WO2006106558A2 - Use of nothobranchius furzeri as a model system for the characterisation of genes that control ageing - Google Patents

Use of nothobranchius furzeri as a model system for the characterisation of genes that control ageing Download PDF

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WO2006106558A2
WO2006106558A2 PCT/IT2006/000238 IT2006000238W WO2006106558A2 WO 2006106558 A2 WO2006106558 A2 WO 2006106558A2 IT 2006000238 W IT2006000238 W IT 2006000238W WO 2006106558 A2 WO2006106558 A2 WO 2006106558A2
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longevity
populations
strains
species
genes
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Alessandro Cellerino
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Lay Line Genomics S.P.A
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • A—HUMAN NECESSITIES
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    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method for the characterisation and identification of genes or gene products which can modulate the onset of pathologies linked with ageing based on the comparison of various populations of the fish belonging to the family of Cyprinodontids (common name: killifish).
  • Nothobranchius furzeri member of the Cyprinodontid family (common name: killifish) belongs to a group of annual fish whose life expectancy in nature is limited to a few months.
  • the longest survival time of N. furzeri was described and characterised by Valdesalici and Cellerino (2003), and it is less than 12 weeks if bred in standard laboratory conditions at 25 0 C. This is the shortest life expectancy ever recorded for a vertebrate.
  • Nothobranchius furzeri can allow to isolate and identify new genes or products thereof which control longevity and functional ageing in vertebrates.
  • QTL analysis requires the presence of polymorphic markers to be used for linkage analysis.
  • the present invention reports a method to derive a large number of polymorphic DNA markers from TV. furzeri.
  • the present invention relates to genes responsible for functional ageing.
  • Functional ageing is a slow and progressive decrease in the physiological efficiency of all systems of the body: cardio-vascular, skeletal-muscular, respiratory, excretory, thermo-regulatory and nervous (Harman, 2001).
  • the result of this phenomenon which takes place in every human being as age advances, is a progressive decay of all biological functions that lead to a reduction in quality of life and in independence, an increase in susceptibility to all traumas and stresses, to an increased need for chronic treatments and hospitalisation and an increase in the incidence of a vary vast range of pathologies properly defined as pathologies linked to ageing which may affect all organs and systems (Hazzard et al., 2003).
  • an object of the present invention is a method for identifying and/or characterising genes or products thereof able to modulate longevity and/or expression of at least one age-related parameter in vertebrates, comprising the steps of: a) selecting at least two different populations or strains of a species of the family of Cyprinodontid (common name killif ⁇ sh) which show differences in longevity and/or in the expression of at least one age-related parameter; b) characterizing at least two polymorphic DNA markers which can distinguish said populations or strains; c) cross breeding two individuals of each of one of said different populations or strains, obtaining a progeny Fl; d) cross breeding the progeny Fl, obtaining a progeny F2; e) analysing the phenotype related to longevity and/or to expression of at least one age- related parameter in each individual of the progeny F2; f) identifying
  • the populations or strains belong to the species Nothobranchius furzeri, Nothobranchius rachovii or Nothobranchius eggersi. More preferably, the populations or strains are hybrids of Nothobranchius furzeri with Nothobranchius kunthae, or any other species or hybrids of Nothobranchius for which at least two populations of different longevities exist. Even more preferably, the populations or strains with a lower longevity have a longevity of 10 to 14 weeks and the populations or strains with a longer longevity have a longevity of 26 to 40 weeks.
  • the populations or strains belonging the species Nothobranchius furzeri having a lower longevity are obtainable in the National Park of Gona Re Zhou, Moscow at the co-ordinates: 21 40.2 S, 31 2.4 E during the months from January to March. More preferably, the populations or strains belonging to the species Nothobranchius rachovii having a lower longevity are obtainable in the Limpopo plain, Mozambique, at the co-ordinates: 23 88.52S, 32 36.01 E during the months from March to April.
  • the polymorphic DNA markers are a single nucleotide polymorphisms (SNPs).
  • the age-related parameter belongs to the following group: accumulation of lipofuscin in the brain and/or the liver, expression of B-galactosidase associated to senescence, reduction in spontaneous locomotory activity or cognitive decay. It is a further object of the invention a method for identifying and/or characterising genes or products thereof able to modulate longevity and/or expression of at least one age-related parameter in vertebrates, comprising the steps of: a) cloning a killifish gene capable of modulating longevity and/or expression of at least one age-related parameter identified with the method of claim 1 ; b) isolating by hybridization and/or cloning techniques genes in at least one other vertebrate species.
  • the other vertebrate species is a fish species. More preferably, the other vertebrate species is a mammalian species.
  • the meaning of species is purely taxonomic (morphological species) and means a group of animals which present a series of diagnostic morphological traits which allows to assign them to a previously-described species. It is important to emphasize in the framework of the present invention that individuals of two morphologically-distinct but closely-related species can interbreed and that such hybrids can be important for QTL analysis (Peichel et al., 2001; Shapiro et al., 2004; Colosimo et al., 2005; Kimmel et al., 2005) .
  • a population within a species meansthe set of animals which inhabit a specific pool in the wild.
  • a strain means the progeny of a group of animals bred in captivity in reproductive isolation.
  • the individuals which are founders of a strain come from a specific population, but as a consequence of inbreeding, random drifts, genetic bottlenecks and selective breeding, different strains, all coming from the same population, can evolve a different phenotypes.
  • Figure 1 Survival of various strains of Nothobranchius fiirzeri.
  • the survival curve of Fl animals was recorded using the method described by Valdesalici and Cellerino (2003).
  • the y-axis shows the percentage of living animals.
  • the x-axis shows age in weeks.
  • GRZ indicates the strain studied by Valdesalici and Cellerino (2003).
  • the wild populations can be found as indicated in the method section.
  • the differences between the survival curves of the various strains were calculated using the Kolmogoroff Smirnoff test (Statistica ®, Statsoft).
  • the difference between MZM3 and MZM6 or MZM8 is significant p ⁇ 0.01.
  • FIG. 1 The difference between the strain GRZ and all other strains is significant pO.0001.
  • Figure 2 Survival of the hybrids Fl between different strains of Nothobranchius fiirzeri. The survival curve was recorded using the method described by Valdesalici and Cellerino (2003). The y-axis shows the percentage of living animals. The x-axis shows age in weeks. Survivorship in two hybrid Fl generations derived from the cross between MZM03 wild- derived line with the GRZ inbred line is shown. In the legend the female is put first, i.e. MZM03xGRZ is the cross of female MZM03 with male GRZ inbred. GRZ and MZM03 survivorships are the same as in Fig. 1.
  • the y-axis shows the percentage of living animals.
  • the x-axis shows age in weeks.
  • 3 wild- derived strains of Nothobranchius furzeri (MZMl Op LF2, MZMlOgr and MZM0403) were compared with the Nothobranchius furzeri GRZ inbred strain.
  • MZM10pLF2 identifies the fishes obtained from the crossing of the strain MZM-8/10 x MZM-8/10 after 10 months incubation. Its survivorship does not differ from the GRZ inbred line.
  • MZMlOgr identifies the fishes obtained from the crossing of the strain MZM-8/10 x MZM-S/10 but hatched after 2 months incubation.
  • MZM0403 is the F2 generation obtained from the strain MZM- 3 x MZM-3. MZMlOgr and MZM0403 have a significantly higher longevity than GRZ inbred strain (Logrank test p ⁇ 0.001).
  • Figure 4 Age-dependent survival of F2 strains of Nothobranchius rachovii. The y-axis shows the percentage of living animals. The x-axis shows age in days.
  • Nothobranchius furzeri 1) Supply and breeding of Nothobranchius furzeri.
  • the fish GRZ descend from fish collected in 1968 as described (Jubb, 1971). They originate in the National Park of Gona Re Zhou (Zimbabwe), alluvial plane of the Gulene river, co-ordinates: 21 40.2 S ; 31 2.4 E .
  • the fish were collected using large fords and sieving water without sight, given the considerable turbidity of these pools.
  • the fish are common and easy to capture in their typical location during the months of January-March if that year the rains were sufficient to fill the pools.
  • Nothobranchius fur ⁇ eri of the Gona Re Zhou strain can be bought from Kenjiro
  • the fish were maintained in a facility comprising sets of 60 litre tanks with centralised filtering, UV germicide lamp and micro-filtration (Aqua, Lucca), to make water quality homogeneous.
  • MZM3 originates from the Limpopo plain, about 300 km away from Gona Re Zhou : 23 88.52 S; 32 36.01 E.
  • MZM6 and MZM8-10 were collected about half-way between Gona Re Zhou and the
  • Nothobranchius rachovii MZM-3 Nothobranchius rachovii MZM-3:
  • Nothobranchius rachovii MOZ-04/10 Nothobranchius rachovii MOZ-04/10:
  • Nothobranchius kunthae originates from the swamps around the city of Beira in Mozambique:
  • the Gona Re Zhou strain was crossed with the strain MZM3.
  • the hybrid were created simply by placing males and females of the different strains in the same tanks.
  • a male GRZ was crossed with a female MZM3 and a female GRZ was crossed with a male MZM3.
  • degenerated primers were constructed, complementary to the regions of maximum conservation based on the sequences of Zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis) and fugu (Takifugu n ⁇ ipes) that are available (Genomic BLAST, http://www.ncbi.nlm.nih.gov/sutils/ genom treccgi) for each of these genes (Table II).
  • the oligonucleotide sequences used are the following, the legend of the degenerated positions is according to the code IUPAC (see for example http://bioinformatics.org/sms/iupac.html * ):
  • IFGRl and IR are amplified using the same primers and are distinguished by the length of the amplified fragment and p66shc and N-shc are also amplified using the same primer are distinguished by the length of the amplified fragment
  • N.fur ⁇ eri GRZ (Seq ID 28) TGATATCGAGTACTTCAGACGGGACCCTAGACCCTTTTTCAAGTTTGCTAAGGAGATCTACCCC GGTCAGTTCCAACCTTCACCCTGCCACAGGTTCATTTCGATGTTAGACAAGCAAGAGAAGCTGC TACGCAATTACACACAAAACATCGACACGTTGGAGCAAGTGGCTGGAGTTCAGAGGATCATCC AGTGTCACGGGTCCTTCGCAACTGCGTCCTGTCTTGTTTGTAAACAAAAAGTGGATTGTGAAGC TATAAGGGAAGATGTCTTTAATCAGGTTGTTCCTCGTTGTCTGAGGTGTCCGGATATTCCTCTGG CAATCATGAAACCTGACATCGTCTTTTTTGGAGAACCTACCAGAAATGTTCCACAGA
  • N.furzeri GRZ (Seq ID 40) CTCAAACGAGAAGATTTCTGGTCCAGGAGTCACATACATTGTGAAGTATCTGGGCTGCATCGAA GTCCTGCGGTCTATGAGATCCCTGGATTTCACCACTAGGTCACAAATAACACGGGAAGCCATCA GCTTGTTGAGTGAAGCTGTTCCTGGAACCAAAGGAGCACCGAGGAAGAGGAAGCCACCGTCTA AAGCTCTGTCCAGCATTTTGGGCAAGAGCAACCTCCAGTTTGCCGGCATGTCCATCAACCTTAA TATCTCCACCTGTAGTCTCAACTTGATGACTCGTGACTGCAAACAGATCATAGCCAACCATCAC ATGCAGTCCATCTCCTTTGCATCAGGTGGAGACCCTGACACGACGGATTATGTTGCCTATGTAG CAAAGGACCCCGTCAACAGAAGAGCTTGTCACATCCTTGAGTGCCCTGATGGATTGGCTCAGGA TGTC
  • N.furzeri MZM3 (Seq ID 41) CTCAAACGAGAAGATTTCTGGTCCAGGAGTCACATACATTGTGAAGTATCTGGGCTGCATCGAA GTCCTGCGGTCTATGAGATCCCTGGATTTCACCACTAGGTCACAAATAACACGGGAAGCCATCA GCTTGTTGAGTGAAGCTGTTCCTGGAACCAAAGGAGCACCGAGGAAGAGGAAGCCACCGTCTA AAGCTCTGTCCAGCATTTTGGGCAAGAGCAACCTCCAGTTTGCCGGCATGTCCATCAACCTTAA TATCTCCACCTGTAGTCTCAACTTGATGACTCGTGACTGCAAACAGATCATAGCCGACCATCAC ATGCAGTCCATCTCCTTTGCATCAGGTGGAGACCCTGACACGACGGATTATGTTGCCTATGTAG CAAAGGACCCCGTCAACAGAAGAGCTTGTCACATCCTTGAGTGCCCTGATGGATTGGCTCAGGA TGTC
  • N.fiirzeri MZM3 (Seq ID 44) GGCCGGGTTTGCTGGAGACGaTGCTCCTCGTGCCGTCTTCCCCTCCATCGTGGGCCGTCCCAGGC ACCAGGGTGTGATGGTGGGCATGGGgCAGAAGGACAGCTaCGTAGGAGACGAGGCCCAGAGTA AAAGAGGCATCCTGACCCTGAAGTACCCCATCGAGCACGgCAtCGTCACCAACTGGGATGACAT GGAAGATCTGGCATCATACCTTCTACAACGAGCTCCGTGTGGCTCCTGAGGAGCATCCTGTC CTCCTGACAGAGGCTCCTCTCAACCCCAAAGCTAACCGGGAGAAGATGACCCAGATCATGTTTG AGACCTTCAACACACCTGCCATGTACGTGGCCATCCAGGCTGTGCTATCTCTGTATGCTTCTGGA CGAACCACAGGTATCGTGATGGACTCTGGAGATGGTGTCAGCTGTGCTATCTCTGTATGCTTCTGGA CGAACCACAGGTATCGTGAT
  • the QTL analysis requires crossing two populations or strains of the same species which have different phenotypes, the phenotypes having a genetic basis.
  • the two populations or strains can be N. furzeri GRZ and N. furzeri MZM-3 and the difference to analyse is life expectancy and/or the expression of an age-related parameter (such as accumulation of lipofuscin in the brain and/or the liver, expression of B-galactosidase associated to senescence, reduction in spontaneous locomotory activity or cognitive decay).
  • the same method can be applied to other populations or strains of Nothobranchius furzeri, or populations or strains of other species of Nothobranchius or hybrids between two closely-related species of Nothobranchius. Such populations differ in life expectancy because they come from regions with different duration of the rainy season.
  • the limiting factor of success for a QTL analysis is the availability of different populations or strains with measurable phenotypic differences. Once two or more populations are identified, three steps are necessary:
  • a high number (at least 100) of genetic markers are identified which are polymorph and have different alleles in the two strains of origin.
  • markers can be sequences of microsatellite DNA, bands of Amplified Fragment Length Polymorphism (AFLP) or Single Nucleotide Polymorphism (SNPs).
  • AFLP Amplified Fragment Length Polymorphism
  • SNPs Single Nucleotide Polymorphism
  • the sequences of microsatellite DNA microsatellite can be identified with a bio-computerised analysis of banks of ESTs (Serapion et al., 2004).
  • the AFLP method has been widely used for genetic analysis in fish of interest for aquaculture and it is based on the amplification of fragments generated by restriction enzyme pairs and then their separation on sequence gel.
  • the SNPs instead identify sequence differences of a single nucleotide which can be identified, for example, comparing homologous EST sequences in the two populations or in two individuals (He et al., 2003)
  • the use of SNP to create linkage maps is becoming increasingly popular in genomic research and was used for example to create a high-density map of the Japanese Medaka (Khorasani et al., 2004).
  • linkage maps can take great advantage of the synteny of related genomes.
  • a high-density map of the Japanese Medaka genome was created by exploiting its synteny with the genome of the puffer fish (Khorasani et al., 2004).
  • Japanese medaka and Nothobranchiiis furzeri are phylogenetically more related than Medaka and pufferfish (www.fishbase.org).
  • Medaka is not available in its entirety, but through a BLAST server.
  • the Medaka sequences contain a memorized "n".
  • the length of the memorized "n" is arbitrary and is meant to indicate the position of the local interruption in the homology between the two species.
  • the phenotype is quantified (life duration, cognitive deficit, motor activity or other parameters of ageing) and at the same time all polymorphic DNA markers that distinguish the two parental strainsare analysed. Because of the crossing-over and of the Mendelian segregation, the chromosomes of every individual will be a mosaic of fragments originating from one and from the other strain.
  • Microsatellite genotyping can take place by direct sequencing, by Southern Blot or by PCR (Perry et al., 2001; Robison et al., 2001; O'Malley et al., 2003; Somorjai et al., 2003; Cnaani et al., 2004b).
  • the genotyping of the AFLP takes place after electrophoretic run on sequence gel (Liu et al., 1998; Liu et al., 2003).
  • the genotyping of SNPs can take place by direct sequencing, using MALDI-TOF mass spectrometry (Vignal et al., 2002) or commercial kits (Promega, READIT® SNP Genotyping System cat.
  • MD 1290 can be performed in outsourcing by companies which provide special services for large-scale genotyping (for example http://bmr.cribi.unipd.it/).
  • Analysing the correlation between the measured phenotype and the genotype of all polymorphic DNA markers in all individuals of the generation F2 two markers are identified whose genotype has the highest correlation with the observed phenotype. These two markers enclose a chromosome region that controls the phenotype of interest.
  • the techniques to be used to analyse the genotypes and to study the genotype/phenotype correlation are standard and amply described by textbooks and manuals (Liu, 1997; Camp and Cox, 2002). The essential requirements to be able to apply this methodology are:
  • RNA and synthesise cDNA were crossed and after obtaining over 100 fertile eggs were frozen to extract RNA and synthesise cDNA using standard methodologies (Sambroock et al., 2001).
  • the method used to identify markers able to distinguish the two strains is based on the presence of single nucleotide polymorphisms (SNPs) within expressed sequence tags (ESTs) and is substantially identical to the method used to distinguish the two species of catfish Ictahirus punctatus and Ictalimis furcatus, two fish with commercial interest in aquaculture (He et al., 2003).
  • ESTs are being sequenced in the two banks of cDNA of GRZ and MZM-3. As described by He et al.
  • a sample of dorsal fin is drawn (fins regenerate rapidly in fish) to extract the genomic DNA according to standard techniques (Sambroock et al., 2001) and the phenotype of interest for every animal (date of death) is recorded.
  • the genotyping of SNPs can be effected rapidly, using commercial kits (Promega, READIT® SNP Genotyping System cat. MD 1290) or can be performed in outsourcing by companies which provide special services for large-scale genotyping (for example http://bmr.cribi.unipd.it/).
  • the mathematical methods and the programs for analysing genotype/phenotype correlations are described in manuals (Liu, 1997; Camp and Cox, 2002).
  • Fig. 1 compares the life expectancy of the strain of N. furzeri GRZ studied by Valdesalici and Cellerino (2003) with the generation Fl of animals descending from three different strains of N. furzeri: MZM3, MZM6 and MZM8-10.
  • the three strains showed a longer life expectancy when compared to N. furzeri GRZ.
  • the strain MZM3 has a maximum life expectancy of 36 weeks, whilst the strains MZM6 and MZM8-10 have an intermediate life expectancy between GRZ and MZM3 of 28 weeks.
  • MZM3female x GRZmale and GRZfemale x MZM3male Life expectancy of both hybrid strains is signifinatly higher than the life expectancy of the GRZ strain and significantly shorter than the life expectancy of the MZM3 parental strain(Fig. 2). This result shows that the difference in life expectancy observed have a genetic basis.
  • the alleles relating to the rapid ageing phenotype of GRZ and the alleles relating to the long- lived phenotype of MZM-3 are co-dominant and their combination in heterozygosis gives origin to an intermediate phenotype.
  • the eggs were hatched for the first time 2 months after being deposited.
  • the median lifespan of the fish which hatched was 20 weeksfor MZMl Ogr and 23 weeks for MZM0403 (Fig. 3).
  • Many MZM-8/10 x MZM-8/10 eggs were still undeveloped after 2 months, so the eggs were put back into incubation and were wetted again after 10 months.
  • These eggs gave rise to adults, MZM10pLF2, with a median lifespan of only 9 weeks, comparable to that of the GRZ strain studied by Valdesalici e Cellerino (2003) (Fig. 3). This clearly shows that two distinct phenotypes (long vs. short life expectancy) were observed among the F2 strain originating form the crossing MZM-8/10 x MZM-8/10.
  • Table III Frequency and position of single nucleotide sequence differences in a set of selected genes between the GRZ strain of Nothobranchius furzeri, the MZM-3 strain of Nothobranchhis furzeri and Nothobranchius kunthae.
  • the first column reports the gene.
  • the second column the primers used for amplification (sequences are reported in Materials and Methods).
  • the third column the size of the amplifies sequence.
  • the fourth column the number of SNPs in the comparision between GRZ and MZM-3.
  • the fifth column the position of the SNPs.
  • the sixth column the number of SNPs in the comparison between GRZ andN. kunthae.
  • the seventh column the position of the SNPs.
  • expressed genes are a source of SNPs which can distinguish the strains of speciesto be used for QTL analysis.
  • the present results were obtained studying an example of 10 genes. The same procedure can be applied to a larger number of expressed genes to derive a larger number of markers needed for QTL analysis. b) Homology with genomic sequences from Japanese Medaka
  • Table IV Percentage of homology of partial sequences of Nothobranchius furzeri GRZ genes with sequences retrieved from the genome of the Japanese medaka (Ori ⁇ yas latipes).

Abstract

The present invention relates to a method for the characterisation and identification of genes or gene products which can modulate the onset of pathologies linked with ageing based on the comparison of various populations of the fish belonging to the family of Cyprinodontids (common name: killifish).

Description

Use of Nothobranchius furzeri as a model system for the characterisation of genes that control ageing
TECHNICAL FIELD OF THE INVENTION The present invention relates to a method for the characterisation and identification of genes or gene products which can modulate the onset of pathologies linked with ageing based on the comparison of various populations of the fish belonging to the family of Cyprinodontids (common name: killifish).
PRIOR ART
Nothobranchius furzeri, member of the Cyprinodontid family (common name: killifish) belongs to a group of annual fish whose life expectancy in nature is limited to a few months. The longest survival time of N. furzeri was described and characterised by Valdesalici and Cellerino (2003), and it is less than 12 weeks if bred in standard laboratory conditions at 25 0C. This is the shortest life expectancy ever recorded for a vertebrate.
Nothobranchius furzeri can allow to isolate and identify new genes or products thereof which control longevity and functional ageing in vertebrates.
It is possible to identify genes that control longevity in vertebrates analysing families or populations with particularly high longevity (Puca et al., 2001; Geesaman et al., 2003). When populations with different longevity are available, it is possible to identify the genes responsible for this difference through cross breeding and the analysis of genetic markers. This methodologies, known as quantitative trait loci (QTL) is described in detail and it has been used to isolate genes that control longevity in natural populations of Drosophila (Luckinbill and Golenberg, 2002; Geiger-Thornsberry and Mackay, 2004; Pasyukova et al., 2004). QTL techniques are also available for fish and have been used to isolate the genes that control different phenotypes of use in aquaculture (Perry et al., 2001; O'Malley et al.? 2003; Somorjai et al., 2003; Cnaani et al., 2004; Zimmerman et al., 2004). Further, comparison of natural populations of stickleback (Gasterosteleus sp.) has revealed the genes responsible for the morphological differences between these populations (Shapiro et al., 2004; Colosimo et al., 2005; Kimmel et al., 2005). The present invention reports the isolation of natural populations of N. furzeri and TV. rachovii which, because of difference in the risk of death in their habit due to the different length of the rainy season and hence to the different duration of the pools where they live, have evolved highly different life expectancy values. Moreover, the present reports the phenotype related to the survival of the crossings between these populations and the strain studied by Valdesalici and Cellerino (2003) and that segregation of two different phenotypes (short lifespan and long lifespan) is observed in the F2 generation of a laboratory strain which derives from wild-caught fish different from the strain analysed by Valdesalici and Cellerino (2003). These data show that the basis for these differences is hereditary. The analysis of these crossings through QTL can lead to the isolation of new genes that control ageing.
QTL analysis requires the presence of polymorphic markers to be used for linkage analysis. The present invention reports a method to derive a large number of polymorphic DNA markers from TV. furzeri.
DISCLOSURE OF THE INVENTION
The present invention relates to genes responsible for functional ageing. Functional ageing is a slow and progressive decrease in the physiological efficiency of all systems of the body: cardio-vascular, skeletal-muscular, respiratory, excretory, thermo-regulatory and nervous (Harman, 2001). The result of this phenomenon, which takes place in every human being as age advances, is a progressive decay of all biological functions that lead to a reduction in quality of life and in independence, an increase in susceptibility to all traumas and stresses, to an increased need for chronic treatments and hospitalisation and an increase in the incidence of a vary vast range of pathologies properly defined as pathologies linked to ageing which may affect all organs and systems (Hazzard et al., 2003). It is important to emphasise that their slow, progressive nature is the distinctive characteristic of these pathologies, which therefore can only be studied in animal models where the course of the symptoms is gradual and progressive (obviously, rapidity in the onset of the symptoms must be related to the rapidity of the life cycle of the analysed species) and it is observed only during the late stages of the life of the organism. Two examples of pathologies linked to ageing are sarcopenia and slight cognitive deficit (Hazzard et al., 2003) Therefore, an object of the present invention is a method for identifying and/or characterising genes or products thereof able to modulate longevity and/or expression of at least one age-related parameter in vertebrates, comprising the steps of: a) selecting at least two different populations or strains of a species of the family of Cyprinodontid (common name killifϊsh) which show differences in longevity and/or in the expression of at least one age-related parameter; b) characterizing at least two polymorphic DNA markers which can distinguish said populations or strains; c) cross breeding two individuals of each of one of said different populations or strains, obtaining a progeny Fl; d) cross breeding the progeny Fl, obtaining a progeny F2; e) analysing the phenotype related to longevity and/or to expression of at least one age- related parameter in each individual of the progeny F2; f) identifying a pair of polymorphic DNA markers in the progeny F2 that co-segregate with each phenotype; g) identifying a genomic region whose boundaries are defined by said pair of polymorphic DNA markers; h) identifying in said genomic region a gene capable of modulating longevity and/or at least one age-related parameter in said killifish species.
Preferably the populations or strains belong to the species Nothobranchius furzeri, Nothobranchius rachovii or Nothobranchius eggersi. More preferably, the populations or strains are hybrids of Nothobranchius furzeri with Nothobranchius kunthae, or any other species or hybrids of Nothobranchius for which at least two populations of different longevities exist. Even more preferably, the populations or strains with a lower longevity have a longevity of 10 to 14 weeks and the populations or strains with a longer longevity have a longevity of 26 to 40 weeks. Preferably, the populations or strains belonging the species Nothobranchius furzeri having a lower longevity are obtainable in the National Park of Gona Re Zhou, Zimbabwe at the co-ordinates: 21 40.2 S, 31 2.4 E during the months from January to March. More preferably, the populations or strains belonging to the species Nothobranchius rachovii having a lower longevity are obtainable in the Limpopo plain, Mozambique, at the co-ordinates: 23 88.52S, 32 36.01 E during the months from March to April. Preferably, the polymorphic DNA markers are a single nucleotide polymorphisms (SNPs). More preferably, the age-related parameter belongs to the following group: accumulation of lipofuscin in the brain and/or the liver, expression of B-galactosidase associated to senescence, reduction in spontaneous locomotory activity or cognitive decay. It is a further object of the invention a method for identifying and/or characterising genes or products thereof able to modulate longevity and/or expression of at least one age-related parameter in vertebrates, comprising the steps of: a) cloning a killifish gene capable of modulating longevity and/or expression of at least one age-related parameter identified with the method of claim 1 ; b) isolating by hybridization and/or cloning techniques genes in at least one other vertebrate species.
Preferably, the other vertebrate species is a fish species. More preferably, the other vertebrate species is a mammalian species. In the present invention the meaning of species is purely taxonomic (morphological species) and means a group of animals which present a series of diagnostic morphological traits which allows to assign them to a previously-described species. It is important to emphasize in the framework of the present invention that individuals of two morphologically-distinct but closely-related species can interbreed and that such hybrids can be important for QTL analysis (Peichel et al., 2001; Shapiro et al., 2004; Colosimo et al., 2005; Kimmel et al., 2005) . A population within a species meansthe set of animals which inhabit a specific pool in the wild. A strain means the progeny of a group of animals bred in captivity in reproductive isolation. Typically, the individuals which are founders of a strain come from a specific population, but as a consequence of inbreeding, random drifts, genetic bottlenecks and selective breeding, different strains, all coming from the same population, can evolve a different phenotypes.
The present invention shall now be described in non limiting examples thereof, with particular reference to the following figures: Figure 1: Survival of various strains of Nothobranchius fiirzeri. The survival curve of Fl animals was recorded using the method described by Valdesalici and Cellerino (2003). The y-axis shows the percentage of living animals. The x-axis shows age in weeks. GRZ indicates the strain studied by Valdesalici and Cellerino (2003). The wild populations can be found as indicated in the method section. The differences between the survival curves of the various strains were calculated using the Kolmogoroff Smirnoff test (Statistica ®, Statsoft). The difference between MZM3 and MZM6 or MZM8 is significant p<0.01. The difference between the strain GRZ and all other strains is significant pO.0001. Figure 2: Survival of the hybrids Fl between different strains of Nothobranchius fiirzeri. The survival curve was recorded using the method described by Valdesalici and Cellerino (2003). The y-axis shows the percentage of living animals. The x-axis shows age in weeks. Survivorship in two hybrid Fl generations derived from the cross between MZM03 wild- derived line with the GRZ inbred line is shown. In the legend the female is put first, i.e. MZM03xGRZ is the cross of female MZM03 with male GRZ inbred. GRZ and MZM03 survivorships are the same as in Fig. 1. Survivorship of MZM03xGRZ is higher than GRZ (Logrank test p<0.001) and lower than MZM-3 (Logrank test p<0.05). Survivorship of GRZxMZM-3 is higher than GRZ (Logrank test p<0.001) and lower than MZM-3 (Logrank test pO.Ol). Figura 3: Age-dependent survival of F2 strains of Nothobranchius fnrzeri.
The y-axis shows the percentage of living animals. The x-axis shows age in weeks. 3 wild- derived strains of Nothobranchius furzeri (MZMl Op LF2, MZMlOgr and MZM0403) were compared with the Nothobranchius furzeri GRZ inbred strain. MZM10pLF2 identifies the fishes obtained from the crossing of the strain MZM-8/10 x MZM-8/10 after 10 months incubation. Its survivorship does not differ from the GRZ inbred line. MZMlOgr identifies the fishes obtained from the crossing of the strain MZM-8/10 x MZM-S/10 but hatched after 2 months incubation. MZM0403 is the F2 generation obtained from the strain MZM- 3 x MZM-3. MZMlOgr and MZM0403 have a significantly higher longevity than GRZ inbred strain (Logrank test p<0.001). Figure 4: Age-dependent survival of F2 strains of Nothobranchius rachovii. The y-axis shows the percentage of living animals. The x-axis shows age in days. The founders of strain MZM-3 (N=34) were collected in the same pool as the strain MZM-3 of Nothobranchius furzeri. The founders of strain MOZ-04/10 (N=41) were collected from the city of Quelimane in Mozambique as indicated in the method section, Differences in lifespan are statistically significant (Logrank test pO.001).
MATERIALS AND METHODS
1) Supply and breeding of Nothobranchius furzeri. a) Wild populations and captive strains of Nothobranchius furzeri The fish GRZ descend from fish collected in 1968 as described (Jubb, 1971). They originate in the National Park of Gona Re Zhou (Zimbabwe), alluvial plane of the Gulene river, co-ordinates: 21 40.2 S ; 31 2.4 E . The fish were collected using large fords and sieving water without sight, given the considerable turbidity of these pools. The fish are common and easy to capture in their typical location during the months of January-March if that year the rains were sufficient to fill the pools. Moreover, Nothobranchius fur∑eri of the Gona Re Zhou strain can be bought from Kenjiro
Tanaka (http://www.ne.Jp/asahi/medaka-ken/k.t/frarnepage3-10.html).
The opening of the eggs, larvae growth and reproduction are described in Valdesalici and
Cellerino (2003). The fish were maintained in a facility comprising sets of 60 litre tanks with centralised filtering, UV germicide lamp and micro-filtration (Aqua, Lucca), to make water quality homogeneous.
The other strains of TV. furzeri originate from a less arid area than the Gona Re Zhou
National Park (Zimbabwe) and were collected with the same method between 15 march and 2 April 2004 in Mozambique at the following sites:
MZM3 originates from the Limpopo plain, about 300 km away from Gona Re Zhou : 23 88.52 S; 32 36.01 E.
MZM6 and MZM8-10 were collected about half-way between Gona Re Zhou and the
Limpopo plain:
MZM6 coordinates:
22 30.49 S; 32 33.03 E. MZM8-10 coordinates:
22 21.81 S; 32 44.39 E. b) Natural populations and captive strains of Nothobranchius rachovii
Nothobranchius rachovii MZM-3:
It originates from the Limpopo plain, about 300 km away from Gona Re Zhou : 23 88.52 S; 32 36.01 E.
Nothobranchius rachovii MOZ-04/10:
It originates from the swamps around the city of Quelimane, Mozambique :
18 14.2 S; 35 47.2 E.
Both strains are commercially availableyhwz Didier Pillet http://taws.club.fr/Liste.htm. c) Strains of Nothobranchius kunthae
Nothobranchius kunthae originates from the swamps around the city of Beira in Mozambique:
19 50.5 S; 34 52.3 E They are commercially available under the taxonomically uncorrect name of Nothobranchins orthonotus "NJianghcmu" MT03/03 from Kenjiro Tanaka, http://www.ne.Jp/asahi/medaka-ken/k.t/page03-10-14.htni or Didier Pillet http://taws.club.fr/Liste.htm d) Hybrids between different strains of Nothobranchius furzeri
To characterise whether the genes responsible for the difference in life expectancy between natural populations of N. furzeri are recessive, dominant or co-dominant, the Gona Re Zhou strain was crossed with the strain MZM3. The hybrid were created simply by placing males and females of the different strains in the same tanks. In the case of the hybrid GRZ x MZM3, a male GRZ was crossed with a female MZM3 and a female GRZ was crossed with a male MZM3.
The methods used to maintain these strains, their reproduction and growth are identical to those described by Valdesalici and Cellerino (2003).
2) Cloning aging-related genes in Nothobranchius furzeri and Nothobranchius kunthae
The authors verified the presence and the expression of the aging-related genes listed in Table I in different strains of Nothobranchius furzeri (GRZ) and in Nothobranchius kunthae.
Figure imgf000008_0001
Table I: aging-relalted genes studies
For this purpose, degenerated primers were constructed, complementary to the regions of maximum conservation based on the sequences of Zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis) and fugu (Takifugu nώipes) that are available (Genomic BLAST, http://www.ncbi.nlm.nih.gov/sutils/ genom treccgi) for each of these genes (Table II). The oligonucleotide sequences used are the following, the legend of the degenerated positions is according to the code IUPAC (see for example http://bioinformatics.org/sms/iupac.html*):
Gene Primer Forward Primer Reverse igflr GMSTTC ARBTGYCACCA YGTGGT (Seq DD 1) TTVARDGACTCNGGNGMCATCCA (Seq ID 2) ir GMSTTCARBTGYCACCAYGTGGT (Seq ID 1) TTVARDGACTCNGGNGMCATCCA (Seq ID 2) p66shc AARCCYWCVMGDGGMTGGYTGCA (Seq ID 3) AAVGCCTGNCCDATKGTRCTGAT (Seq ID 4) sirtl CCAGAYCCTCAAGAVATGTT (Seq ID 5) TCRTYTTTRTCMTRCTTCATGGC (Seq ID6) mtp AHGARATGAAYAARTACATGCT (Seq ID 7) TYTTGGACATSARRTCRCTGTA (Seq ID 8) myostatin TGGCARAGYATMGAYCTVAARCARGTG (Seq ID 9) ATGCATCYSTCTCAGATYGBGCT (Seq ID 10) foxo3a/l GGAACGCVTGGGGMAACTAYTCCTA (Seq ID 1 1) TCATCCAGCTCCAGGTTAGCCAA (Seq ID 12) foxo3a/2 ACHAAYTCYAAYGCCAGCAC (Seq ID 13) TTYTCCTGGATGGTYTGCAT (Seq ED 14) foxo3a/3 TΠTCAGCCCTTCTTCCATCA (Seq ID 15) GARTCAAARCTGAGSTCCAGGC (Seq ID 16) n-shc AARCCYWCVMGDGGMTGGYTGCA (Seq DD3) AAVGCCTGNCCDATKGTRCTGAT (Seq ID 4) TTAGAAGCAYTTVCKGTGGACGATGG (Seq BD beta-actin GTTGACAAYGGMTCYGGTATGTGCAA (Seq ID 17) 18)
Table II: Genes and corresponding primers used in the present invention conditions PCR :
5 min 94 °C
5 cycles 94 0C 45 s, 43 0C 30 s, 72 0C 30 s
25 cycles 94 0C 45 s, 48 °C 30 s, 72 °C 30 s
15 min 72°C
The amplifications gave rise to the following sequences. Please note that IFGRl and IR are amplified using the same primers and are distinguished by the length of the amplified fragment and p66shc and N-shc are also amplified using the same primer are distinguished by the length of the amplified fragment
Gene igflr:
N.furzeri GRZ: (Seq E) 19)
GGAGAGCATTTCCTTCTACGTTTCGCCACCTAAACGAGATGACGTTGTTGCACTCTACTTGATCA
TCCTACTTCCCATCCTAGCGACTATCTTAATTGTCGGCCTCACCATCATTGCCTTCTTTATCAACA
AAAAGAGGAACAACAACAGACTCGGGAATGGAGTTCTTTATGCATCTGTCAACCCAGAGTACA
TCAGCGCTGCTGAAATGTACACACCGGATGAGTGGGAGGTGGCCAGGGAGAAGATCACCATGC
ACAAAGAGCTGGGTCAGGGCTCCTTCGGCATGGTGTACGAAGGCATCGCGAAAGGCGTTGTTA
AGGACGAGCCTGAGACGCGGGTGGCCATCAAGACTGTCAATGAGTCGGCCAGCATGAGGGAGC
GGATAGAGTTTTTAAACGAGGCGTCTGTCATGAAGGAG
N.furzeri MZM3: (Seq ID 20)
GGAGAGCATTTCCTTCTACGTTTCGCCACCTAAACGAGATGACGTTGTTGCACTCTACTTGATCA TCCTACTTCCCATCCTAGCGACTATCTTAATTGTCGGCCTCACCATCATTGCCTTCTTTATCAACA AAAAGAGGAACAACAACAGACTCGGGAATGGAGTTCTTTATGCATCTGTCAACCCAGAGTACA
TCAGCGCTGCTGAAATGTACACACCGGATGAGTGGGAGGTGGCCAGGGAGAAGATCACCATGC
ACAAAGAGCTGGGTCAGGGCTCCTTCGGCATGGTGTACGAAGGCATCGCGAAAGGCGTTGTTA
AGGACGAGCCTGAGACGCGGGTGGCCATCAAGACTGTCAATGAGTCGGCCAGCATGAGGGAGC
GGATAGAGTTTTTAAACGAGGCGTCTGTCATGAAGGAG
N. kunthae: (Seq ID 21)
GGAGAGCATTTCCTTCTACGTTTCGCCACCTAAACGAGATGACGTTGTTGCACTCTACTTGATCA TCCTACTTCCCATCCTAGCGACTATCTTAATTGTCTGCCTCACCATCATTGCCTTCTTTATCAACA AAAAGAGGAACAACAACAGACTCGGGAATGGAGTTCTTTATGCATCTGTCAACCCAGAGTACA TCAGCGCTGCTGAAATGTACACACCGGATGAGTGGGAGGTGGCCAGGGAGAAGATCACCATGC ACAAAGAGCTGGGTCAGGGCTCCTTCGGCATGGTGTACGAAGGCATCGCGAAAGGCGTTGTTA AGGACGAGCCTGAGACGCGGGTGGCCATCAAGACTGTCAATGAGTCGGCCAGCATGAGGGAGC GGATAGAGTTTTTAAACGAGGCGTCTGTCATGAAGGAG
Gene ir:
N. furzeή GRZ: (Seq ID 22)
CGAACCTACTTACTTCTATGTTCAGGATGCAAGCGATCCCCTCTACATTGTGAAGATCATCATCG GGCCAATCATCTGCGTGGTTCTGGTGCTGTTTATGGCCGTTGTGGGATTTTTCATGTTCAAGAAA AATCAAACCCAAGGGCCTAGCGGTCCCATTTATGCCTCTTCAAACCCTGAGTATCTCAGCACTA ATGATGTGTATGAGGAGGACGAGTGGGAGGTACCCCGTGATAAGAtCGCCATCCTGAGGGAGCT GGGTCAGGGCTCGTTTGGGATGGTCTACGAAGGCATTGCAAAGGACATTGTGAAGGGCGAGGG CGAAACACGCGTCGCAGTGAAAACTGTGAACGAGTCGGCCAGCCTGAGGGAGAGAATCGAGTT CCTGAACGAGGCCTCGGTCATGAAGGCC
N. fiir∑eή MZM3: (Seq ED 23)
CGAACCTACTTACTTCTATGTTCAGGATGCAAGCGATCCCCTCTACATTGTGAAGATCATCATCG GGCCAATCATCTGCGTGGTTCTGGTGCTGTTTATGGCCGTTGTGGGATTTTTCATGTTCAAGAAA AATCAAACCCAAGGGCCTAGCGGTCCCATTTATGCCTCTTCAAACCCCGAGTATCTCAGCACTA ATGATGTGTATGAGGAGGACGAGTGGGAGGTACCCCGTGATAAGAtCGCCATCCTGAGGGAGCT GGGTCAGGGCTCGTTTGGGATGGTCTACGAAGGCATTGCAAAGGACATTGTGAAGGGCGAGGG CGAAACACGCGTCGCAGTGAAAACTGTGAACGAGTCGGCCAGCCTGAGGGAGAGAATCGAGTT CCTGAACGAGGCCTCGGTCATGAAGGCC
N. kunthae: (Seq K) 24)
CGAACCTACTTACTTCTATGTTCAGGATGCAAGCGATCCCCTCTACATTGTGAAGATCATCATCG GGCCAATCATCTGCGTGGTTCTGGTGCTGTTTATGGCCGTTGTGGGaTTTTTCATGTTCAAGAAA AATCAAACCCAAGGGcCTAGCGGTCCCATTTATGCCTcTTcAAACCCTGAGTATCTCAGCACTAA TGATGTGTATGAGGAGGACGAGTGGGAGGTACCCCgtGATAAGAtCGCCATCCTgAGGGaGCTGG GtCAGGGCTCGTTTGGGaTGGTCTACGAAGGCATTgCAAAGGACATTGTGAAGGGCGAGGGCGaA ACACGCGTAgcAGTGAaAACTgTAAACgAGTCGGCCAGCCTGAGGGAGAGAATCGAGTTCCTGAA CGAGGcCTCGGTCAtGAAGGCC
Gene p66shc:
N.furzeή GRZ: (Seq ED 25)
TTCTGACAACGTGATCAGCACCACGGGAGTTTCCTATACTGTTCGGTACATGGGTTGTGTGGAG GTGCTACAGTCAATGAGAGCACTGGACTTCAACACCAGAACTCAGGTCACCAGGGAAGCTATC
TCTGTGGTGTGTGAAGCGGTACCTGGAGCCAAAGGAGCCCGCAGGAGAAAGCCCGCCCCTCGC
GGTTTGATGTCCATCTTGGGAAAGAGTAACTTGCAGTTTGCGGGCATGACAATCAACCTCACCA TCTCGACCAGCAGCCTCAATCTGCTGGCTTCTGACTGCAAAGAGATTATCGCCAATCATCACAT GCAGTCCATCTCCTTTGCTTCAGGAGGAGACCCAGATACAGCTGAGTACGTAGCATATGTGGCC AAAGATCCAGTCAACCACAGAGCTTGTCACATCCTGGAGTGCTCAGAGGGTTTAGCCCAGGAG GTC
N.fiirzeri MZM3: (Seq ID 26)
TTCTGACAACGTGATCAGCACCACGGGAGTTTCCTATACTGTTCGGTACATGGGTTGTGTGGAG GTGCTACAGTCAATGAGAGCACTGGACTTCAACACCAGAACTCAGGTCACCAGGGAAGCTATC TCTGTGGTGTGTGAAGCGGTACCTGGAGCCAAAGGAGCCCGCAGGAGAAAGCCCGCCCCTCGC GGTTTGATGTCCATCTTGGGAAAGAGTAACTTGCAGTTTGCGGGCATGACAATCAACCTCACCA TCTCGACCAGCAGCCTCAATCTGCTGGCTTCTGACTGCAAAGAGATTATCGCCAATCATCACAT GCAGTCCATCTCCTTTGCTTCAGGAGGAGACCCAGATACAGCTGAGTACGTAGCATATGTGGCC AAAGATCCAGTCAACCACAGAGCTTGTCACATCCTGGAGTGCTCAGAGGGTTTAGCCCAGGAG GTC
N. kunthae: (Seq ID 27)
TTCTGACAACGTGATCAGCACCACTGGAGTTTCCTATACTGTTCGGTACATGGGTTGTGTGGAG GTGCTACAGTCAATGAGAGCACTGGACTTCAACACCAGAACTCAGGTCACCAGGGAAGCTATC TCTGTGGTGTGTGAAGCGGTACCTGGAGCCAAAGGAGCCCGCAGGAGAAAGCCCGCCCCtCGCG GTTTGATGTCCATCTTGGGAAAGAGTAACTTGCAGTTTGCAGGCATGACAATCAACCTCACCAT CTCGACCAGCAGCCTCAATCTGCTGGCTTCTGACTGCAAAGAGATTATCGCCAATCATCACATG CAGTCCATCTCCTTTGCTTCAGGAGGAGACCCAGATACAGCTGAGTACGTAGCATATGTGGCCA AAGATCCAGTCAACCACAGAGCTTGTCACATCCTGGAGTGCTCAGAGGGTTTAGCCCAGGAGGT C
Gene sirtl :
N.fur∑eri GRZ: (Seq ID 28) TGATATCGAGTACTTCAGACGGGACCCTAGACCCTTTTTCAAGTTTGCTAAGGAGATCTACCCC GGTCAGTTCCAACCTTCACCCTGCCACAGGTTCATTTCGATGTTAGACAAGCAAGAGAAGCTGC TACGCAATTACACACAAAACATCGACACGTTGGAGCAAGTGGCTGGAGTTCAGAGGATCATCC AGTGTCACGGGTCCTTCGCAACTGCGTCCTGTCTTGTTTGTAAACAAAAAGTGGATTGTGAAGC TATAAGGGAAGATGTCTTTAATCAGGTTGTTCCTCGTTGTCTGAGGTGTCCGGATATTCCTCTGG CAATCATGAAACCTGACATCGTCTTTTTTGGAGAGAACCTACCAGAAATGTTCCACAGA
N.fiirzeri MZM3: (Seq TD 29)
TGATATCGAGTACTTCAGACGGGACCCTAGACCCTTTTTCAAGTTTGCTAAGGAGATCTACCCC GGTCAGTTCCAACCTTCACCCTGCCACAGGTTCATTTCGATGTTAGACAAGCAAGAGAAGCTGC TACGCAATTACACACAAAACATCGACACGTTGGAGCAAGTGGCTGGAGTTCAGAGGATCATCC AGTGTCACGGGTCCTTCGCAACTGCGTCCTGTCTTGTTTGTAAACAAAAAGTGGATTGTGAAGC TATAAGGGAAGATGTCTTTAATCAGGTTGTTCCTCGTTGTCTGAGGTGTCCGGATATTCCTCTGG CAATCATGAAACCTGACATCGTCTTTTTTGGAGAGAACCTACCAGAAATGTTCCACAGA
N. kunthae: (Seq ID 30) TGATATCGAGTACTTCAGACGGGACCCTAGACCCTTTTTCAAGTTTGCTAAGGAGATCTACCCC GGTCAGTTCCAACCTTCACCCTGCCACAGGTTCATTTCGATGTTAGACAAGCAAGAGAAGCTGC TACGCAATTACACACAAAACATCGACACGTTGGAGCAAGTGGCTGGAGTTCAGAGGATCATCC AGTGTCACGGGTCCTTCGCAACTGCATCCTGTCTGGTTTGTAAACAAAAAGTGGATTGTGAAGC TATAAGGGAAGATGTCTtTAATCAGGTtGTTCCTCGTTGTCTGAGGTGTCCGGATATTCCTCTGGC AATCATGAAACCTGACATCGTCTTtTTTGGAGAGAACCTACCAGAAATGTTCCACAGA
Gene mtp: N. furzeri GRZ: (Seq ID 31)
GTCAAAGATCCAGGACATCCTTCGCTTCGAAATGCCTGCCAGCAAAGTTATTCAACAAGCTATG
AAAGACATGATTTCCCACAACTATAACCGCTTTGCCAAAGTCGGTTCATCGTCTGCATTCTCGG
GTTTCATGGCACGATCTGCTGATCTGACCTCCACCTACAGTTTGGACATCCTGTATTCAGGCTCT GGGATCATGAGAAGCAGCAACATGAATATTTATGGTTCTAGTAATGGAGCAATGCTACATGGA CTACAGGTGGCGATTGAGGCTCAAGGTCTGGAGTCTCTGATTGCTGCGACACCAGACGCGGGG GAGGAGGACCTGGAGTCATTCGCCGGCATGTCAGCTCTGCTCTTTGATGTCCAGCTGCGACCGG TCACGTTTTTCAAGGGT
N.furzeri MZM3: (Seq ID 32)
GTCAAAGATCCAGGACATCCTTCGCTTCGAAATGCCTGCCAGCAAAGTTATTCAACAAGCTATG AAAGACATGATTTCCCACAACTATAACCGCTTTGCCAAAGTCGGTTCATCGTCTGCATTCTCGG GTTTCATGGCACGATCTGCTGATCTGACCTCCACCTACAGTTTGGACATCCTGTATTCAGGCTCT GGGATCATGAGAAGCAGCAACATGAATATTTATGGTTCTAGTAATGGAGCAATGCTACATGGA CTACAGGTGGCGATTGAGGCTCAAGGTCTGGAGTCTCTGATTGCTGCGACACCAGACGCGGGG GAGGAGGACCTGGAGTCATTCGCCGGCATGTCAGCTCTGCTCTTTGATGTCCAGCTGCGACCGG TCACGTTTTTCAAGGGT N. kunthae: (Seq ID 33)
GTCAAAGATCCAGGACATCCTTCGCTTCGAAATGCCTGCCAGCAAAGTTATTCAACAAGCTATG AAAGACATGATTTCCCACAACTATAACCGCTTTGCCAAAGTCGGTTCATCGTCTGCATTCTCGG GTTTCATGGCACGATCTGCTGATCTGACCTCCACCTACAGTTTGGACATCCTGTATTCAGGCTCT GGGATCATGAGAAGCAGCAACATGAATATTTATGGTTCTAGTAATGGAGCAATGCTACATGGA CTACAGGTGGCGATTGAGGCTCAAGGTCTGGAGTCTCTGATTGCTGCGACACCAGATGCGGGGG AGGAGGACCTGGAGTCATTCGCCGGCATGTCAGCTCTGCTCTTTGATGTCCAGCTGCGACCGGT CACGTTΠTCAAGGGT
Gene myostatin:
N.furzeri GRZ: (Seq ID 34)
CTGGCCGTGTGGCTGCGGCAGCCGGAGACCAACTGGGGCATCGAGATCAACGCTTTCGACTCCC
GGGGGAATGATTTAGCCGTGACCTCCACAGAGCCTGGAGAGGAGGGACTGCAACCGTTCATGG AGGTTAAAATCTCTGAGGGCCCCAAGCGTGTCAGGAGAGACTCGGGTCTGGACTGTGATGAGA ACTCTCCGGAGTCCCGGTGCTGCCGCTACCCCCTTACGGTGGACTTTGAGGATTTTGGTTGGGAC TGGATTATTGCCCCAAAGCGCTACAAAGCCAACTATTGCTCTGGGGAATGTGAATACATGCACC TGCAGAAGTACCCCCACACTCACCTAGTGAACAAAGCCAACCCCAGAGGGACCGCAGGCCCCT GCTGTACCCCCACCAAGATGTCGCCCATCAACATGCTCTACTTTAACCGCAAAG
N.furzeri MZM3: (Seq ID 35)
CTGGCCGTGTGGCTGCGGCAGCCGGAGACCAACTGGGGCATCGAGATCAACGCTTTCGACTCCC GGGGGAATGATTTAGCCGTGACCTCCACAGAGCCTGGAGAGGAGGGACTGCAACCGTTCATGG AGGTTAAAATCTCTGAGGGCCCCAAGCGTGTCAGGAGAGACTCGGGTCTGGACTGTGATGAGA ACTCTCCGGAGTCCCGGTGCTGCCGCTACCCCCTTACGGTGGACTTTGAGGATTTTGGTTGGGAC TGGATTATTGCCCCAAAGCGCTACAAAGCCAACTATTGCTCTGGGGAATGTGAATACATGCACC TGCAGAAGTACCCCCACACTCACCTAGTGAACAAAGCCAACCCCAGAGGGACCGCAGGCCCCT GCTGTACCCCCACCAAGATGTCGCCCATCAACATGCTCTACTTTAACCGCAAAG N. kunthae: (Seq ID 36)
CTGGCCGTGTGGCTGCGGCAGCCGGAGACCAACTGGGGCATCGAGATCAACGCTTTCGACTCCC GGGGGAATGATTTAGCCGTGACCTCCGCAGAGCCTGGAGAGGAGGGACTGCAACCGTTCATGG AGGTTAAAATCTCTGAGGGCCCCAAGCGTGTCAGGAGAGACTCGGGTCTGGACTGTGATGAGA ACTCTCCGGAGTCCCGGTGCTGCCGCTACCCCCTTACGGTGGACTTTGAGGATTTTGGTTGGGAC TGGATTATTGCCCCAAAGCGCTACAAAGCCAACTATTGCTCTGGGGAGTGTGAATACATGCACC TGCAGAAGTACCCCCACACTCACCTAGTGAACAAAGCCAACCCCAGAGGGACCGCAGGTCCTT GCTGTACCCCCACCAAGATGTCGCCCATCAACATGCTCTACTTTAACCGCAAAG
Gene foxo3a:
N.furzeri GRZ: (Seq ID 37)
TGCGGACCTGATCACCCAGGCCATCGAGAGCTCCCCCGAGAAGAGGCTGACCTTGTCCCAAATc
TAtGACTGgATGGTGAGGTCTGTGCCATATTTcAAgGaCAAAGGCGACAgcAACAGCTCAGCTGGC TGGAAGAATTCTATCCGACAcAACTTATCCCTTCATAGTCGGTTTGTGAAAGTCCAgAATGAAGG AACGGGGAAAAGCTCCTGGTGGATGGTcAACCCAGAGGGGGGTAAAGGaGGcAAAGCTCCGCGT CGACGGGCAGTTTcTaTGGACAACAGCAAATAcATTAAAgGAGCCCGAGGACGGGCCACCAAGA AGAAGGcCTCCTTACAgGCCACCCAGGACGGCAGCTcTgAGAGCTCCTCAAgCCTCTcCAAGTGGa CCGGAAGTcCCACCTcGCGcACGAGCGACGAACTGGACGCCTGGaCAgaCTTCCGCTCTCGAACCA AtTCCAAcGCCAGCACACTCAgCGGcCGTCTgTCTCCGATCACTCAGCGGCCGTCTGTcTCCGATCT TGGCTAACCTGGAGCTGGATGAGGTTCCCGATGATGACTCCCCCCTCTCTCCGATGCTGTACTCC AGCCCCAGCAGCATGTCTCCGTCCACAGGACCCACAGGACTCTCCGACCTGGCAGGTACTATG AACCTCAACGATGGCCTGTCGGAcAACCTGATGGATGACCTTTTGGACAATATcAGCTTGACGGC ATCTCAGCAGCCGCCCCCTGGaGAGGAAGACCCTGGATCCAACGGTCATGGGGCGTCAGTGTTT ACCTTCAGCTGCTCAGGCGGCGCTCTGGGTAGTCCTTCTGGaAGCTACGtGGCCAATCCTCTTTTC AGCCCTTCTTCCATCACAAGTCTCCGCCAGTCGCCCCAAGTCTcCGccAGTcGcCCATGcAgACCAT cCAGGAGAACAAGcAGACTACCTTCACTTGTGTCTCACATTTCAGTGACCACCAGACCCTgCAGG ATCTGCTGAGCCTCgACTCCCATAGtCCCAACAACGTCATGCTAACCCAGTCCGATCCTCTTATGT CAcAGACCAGCACCGCCATCACCCTGCAGAACTCCCGCCGAAACGCCATGCTGCTCCGCAAGGA CCACATTCTGCTGAGTCCCACCAgTGcAAGCCAGGcCCAAGGCTCTTCAGTGCCTGGTTgGCAAG CTGGCTTGTCAACCCCAGACAGCGaGGCGGGcCGgTCCGATACCAAGCCGcCGcTCCTGAagTCTC CCAGCAAGAACACTTCTATGCAGCTGaGCTCTGGTTTGTCAGTTCAGGACCGCTTCCCCGCCGAt CTGGATCTCGACGtGTTCAATGGCAGCctaGAGTGcGACATGGaCTCCATCATCCGTAACGaATTGA TGGACGCAGACT
N.furzeri MZM3: (Seq ID 38)
TgCGGACCTGGTCACCCAGGCCAtCGAGAGCTCCCCCGAGAAGAGGCTGACCTTGTCCCAAATCT
ATGACTGGATGGTGAGGTCTGTGCCATATTTCAAGGACAAAGGCGACAGcAACAGCTCAGCTGG CTGGAAGAAtTCTAtCcGACACAACTTATCCCTTCATAGTCGGTTtGTGAAAGtCCAGAATGAAGG GACGGGGAAAAGCTCCtGGTGGATGGTCAACCCAGAGGGGGGTAAAGGAGGCAAAGCTCCGCG TCGACGGGCAGTTTCTATgGACAACAGCAAATACATTAAAGGAGCCCgaGGACGGGCCACCAAG AAGAAGGCCTCCTTACAGGCCACCCAGGACGGCAGCTCTGAGAGCTCcTCAAGCcTCTCCAAGT GGACCGGAAGTCCCACCTCGCGCAcgAGCGACGAACTGGACGCCTGGACAgACTTCCGCTCTCG AACCAATTCCAACGcCAGCACACTCAGcGGCCGTCTGTCTCCGATCACTCAGCGGCCGTCTGTcT CCGATCTTGGCTAACCTGGAGCTGGATGAGGTTCCCGATGATGACTCCCCCCTCTCTCCGATGCT GTACTCCAGCCCCAGCAGCATGTCTCCGTCCACAGGACCCACAGGACTCTCCGACCTGGCAGGT ACTATGAACCTCAACGATGGCCTGTCGGAcAACCTGATGGATGACCTTTTGGACAATATcAGCTT GACGGCATCTCAGCAGCCGCCCCCTGGaGAGGAAGACCCTGGATCCAACGGTCATGGGGCGTCA GTGTTTACCTTCAGCTGCTCAGGCGGCGCTCTGGGTAGTCCTTCTGGaAGCTACGtGGCCAATCCT CTTTTCAGCCCTTCTTCCATCACAAGTCTCCGCCAGTCGCCCCAAGTCTcCGccAGTcGcCCATGcA gACCATcCAGGAGAACAAGcAGACTACCTTCACTTGTGTCTCACATTTCAGTGACCACCAGACCC TgCAGGATCTGCTGAGCCTCgACTCCCATAGtCCCAACAACGTCATGCTAACCCAGTCCGATCCT CTTATGTCAcAGACCAGCACCGCCATCACCCTGCAGAACTCCCGCCGAAACGCCATGCTGCTCC GCAAGGACCACATTCTGCTGAGTCCTACCAgTGcAAGCCAGGcCCAAGGCTCTTCAGTGCCTGGT TgGCAAGCTGGCTTGTCAACCCCAGACAGCGaGGCGGGcCGgTCCGATACCAAGCCGcCGcTCCT GAagTCTCCCAGCAAGAACACTTCTATGCAGCTGaGCTCTGGTTTGTCAGTTCAGGACCGCTTCCC CGCCGAtCTGGATCTCGACGtGTTCAATGGCAGCctaGAGTGcGACATGGaCTCCATCATCCGTAAC GaATTGATGGACGcAGACT
N. kunthae: (Seq ID 39)
TGCGGACCTGATCACCCAGGCCATCGAGAGCTCCCCCGAGAAGAGGCTGACCTTGTCCCAAATc TAtGACTGgATGGTGAGGTCTGTGCCATATTTcAAgGaCAAAGGCGACAgcAACAGCTCAGCTGGC TGGAAGAATTCTATCCGACAcAACTTATCCCTTCATAGTCGGTTTGTGAAAGTCCAgAATGAAGG AACGGGGAAAAGCTCCTGGTGGATGGTcAACCCAGAGGGGGGTAAAGGaGGcAAAGCTCCACGT CGACGGGCAGTTTcTaTGGACAACAGCAAATAcATTAAAgGAGCCCGAGGACGGGCCACCAAGA AGAAGGcCTCCTTACAgGCCACCCAGGACGGCAGCTcTgAGAGCTCCTCAAgCCTCTcCAAGTGGa CCGGAAGTcCCACCTcGCGcACGAGCGACGAACTGGACGCCTGGaCAgaCTTCCGCTCTCGAACCA AtTCCAAcGCCAGCACACTCAgCGGcCGTCTgTCTCCGATCACTCAGCGGCCGTCTGTCTCCGATC TTGGCTAACCTGGAGCTGGATGAGGTTCCCGATGATGACTCCCCCCTCTCTCCGATGCTGTACTC CAGCCCCAGCAGCATGTCTCCGTCCACAGGACCCACAGGACTCTCCGACCTGGCAGGTACTATG AACCTCAACGATGGCCTGTCGGACAACCTGATGGATGACCTTTTGGACAATATCAGCTTGACGG CATCTcAGCAGCCACCCCCTGGAGAGGAAGACCCTGGATCCAACGGTCATGGGGCGTCAGTGTT TACCTTCAGCTGCTCAGGCGGCGCTCTGGGTAGTCCTTCTGGAAGCTACGTGGCCAATCCTCTTT TCAGCCCTTCTTCCATCACAAGTCTCCGCCAGTCGCCCCAAGTCTcCGccAGTcGcCCATGcAgACC ATcCAGGAGAACAAGcAGACTACCTTCACTTGTGTCTCACATTTCAGTGACCACCAGACCCTgCA GGATCTGCTGAGCCTCgACTCCCATAGtCCCAACAACGTCATGCTAACCCAGTCCGATCCTCTTA TGTCAcAGACCAGCACCGCCATCACCCTGCAGAACTCCCGCCGAAACGCCATGCTGCTCCGCAA GGACCACATTCTGCTGAGCCCCACCAgTGcAAGCCAGGcCCAAGGCTCTTCAGTGCCTGGTTgGC AAGCTGGCTTGTCAACCCCGGACAGCGaGGCGGGcCGgTCCGATACCAAGCCGcCGcTCCTGAagT CTCCCAGCAAGAACACTTCTATGCAGCTGaGCTCTGGTTTGTCAGTTCAGGACCGCTTCCCCGCC GAtCTGGATCTCGACGtGTTCAATGGCAGCctaGAGTGcGACATGGaCTCCATCATCCGTAACGaAT TGATGGACGCAGACT
Gene n-shc:
N.furzeri GRZ: (Seq ID 40) CTCAAACGAGAAGATTTCTGGTCCAGGAGTCACATACATTGTGAAGTATCTGGGCTGCATCGAA GTCCTGCGGTCTATGAGATCCCTGGATTTCACCACTAGGTCACAAATAACACGGGAAGCCATCA GCTTGTTGAGTGAAGCTGTTCCTGGAACCAAAGGAGCACCGAGGAAGAGGAAGCCACCGTCTA AAGCTCTGTCCAGCATTTTGGGCAAGAGCAACCTCCAGTTTGCCGGCATGTCCATCAACCTTAA TATCTCCACCTGTAGTCTCAACTTGATGACTCGTGACTGCAAACAGATCATAGCCAACCATCAC ATGCAGTCCATCTCCTTTGCATCAGGTGGAGACCCTGACACGACGGATTATGTTGCCTATGTAG CAAAGGACCCCGTCAACAGAAGAGCTTGTCACATCCTTGAGTGCCCTGATGGATTGGCTCAGGA TGTC
N.furzeri MZM3: (Seq ID 41) CTCAAACGAGAAGATTTCTGGTCCAGGAGTCACATACATTGTGAAGTATCTGGGCTGCATCGAA GTCCTGCGGTCTATGAGATCCCTGGATTTCACCACTAGGTCACAAATAACACGGGAAGCCATCA GCTTGTTGAGTGAAGCTGTTCCTGGAACCAAAGGAGCACCGAGGAAGAGGAAGCCACCGTCTA AAGCTCTGTCCAGCATTTTGGGCAAGAGCAACCTCCAGTTTGCCGGCATGTCCATCAACCTTAA TATCTCCACCTGTAGTCTCAACTTGATGACTCGTGACTGCAAACAGATCATAGCCGACCATCAC ATGCAGTCCATCTCCTTTGCATCAGGTGGAGACCCTGACACGACGGATTATGTTGCCTATGTAG CAAAGGACCCCGTCAACAGAAGAGCTTGTCACATCCTTGAGTGCCCTGATGGATTGGCTCAGGA TGTC
N. kunthae: (Seq ID 42) CTCAAACGAGAAGATTTCTGGTCCAGGAGTCACATACATTGTGAAGTATCTGGGCTGCATCGAA GTCCTGCGGTCCATGAGATCCCTGGATTTCACCACTAGGTCACAAATAACACGGGAAGCCATCA GCTTGTTGAGTGAAGCTGTTCCTGGAACCAAAGGAGCACCGAGGAAGAGGAAGCCACCGTCTA AAGCTCTGTCCAGCATTTTGGGCAAGAGCAACCTCCAGTTTGCCGGCATGTCCATCAACCTTAA TATCTCCACCTGTAGTCTCAACTTGATGACTCGTGACTGCAAACAGATCATAGCCAACCATCAC ATGCAGTCCATCTCCTTTGCATCAGGTGGAGACCCTGACACGACGGATTATGTTGCCTATGTAG CAAAGGACCCTGTCAACAGAAGAGCTTGTCACATCCTTGAGTGCCCTGATGGATTGGCTCAGGA TGTC
Gene beta-actin:
N.furzeri GRZ: (Seq ID 43)
GGCCGGGTTTGCTGGAGACGaTGCTCCTCGTGCCGTCTTCCCCTCCATCGTGGGCCGTCCCAGGC
ACCAGGGTGTGATGGTGGGCATGGGgCAGAAGGACAGCTaCGTAGGAGACGAGGCCCAGAGTA AAAGAGGCATCCTGACCCTGAAGTACCCCATCGAGCACGgCAtCGTCACCAACTGGGATGACAT GGAGAAGATCTGGCATCATACCTTCTACAACGAGCTCCGTGTGGCTCCTGAGGAGCATCCTGTC CTCCTGACAGAGGCTCCTCTCAACCCCAAAGCTAACCGGGAGAAGATGACCCAGATCATGTTTG AGACCTTCAACACACCTGCCATGTACGTGGCCATCCAGGCTGTGCTATCTCTGTATGCTTCTGGA CGAACCACAGGTATCGTGATGGACTCTGGAGATGGTGTCAGCCATACTGTCCCCATCTACGAGG GCTACGCTCTGCCTCACGCCATCCTCCGTCTGGACCTGGCTGGCAGAGATCTGACGGACTACCT GATGAAGATCCTGACTGAGAGAGGCTACAGCTTCACCACCACTGCTGAGCGTGAGATTGTCCGT GACATCAAGGAGAAGCTCTGCTACGTGGCTCTAGACTTTGAGCAGGAGATGCAGACCGCAGCC TCCTCTTCCTCCCTGGAGAAGAGCTACGAGCTTCCTGATGGACAGGTCATCACCATCGGCAACG AGAGGTTCCGCTGCCCGGAGGCGCTCTTCCAGCCTTCCTTCATCGGTATGGAGTCTGCTGGGAT CCACGAGACCACCTACAACAGCATCATGAAGTGTGACGTGGACATCCGTAAGGACCTGTACGC CAACACGGTTCTGTCCGGTGGAACCACCATGTACCCTGGAATCGCTGACCGCATGCAGAAGGA GATCACCGCCCTGGCCCCTCCCACCATGAAGATCAAGATCATTGCTCCCCCCGAGAGGAAGTAC TCCGTCTGGATCGGTGGCTCCATCCTGGCCTCCCTCTCCACCTTCCAGCAGATGTGGATCAGCAA ACAGGAGTACGACGAGTCCGgCCCCG
N.fiirzeri MZM3: (Seq ID 44) GGCCGGGTTTGCTGGAGACGaTGCTCCTCGTGCCGTCTTCCCCTCCATCGTGGGCCGTCCCAGGC ACCAGGGTGTGATGGTGGGCATGGGgCAGAAGGACAGCTaCGTAGGAGACGAGGCCCAGAGTA AAAGAGGCATCCTGACCCTGAAGTACCCCATCGAGCACGgCAtCGTCACCAACTGGGATGACAT GGAGAAGATCTGGCATCATACCTTCTACAACGAGCTCCGTGTGGCTCCTGAGGAGCATCCTGTC CTCCTGACAGAGGCTCCTCTCAACCCCAAAGCTAACCGGGAGAAGATGACCCAGATCATGTTTG AGACCTTCAACACACCTGCCATGTACGTGGCCATCCAGGCTGTGCTATCTCTGTATGCTTCTGGA CGAACCACAGGTATCGTGATGGACTCTGGAGATGGTGTCAGCCATACTGTCCCCATCTACGAGG GCTACGCTCTGCCTCACGCCATCCTCCGTCTGGACCTGGCTGGCAGAGATCTGACGGACTACCT GATGAAGATCCTGACTGAGAGAGGCTACAGCTTCACCACCACTGCTGAGCGTGAGATTGTCCGT GACATCAAGGAGAAGCTTTGCTACGTGGCTCTAGACTTTGAGCAGGAGATGCAGACCGCAGCC TCCTCTTCCTCCCTGGAGAAGAGCTACGAGCTTCCTGATGGACAGGTCATCACCATCGGCAACG AGAGGTTCCGCTGCCCGGAGGCGCTCTTCCAGCCTTCCTTCATCGGTATGGAGTCTGCTGGGAT CCACGAGACCACCTACAACAGCATCATGAAGTGTGACGTGGACATCCGTAAGGACCTGTACGC CAACACGGTTCTGTCCGGTGGAACCACCATGTACCCTGGAATCGCTGACCGCATGCAGAAGGA GATCACCGCCCTGGCCCCTCCCACCATGAAGATCAAGATCATTGCTCCCCCCGAGAGGAAGTAC TCCGTCTGGATCGGTGGCTCCATCCTGGCCTCCCTCTCCACCTTCCAGCAGATGTGGATCAGCAA ACAGGAGTACGACGAGTCCGgCCCCG
N. kvmthae: (Seq ID 45)
GGCCGGGTTTGCTGGAGACGaTGCTCCTCGTGCCGTCTTCCCCTCCATCGTGGGCCGTCCTAGGC
ACCAGGGTGTGATGGTGGGCATGGGgCAGAAGGACAGCTaCGTAGGAGACGAGGCCCAGAGTA AAAGAGGCATCCTGACCCTGAAGTACCCCATCGAGCACGgCAtCGTCACCAACTGGGATGACAT GGAGAAGATCTGGCATCATACCTTCTACAACGAGCTCCGTGTGGCTCCTGAGGAGCATCCTGTC CTCCTGACAGAGGCTCCTCTCAACCCCAAAGCTAACCGGGAGAAGATGACCCAGATCATGTTTG AGACCTTCAACACACCTGCCATGTACGTGGCCATCCAGGCTGTGCTATCTCTGTATGCTTCTGGA CGAACCACAGGTATCGTGATGGACTCTGGAGATGGTGTCAGCCATACTGTCCCCATCTACGAGG GCTACGCTCTGCCTCACGCCATCCTCCGTCTGGACCTGGCTGGCAGAGATCTGACGGACTACCG ATGAAGATCCTGACTGAGAGAGGCTACAGCTTCACCACCACTGCTGAGCGTGAGATTGTCCGTG ACATCAAGGAGAAGCTCTGCTACGTGGCTCTAGACTTTGAGCAGGAGATGCAGACCGCAGCCT CCTCTTCCTCCCTGGAGAAGAGCTACGAGCTTCCTGATGGACAGGTCATCACCATCGGCAACGA GAGGTTCCGCTGCCCGGAGGCGCTCTTCCAGCCTTCCTTCATCGGTATGGAGTCTGCTGGGATCC ACGAGACCACCTACAACAGCATCATGAAGTGTGACGTGGACATCCGTAAGGACCTGTACGCCA ACACGGTTCTGTCCGGTGGAACCACCATGTACCCTGGAATCGCTGACCGCATGCAGAAGGAGAT CACCGCCCTAGCCCCTCCCACCATGAAGATCAAGATCATTGCTCCCCCCGAGAGGAAGTACTCC GTCTGGATCGGTGGCTCCATCCTGGCCTCCCTCTCCACCTTCCAGCAGATGTGGATCAGCAAAC AGGAGTACGACGAGTCCGgCCCCG
3) QTL analysis
The QTL analysis requires crossing two populations or strains of the same species which have different phenotypes, the phenotypes having a genetic basis. In our case, the two populations or strains can be N. furzeri GRZ and N. furzeri MZM-3 and the difference to analyse is life expectancy and/or the expression of an age-related parameter (such as accumulation of lipofuscin in the brain and/or the liver, expression of B-galactosidase associated to senescence, reduction in spontaneous locomotory activity or cognitive decay). The same method can be applied to other populations or strains of Nothobranchius furzeri, or populations or strains of other species of Nothobranchius or hybrids between two closely-related species of Nothobranchius. Such populations differ in life expectancy because they come from regions with different duration of the rainy season. The limiting factor of success for a QTL analysis is the availability of different populations or strains with measurable phenotypic differences. Once two or more populations are identified, three steps are necessary:
1) Identification of polymorphic DNA markers which can differentiate the populations or strains; 2) Crossing two individuals of different populations or strains, originating the generation Fl which is subsequently crossed with itself, originating the generation F2. In F2 generation mendelian segregation of the alleles takes place;
3) Characterisation of phenotype/genotype correlation for each of the F2 hybrids to identify a set of polymorphic DNA markers which shows the highest correlation with the observed phenotype.
The techniques 1) and 3) are described in more detail:
Step 1) Identification of polymorphic DNA markers.
To differentiate the genomic regions corresponding to two parental strains, a high number (at least 100) of genetic markers areidentified which are polymorph and have different alleles in the two strains of origin. These markers can be sequences of microsatellite DNA, bands of Amplified Fragment Length Polymorphism (AFLP) or Single Nucleotide Polymorphism (SNPs). The sequences of microsatellite DNA microsatellite can be identified with a bio-computerised analysis of banks of ESTs (Serapion et al., 2004). The AFLP method has been widely used for genetic analysis in fish of interest for aquaculture and it is based on the amplification of fragments generated by restriction enzyme pairs and then their separation on sequence gel. The SNPs instead identify sequence differences of a single nucleotide which can be identified, for example, comparing homologous EST sequences in the two populations or in two individuals (He et al., 2003) The use of SNP to create linkage maps is becoming increasingly popular in genomic research and was used for example to create a high-density map of the Japanese Medaka (Khorasani et al., 2004). To demonstrate that a large number of SNPs that differentiate between populations, strains or species can be easily obtained by comparing the sequence of expressed genes across species and populations, the partial sequence of the genes studied as reported in table I was compared between Nothobranchins furzeri GRZ, Nothobranchiiis furzeri MZM-3 and Nothobranchins kunthae.
The creation of linkage maps can take great advantage of the synteny of related genomes. For example, a high-density map of the Japanese Medaka genome was created by exploiting its synteny with the genome of the puffer fish (Khorasani et al., 2004). Japanese medaka and Nothobranchiiis furzeri are phylogenetically more related than Medaka and pufferfish (www.fishbase.org). To measure the sequence homology between expressed sequences of Nothobranchius furzeri GRZ and Japanese Medaka, the sequences of studied gene indicated in Table I were BLASTed against the available genomic sequences of Medaka (http://dolphin.lab.nig.ac.ip/seqcenter/srch db/search medaka blastdb.php). The nucleotide and amino acid sequences of Medaka (Oiyzias Iatipes), homologue to the genes of Nothobranchius furzeri GRZ cloned in the present invention, are reported below. These sequences have been used to calculate the percent of identity reported in Table . It should be noted that the genome of Medaka is not available in its entirety, but through a BLAST server. In some fragments where the sequences of Nothobranchius do not find an homologue in Medaka, the Medaka sequences contain a serie of "n". The length of the serie of "n" is arbitrary and is meant to indicate the position of the local interruption in the homology between the two species. Nucleotide and amino acid sequences of Medaka (Oryzias Iatipes), homologue to the corresponding Nothobranchiiis furzeri GRZ genes : beta-actin: (Seq JD 46) gccggattcgctggagacgatgcccctcgtgctgtctttccctccatcgttggtcgccccaggcaccagggtgtcatggtgggtatg ggccagaaagacagctacgtaggtgatgaagcccagagcaagaggggtatcctgaccctgaagtatcccattgagcacggtatt gttaccaactgggatgacatggagaagatctggcaccacaccttctacaatgagctgagaattgcccctgaggagcaccctgtcct gctcactgaagcccccctgaaccccaaagccaacagggagaagatgacccagatcatgtttgagaccttcaacagccctgccatg tacgttgccatccaggctgtgctgtccctgtatgcctctggtcgtaccactggtatcgtcatggactctggtgatggtgtgacccacac agtgcccatctacgagggctacgctctgccccacgccatcctgcgtctggacttggccggccgcgaccttacagactacctcatga agatcctgacggagcgtggctactccttcaccaccacagccgagagggaaattgtccgtgacatcaaggagaagctgtgctacgt cgccctggacttcgagcaggagatgggcaccgctgcctcctcttcctccctggagaagagctatgagctgcctgacggacaggtc atcaccattggcaatgagaggttccgttgcccagaggccctcttccagccttccttcmrniinniinnnatccatgagaccacctaca acagcatcatgaagtgtgatgttgatatccgtaaggatctgtacgccaacactgtgctgtctggaggtaccaccatgtaccctggaat cgcagacagaatgcagaaggagatcacagccctggccccatccaccatgaagatcaagatcattgccccaccagagcgtaaata ctctgtctggattggaggctccatcctggcctctctgtccaccttccagcagatgtggatcagcaagcaggagtacgatgagtctgg cccc
(Seq ID 47) 7
AGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEH GWTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANREKMTQIMFETFN
TP AMYV AiQAVLSLY ASGRTTGΓVMDSGDGVTHTVPΓYEGYALPHAILRLDLAGR
DLTDYLMKILTERGYSFTTTAEREIVRDIKEKXCYV ALDFEQEMGTAASSSSLEKS YELPDGQVITIGNERFRCPEALFQPSFLGMESCGIHETTFNSIMKCDVDIRKDLYAN TVLSGGTTMYPGIADRMQKEITALAPTTMKIKIIAPPERKYSVWIGGSILASLSTFQ QMWISKQEYDESGP
foxo3a: (Seq E) 48) nnnniiniTiinnagaattccatccggcacaacctgtccctccacagccgttttgtcaaagtccagaacgaaggaactgggaaaagc tcctggtgggtggtcaaccctgaaggtgggaaaggcgggaaggccccccgcaggcgggctgtttccatggacaacagcaagta catgaagggggctcgggggcgcgccaccaaaaagaaggcgtcgctgcaggcggcccaggacggcagctccgagagctcgtc cagcctctctaagtggaccggcagcccaagcagcgacgagctggacgcctggacggacttccgttcccggaccaactccaacg ccagcacgctcagcggccgcctgtctcccatcctcgccaacctggagctggacgaggttcccgacgacgactcccccctctccc ccatgctgtactccagccccagcagcatgtccccgtccaccggccccacgggactctcggacctggccggaaccatgaacctga acgacgggctgtcggacaacctgatggacgacctcttggacaacatcagcctgacggcgtcccagcagcctcctcctggagagg aagaccccggcgccagcgctcagggggggtctgtgtttacattcagctgccccggtggtggtttaggaagtccatctgggaccta cgggtccagccctctgttcagccccccctccctgacaggcctgcggccgtcccccatgcaaaccatccaggagaacaagcag accaccttcaciinnnnnruinnacaacgtcctgctcacccagtccgaccccctgatgtcgcaggcgagcgccgccatggccctg cagaactcccgccgnmiiinnnnimccgacctggacctggacgtgtttgacggcagcctggagtgcgacatggaciinnnnnn nnn
(Seq ID 49) NNNNNNNNNNQNSIRHNLSLHSRFVKVQNEGTGKSSWWVVNPEGGKGGKAPRR RAVSMDNSKYMKGARGRATKKKASLQAAQDGSSESSSSLSKWTGSPSSDELDAW TDFRSRTNSNASTLSGRLSPILANLELDEVPDDDSPLSPMLYSSPSSMSPSTGPTGLS DLAGTMNLNDGLSDNLMDDLLDNISLTASQQPPPGEEDPGASAQGGSVFTFSCPG GGLGSPSGTYGSSPLFSPPSLTGLRPSPMQTIQENKQTTFTSPFSDHQSLLDSPSHNV LLTQSDPLMSQASAAMALQNSRRNLFRKDALLVTHGGSAQSVALWQAGSSACDG DGGRVD AKQLKSPSKLAGPDLDLDVFDGSLECDMDVΓVRNELMEAD
(Seq ID 50) nnrnimirLnnnaggaacagcaacagactgggcaatggagtcctgtatgcatccgtcaacccagagtacatnimnniinniintgt acactccagacgaatgggaggtggctcgagagaagatcaccatgcacaaggagctgggtcagggctcttttggcatggtgtacg agggcctcgcaaagggcgtcgtcaaggatgaacctgagacacgggtggccatcaagacggtcaatgaatcggccagcatgag ggagcggatcgagtttttgaacgaggcgtcagtcatgaaggag (Seq ID 51)
ESIYFYWPPK-RDDDVTFYLSIIIPIIVTLFIASLITILFFINKKRNSNRLGNGVLYASVN PEYISAAEVYTPDEWEVAREKITMHKELGQGSFGMVYEGLAKGVVKDEPETRVAI KTVNESASMRERIEFLNEASVMKE
in
(Seq ID 52) iτnnnnminnngtgaagatcatcatcgggccgatcatctgcnnniτnniinnncaaacccagggtcccagcggacccatctacg cctcctccaaccctgagtatctcagnnniinnnnnngtgtacgaggaggacgagtgggaggttccccgggacaagatccacatc ctgagggagctgggccagggctcctttgggatggtctatgaaggcctcgccaaagacatcgtgaagggggagggggagacgc acgtcgccgtgaagacggtgaacgagtccgccagcctgcgggagaggatcgagttcctgaacgaagcgtcggtcatgaaggcc
(Seq ID 53)
NNNNNNNNNNDPLYWKΠIGPIICFVLLLLVSV AGFWFKKSQTQGPSGPIYASSN PEYLSAND VYΕEDEWEVPRDKffllLRELGQGSFGMVYEGLAKDIVKGEGETHVAV KTVNESASLRERIEFLNEASVMKA
m
(Seq ID 54) imniiiinnnnnacctccacatacagtttggacatcctgtattcaggctctgggatcctgagaagaagcaacatgaatatttatggtg ctagtaaagatgcaatgcttcatggaminnnnnnnncaggtggcgattgaggcccagggtctggagtccctcattgctgcaacg cctgatgaaggagaggaagagctggagtcgtttgctgggatgtcagctctactgtttgatgttcagcttcgccctgtcac tttcttcaannnnnnnnnn
(Seq ID 55)
NNNNNNNNNNSPFLAGSGDLTSTYSLDILYSGSGILRRSNMNΓYGASKD AMLHGL QVAIEAQGLESLIAATPDEGEEELESFAGMSALLFDVQLRPVTFFKG
mui f:
(Seq ID 56) nnnnmmiinnatctgccccatctgcctggagatgttcaccaagccggtggtcatcctgccatgtcaacacaacctgtgccgcaag tgtgccaacgatgtiirumnnnnnncaggcctccaacccctacctgtctgcgaggagcggctccaccgtgacctcaggggggc gtttccgctgcccttcctgccgccatgaagtggttctggannnnnnnnnn
(Seq ID 57)
QLSCPICLEMFTKPWILPCQHNLCRSCASDLYDSRNPYRYSGGVFRCPTCRFEW LDRHGVHGLQRNLL
myostatin:
(Seq ID 58) iTnnniTiinnnntgcgaagttcggcagcacatcaaaaccatgcggttaaacgcaatcaagtcgcagatactgagtaagctgcgcat gagagaagcccccaacatcagccgtgacacggtgaaccaactcctgcccaaagcgccgccgctgcagcaacttctcgaccagt acgacgtgctggcagacgacagcatggacgctgtcgcggaggaggacgacgagcatgcatctacggagacaattatgttgatg gcnnnnnnniinngcgcagctgtgggtgcatctgcgcccggcggacgaggcgaccaccgtcttcctgcagatctctcgcttgat gccggtgacggacggaaacaggcacatcgtgcgtatccgctccttgaagatcgacgtgagagccgggctcagctcttggcagag tatagatgtgaaacaagtattggctgtgtgggttcggcagcccgaaaccaactggggcatcgaaatcaacgccttcgattccagag gaaatgacttagccgtcacctcagcagaaccaggagagcaaggactgcaaccgttcattgaggtgaagatctcagagggcccca ggcgtgccaggagagactctggcttggattgcgatgagaactccccagagtcccgttgctgccgctacccactcacggtggacttt gaggacttcggttgggactggattattgccccaaagcgctacaaggccaactattgttctggggagtgtgagtacatgtacttgcag aagtacccacatacccacctggtgaacaaggccaatcccaggggcactgcaggtccctgttgcacacccaccaagatgtcgccc atcaacatgctctactttaaccgcaaag
(Seq ID 59) NNNNNNNNNNLTALGPWLGDQETQQQPSATSAAD AEQCATCEVRQHIKTMRLN AIKSQILSKLRMREAPNISRDTVNQLLPKAPPLQQLLDQYDVLADDSMDAVAEED DEHASTETMLMATENNNNNNNNNNVQVDGEPKCCLFSFAQKFY ASRTVRAQLW
VHLRP ADEATTVFLQisRLMP VTDGNRHIVRIRSLKID VRAGLSSWQSΠDVKQVLA VWVRQPETNWGIEINAFDSRGNDLAVTSAEPGEQGLQPFIEVKISEGPRRARRDSG
LDCDENSPESRCCRYPLTVDFEDFGWD WΠAPKRYKANYCSGECEYMYLQKYPHT
HLVNKANPRGTAGPCCTPTKMSPINMLYFNRK
n-shc: (Seq ID 60)
ctcaaacgagaagatttctggtccaggagtcacatacatrnirniniinnnnagtatctggggtgcatagaagtcttgcggtcgatga gatccctggatttcacaacaagatcacaaatmirinminnnngggaagccataagtctggtgtgtgaagctgttcctgggaccaaa ggagcnniΗiiΗinnnnaaagctctttccaatattttgggaaagagtaacctgcagtttgctgggatgtctataaacctaaatatctcc accagtagcctcaacttgatgacccccgactgcaaacagatcatagccaaccatcatatgcagtccatctcttttgcatcaggtgga gannimnnminntgttgcctatgtagcaaaagaccctgtgaacagaagagnnnnnnnnnn
(Seq ID 61)
SNEKISGPGVTYIVKYLGCIEVLRSMRSLDFTTRSQITRE AISLVCEAVPGTKGALR KKKPPSKALSNILGKSNLQFAGMSINLNISTSSLNLMTPDCKQQIIANHHMQSISFAS GGDPDTTDFV AYVAKDPVNRRACHILECADGLAQDV
pθόshc:
(Seq ID 62) nnunnnnniiiingtacatgggttgtgtagaggtgctacagtcaatgcgggcgctggacttcaacaccagaacacaagtcaccagg gaagctatcgctgtggtgtgtgaggcagtacccggagccaaaggcgcccgccgaagaaagnnnnnnnnnntgggaaagagt aacttgcagtttgcaggcatgccaatcagcctcaccatctcaaccagcagcctaaacctgctagcttctgactgcaaaagattattgc caatcatcacatgcagtccatatcctttgcctctggaggagacccagatacagcagagtacgtagcatatgtggccaaagacccag tcaaccatagagtgtcacatcctggagtgcacagagggtttagcnnnnnnnniTii
(Seq ID 63)
NNNNNNNNNNQYMGCVEVLQSMRALDFNTRTQVTREAIAVVCEAVPGAKGARR RQPSSRCLTSILGKSNLQFAGMPISLTISTSSLNLLASDCKQIIANHHMQSISFASGGD PDTAEYVAYVAKDPVNHRACHILECTEGLAQEV
sirtl :
(Seq ID 64) nmirinminnnatcgaatacttccgacgagaccccagaccgtttttcaagtttgctaaaggagatcttccccgggcagttccagcc gtctccctgccacagatttatcgctatgctggacaagcaggagaagctgctgcgcaattacacacaaaacatcgacacgctggaac aagtggctggagtgcagaaaatcatccagtgtcacggrmnnnnniiniitcctgtctcgtttgtaaacacaaagtggattgtgaggc tataagggaggacatttttaatcaggttgttcctcggtgtccgcgctgttcggacattcctctggccatcatgaaaccggacattgtgtt ttttggagagaacctcccagagatgttccacaga
(Seq ID 65) DEE YFRRDPRPFFKFAKEIFPGQFQPSPCHRFIAMLDKQEKLLRNYTQNIDTLEQVA GVQKIIQCHGGSF ATASCLVCKHKVDCEAIREDIFNQWPRCPRCSDffLAIMKPDI VFFGENLPEMFHR
Step 3) Genotvpe/phenotvpe correlation
In every individual of F2, the phenotype is quantified (life duration, cognitive deficit, motor activity or other parameters of ageing) and at the same time all polymorphic DNA markers that distinguish the two parental strainsare analysed. Because of the crossing-over and of the Mendelian segregation, the chromosomes of every individual will be a mosaic of fragments originating from one and from the other strain.
Microsatellite genotyping can take place by direct sequencing, by Southern Blot or by PCR (Perry et al., 2001; Robison et al., 2001; O'Malley et al., 2003; Somorjai et al., 2003; Cnaani et al., 2004b). The genotyping of the AFLP takes place after electrophoretic run on sequence gel (Liu et al., 1998; Liu et al., 2003). The genotyping of SNPs can take place by direct sequencing, using MALDI-TOF mass spectrometry (Vignal et al., 2002) or commercial kits (Promega, READIT® SNP Genotyping System cat. MD 1290) or can be performed in outsourcing by companies which provide special services for large-scale genotyping (for example http://bmr.cribi.unipd.it/). Analysing the correlation between the measured phenotype and the genotype of all polymorphic DNA markers in all individuals of the generation F2, two markers are identified whose genotype has the highest correlation with the observed phenotype. These two markers enclose a chromosome region that controls the phenotype of interest. The techniques to be used to analyse the genotypes and to study the genotype/phenotype correlation are standard and amply described by textbooks and manuals (Liu, 1997; Camp and Cox, 2002). The essential requirements to be able to apply this methodology are:
1) having at least two genetically distinct populations or strains with phenotype differences;
2) being able to cross individuals belonging these populations or strains to reach the generation F2; 3) to have a system which allows to derive a large number of polymorphic markers. Four unexpected results are part of the present invention: i) distinct populations of Nothobranchius furzeri, because of differences in their natural habitat, exhibit large differences in their life expectancy. These populations can be crossed together and the progenies exhibit an intermediate phenotype. ii) a segregation between a strain with very short lifespan and a strain with intermediate lifespan, both originating from the same populations observed in the F2 generation. iii) polymorphic DNA markers can be derived by comparing the sequence of expressed genes across populations. iv) there is a high degree of sequence identity between the sequence of expressed genes in Nothobranchius fur∑eri and in Japanese Medaka. This result allows to create a linkage map based on expressed sequences by exploiting the synteny of related genomes. By way of non exhaustive example of the possibilities of this system, a method for performing QTL analysis in Nothobranchius fur∑eri is described.
A male of GRZ and a female of MZM-3 were crossed and after obtaining over 100 fertile eggs were frozen to extract RNA and synthesise cDNA using standard methodologies (Sambroock et al., 2001). The method used to identify markers able to distinguish the two strains is based on the presence of single nucleotide polymorphisms (SNPs) within expressed sequence tags (ESTs) and is substantially identical to the method used to distinguish the two species of catfish Ictahirus punctatus and Ictalimis furcatus, two fish with commercial interest in aquaculture (He et al., 2003). ESTs are being sequenced in the two banks of cDNA of GRZ and MZM-3. As described by He et al. (2003), the comparison between the homologous sequences of EST in the two cDNA banks is a certain source of numerous SNPs. These SNPs represent the markers to conduct QTL analysis of the generation F2 derived from the crossing of GRZ x MZM-3.
For every animal F2 a sample of dorsal fin is drawn (fins regenerate rapidly in fish) to extract the genomic DNA according to standard techniques (Sambroock et al., 2001) and the phenotype of interest for every animal (date of death) is recorded. The genotyping of SNPs can be effected rapidly, using commercial kits (Promega, READIT® SNP Genotyping System cat. MD 1290) or can be performed in outsourcing by companies which provide special services for large-scale genotyping (for example http://bmr.cribi.unipd.it/). The mathematical methods and the programs for analysing genotype/phenotype correlations are described in manuals (Liu, 1997; Camp and Cox, 2002). RESULTS
1) Life expectancy in the Fl generation of different wild-derived strains of N. furzeri a) Life expectancy of various strainsof N. furzeri
Fig. 1 compares the life expectancy of the strain of N. furzeri GRZ studied by Valdesalici and Cellerino (2003) with the generation Fl of animals descending from three different strains of N. furzeri: MZM3, MZM6 and MZM8-10. The three strains showed a longer life expectancy when compared to N. furzeri GRZ. The strain MZM3 has a maximum life expectancy of 36 weeks, whilst the strains MZM6 and MZM8-10 have an intermediate life expectancy between GRZ and MZM3 of 28 weeks. These results are as expected from evolutionary theories of ageing (Kirkwood and Holliday, 1979; Stearns et al., 1998). b) Life expectancy of the hybrids between various strains of N. furzeri
Two different Fl crosses were performed to study the heritability of lifespan in N. furzeri
MZM3female x GRZmale and GRZfemale x MZM3male. Life expectancy of both hybrid strains is signifinatly higher than the life expectancy of the GRZ strain and significantly shorter than the life expectancy of the MZM3 parental strain(Fig. 2). This result shows that the difference in life expectancy observed have a genetic basis. The alleles relating to the rapid ageing phenotype of GRZ and the alleles relating to the long- lived phenotype of MZM-3 are co-dominant and their combination in heterozygosis gives origin to an intermediate phenotype. The difference between the MZM3 female x GRZmale and GRZfemale x MZM3male is statistically significant (Log-Rank test p<0.05) suggesting that sex-linked genes might be implicated in the modulation of lifespan. c) Lifespan of the F2 generation of wild-derived strains of 'N. furzeri
The F2 generation of wild-derived strains MZM-8/10 and MZM-3 were analysed to detect whether mendelian segregation of lifespan takes place within one strain.
The eggs were hatched for the first time 2 months after being deposited. The median lifespan of the fish which hatched was 20 weeksfor MZMl Ogr and 23 weeks for MZM0403 (Fig. 3). Many MZM-8/10 x MZM-8/10 eggs were still undeveloped after 2 months, so the eggs were put back into incubation and were wetted again after 10 months. These eggs gave rise to adults, MZM10pLF2, with a median lifespan of only 9 weeks, comparable to that of the GRZ strain studied by Valdesalici e Cellerino (2003) (Fig. 3). This clearly shows that two distinct phenotypes (long vs. short life expectancy) were observed among the F2 strain originating form the crossing MZM-8/10 x MZM-8/10. Since all eggs originated from the same parents, the difference in lifespan observed is considered genetic and controlled by one or few genes. d*) Lifespan of the Fl generation of wild-derived strains of N.rachovii N rachovii is another species with a widespread geographic distribution which has large- scale differences in precipitations. The authors compared the lifespan of two strains of N. rachovii: the strain MZM-3 which originates from a population which lives sympatrically with N. fiirzeri MZM-3 and the strain MOZ-04/10, which originates from a population living in the city of Quelimane (Fig. 4). Median lifespan is 16 weeksfor N. rachovii MZM- 3 and 23 for N. rachovii MOZ-04/10, the difference in lifespan observed was statistically significant.
2) Identification of polymorphic DNA markers a) Polymorphic DNA markers in expressed sequences
To estimate the rate of sequence variation in expressed genes across genes and strains, partial sequences from 9 aging-related genes were obtained by PCR. These sequences covered a total of 5378 bp. The same sequences were amplified from N. furzeri GRZ, N. furzeri MZM-3 and N. kunthae. The number and position of the SNPs are reported in Table III.
Table III: Frequency and position of single nucleotide sequence differences in a set of selected genes between the GRZ strain of Nothobranchius furzeri, the MZM-3 strain of Nothobranchhis furzeri and Nothobranchius kunthae. The first column reports the gene. The second column the primers used for amplification (sequences are reported in Materials and Methods). The third column the size of the amplifies sequence. The fourth column the number of SNPs in the comparision between GRZ and MZM-3. The fifth column the position of the SNPs. The sixth column the number of SNPs in the comparison between GRZ andN. kunthae. The seventh column the position of the SNPs.
The author found that 4 out of 9 of MZM sequences presented at least one nucleotide difference with an average difference of one nucleotide every 896 when compared to GRZ sequences. In addition, all N. kunthae sequence revealed at least one nucleotide difference with an average difference of one nucleotide every 267 also when compared to GRZ sequences.
Based on the present results, it is demonstrated that expressed genes are a source of SNPs which can distinguish the strains of speciesto be used for QTL analysis. The present results were obtained studying an example of 10 genes. The same procedure can be applied to a larger number of expressed genes to derive a larger number of markers needed for QTL analysis. b) Homology with genomic sequences from Japanese Medaka
Construction of a linkage map can be greatly expedited by exploiting synteny with closely- related and already assembled genomes (Khorasani et al., 2004). The author therefore tested whether homologues of the sequences of the genes studied as presented in Table I could be retrieved from the Medaka genome
(http://dolphin.lab.nig.ac.jp/seqcenter/srch db/search medaka blastdb.php). All sequences showed a clear match and their position in the Medaka physical map was indicated. The sequence identity between N. furzeri GRZ and Medaka for all the genes is reported in Table IV.
Figure imgf000025_0001
Table IV: Percentage of homology of partial sequences of Nothobranchius furzeri GRZ genes with sequences retrieved from the genome of the Japanese medaka (Ori∑yas latipes).
Average sequence identity is 87%.
These results demonstrate that the homologous of N. furzeri expressed sequences can be easily located on the physical map of the Medaka genome.
CONCLUSIONS
The existence of populations of N. furzeri with different life expectancies provides the possibility of isolating genes that control ageing in vertebrates. 2
BIBLIOGRAPHY
Camp NJ, Cox A. 2002. Quantitative Trait Loci: Methods and Protocols. Totowa, NJ:
Humana Press. Chaves LD, Rowe JA, Reed KM. 2005. Genome 48:12-17. Cnaani A et al., 2004. MoI Genet Genomics 272:162-172. Colosimo PF et al., 2005. Science 307:1928-1933. Fahrenkrug SC et al., 2002. Anim Genet 33:186-195. Geesaman BJ et al., 2003. Proc Natl Acad Sci U S A 100:14115-14120. Geiger-Thornsberry GL, Mackay TF. 2004. Mech Ageing Dev 125:179-189. Grivet L et al., 2003. Theor Appl Genet 106:190-197. Harman D. 2001. Ann N Y Acad Sci 928:1-21.
Hazzard WR et al., 2003. Principles of Geriatric Medicine and Gerontology. New York,
USA: McGraw-Hill Professional. He C, Chen L, Simmons M, Li P, Kim S, Liu ZJ. 2003. Anim Genet 34:445-448. Jubb RA. 1971. Journal of the American Killifish Association 8:12-19. Khorasani MZ et al., 2004. Mech Dev 121:903-913. Kimmel CB et al., 2005. Proc Natl Acad Sci U S A 102:5791-5796. Kirkwood TB, Holliday R. 1979. Proc R Soc Lond B Biol Sci 205:531-546.
Liu B-H. 1997. Statistical Genomics: Linkage, Mapping, and QTL Analysis. CRC Press. New York, USA.
Liu Z, Karsi A, Li P, Cao D, Dunham R. 2003. Genetics 165:687-694.
Liu Z, Nichols A, Li P, Dunham RA. 1998. MoI Gen Genet 258:260-268.
Luckinbill LS, Golenberg EM. 2002. Genetica 114:147-156.
Nonneman DJ, Rohrer GA. 2003. Anim Genet 34:42-46. O'Malley KG, Sakamoto T, Danzmann RG, Ferguson MM. 2003. J Hered 94:273-284.
Pasyukova EG, Rosliina NV, Mackay TF. 2004. Ageing Cell 3:297-307.
Peichel CL, et al., 2001. Nature 414:901-905.
Perry GM, Danzmann RG, Ferguson MM, Gibson JP. 2001. Heredity 86:333-341.
Picoult-Newberg L et al., 1999. Genome Res 9:167-174. Puca AA, Daly MJ et al., 2001. Proc Natl Acad Sci U S A 98:10505-10508.
Robison BD, Wheeler PA, Sundin K, Sikka P, Thorgaard GH. 2001. J Hered 92:16-22.
Sambroock J, et al. 2001. Molecular Cloning: A Laboratory Manual (3-Volume Set). Cold
Spring Harbor, USA: Cold Spring Harbor Laboratory Press. Serapion J, Kucuktas H, Feng J, Liu Z. 2004. Mar Biotechnol (NY) 6:364-377. Shapiro MD, et al., 2004. Nature 428:717-723. Smith EJ et al., 2001. J Hered 92:1-8. Somorjai IM, Danzmann RG, Ferguson MM. 2003. Genetics 165:1443-1456. Steams SC, Ackermann M, Doebeli M. 1998. Exp Gerontol 33:785-792. Valdesalici, S. & Cellerino, A. 2003 Proc R Soc Lond B Biol Sci 270 Suppl 2, S 189-91. Vignal A, Milan D, SanCristobal M, Eggen A. 2002. Genet SeI Evol 34:275-305. Zimmerman AM et al., 2004. Immunogenetics 55:825-835.

Claims

1. A method for identifying and/or characterising genes or products thereof able to modulate longevity and/or expression of at least one age-related parameter in vertebrates, comprising the steps of: a) selecting at least two different populations or strains of a species of the family of Cyprinodontid (common name killifish) which show differences in longevity and/or in the expression of at least one age-related parameter; b) characterizing at least two polymorphic DNA markers which can distinguish said populations or strains; c) cross breeding two individuals of each of one of said different populations or strains, obtaining a progeny Fl ; d) cross breeding the progeny Fl, obtaining a progeny F2; e) analysing the phenotype related to longevity and/or to expression of at least one age- related parameter in each individual of the progeny F2; f) identifying a pair of polymorphic DNA markers in the progeny F2 that co-segregate with each phenotype; g) identifying a genomic region whose boundaries are defined by said pair of polymorphic
DNA markers; h) identifying in said genomic region a gene capable of modulating longevity and/or at least one age-related parameter in said killifish species.
2. The method according to claim 1 wherein the populations or strains belong to the species Nothobranchius furzeri, Nothobranchins rachovii or Nothobranchiiis eggersi
3. The method according to claim 1 wherein the populations or strains are hybrids of Nothobranchius furzeri with Nothobranchius kunthae, or any other species or hybrids of Nothobranchius for which at least two populations of different longevities exist.
4. The method according to claim 2 wherein the populations or strains with a lower longevity have a longevity of 10 to 14 weeks and the populations or strains with a longer longevity have a longevity of 26 to 40 weeks.
5. The method according to claim 4, wherein the populations or strains belonging the species Nothobranchius furzeri having a lower longevity are obtainable in the National
Park of Gona Re Zhou, Zimbabwe at the co-ordinates: 21 40.2 S, 31 2.4 E during the months from January to March.
6. The method according to claim 3 wherein the populations or strains belonging to the species Nothobranchius rachovii having a lower longevity are obtainable in the Limpopo plain, Mozambique, at the co-ordinates: 23 88.52S5 32 36.01 E during the months from March to April.
7. The method according to claim 1 wherein the polymorphic DNA markers are a single nucleotide polymorphisms (SNPs).
8. The method according to claim 1, wherein the age-related parameter belongs to the following group: accumulation of lipofuscin in the brain and/or the liver, expression of B- galactosidase associated to senescence, reduction in spontaneous locomotory activity or cognitive decay.
9. A method for identifying and/or characterising genes or products thereof able to modulate longevity and/or expression of at least one age-related parameter in vertebrates, comprising the steps of: a) cloning a killifish gene capable of modulating longevity and/or expression of at least one age-related parameter identified with the method of claim 1 ; b) isolating by hybridization and/or cloning techniques genes in at least one other vertebrate species.
10. The method for identifying and/or characterising genes or products thereof able to modulate longevity and/or expression of at least one age-related parameter according to claim 9 wherein the other vertebrate species is a fish species.
11. The method for identifying and/or characterising genes or products thereof able to modulate longevity and/or expression of at least one age-related parameter according to claim 9 wherein the other vertebrate species is a mammalian species.
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