolution, and belie the textbook interpretation that non-coding microsatellite length variation lacks functional consequences. In developing the invention, Applicants reasoned that a regulatory effect similar to that found with respect to microsatellite repeat sequences that formed Z-DNA and repressed prl 1 in the rat (Naylor L. H. and Clarke E. M.
Nucleic Acids Res
18: 1595-1601, 1990) may be found in other species even though microsatellite length variation is generally thought to lack functional consequences. In the present invention Applicants investigated whether any regulatory effect might be present in tilapia with respect to microsatellite length of the prl 1 promoter, and whether individual differences in microsatellite length might affect prl 1 expression in vivo and might contribute to the known variance in salt tolerance and growth of tilapia at different salinities.
In fact, Applicants found that the differences in length of the simple sequence repeat stretch of DNA in the tilapia prolactin promoter do elicit changes in the quantity of prolactin that is produced from the pituitary. This variation in prolactin expression is associated with divergent growth rates of individuals in different salt conditions. Applicants found with tilapia that individual variation in microsatellite length does affect prl 1 expression in vivo and contributes to the known variance in salt tolerance and growth. Knowing the optimal genotype/salinity combinations lead to the methods of the present invention in which much better growth rates and reduced growth time to market size can be achieved with tilapia. In the methods of the invention, the prl 1 genotypes of fish are determined, then fish of a desire
Kocher Thomas David
Streelman Jeffrey Todd
Devine Millimet & Branch PA
Gellner Jeffrey L.
Kohler Kristin
Poon Peter M.
Remus Paul
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