Atlantic cod (Gadus morhua) hemoglobin genes: multiplicity and polymorphism

Abstract

Background: Hemoglobin (Hb) polymorphism, assessed by protein gel electrophoresis, has been used almost exclusively to characterize the genetic structure of Atlantic cod (Gadus morhua) populations and to establish correlations with phenotypic traits such as Hb oxygen binding capacity, temperature tolerance and growth characteristics. The genetic system used to explain the results of gel electrophoresis entails the presence of one polymorphic locus with two major alleles (HbI-1; HbI-2). However, vertebrates have more than one gene encoding Hbs and recent studies have reported that more than one Hb gene is present in Atlantic cod. These observations prompted us to re-evaluate the number of Hb genes expressed in Atlantic cod, and to perform an in depth search for polymorphisms that might produce relevant phenotypes for breeding programs.

Results: Analysis of Expressed Sequence Tags (ESTs) led to the identification of nine distinct Hb transcripts; four corresponding to the alpha Hb gene family and five to the beta Hb gene family. To gain insights about the Hb genes encoding these transcripts, genomic sequence data was generated from heterozygous (HbI-1/2) parents and fifteen progeny; five of each HbI type, i.e., HbI-1/1, HbI-1/2 and HbI-2/2. beta Hb genes displayed more polymorphism than alpha Hb genes. Two major allele types (beta1A and beta1B) that differ by two linked non-synonymous substitutions (Met55Val and Lys62Ala) were found in the beta1 Hb gene, and the distribution of these beta1A and beta1B alleles among individuals was congruent with that of the HbI-1 and HbI-2 alleles determined by protein gel electrophoresis. RT-PCR and Q-PCR analysis of the nine Hb genes indicates that all genes are expressed in adult fish, but their level of expression varies greatly; higher expression of almost all Hb genes was found in individuals displaying the HbI-2/2 electrophoretic type.

Conclusion: This study indicates that more Hb genes are present and expressed in adult Atlantic cod than previously documented. Our finding that nine Hb genes are expressed simultaneously in adult fish suggests that Atlantic cod, similarly to fish such as rainbow trout, carp, and goldfish, might be able to respond to environmental challenges such as chronic hypoxia or long-term changes in temperature by altering the level of expression of these genes. In this context, the role of the non-conservative substitution Lys62Ala found in the beta1 Hb gene, which appears to explain the occurrence of the HbI-1 and HbI-2 alleles described by gel electrophoresis, and which was found to be present in other fish such as eel, emerald rockcod, rainbow trout and moray, requires further investigation.

Figure 1

The expression of the nine Hb genes in individuals of the three HbI electrophoretic types. Q-PCR analysis was performed using the ΔΔCt method and ubiquitin as a reporter gene. Data for every gene and HbI type was normalized relative to the gene and HbI type with the lowest expression (i.e. the α4 Hb gene, the sample representing the HbI electrophoretic type 2/2).

Figure 2

Phylogenetic Bayesian tree of the α hemoglobins from different teleost fish. The statistical support for internal nodes was determined by posterior probabilities (Bayesian inference) and bootstrap analyses (Maximum Likelihood and Neighbor-Joining analyses) and is shown, in this order, at the corresponding branches. Values ≤ 50% are indicated by (-). The tree resulting from ML and NJ analysis gave similar topology. The nomenclature of the Hb groups is from Maruyama et al [33].

Figure 3

Phylogenetic Bayesian tree of the β hemoglobins from different teleost fish. The statistical support for internal nodes was determined by posterior probabilities (Bayesian inference) and bootstrap analyses (Maximum Likelihood and Neighbor-Joining analyses) and is shown, in this order, at the corresponding branches. Values ≤ 50% are indicated by (-). The tree resulting from ML and NJ analysis gave similar topology. The nomenclature of the Hb groups in from Maruyama et al [33].

Figure 4

Alignment of the five Atlantic cod β Hbs and of the β Hbs from rainbow trout (O. mykiss), eel (A. anguilla) and emerald rockcod (T. bernacchii) displaying the substitution Lys62Ala. In Atlantic cod, the two major alleles β1A and β1B display in position 62 the amino acids Lys (K) and Ala (A), respectively. In the other Atlantic cod Hbs position 62 is occupied by Ala (β2-4 Hbs) or Asn (N; β5 Hb). The other non-conservative substitution identified in the β1 chain of Atlantic cod, Met55Val, seems to have a distribution restricted to this species.