The Journal of Heredity 2001:92(3)
© 2001 The American Genetic Association 92:287-290
Brief Communication |
A Cryptic RRY(i) Microsatellite From Atlantic Salmon (Salmo salar): Characterization and Chromosomal Location
From the Departmento de Biologia Funcional, Facultad Medicina, Universidad de Oviedo, Julian Claveria s/n, 33006 Oviedo, Spain.
Address correspondence to Jose Luis Martinez at the address above or e-mail: jlmf{at}correo.uniovi.es
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Abstract |
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In this article we describe the isolation and characterization of a cryptic RRY(i) microsatellite from an Atlantic salmon genomic cosmid library. The chromosomal location of the microsatellite-containing cosmid was performed by fluorescent in situ hybridization (FISH) showing a single-locus signal located on an interstitial position of an acrocentric pair. The suitability of this type of microsatellite marker for population genetic analysis and for the development of a genetic map in this species is discussed. In addition, the usefulness of cosmid libraries for physical mapping of microsatellite markers and therefore for the integration of physical and genetic maps is pointed out.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Most eukaryotic genomes contain a considerable number of repetitive noncoding sequences that exist as both dispersed copies and tandem arrays. Microsatellites (tandemly repeated motifs of 1–5 bp) belong to this second category.
Microsatellite loci can be defined by their specific flanking sequences showing a high degree of length polymorphism (Weber 1990), which can be analyzed by the polymerase chain reaction (PCR) followed by sizing on polyacrylamide gels (Weber and May 1989). This polymorphism, joined with their even and apparently random distribution in the genome, makes microsatellite loci very useful as markers for genetic mapping and identity control and they have been used for the development of high-resolution genetic maps of species such as human and mouse (Chapman and Nadeau 1992; Weissenbach et al. 1992). Low-resolution genetic marker maps, also based on microsatellite markers, are being developed in a wide variety of commercially important species, such as pig, chicken, cattle, rainbow trout, tilapia, and flat oyster (Buchanan et al. 1993; Kocher et al. 1998; Moran 1993; Naciri et al. 1995; Rohrer et al. 1994; Young et al. 1998).
Genetic linkage maps are complemented with physical mapping, which enables the assignment of linkage groups to specific chromosomes (Ellegren et al. 1994; Toldo et al. 1993). The development of fluorescent in situ hybridization (FISH) using microsatellite-containing cosmids as probes has been an important advance and has been used in different map projects (Dickens et al. 1999; Fischer et al. 1996; Toldo et al. 1993). This method is of particular importance in species, like fishes, whose karyotypes are not standardized, since FISH can simultaneously allow chromosome identification and genetic data integration.
In this work we describe the isolation and characterization of a single trinucleotide locus microsatellite from an Atlantic salmon (Salmo salar) cosmid library. We also report the chromosomal location of the microsatellite-containing cosmid clone on the Atlantic salmon chromosome complement.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Isolation and Characterization of the Locus Microsatellite
A cosmid genomic library has been constructed in superCosI according to manufacturer's instructions (Stratagene, La Jolla, CA). A (GAC)6 oligonucleotide was kinased with (
32P). Positive clones were isolated and DNA extracted by the standard alkali lysis. Cosmid DNA was digested with several restriction enzymes and analyzed by Southern blotting. Positive restriction fragments smaller than 1.3 kb were subcloned into pUC and sequenced with the Sequenase 2.0 sequencing kit (Amersham, Sweden). Clone SS10 was chosen for microsatellite analysis after being mapped by FISH. Two primers flanking the trinucleotide repeat were designed for PCR amplification of this microsatellite (submitted to the EMBL, accession number AJ012206).
FISH
Chromosome obtention. Metaphase chromosomes were obtained from lymphocyte cultures. Two to 3 ml of venous blood was extracted from the dorsal vein of several Atlantic salmon adults and stored in heparinized tubes. Lymphocytes were purified and cultured according to standard procedures. Cultures were incubated at 19°C for 5 days. Six hours before harvesting, colchicine was added to a final concentration of 0.01 µg/ml. Cells were treated with 0.5% KCl and fixed in methanol:acetic acid (3:1). Slides were prepared according to standard procedures.
Probes
The chromosomal location of locus SS10, characterized in this study, was established using as probe the whole microsatellite-containing cosmid clone labeled with biotin 16-dUTP by nick translation according to the manufacturer's recommendations (Roche Diagnostics).
Chromosome slides were pretreated with RNase and pepsin as described by Wiegant et al. (1991). Repetitive sequences were suppressed by prehybridizing 100 ng of the labeled probe with 100 µg of sonicated salmon testes DNA. After overnight hybridization at 37°C, the slides were washed for 10 min at 42°C in 50% formamide 2x SSC and then washed twice for 5 min in 0.1x SSC at 50°C. Detection of signals was performed according to Pendás et al. (1993). Images were obtained using a Zeiss axioscope epifluorescent microscope equipped with a CCD camera (Photometrics).
Microsatellite Analyses
PCR amplifications were carried out using the GeneAmp PCR System 2400 from Perkin-Elmer Cetus, with samples containing approximately 50 ng Atlantic salmon DNA, 10 mM Tris-HCl pH 8.8, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 20 pmol of each primer, 1 U Dynazyme II DNA polymerase (Finnzymes Oy), and 250 µM dNTP in a final volume of 20 µl. PCRs were performed with an initial denaturing step (5 min at 95°C) followed by 35 cycles consisting of denaturation at 95°C for 20 s, annealing at 57°C for 20 s, and extension at 72°C for 20 s. The final extension was at 72°C for 5 min. PCR products were run on 5.6% acrylamide, 5.6 M urea denaturing gels, and detected by silver staining using the DNA Silver Staining System (Promega). The sizes of allele products were estimated by comparison with pUC sequence reactions.
Mendelian inheritance of this microsatellite locus was tested in two half-sib families. To obtain a first estimate of its variability, 30 wild adults caught in the Esva River (Spain) were analyzed.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The detailed sequence analysis of the positive clone SS10 showed that the repeat motif present in this clone was composed of two different interspersed trinucleotide sequences (AAC and GAC), the longest single triple array being seven GAC repeats. According to Jacobson et al. (1993), this sequence can be considered a "cryptic repeat" because the nature of the long tandem repeat is only appreciated when the sequence is categorized into purines and pyrimidines. When this transformation was done to our sequence, we obtained a cryptic repeat consisting of 82 triplets (cRRY(82)). In human, mouse, and yeast sequences, cryptic RRY are abundant and, like simple RRY, are nonrandomly distributed with respect to both sequence and location, being the trinucleotides GGC or AGC predominant within human cRRY(i) (Gostout et al. 1993) and preferentially located in coding and 5' un[chtrans[chlated regions (Ricke et al. 1995). Whether these cryptically simple regions within genes are important for the function of the gene product or represent relatively weakly selected parts of the gene remains unclear.
When the two half-sib Atlantic salmon were analyzed using this cRRY microsatellite, we observed a perfect codominant single-locus Mendelian inheritance (Figure 1). In the sample from the Esva River, seven alleles were detected, ranging from 380 to 456 bp in length. The 434 bp allele showed the highest frequency (0.55), far from the others which showed frequencies of 0.21 (425 bp allele), 0.1 (380 bp allele), 0.06 (413 bp allele), 0.03 (391 and 456 bp alleles), and 0.02 (409 bp allele). The heterozygosity observed for this population was 0.63. In all cases only one or two alleles per individual were observed.
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Most of the Atlantic salmon microsatellites characterized in other works are comprised of two base pair repeat units, usually (GT)n or (GA)n motives. A disadvantage of dinucleotide repeat polymorphisms is that in acrylamide gels, each allele is revealed as several shadow bands that sometimes obscure the position of other allelic fragments, which makes genotyping difficult or impossible: for example, it is difficult to differentiate heterozygotes from homozygotes for alleles differing in length by only two nucleotides.
In this work, and despite the size of the repeat segment in this polymorphic locus (longer than 380 bp), all the alleles at the cRRY microsatellite locus could be identified unambiguously and no stutter bands were observed (Figure 1). This could be due to the complexity of the sequence, which can prevent the substantial polymerase stuttering that is commonly seen when more monotonous tandem repeats [e.g., (GT)n] are amplified by PCR (Gostout et al. 1993). Similar results have been observed by other authors in different species (Edwards et al. 1992; Francisco et al. 1996; Naish 1998; O'Reilly et al. 1996). The unambiguous allele sizing of trinucleotide and tetranucleotide core motives in comparison with dinucleotide core sequences leads us to consider these types of microsatellite loci to be more suitable genetic markers for population analyses.
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As expected from the Mendelian monogenic inheritance detected in the two studied families, the FISH of the whole microsatellite-containing cosmid clone shows a single-locus signal in most of the cells analyzed. The signals were located on an interstitial position of an acrocentric chromosome pair (Figure 2). Accordingly we believe that this microsatellite can be used for anchoring the developing genetic and physical map in Atlantic salmon. As previously reported by Lundin et al. (1999) and Martinez et al. (1999), the use of cosmid libraries in Atlantic salmon for isolation and characterization of molecular markers allows the integration of physical and genetic maps and also the identification of the different chromosome pairs.
Looking at the results obtained in this work, we conclude that the isolation of microsatellite markers from cosmid clones is a useful tool for the development of the genetic and physical map in species like fish, with poorly standardized karyotypes.
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Acknowledgments |
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We are grateful to Dr. A. M. Pendas for technical assistance and to Dr. J. B. Taggart for the half-sib families. Jose Luis Martinez received a fellowship from the Regional Government (Consejeria de Agricultura) of the Principado de Asturias. This work was supported by the Spanish DGICYT (PB98-1570).
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Footnotes |
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Corresponding Editor: Bernie May
Received January 18, 2000
Accepted January 15, 2001
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References |
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