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  • Short Report
  • Open Access

Integrating the markers Pan I and haemoglobin with the genetic linkage map of Atlantic cod (Gadus morhua)

BMC Research Notes20103:261

  • Received: 21 June 2010
  • Accepted: 15 October 2010
  • Published:



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 [1]. 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 [5], age and seasonality of sexual maturation [6] or annual mortality [7]. Similarly, growth rate [8], 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 [1113] 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

We used a curated Illumina GoldenGate panel containing 1536 SNPs [12], including three SNPs identified in the Hb β1 gene, two SNPs specific to the Hb β3 gene and one characteristic for the Hb β4 gene (details in Additional file 1). These SNPs were derived from polymorphisms detected by sequencing nine Atlantic cod Hb genes in the parents and 15 progeny of a family, B30, that was determined to be heterozygous (1/2) at the HbI locus [16]. Western Atlantic cod populations are characterized by a low frequency of allele 1 at the HbI locus [19, 20]. Family B30 was one of the few families heterozygous at this locus produced by the breeding program of Atlantic Cod Genomics and Broodstock Development Project (CGP) [11]; parents and 93 progeny from this family were used to map these loci. Illumina GoldenGate probe design failed for several of the Hb SNPs, therefore additional Hb polymorphisms were identified and used to develop assays for the KASPar SNP genotyping system (Additional file 2). A genetic linkage map was constructed using JoinMap®4 [21]. The genotypes for progeny were converted to CP codes based on parental genotypes and mapping was performed using a LOD cut-off value of 5.0 and Kosambi's mapping function [21]. The 23 linkage groups obtained for family B30 were compared to the linkage groups previously reported by Hubert et al. 2010 [12]; a 1:1 correspondence between linkage groups (LGs) was confirmed and the corresponding LGs were merged (Figures 1, 2, 3; more details about mapping can be found in Hubert et al. 2010 and Additional file 1). The 23 linkage groups observed for family B30 (and those described in Hubert et al. 2010) matches the number of haploid chromosomes reported for the Atlantic cod by Fan and Fox [22]. The combined results from GoldenGate and KASPar assays allowed the mapping of all Hb genes analysed: Hb genes α1, α4, β1 and β5 were mapped on LG 2 while Hb genes α3 and β2, β3 and β4 were mapped on LG 18 (Figures 1 and 3). Sequencing data from the Hb α2 gene, which was not polymorphic in the B30 family, indicate that this gene is situated immediately adjacent to the Hb β3 gene [23, 24]; therefore the cluster of Hb genes located on LG 18 contains at least five different Hb genes. The mapping of Atlantic cod Hb genes is in agreement with other studies on fish and other vertebrates which indicate that Hb loci are placed on two linkage groups/two different chromosomes [25, 26]. Allele frequencies at the Hb β1 locus from several North Atlantic cod populations (Additional file 2) were similar to data collected by Hb allozyme electrophoresis [19, 20] suggesting that Hb β1, or other closely linked genes, might underlie QTLs.
Figure 1
Figure 1

Genetic linkage map for Atlantic cod (linkage groups 1-8). The first eight of the 23 major linkage groups are shown. The 23 major linkage groups have been numbered CGP 1 to 23 as in Hubert et al. 2010, to distinguish them from the linkage groups generated by Moen et al. 2009. Distances in centimorgans (Kosambi cM) are indicated on the left of each linkage group, with SNP identifiers on the right. Pan I and Hb loci are highlighted in bold, while SNPs common to Moen et al. 2009 are both bold and italicized.

Figure 2
Figure 2

Genetic linkage map for Atlantic cod (linkage groups 9-16). Eight of the 23 major linkage groups are shown. The 23 major linkage groups have been numbered CGP 1 to 23 as in Hubert et al. 2010, to distinguish them from the linkage groups generated by Moen et al. 2009. Distances in centimorgans (Kosambi cM) are indicated on the left of each linkage group, with SNP identifiers on the right. SNPs common to Moen et al. 2009 are highlighted in bold and italicized.

Figure 3
Figure 3

Genetic linkage map for Atlantic cod (linkage groups 17-23). Seven of the 23 major linkage groups are shown. The 23 major linkage groups have been numbered CGP 1 to 23 as in Hubert et al. 2010, to distinguish them from the linkage groups generated by Moen et al. 2009. Distances in centimorgans (Kosambi cM) are indicated on the left of each linkage group, with SNP identifiers on the right. Hb loci are highlighted in bold while SNPs common to Moen et al. 2009 are both bold and italicized.

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 [11] 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 [17]. Family B87, one of the two families used in the generation of the initial map [12] 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 [12]. 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 [21]. Another map, produced by the third round of mapping calculations [21] 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 [21].

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. [12]) with the genetic linkage map data produced by a different group [14]. To identify sequences that were mapped by both, CGP (this paper and Hubert et al. [12]) and Moen et al. [14] we performed a Blastn search of an in-house made database containing the ESTs listed by Moen et al. [14] 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 [14]. 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. [14] 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 [16]) 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

Authors’ Affiliations

Genome Atlantic, NRC Institute for Marine Biosciences, Halifax, NS, Canada


  1. 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
  2. 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
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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
  10. 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
  11. 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
  12. 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
  13. 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
  14. 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
  15. 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
  16. 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
  17. 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
  18. 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
  19. 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
  20. 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
  21. Van Ooijen JW: JoinMap® 4. Software for the calculation of genetic linkage maps in experimental populations. 2006, Wageningen, Netherlands: Kyazma B.VGoogle Scholar
  22. 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
  23. 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
  24. 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
  25. 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
  26. 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
  27. 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
  28. 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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