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Genetic characterization of insulin-like growth factor-I
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(IGF-I) in Mekong giant catfish (Pagasianodon gigas) and
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striped catfish (Pagasianodon hypophthalmus)
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IGF-I in Mekong giant catfish and striped catfish
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Nantaporn Sutthi1, Doungporn Amornlerdpison1, Supamit
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Mekchay2, Nares Trakooljul3, Siriluck Ponsuksili3, Klaus
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Wimmers3 and Kriangsak Mengumphan1*
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Faculty of Fisheries Technology and Aquatic Resources, Maejo University,
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Chiang Mai, 50290, Thailand. Tel. 0-5387-3470 Fax. 0-5349-8178;
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E-mail: kriang1122sak@gmail.com
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Department of Animal and aquatic science, Faculty of Agriculture,
Chiang Mai University, Chiang Mai, 50200, Thailand.
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Leibniz Institute for Farm Animal Biology, Dummerstorf 18196, Germany
* Corresponding author
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Abstract
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Insulin-like growth factor-I (IGF-I) plays an important role in
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development and growth of multiple species including fishes. IGF-I may also
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contribute to high growth traits of an endangered species and the largest
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fresh-water fish, the Mekong giant catfish (Pangasianodon gigas) as well as
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an
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hypophthalmus). Nevertheless, genetic information of IGF-I in these two
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species has not been available. Thus, our study goal was sequence isolation
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and comparative sequencing of IGF-I of the two species to obtain genetic
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information and identify single nucleotide polymorphisms. Degenerate
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primers were designed from a highly conserved region of a closely related
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species, channel catfish (Ictalurus punctatus). A fragment of 146 bp was
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amplified, cloned and sequenced from Mekong giant catfish and striped
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catfish DNA samples. A sequence comparison showed 99% similarity
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between Mekong giant catfish and striped catfish. Single nucleotide
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polymorphism (SNP) was identified g.80C > T in P. gigas, but did not change
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the amino acid prediction (Leucine). 98 % of the sequence was similar to the
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channel catfish reference with two synonymous substitutions, g.94 A > G and
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g.97 A > G. This is the first report on genetics of the P. gigas and P.
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hypophthalmus IGF-I. The results provide essential information for genetic
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study and further understanding functional roles of IGF-I in Mekong giant
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catfish and striped catfish.
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economically
important
species,
striped
catfish
(Pangasianodon
3
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Keywords: insulin-like growth factor-I, Pangasianodon gigas, P.
hypophthalmus, SNP
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Introduction
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Insulin-like growth factor-I (IGF-I) is a peptide hormone structurally
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similar to proinsulin and plays an important role in mammalian growth and
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development (Daughaday et al., 1989). In teleost IGF-I is primarily expressed in
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liver, but it is also produced in variety of tissues (Maures et al., 2002). Moreover,
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IGF-I also has a role in regulating postnatal growth in mammals and fish (Florini
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et al., 1996; Duan, 1997; Reinecke and Collet, 1998; Moriyama et al., 2000). The
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production of IGF-I depends on growth hormone and other endocrine factors,
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such as thyroid hormone (Schmid et al., 2003) and/or estrogen (Riley et al.,
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2004). In several fish species, blood and tissue IGF-I levels positively correlate
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with dietary ration, protein content, and body growth rate (Beckham et al., 2004;
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Duan, 1998; Pérez-Sánchez et al., 1995). The blood IGF-I also increase during the
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growing season (Mingarro et al., 2002) and the level is stimulated by an increase
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temperature (Beckham et al., 1998) and day length (McCormick et al., 2000), and
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has been associated with metabolism (Castillo et al., 2004), development (Greene
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and Chen, 1999; Pozios et al., 2001), reproduction (Maestro et al., 1997), and
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osmoregulation in seawater (McCormick, 2001).
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The highly conserved IGF system is generally composed of the ligands
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IGF-I and IGF-II, their receptors, and IGF binding proteins (Reinecke and collet,
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1998). Mature IGF-I peptides share high levels of amino acid sequence similarity
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between vertebrates (Duan, 1997). Not only sequence similarity of amino acids,
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but also the gene expression and function have been demonstrated to be conserved
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between fishes and mammals (Duan, 1997) possibly via evolutionarily conserved
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polypeptides (Duan, 1997, 1998). Although the nucleotide sequence is known in
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Genbank databases for several fish IGF-I such as Ictalurus punctatus (Clay et al.,
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2005), Oreochromis niloticus (Wang et al., 2008), Danio rerio (See et al., 2014),
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the information is limited for Pangasius catfish species. The Pangasius catfishes
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such as Mekong giant catfish (Pagasianodon gigas) and striped catfish
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(Pagasianodon hypophthalmus) are ecologically and economically important
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freshwater catfish in Thailand. The Mekong giant catfish is the largest freshwater
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fish which could reach a weight up to 300 kilograms (Roberts and Vidthayanon,
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1991; Zanden et al., 2004). Despite their ecological and economic importance,
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genetic information is still limited only P. gigas mitochondrial genome (Jondeung
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et al., 2007) and growth hormone (Lemaire et al., 1994) that are accessible yet.
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IGF-I has gained our attentions since it may functionally contribute to the high
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growth potential and performance of the Mekong giant catfish based on large
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body size and weight. Therefore, in this study we aimed to identify DNA
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sequence and polymorphisms of IGF-I in P. gigas and P. hypophthalmus using
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comparative sequencing approach and degenerate primers designed from the most
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conserved exon 3 and 4 of channel catfish (I. punctatus) (Clay et al., 2005;
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Peterson et al., 2005).
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Materials and Methods
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Fish samples
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A total of 40 individual catfish of P. gigas (n = 20) and P. hypophthalmus
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(n = 20) were obtained from Chiangrai and Chiangmai Inland Fisheries Research
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and Development Center, Department of Fisheries, Thailand.
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DNA Isolation
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Genomic DNA was extracted from fin samples using phenol-chloroform
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extraction procedure slightly modified from the protocol described by Harris et al.
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(1991). The samples were minced and digested at 37 °C overnight in 700L of
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TNES-urea buffer (1mM Tris-HCL pH 8.0, 6 M NaCl, 0.5M EDTA pH 8.0, 10%
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sodium dodecyl sulphate, and 4M urea) containing 4L of proteinase K solution
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(20 g/mL) (Invitrogen, USA). An equal volume of phenol-chloroform (1:1 v/v)
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was then added and mixed until an emulsion formed. Separation of the DNA
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contained aqueous phase was achieved by centrifugation at 10,000 rpm for 10
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min. The phenol-chloroform extraction step was repeated twice. DNA was
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precipitated with 1/10 of the volume of Sodium acetate (3.0 M, pH 5.2) and an
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equal volume of isopropanol, washed twice with 70% ethanol and followed by air
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dry. Finally, the DNA was dissolved in a Tris-EDTA buffer and stored at – 20
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°C. The DNA concentration was determined using a spectrophotometer
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(NanoDrop 2000, Thermo Fisher Scientific Inc.) and assessed for integrity on 1%
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agarose-gel electrophoresis.
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Cloning and Sequencing
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A sequence alignment of IGF-I across multiple fishes showed a highly
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conserved region corresponding to exon 3-4 of the channel catfish as shown
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Figure 1. Degenerate primers were designed from the channel catfish (Ictalurus
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punctatus)
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TTGCACAACCGTGGCATCG-3´
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TTTGGTGTTTTGGGCGTGTC-3´ (reverse primer). Each of eight DNA pools
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was generated from five individual of P. gigas or P. hypophthalmus (total n=40)
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for a comparative sequencing experiment. A PCR fragment was amplified using a
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touchdown PCR condition: 94 C for 3 min, followed by 12x cycles of [94 C for
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30 s; 60 - 52C for 30 s (minus 0.5 C for each cycle); 72C for 1 min] and 20x
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cycles of [94 C for 30 s; 52C for 30 s; 72C for 1 min], and final extension of
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72C for 5 min. The PCR product was purified and ligated into the pGEM®-T
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vector (Promega, USA). The recombinant DNA was transformed into competent
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cells (DH5α strain) and the blue-white screening method was used to identify a
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positive clone harboring the insert DNA fragments according to the
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manufacturer’s protocol. Plasmid DNA was extracted from bacteria culture using
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GenElute™ Plasmid Miniprep Kit (Qiagen) for sequence analysis. Nucleotide
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sequence was determined using CEQ 8000 Genetic Analysis System (Beckman
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Coulter) using Dye Terminator Cycle Sequencing (GenomeLab™ DTCS) Quick
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Start Kit (Beckman Coulter).
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Sequence analysis
sequence
(DQ
088971).
The
(forward
primer
sequences
primer)
and
are
5´5´-
7
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Sequence alignment was performed using basic local alignment search
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tool namely BLAST at the NCBI webpage. The search analysis blast the query
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sequence against all sequences deposited across the database.
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Results and Discussion
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Partial sequence of P. gigas and P. hypophthalmus IGF-I
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The degenerate primer designed from channel catfish IGF-I corresponding
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to exon 3 and 4 were able to amplify a PCR fragment in all Mekong giant catfish
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(P. gigas) and striped catfish (P. hypophthalmus) DNA samples (Figure 2). The
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fragment length was 146 bp which was the same as in channel catfish. An
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alignment of sequences derived from all 8 DNA pools revealed the sequence
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similarity between Mekong giant catfish and striped catfish of 99 % which 145
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out of 146 bp were identical. Comparing between a consensus sequence of
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Mekong giant catfish and striped catfish and the reference of channel catfish IGF-
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I showed 98% similarity (144/146bp) among these three species.
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Single nucleotide polymorphism detection
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A sequence comparison of IGF-I cloned from different DNA pools of P.
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gigas and P. hypophthalmus showed sequence difference at the position g.60C >
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T as shown in Figure 3. Two clones derived from the DNA pool number 2 and 4
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of Mekong giant catfish showed T allele, while the other two clones from DNA
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pool 1 and 3 of the same species revealed C allele indicating a heterozygosity of
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the locus in Mekong giant catfish. Amino acid prediction suggested that the SNP
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is non-sense, both alleles code for Leucine. In striped catfish, all four clones
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sequenced identified only the C allele suggesting a non-polymorphic likelihood of
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the locus in striped catfish. However, genotyping in a large population is
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warranted. In addition, comparing a consensus sequence of Mekong giant catfish
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and Strip catfish IGF-I with that of the channel catfish identified two sequence
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variations at g.75A > G and g.78A > G positions (Figure 3). These two SNPs
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were synonymous substitutions and did not change the amino acid prediction for
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Alanine and Proline.
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In this report, partial sequence of IGF-I of Mekong giant catfish and
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striped catfish was isolated and SNPs within this genomic region were reported.
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The results suggested that this genomic region is highly conserved between
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Mekong giant catfish, striped catfish and channel catfish. A synonymous SNP was
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identified at the position g.60C > T in P. gigas. Although the SNP does not
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change the amino acid sequence and is unlikely to change functions of the protein,
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the SNP may be closely linked to yet uncharacterized causative SNPs. Therefore
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the SNP may be useful as a valuable genetic marker for association study in a
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segregated population. Genomic and genetic information of Pangasiid catfish are
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still very limited. This is also true for IGF-I. A polymorphic CT/GA microsatellite
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close to the end of intron 1 of IGF-I has been previously reported in catfish. It has
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been shown that the catfish IGF-I signal peptide was quite different from other
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fishes, avian, and mammalian species (Chay et al., 2005). Characterization of a
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relevant gene, growth hormone (GH) in striped catfish suggests that the gene is
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highly conserved compared to vertebrates. Moreover, a high degree of similarity
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of the GH amino acid sequence between Mekong giant catfish and channel catfish
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has been demonstrated (Poen, 2009). The GH in striped catfish and Mekong giant
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catfish are closely related based on the similarity of amino acid sequence. A
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substitution of asparagine and serine at the position 163 has been previously
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reported between Mekong giant catfish and striped (Poen, 2009).
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For functional study of the gene, an expression of IGF-I has been reported
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in multiple fish species including embryos and larvae of sea bream (Sparus
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aurata) (Radaelli et al., 2003), rainbow trout (Perrot and Funkenstein, 1999), and
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common carp (Cyprinus carpio) (Tse et al., 2002). Interestingly, the IGF-I
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transcript has been detected as early as in unfertilized eggs in development stage
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of seabream (Fukenstein et al., 1996) and rainbow trout (Greene and Chen, 1997).
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However, it is only detected after hatching and during embryogenesis in rabbitfish
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(Siganus guttatus) (Ayson et al., 2002). A study of the IGF-I gene in Chilean
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flounder (Paralichthys adspersus) suggests a high degree of conservation of the
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protein family during vertebrate evolution (Elies et al., 1999; Nakao et al., 2002).
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Moreover, IGF-I has been reported to be expressed in liver higher than in muscle,
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brain, heart, and kidney (Chay et al., 2005).
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Conclusions
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Using degenerate primers designed from highly conserved region of Ictalurus
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punctatus IGF-I, partial genomic DNA sequence (146 bp) of the P. gigas and P.
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hypophthalmus IGF-I were cloned and sequenced. A comparative sequencing
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identified a SNPs at g.60C > T position between Mekong giant catfish and striped
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catfish. Comparing the isolated sequence and the channel catfish reference
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showed two synonymous substitution, g.75A > G and g.78A > G suggesting a
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high conservation, at least at this genomic location between these species. The
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identified SNPs may be used as a genetic marker for a trait-association study in
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segregating populations.
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Acknowledgements
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We are grateful to Thailand Research Fund (TRF) for financial support through
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the Royal Golden Jubilee Ph.D. Program (PHD/ 0149/ 2554). We are grateful to
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the Plabuk Integration Knowledge Base, Faculty of Fisheries Technology and
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Aquatic Resources, Maejo University for providing fish samples. We wish to
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express our gratitude to the Centre of Excellence on Agricultural Biotechnology,
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Science and Technology Postgraduate Education and Research Development
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Office, Commission on Higher Education (AG-BIO/PERDO-CHE), Ministry of
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Education, as well as the Project of the Service and Technology Transfer of
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Agricultural Biotechnology Centre, Faculty of Agriculture, Chiang Mai
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University, for providing research facilities.
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References
218
Ayson, F.G., de Jesus, E.G., Moriyama, S., Hyodo, S., Funkenstein, B., Gertler,
219
A. and Kawauchi, H. (2002). Differential expression of insulin-like growth
11
220
factor I and II mRNAs during embryogenesis and early larval development in
221
rabbitfish, Siganus guttatus. Gen. Comp. Endocrinol., 126(2): 165–174.
222
Beckham, B.R., Larsen, D.A., Moriyama, S., Lee-Pawilak, B. and Dickhoff,
223
W.W. (1998). Insulin-like growth factor-1 and environmental modulation of
224
growth during smoltication of spring chinook salmon (Oncorhynchus
225
tshawytscha). Gen. Comp. Endocrinol., 109: 325–335.
226
Beckham, B.R., Shimizu, M., Gadberry, BA. and Cooper, K.A. (2004). Response
227
of the somatotropic axis of juvenile coho salmon to alterations in plane of
228
nutrition with an analysis of the relationships among growth rate and
229
circulating IGF-I and 41 kDa IGFBP. Gen. Comp. Endocrinol., 135(3): 334–
230
344.
231
Castillo, J., Codina, M., Martí nez, M.L., Navarro, I. and Gutié rrez, J. (2004).
232
Metabolic and mitogenic effects of IGF-I and insulin on muscle cells of
233
rainbow trout. Am. J. Physiol. Regul. Integr. Comp. Physiol., 286(5): 935–
234
941.
235
Clay, L.A., Wang, S.Y., Wolters, W.R., Peterson, B.C. and Waldbieser, G.C.
236
(2005). Molecular characterization of the insulin-like growth factor-I (IGF-I)
237
gene in channel catfish (Ictalurus punctatus). Biochim. Biophys. Acta.,
238
1731(3): 139-148.
239
Daughaday, W.H. and Rotwein, P. (1989). Insulin-like growth factors I and II.
240
Peptide, messenger ribonucleic acid and gene structures, serum, and tissue
241
concentrations. Endoc.r Rev., 10(1): 68–91.
12
242
243
244
245
Duan, C. (1997). The insulin-like growth factor system and its biological actions
in fish. Amer. Zool., 37: 491–503.
Duan, C. (1998). Nutritional and developmental regulation of insulin-like growth
factors in fish. J. Nutr., 128: 306–314.
246
Eliѐs, G., Duval. H., Bonnec, G., Wolff, J., Boeuf, G. and Boujard, D. (1999).
247
Insulin and insulin-like growth factor-1 receptors in an evoluted fish, the
248
turbot: cDNA cloning and mRNA expression. Mol. Cell. Endocrinol., 158:
249
173-185.
250
Florini, J.R., Ewton, D.Z. and Coolican, S.A. 1996. Growth hormone and the
251
insulin-like growth factor system in myogenesis. Endocr. Rev., 17(5): 481–
252
517.
253
Fukenstein, B., Shemer, R., Amuly, R. and Cohen, I. (1996). Nucleotide sequence
254
of the promoter region of Sparus aurata insulin-like growth factor I gene and
255
expression of IGF-I in eggs and embryos. Mol. Mar. Biol. Biotechnol., 5(1):
256
43–51.
257
Greene, M.W. and Chen, T.T., (1997). Temporal expression pattern of insulin-like
258
growth factor mRNA during embryonic development in a teleost, rainbow
259
trout (Onchorynchus mykiss). Mol. Mar. Biol. Biotechnol., 6(2): 144–151.
260
Greene, M.W. and Chen, T.T., (1999). Quantitation of IGF-I, IGF-II, and multiple
261
insulin receptor family member messenger RNAs during embryonic
262
development in rainbow trout. Mol. Reprod. Dev., 54(4): 348–361.
13
263
Harris, A.S., Bieger, S., Doyle, R.W. and Wright, J.M. (1991). DNA
264
fingerprinting of tilapia, Oreochromis niloticus, and its application to
265
aquaculture genetics. Aquaculture, 92: 157-163.
266
Jondeung, A., Sangthong, P. and Zardoya, R. (2007). The complete mitochondrial
267
DNA sequence of the Mekong giant catfish (Pangasianodon gigas), and the
268
phylogenetic relationships among Siluriformes. Gene, 387(1-2): 49–57.
269
Lemaire, C., Warit, S. and Panyim, S., (1994). Giant catfish Pangasianodon gigas
270
growth hormone-encoding cDNA: cloning and sequencing by one-sided
271
polymerase chain reaction. Gene, 149(2): 271-276.
272
Maestro, M.A., Planas, J.V., Moriyama, S., Gutiérrez, J., Planas, J. and Swanson,
273
P. (1997). Ovarian receptors for insulin and insulin-like growth factor I (IGF-
274
I) and effects of IGF-I on steroid production by isolated follicular layers of
275
the preovulatory coho salmon ovarian follicle. Gen. Comp. Endocrinol.,
276
106(2): 189–201.
277
Maures, T., Chan, S.J., Xu, B., Sun, H., Ding, J., and Duan, C. (2002). Structural,
278
biochemical, and expression analysis of two distinct insulin-like growth
279
factor I receptors and their ligands in zebrafish. Endocrinology, 143(5):
280
1858– 1871.
281
McCormick, S.D., Moriyama, S. and BjÖ rnsson, B.T. (2000). Low temperature
282
limits photoperiod control of smolting in atlantic salmon through endocrine
283
mechanisms. Am. J. Physiol. Regul. Integr. Comp. Physiol., 278(5): R1352–
284
1361.
14
285
286
McCormick, S.D. (2001). Endocrine control of osmoregulation in teleost fish.
Amer. Zool., 41(4): 781–794.
287
Mingarro, M., Vega-Rubin de Celis, S., Astola, A., Pendón, C., Valdivia, M.M.
288
and Pérez-Sánchez, J. (2002). Endocrine mediators of seasonal growth in
289
gilthead sea bream (Sparus aurata): the growth hormone and somatolactin
290
paradigm. Gen. Comp. Endocrinol., 128(2): 102–111.
291
Moriyama, S., Ayson, F.G. and Kawauchi, H. (2000). Growth regulation by
292
insulin-like growth factor-I in fish. Biosci. Biotechnol. Biochem., 64(8):
293
1553– 1562.
294
Nakao, N., Tanaka, M., Higashimoto, Y. and Nakashima, K. (2002). Molecular
295
cloning, identification and characterization of four distinct receptor subtypes
296
for insulin and IGF-I in Japanese flounder, Paralichthys olivaceus. J.
297
Endocrinol., 173(2): 365-375.
298
Perrot, V. and Funkenstein, B. (1999). Cellular distribution of insulin-like growth
299
factor II (IGF-II) mRNA and hormone regulation of IGF-I and IGF-II mRNA
300
expression in rainbow trout testis (Oncorhynchus mykiss). Fish. Physiol.
301
Biochem., 20: 219– 229.
302
Pérez-Sá nchez, J., Marti-Palanca, H. and Kaushik, S.J. (1995). Ration size and
303
protein intake affect circulating growth hormone concentration, hepatic
304
growth
305
immunoreactivity in a marine teleost, the gilthead sea bream (Sparus aurata).
306
J. Nutr., 125(3): 546–552.
hormone
binding
and
plasma
insulin-like
growth
factor-I
15
307
Peterson, B.C., Bosworth, B.G. and Bilodeau, A.L. (2005). Differential gene
308
expression of IGF-I, IGF-II, and toll-like receptors 3 and 5 during
309
embryogenesis in hybrid (channel x blue) and channel catfish. Comp.
310
Biochem. Physiol. A Mol. Integr. Physiol. 141(1): 42-47.
311
Poen, S. (2009). Cloning, over-expression and characterization of growth
312
hormone from striped catfish (Pangasianodon hypopthalmus), [MSc. thesis].
313
Submitted to Graduate School, Kasetsart University. Bangkok, Thailand,
314
204p.
315
Pozios, K.C., Ding, J., Degger, B., Upton, Z. and Duan, C. 2001. IGFs stimulate
316
zebrafish cell proliferation by activating MAP kinase and PI3-kinase-
317
signaling pathways. Am. J. Physiol. Regul. Integr. Comp. Physiol., 280(4):
318
1230–1239.
319
Radaelli, G., Domeneghini, C., Arrighi, S., Bosi, G., Patruno, M. and Funkenstein,
320
B. (2003). Localization of IGF-I, IG-I receptor, and IGFBP-2 in developing
321
Umbrina cirrosa (Pisces: Osteichthyes). Gen. Comp. Endocrinol., 130(3):
322
232–244.
323
324
Reinecke, M. and Collet, C. (1998). The phylogeny of the insulin-like growth
factors. Int. Rev. Cytol., 183: 1 –94.
325
Riley, L.G., Hirano, T. and Grau, E.G. (2004). Estradiol-17beta and
326
dihydrotestosterone differentially regulate vitellogenin and insulin-like
327
growth factor-I production in primary hepatocytes of the tilapia Oreochromis
328
mossambicus. Comp. Biochem. Physiol. C Toxicol. Pharmacol., 138(2): 177–
329
186.
16
330
Roberts, T.R. and Vidthayanon, C. (1991). Systematic revision of the Asian
331
catfish family Pangasiidae, with biological observations and descriptions of
332
three new species. Proc. Acad. Nat. Sci. Philadelphia., 143: 97-144.
333
Schmid, A.C., Lutz, I., Kloas, W. and Reinecke, M. (2003). Thyroid hormone
334
stimulates hepatic IGF-I mRNA expression in a bony fish, the tilapia
335
Oreochromis mossambicus, in vitro and in vivo. Gen. Comp. Endocrinol.,
336
130(2): 129–134.
337
See, K., Yadav, P., Giegerich, M., Cheong, P.S., Graf, M., Vyas, H., Lee, S.G.,
338
Mathavan, S., Fischer, U., Sendtner, M. and Winkler, C. (2014). SMN
339
deficiency alters Nrxn2 expression and splicing in zebrafish and mouse
340
models of spinal muscular atrophy. Hum. Mol. Genet., 23(7): 1754-1770.
341
Tse, M.C., Vong, Q.P., Cheng, C.H., Chan, K.M. (2002). PCR-cloning and gene
342
expression studies in common carp (Cyprinus carpio) insulin-like growth
343
factor-II. Biochim. Biophys. Acta., 1575: 63–74.
344
Wang, D.S., Jiao, B., Hu, C., Huang, X., Liu, Z. and Cheng, C.H. (2008).
345
Discovery of a gonad-specific IGF subtype in teleost. Biochem. Biophys.
346
Res. Commun., 367(2): 336-341.
347
348
349
350
351
Zanden, M.J.V., Hogan, Z., Moyle, P., May, B., and Baird, I. (2004). The
imperiled giants of the Mekong. Am. Sci., 92: 228–237.
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Figure 1. conserved region (red color) of IGF-I among closely related fish
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species; Ictalurus punctatus (DQ 088971), Cyprinus carpio
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(D83271), Danio rerio (BC114262), Ctenopharyngodon idella
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(EU051323), Carassius auratus (GU583648), Elopichthys bambusa
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(JF712623), Procypris rabaudi (EU787399) and Spinibarbus sinensis
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(DQ449024).
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Figure 2. PCR amplification of IGF-I in pooled DNA of P. gigas lane 1-4)
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and P. hypophthalmus (lane 5-8). Lane M represents for 100 bp
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markers and lane C for water negative control.
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Figure 3. Sequence conservation and single nucleotide variation of a 146 bp
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fragment of IGF-I. Pooled DNA sequencing in Pagasianodon gigas
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and P. hypophthalmus showed the g.60C > T SNP. Comparing with
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the channel catfish reference identified two SNPs, g.75A > G and
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g.78A > G.
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