- Short Report
- Open Access
Integrating the markers Pan I and haemoglobin with the genetic linkage map of Atlantic cod (Gadus morhua)
© Bowman et al; licensee BioMed Central Ltd. 2010
- Received: 21 June 2010
- Accepted: 15 October 2010
- Published: 15 October 2010
Haemoglobin (Hb) and pantophysin (Pan I) markers have been used intensively in population studies of Atlantic cod (Gadus morhua) and in the analysis of traits such as temperature tolerance, growth characteristics and sexual maturation. We used an Illumina GoldenGate panel and the KASPar SNP genotyping system to analyse SNPs in three Atlantic cod families, one of which was polymorphic at the Hb β1 locus, and to generate a genetic linkage map integrating Pan I and multiple Hb loci.
Data generated allowed the mapping of nine Hb loci, the Pan I locus, and other 122 SNPs onto an existing linkage genetic map for Atlantic cod. Four Hb genes (i.e. α1, α4, β1 and β5) have been mapped on linkage group (LG) 2 while the other five (i.e. α2, α3, β2, β3 and β4) were placed on LG18. Pan I was mapped on LG 1 using a newly developed KASPar assay for a SNP variable only in Pan IA allelic variants. The new linkage genetic map presented here comprises 1046 SNPs distributed between 23 linkage groups, with a length of 1145.6 cM. A map produced by forcing additional loci, resulting in a reduced goodness-of-fit for mapped markers, allowed the mapping of a total of 1300 SNPs. Finally, we compared our genetic linkage map data with the genetic linkage map data produced by a different group and identified 29 shared SNPs distributed on 10 different linkage groups.
The genetic linkage map presented here incorporates the marker Pan I, together with multiple Hb loci, and integrates genetic linkage data produced by two different research groups. This represents a useful resource to further explore if Pan I and Hbs or other genes underlie quantitative trait loci (QTL) for temperature sensitivity/tolerance or other phenotypes.
- Quantitative Trait Locus
- Linkage Group
- KASPar Assay
- Shared SNPs
- Genetic Linkage Data
The Atlantic cod (Gadus morhua) represents one of the most valuable commercial resources for international fisheries . Haemoglobin (Hb), pantophysin (Pan I), and microsatellite markers have been widely used to characterize the genetic diversity of Atlantic cod populations and their dispersal characteristics [2, 3]. In addition, Hb analyses have revealed correlations between allele types and traits such as growth [2, 4] , water temperature preference , age and seasonality of sexual maturation  or annual mortality . Similarly, growth rate , water temperature, salinity and depth appear to have an effect on Pan I allele frequencies [2, 9, 10]. Recently, a large number of single nucleotide polymorphisms (SNPs) have been identified [11–13] and two independent genetic linkage maps have been generated [12, 14]. However, although SNPs have been characterized in both Hb [15, 16] and Pan I [9, 17, 18], the position of these markers on a linkage map has yet to be determined. To overcome this deficit we used a combination of methods, i.e. a Illumina GoldenGate panel (Illumina Inc.) comprising 1536 SNPs and the KASPar SNP genotyping system (KBioscience, UK) to place these genes onto a genetic linkage map.
Mapping the haemoglobin genes
Mapping Pan I
The two main allele variants Pan IA and Pan IB described at the Pan locus can be determined by assessing the polymorphism present at a DraI site located in intron 4 [9, 17, 27]. However, these alleles have very different frequencies within different Atlantic cod stocks, and all candidate families from the CGP  have been determined to be Pan IA homozygotes (Additional file 2). To overcome this problem a new assay was developed using the KASPar SNP genotyping system and the non-synonymous substitution (Glu/Lys) present in exon 4 (G767A), which is variable only in Pan IA allelic variants . Family B87, one of the two families used in the generation of the initial map  was determined to be polymorphic for Pan IA G767A and the Pan I locus was mapped on LG1 (Figure 1; Additional files 1 and 2) using that family.
The addition of family B30 allowed new SNPs to be mapped onto the CGP map
The new map, resulted from the addition of family B30, and of Pan I and Hb loci, contains 1046 mapped SNPs, an increase of 122 SNPs when compared to the genetic linkage map reported earlier by Hubert et al. 2010 . This map was generated by the first round of calculations of the regression mapping algorithm and the positions of all loci were statistically strongly supported . Another map, produced by the third round of mapping calculations  integrates 1300 SNPs, which represents a contribution of 376 new SNPs that could be mapped (Additional file 3). This map was generated to obtain a general idea of where poorer fitting loci reside on the genetic linkage map .
Linkage group correspondence between maps produced by independent research groups
Finally, we aimed to merge the CGP genetic linkage map data (this paper and Hubert et al. ) with the genetic linkage map data produced by a different group . To identify sequences that were mapped by both, CGP (this paper and Hubert et al. ) and Moen et al.  we performed a Blastn search of an in-house made database containing the ESTs listed by Moen et al.  using 124 nucleotides long CGP SNP-containing sequences. EST sequence data used by Moen et al. was retrieved from GenBank using the GenBank accession numbers listed by this group . Using this approach we identified 29 shared SNPs distributed on 10 different linkage groups (Additional file 4). The minimal overlap between the linkage map produced in the present study and that of Moen et al.  might result from ascertainment bias caused by different frequencies of SNPs in NE and NW Atlantic populations of cod and/or because CGP sequenced the 3' ends of ESTs to identify SNPs [11, 12] whereas Moen et al. [13, 14] sequenced the 5' ends of their ESTs to detect polymorphisms.
The genetic linkage map presented here, that includes the marker PanI and multiple Hb loci, represents a useful resource for studying genotype-phenotype relationships, for QTL studies, as well as for population studies. Our data indicate that Hb genes are located on two different linkage groups while Pan I locus was mapped on a third linkage group. Further studies are needed to elucidate which of these genes/linkage groups will correlate with phenotypic traits. The Hb β1 gene, which has been linked to variation in haemoglobin oxygen binding capacity and water temperature preference [15, 28] (although the role of this gene in temperature adaptation is still subject to debate ) was mapped on LG 2. New avenues for physiological and biochemical studies related to temperature and hypoxia tolerance in Atlantic cod and other fishes may result if, through further studies, it can be demonstrated that this gene is associated with relevant QTL.
This study was supported by Genome Canada, Genome Atlantic, and the Atlantic Canada Opportunities Agency through the Atlantic Cod Genomics and Broodstock Development Project and through an NSERC Discovery grant to SB. A complete list of supporting partners can be found at http://www.codgene.ca/partners.php.
- Rosenlund G, Skretting M: Worldwide status and perspective on gadoid culture. ICES J Mar Sci. 2006, 63 (2): 194-197. 10.1016/j.icesjms.2005.11.012.View ArticleGoogle Scholar
- Imsland AK, Jonsdottir ODB: Linking population genetics and growth properties of Atlantic cod. Reviews in Fish Biology and Fisheries. 2003, 13: 1-26. 10.1023/A:1026373509576.View ArticleGoogle Scholar
- Sarvas TH, Fevolden SE: Pantophysin (Pan I) locus divergence between inshore v. offshore and northern v. southern populations of Atlantic cod in the north-east Atlantic. Journal of Fish Biology. 2005, 67: 444-469. 10.1111/j.0022-1112.2005.00738.x.View ArticleGoogle Scholar
- Imsland AK, Foss A, Naevdal G, Johansen T, Folkvord A, Stefansson SO, Jonassen TM: Variations in growth in haemoglobin genotypes of Atlantic cod. Fish Physiol Biochem. 2004, 30 (1): 47-55. 10.1007/s10695-004-6787-5.View ArticleGoogle Scholar
- Petersen MF, Steffensen JF: Preferred temperature of juvenile Atlantic cod Gadus morhua with different haemoglobin genotypes at normoxia and moderate hypoxia. J Exp Biol. 2003, 206 (Pt 2): 359-364. 10.1242/jeb.00111.PubMedView ArticleGoogle Scholar
- Mork J, Giskeødegård R, Sundnes G: Haemoglobin polymorphism in Gadus morhua: genotypic differences in maturing age and within-season gonad maturation. Helgolander Meeresunters. 1983, 36: 313-322. 10.1007/BF01983634.View ArticleGoogle Scholar
- Mork J, Giskeødegård R, Sundnes G: Population genetic studies in cod (Gadus morhua L.) by means of the haemoglobin polymorphism; observations in a Norwegian coastal population. Fiskeridir Skr (Havundersøkelser). 1984, 17: 449-471.Google Scholar
- Jónsdóttir ÓDB, Imsland AK, Daníelsdóttir AK, Marteinsdóttir G: Genetic heterogeneity and growth properties of different genotypes of Atlantic cod (Gadus morhua L.) at two spawning sites off south Iceland. Fish Res. 2002, 55: 37-47. 10.1016/S0165-7836(01)00296-X.View ArticleGoogle Scholar
- Case RAJ, Hutchinson WF, Hauser L, Van Oosterhout C, Carvalho GR: Macro- and micro-geographic variation in pantophysin (PanI) allele frequencies in NE Atlantic cod Gadus morhua. Marine Ecology Progress Series. 2005, 267-278. 10.3354/meps301267. 301Google Scholar
- Pampoulie C, Jakobsdottir KB, Marteinsdottir G, Thorsteinsson V: Are vertical behaviour patterns related to the pantophysin locus in the Atlantic cod (Gadus morhua L.)?. Behav Genet. 2008, 38 (1): 76-81. 10.1007/s10519-007-9175-y.PubMedView ArticleGoogle Scholar
- Bowman S, Hubert S, Higgins B, Stone C, Kimball J, Borza T, Bussey JT, Simpson G, Kozera C, Curtis BA: An integrated approach to gene discovery and marker development in Atlantic cod (Gadus morhua). Mar Biotechnol (NY). 2010Google Scholar
- Hubert S, Higgins B, Borza T, Bowman S: Development of a SNP resource and a genetic linkage map for Atlantic cod (Gadus morhua). BMC Genomics. 2010, 11: 191-10.1186/1471-2164-11-191.PubMed CentralPubMedView ArticleGoogle Scholar
- Moen T, Hayes B, Nilsen F, Delghandi M, Fjalestad KT, Fevolden SE, Berg PR, Lien S: Identification and characterisation of novel SNP markers in Atlantic cod: evidence for directional selection. BMC Genet. 2008, 9: 18-10.1186/1471-2156-9-18.PubMed CentralPubMedView ArticleGoogle Scholar
- Moen T, Delghandi M, Wesmajervi MS, Westgaard JI, Fjalestad KT: A SNP/microsatellite genetic linkage map of the Atlantic cod (Gadus morhua). Anim Genet. 2009, 40 (6): 993-996. 10.1111/j.1365-2052.2009.01938.x.PubMedView ArticleGoogle Scholar
- Andersen O, Wetten OF, De Rosa MC, Andre C, Carelli Alinovi C, Colafranceschi M, Brix O, Colosimo A: Haemoglobin polymorphisms affect the oxygen-binding properties in Atlantic cod populations. P Roy Soc B - Biol Sci. 2009, 276 (1658): 833-841. 10.1098/rspb.2008.1529.View ArticleGoogle Scholar
- Borza T, Stone C, Gamperl AK, Bowman S: Atlantic cod (Gadus morhua) hemoglobin genes: multiplicity and polymorphism. BMC Genet. 2009, 10: 51-10.1186/1471-2156-10-51.PubMed CentralPubMedView ArticleGoogle Scholar
- Pogson GH: Nucleotide polymorphism and natural selection at the pantophysin (Pan I) locus in the Atlantic cod, Gadus morhua (L.). Genetics. 2001, 157 (1): 317-330.PubMed CentralPubMedGoogle Scholar
- Pogson GH, Fevolden SE: Natural selection and the genetic differentiation of coastal and Arctic populations of the Atlantic cod in northern Norway: a test involving nucleotide sequence variation at the pantophysin (Pan) locus. Molecular Ecology. 2003, 12 (1): 63-74. 10.1046/j.1365-294X.2003.01713.x.PubMedView ArticleGoogle Scholar
- Sick K: Haemoglobin polymorphism of cod in the North Sea and the North Atlantic Ocean. Hereditas. 1965, 54 (1): 49-69. 10.1111/j.1601-5223.1965.tb02005.x.PubMedView ArticleGoogle Scholar
- Jamieson A, Birley AJ: The demography of a haemoglobin polymorphism in the Atlantic cod, Gadus morhua L. Journal of Fish Biology. 1989, 35 (sa): 193-204.Google Scholar
- Van Ooijen JW: JoinMap® 4. Software for the calculation of genetic linkage maps in experimental populations. 2006, Wageningen, Netherlands: Kyazma B.VGoogle Scholar
- Fan Z, Fox DP: Robertsonian polymorphism in plaice, Pleuronectes platessa L., and cod, Gadus morhua L., (Pisces Pleuronectiformes and Gadiformes). Journal of Fish Biology. 1991, 38: 635-640. 10.1111/j.1095-8649.1991.tb03152.x.View ArticleGoogle Scholar
- Halldorsdottir K, Arnason E: Organization of a beta and alpha globin gene set in the teleost Atlantic cod, Gadus morhua. Biochem Genet. 2009, 47 (11-12): 817-830. 10.1007/s10528-009-9280-0.PubMedView ArticleGoogle Scholar
- Halldorsdottir K, Arnason E: Multiple linked beta and alpha globin genes in Atlantic cod: a PCR based strategy of genomic exploration. Marine Genomics. 2009, 2: 169-181. 10.1016/j.margen.2009.10.001.PubMedView ArticleGoogle Scholar
- Maruyama K, Yasumasu S, Naruse K, Mitani H, Shima A, Iuchi I: Genomic organization and developmental expression of globin genes in the teleost Oryzias latipes. Gene. 2004, 335: 89-100. 10.1016/j.gene.2004.03.007.PubMedView ArticleGoogle Scholar
- Gillemans N, McMorrow T, Tewari R, Wai AW, Burgtorf C, Drabek D, Ventress N, Langeveld A, Higgs D, Tan-Un K: Functional and comparative analysis of globin loci in pufferfish and humans. Blood. 2003, 101 (7): 2842-2849. 10.1182/blood-2002-09-2850.PubMedView ArticleGoogle Scholar
- Fevolden SE, Pogson GH: Genetic divergence at the synaptophysin (Syp I) locus among Norwegian coastal and north-east Arctic populations of Atlantic cod. Journal of Fish Biology. 1997, 51 (5): 895-908.Google Scholar
- Colafranceschi M, Giuliani A, Andersen O, Brix O, De Rosa MC, Giardina B, Colosimo A: Hydrophobicity Patterns and Biological Adaptation: An Exemplary Case from Fish Hemoglobins. OMICS. 2010Google Scholar
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