A genetic polymorphism in hemoglobins of chinook salmon, Oncorhynchus tshawytscha

UNNI E. H. FYHN' AND RUTH E. WITHLER Department of Fisheries and Oceans, Fisheries Research Branch, Pacific Biological Station,

Nanaimo, B.C., Canada V9R 5K6 Received April 12, 1990

FYHN, U. E. H., and WITHLER, R. E. 1991. A genetic polymorphism in hemoglobins of chinook salmon, Oncorhynchus tshawytscha. Can. J. Zool. 69: 1904-1910.

A genetic polymorphism with three phenotypes is described for the anodally migrating hemoglobins of adult chinook salmon !Onchorhynchus tshawytscha) from British Columbia, Canada. A genetic model with the genotypes DD, DH, and HH is suggested, on the basis of Hardy-Weinberg genotypic frequencies, in samples of adult chinook salmon from three stocks, and on Mendelian genotypic frequencies among progeny of single-parent crosses. Allelic frequencies differed among stocks. The polymorphism may result from a dimorphism in one or both of the tentatively called P-chain loci, with allele D encoding a chain PFf and allele H encoding a chain pF" The two p-loci may be individually regulated. The locus tentatively referred to as a is monomorphic, as are the minimum of three or four loci that encode the globins of the cathodal hemoglobins. The cathodal and anodal hemoglobins had no globins in common.

FYHN, U. E. H., et WITHLER, R. E. 1991. A genetic polymorphism in hemoglobins of chinook salmon, Oncorhynchus tshawytscha. Can. J. Zool. 69 : 1904-1910.

Un polymorphisme gknktique donne lieu ii trois phinotypes d'hkmoglobines migrant vers l'anode chez des Saumons chinooks (Oncorhynchus tsawytscha) adultes de Colombie-Britannique, Canada. Un modkle gknktique avec les gknotypes DD, DH et HH, est proposk, dans lequel les frkquences gknotypiques suivent la loi Hardy-Weinberg dans le cas de kchantillons de saumons appartenant ii trois stocks, et la courbe de Mendel dans le cas de la progkniture de croisements impliquant des parents d'un seul type. Les frkquences allkliques different chez les divers stocks. Le polymorphisme peut rksulter du dimorphisme ii l'un de deux locus ou aux deui, appelks ici provisoirement locus P (P-chain) oh l'allkle D code une chaine PFf et l'allkle H spkcifie une chaine pF'. Les deux locus p peuvent Etre sous contr8les indkpendants. Le locus appelk provisoirement locus a est monomorphe, comme le sont les trois ou quatre locus, au minimum, qui codent les globines des hkmoglobines qui migrent vers la cathode. Les hkmoglobines cathodiques et anodiques n'ont pas de globines en commun.

[Traduit par la rkdaction]

Introduction probably occurred (Schultz 1980). A minimum of six to eight

The hemolysates of the chinook salmon, Oncorhynchus tshawytscha, contain multiple hemoglobin (Hb) components (Buhler and Shanks 1959; Hashimoto and Matsuura 1960; Buhler 1963; Tsuyuki and Gadd 1963; Tsuyuki et al. 1965; Tsuyuki and Ronald 197 1 ; Harrington 1986; Fyhn and Withler 1991a; Fyhn et al. 1991). The components separate as anodally (Hb A) and cathodally (Hb C) migrating bands in high-pH gel electrophoresis. Up to 18 components of oxy- and met-hemoglo- bins have been observed in spawning chinook salmon (Tsuyuki and Ronald 1971). Hemolysates of adults contain a higher number of cathodal components than hemolysates of juveniles (Harrington 1986; Fyhn and Withler 1 9 9 1 ~ ) ; the ontogentic variation in chinook Hbs has been described (Fyhn and Withler 1991a), and is similar to that found in other salmonids (see review by Wilkins 1985).

Salmonid Hb tetramers include two a-like and two P-like globin chains which have been shown in rainbow trout, Salmo gairdneri, to be homologous with the a - and P-chains of human Hb (Bossa et al. 1978; Barra et al. 1983; Petruzzelli et al. 1984). Unlike Hbs of higher vertebrates, the tetramers may contain up to four different polypeptide chains (Wilkins 1970, 1985; Tsuyuki and Ronald 1970, 197 1 ; Ronald and Tsuyuki 197 1 ; Southard et al. 1986). Multiple loci encoding closely related proteins are assumed to result from successive gene duplications, with subsequent structural divergence (Ohno 1970; Buth 1983). In salmonids, additional tetraploidization of the total genome has

'Present address: Department of Fisheries and Marine Biology, University of Bergen, High Technology Centre, N-5020 Bergen, Norway.

structural loci thus encode the globins of extant salmonids, and each locus may be controlled by a separate regulatory gene (Wilkins 1985).

The adaptive value of the multiple Hb components is uncer- tain, although large functional differences have been found among individual Hb components of chinook salmon (Harring- ton 1986) and other salmonids (Hashimoto et al. 1960; Binotti et al. 1971; Brunori et al. 1973; Southard et al. 1986; Sauer and Harrington 1988). Hemoglobins with different oxygen-binding properties may have adaptive value for anadromous fishes that experience differences in oxygen availability as well as in oxygen requirements during their life cycle (Brunori 1975; Giles and Randall 1980).

Many fishes show allelic Hb polymorphism (Sick 196 1 ; Fyhn and Sullivan 1974, 1975; Bonaventura et al. 1975; Fyhn et al. 1979; Perez and Rylander 1985). Hemoglobin polymorphisms have been reported in cathodal components of Arctic charr, Salvelinus alpinus (Giles and Rystephanuk 1989), and cutthroat trout, Salmo clarkii (Braman et al. 1980). To our knowledge, no allelic polymorphism has yet been described in anodal compo- nents of salmonids. In this paper, such a polymorphism among the anodal components in Hbs of adult chinook salmon is presented and its genetic basis is examined by means of globin of analyses and genotypic frequencies in populations and families.

Materials Chinook salmon from British Columbia, Canada, used in the present

study are listed in Table 1. Mature (3-6 years old) adults were sampled form the Big Qualicum and Conuma rivers (on the east and west coasts of Vancouver Island, respectively) and the Quesnel River (a tributary

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TABLE 1. Chinook salmon used in this study

Length Sampling Brood ( j k S D ) ,

Stock date Maturity Year cm N

Big Qualicum River Oct . -Nov. 1987 May 1987 April 1988 May 1988

Conuma River Sept. 1987 Quesnel River Sept. 1987 Kitsurnkalum River Jan.-Feb. 1988 Robertson Creek May 1988 Harrison River May 1988

NOTE: Brood year is the year in which fertilization took place. All samples were from mature (M) or immature, post-smolt (I) salmon. Length is standard body length. N is sample size.

of the upper Fraser River). Immature adults of various stocks reared in captivity were sampled from seapens at the Pacific Biological Station, Nanaimo. Big Qualicum River, Robertson Creek, and Harrison River samples from the 1984 brood year were drawn from single-family crosses (Withler et al. 1987). The 1985( 1) samples from Big Qualicum River consisted of pooled offspring from 1 1 females and 7 males. The 1985(2) samples from Big Qualicum River and those from Kitsum- kalum River were offspring from 13 and 6 separate families, respec- tively. L

Methods Sampling and hemolysate preparation

Standard body length was measured to the nearest millimetre (Table 1). Blood samples were taken by cardiac puncture into ice- cold heparinized syringes. Three to five volumes of ice-cold saline (1.7% NaCl, 1 mM Tris, pH 8) were added to each volume of blood. The erythrocytes were washed 3 times in 10 volumes of saline at 700 X g and 4°C and hemolyzed in 3 4 volumes of ice-cold 1 mM Tris, pH 8.0. The cell debris was pelleted by centrifugation at 28 000 x g for 20 min at 4°C. The hemolysates were stored at 4°C and used for electrophoresis, without further treatment, within 20 h.

High-pH electrophoresis Horizontal, high-pH starch-gel electrophoresis was done according

to Utter et al. (1974) and Fyhn and Withler (19916) with 11.4% gels and Smithies' Tris - boric acid - EDTA Hb buffer, pH 8.54. Electro- phoresis was carried out to 20 mA for 6 h in an incubator of 3-5°C and with an ice pack on top of the gel box. Gels were sliced horizontally and stained with benzidine (Moss and Ingram 1968) or with Brilliant Blue G Quick stain in perchloric acid (McFarland 1977). Quick-stained gels were destained by diffusion, photographed by transmitted light, and stored at 4°C for later scanning. The two staining procedures gave identical banding patterns, so Brilliant Blue G Quick stain was subsequently used routinely as a detector stain for hemoglobins.

Hb A, Hb C, or individual Hb components were isolated by cutting out gel pieces containing the respective Hbs from high-pH gels run for 20 min (Hb A and Hb C) or 8-9 h (individual Hb components), and letting the Hbs diffuse overnight into a minimum of gel buffer.

The percentage of total Hb accounted for by Hb A (%Hb A) in hemolysates was determined spectrophotometrically at 542 nm by the diffusion<yanomet-hemoglobin method of Koch (1982). The correspondence between duplicates was within 3%.

The distribution of Hb within the anodal and cathodal bands was determined from densitometer tracings of the Brilliant Blue G Quick stained gels with 570-nm transmitted light by means of an E-C Apparatus Corporation densitometer connected to a Philip PM 8100 recorder. The planimetry was done manually. The correspondence between repeated scans of the same gel or scans of the same hemolysate on different gels was within 5% except for bands containing less than

5-8% of the Hb when variation in background absorption interfered with the results.

Low-pH urea gel electrophoresis Horizontal, low-pH, 8 M urea gel electrophoresis was carried out

according to Tsuyuki and Ronald (197 1) with the gel buffer at pH 2.9 and electrode buffer at pH 2.0, and according to Wilkins (1970) at pH 3.5 and 3.1. Dithiothreitol was omitted, but P-mercapto ethanol (0.25%) was added to all buffers.

For globin preparation fresh hemoly sates were bubbled with CO and dialyzed overnight against 0.5 mM Tris, pH 8.0, saturated with CO. The Tris buffer was employed because dialysis against deionized water gives a significant amount of precipitation and formation of methemo- globin (Fyhn and Withler 199 16). Globins were prepared by extracting the heme with acid acetone 'at -20°C (Anson and Mirsky 1930). Solutions of Hb A, Hb C, and isolated Hb components were dialyzed, frozen at -80°C, and lyophilized. The Hbs were resuspended in deionized water saturated with CO, and the globins were precipitated by acid acetone. Two hours prior to electrophoresis the globins were resuspended in the urea gel buffer. The urea gels were run for 20-30 h at 1 5 4 0 mA (80-140 V), depending upon the thickness of the gel (3- 8 mm). The gels were sliced and stained overnight in Quick stain or in Brilliant Blue G in trichloroacetic acid (TCA) (10 mL stock solution, 200 mL 12.5% TCA), destained, and photographed by transmitted light.

Because untreated hemolysates and solutions of Hb A, Hb C, or Hb components applied directly to the urea gels gave gel patterns identical with the corresponding globin preparations, a simplified technique with two successive electrophoretic runs was also employed. Thin slices of a prerun high-pH gel containing the various Hb components were cut out and placed directly in the slit of the urea gel. The gel slices were removed from the urea gel after 25 min at 40 mA, and electro'phoresis was continued at 1 5 4 0 mA.

Results High-pH electrophoresis

At pH 8.54 electrophoretic patterns of chinook salmon hemolysates consisted of 8-10 Hb bands (Figs. l a and 2). A group of two to four Hbs migrated anodally (Hb A), whereas five or six Hbs migrated cathodally (Hb C).

The cathodal band pattern was uniform in gels of all hemo- lysates, except that band 1C varied in appearance and intensity even among gels of the same hemolysate. Bands 2C, 3C, and 4C were most prominent and together contained 75-94% of the total Hb C (Table 2). Bands 5C and 6C were low in intensity, but were present in all gels. No differences in Hb distribution among cathodal bands were seen between the brood-year classes.

The anodal bands revealed a polymorphism of three distinct

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TABLE 2. Quantitative distribution (%) of hemoglobin among anodal and cathodal bands of mature (3 and 4 years old), and immature 3-year-old (1984) and 2-year-old (1985) chinook salmon measured by scanning densitometry of starch gels

Anodal Hb Cathodal Hb Year

Phenotype class N 1A 2A 3A 4A 1C 2C 3C 4C 5C 6C

NOTES: Values are given as the mean 2 SD or as individual values. tr, trace amounts.

TABLE 3. Percentages of total hemoglobin that migrated anodally in Big Qualicum River chinook salmon hemolysates of phenotypes I,

11, and I11

Phenotype

Brood year I I1 I11 -

1983-1984 (mature) 60k3.1 (4) 6454.3 (9) 64k2.7 (9) 1984 62 (1) - 62, 66 (2) 1985(1) 70 (1) 62k5.4 (5) 67k3.6 (4) 1985(2) 68,69(2) 66k3.5(8) 69+3.6(7)

NOTE: Values are given as the mean 2 SD or as individual values. Numbers in parentheses show sample size.

patterns, phenotypes 1-111 (Figs. la and 2). The phenotypes were easily distinguished, both visually and by densitometry (Table 2). No intermediate patterns were observed. Electrophoresis of hemolysate mixtures gave additive effects only.

Band 1A of the anodal patterns aligned in all three phenotypes, and bands 2A and 3A aligned in phenotypes I1 and 111. Bands 2A and 3A in phenotype I were slightly more anodic than the corresponding bands of phenotypes I1 and 111. Band 4A was a minor, variable band containing less than 6% of the Hb A in phenotype 111, was faint in phenotype 11, and absent in pheno- type I.

The quantitative distribution of Hb among the three major anodal bands characterized phenotypes I1 and I11 (Fig. 2, Table 2). In phenotype 111, the intensity of band 3A was about twice that of band lA, whereas in phenotype I1 the proportions were reversed. Band 2A was the most prominent band in both phenotypes, representing 40-50% of the Hb A. No difference in Hb distribution was found between brood-year classes of phenotypes I1 and 111.

Phenotype I was characterized by a significantly higher intensity of band 1A relative to 2A, and by the absence of band 3A in spawning fish. In spawning fish the Hb A was shared 9: 1 between bands 1A and 2A. In immature fish the intensities of bands 2A and 3A decreased with increasing age (Table 2, Fig. 3), and these bands aligned with bands in the Hb patterns of juvenile (pre-smolt) chinook salmon hemolysates (Fyhn

FIG. 1. Electrophoretic patterns on starch gels of hemoglobins and globins of immature adult chinook salmon. (a) Hemoglobins of 2-year- old fish (1985(1)) with phenotypes 1-111 (pH 8.54, Brilliant Blue Quick stain). Anode is up. (b) Globins of Hb A and Hb C of a 3-year-old salmon and of human hemoglobin (Hu) (pH 2.0-2.9,8 M urea, Brilliant Blue in TCA). Cathode is up.

and Withler 199 la). In phenotype I, bands 2A and 3A may there- fore represent remnants of juvenile Hbs rather than the true adult bands 2A and 3A of phenotypes I1 and 111.

Hb A accounted for more than 60% of the total Hb in the three phenotypes (Table 3). The %Hb A value decreased ( p < 0.01) in hemolysates of phenotypes I and 111. A similar decrease in %Hb A was not found in hemolysates of phenotype 11.

Globins The globin patterns of total Hb A and Hb C each consisted of

two zones with different mobilities (Fig. 16). The Hb A zones were intermediate in mobility to the Hb C zones. The total hemolysate gave a globin pattern that was the sum of Hb A and Hb C. The two buffer systems tested for low-pH, 8 M urea gels did not differ in resolution or number of globin bands.

Individual Hb A components (bands 1A-3A) produced the

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- 1 - - 0 3A HbA m 0 2 A

, - - - 3C / HbC - 0 - 4 C

FIG. 2. Diagram of hemoglobin phenotypes 1-111 of mature chinook salmon with anodally (Hb A) and cathodally (Hb C) migrating bands on starch gels, pH 8.54.

Direction of migration

FIG. 3. Densitometer scans of the anodal hemoglobin bands (1A-3A) of chinook salmon phenotype I. (a) Two-year-old immature salmon (1985(2) brood year) (b) Three-year-old immature salmon (1984 brood year). (c) Four-year-old mature salmon.

same globin patterns as total Hb A. Only one band was discerni- ble within each zone. Individual components of Hb C produced globin patterns with two bands in the slowest migrating zone, and the distribution of globin between the two bands depended upon the cathodal component tested. A similar variation seemed probable in the fast-migrating zone, but distinct bands could not be resolved.

Gels pH 8.54 - + HbA

A ,

1A 2 A 3 A 4 A

@ 0 I

Gels pH 2.8 8M urea

FIG. 4. Chinook salmon anodal hemoglobin (Hb A) bands on high- pH (8.54) gels and an interpretation of the corresponding globins on low-pH (2.0-3.5), 8 M urea gels. The globins are named according to Tsuyuki and Ronald (197 1). Two a-like and two P-like globin chains are included in each Hb tetramer. ..

from the Harrison River were of phenotypes I and 111, and two from Robertson Creek were both of phenotype 111. Phenotypes I1 and I11 were represented among nine chinook salmon derived from a limited number of parents (two females, four males) from the Kitsumkalum River.

In the samples of mature chinook salmon from the three populations sampled randomly, there was no association between Hb phenotype and size (all p > 0.05), nor did the Hb phenotypes appear to be sex-limited or sex-linked (Table 4). The Hb phenotypes of 48 offspring from four male and four female Big Qualicum River parents mated in a factorial design (brood year 1985(2)) are shown by family in Table 5. All parents produced offspring of phenotypes I1 and 111, and in all families in which more than two offspring were tested, phenotypes I1 and I11 both occurred. A single phenotype I was observed, and it had both phenotype I1 and I11 siblings.

The simplest genetic model able to account for the observed Hb phenotypic distributions in the three wild populations and in the Big Qualicum River families is one with a single-locus, two- allele system. Genotypes DD, DH, and HH can be considered to underlie phenotypes I, 11, and 111, respectively. In this model the frequency of the D allele is higher in the Big Qualicum River population (0.41) than in the ~ o n u m a River (0.13) or Quesnel River (0.08) populations. The genotypic frequencies for all three populations are in accordance with Hardy-Weinberg expecta- tions (x2 = 0.55, df = 1, p > 0.05; X2 = 0.02, df = 1, p > 0.05; X2 = 0.01, df = 1, p > 0.05, respectively).

The Hb phenotypic distributions among and within the 13 half-sib Big Qualicum River families sampled (Table 5) are also consistent with the single-locus model. Although the parents

Phenotypic frequencies and a genetic model were not sampled f o r ~ b phenotype, they can be assigned The distribution of Hb phenotypes among mature chinook putative genotypes that account for the observed distribution of

salmon from Big Qualicum, Conuma, and Quesnel rivers is Hb phenotypes among their progeny. Because one cross (male shown in Table 4. Phenotype I was not observed in the Conuma 4 x female 3) produced progeny of all three Hb phenotypes, River or Quesnel River samples. Two chinook salmon sampled both parents must have been heterozygous (DH) at the Hb locus.

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TABLE 4. Distribution of chinook salmon hemoglobin phenotypes among males and females from three stocks

Hb phenotypes

Stock Maturity Sex I I1 I11 Total

Big Qualicum River Mature F 3 7 6 16 M 1 9 3 13 Total 4(14) 16(55) 9(31) 29

Conuma River Mature F 0 3 7 10 M 0 2 8 10 Total 0 (0) 5 (25) 15 (75) 20

Quesnel River Mature F 0 0 10 10 M 0 3 7 10 Total 0 (0) 3(15) 17(85) 20

Big Qualicum River (families) Immature F 2 10 11 23

M 0 10 16 26 Total 2 20 27 49

NOTE: Numbers in parentheses are percentages.

TABLE 5. Hemoglobin phenotypes observed among offspring of 13 chinook salmon families from the Big Qualicum River (1985(2)),

resulting from a factorial cross of four males and four females b

Male

1 2 3 4

Phenotype: I I1 111 I I1 111 I I1 I11 I I1 I11

Female 1 0 3 2 0 4 1 - - - 0 2 1 2 0 0 2 0 0 1 0 1 2 0 2 0 3 0 6 8 0 0 1 0 1 1 1 1 1 4 0 1 5 - - - 0 0 1 - - -

Total 0 10 17 0 4 3 0 2 4 1 5 2

All other parents were either heterozygous or homozygous for the H allele. For example, the results are consistent with the possibility that males 3 and 4 and females 1, 3, and 4 were heterozygous and the remainder of the parents were homozygous for allele H.

Discussion The present investigation reveals a polymorphism in the

anodally migrating Hbs of the adult chinook salmon. The polymorphism is truly genetic and not caused by nongenetic variation in protein expression. Such variations can be due to factors like ontogenetic changes, environmental differences, extraction and analytical procedures, or storage conditions (Allendorf and Utter 1979), but none of these seem to apply to this polymorphism.

Ontogenetic variation occurs in chinook salmon Hbs with a decrease in mobility of the anodal bands and an increase in the number and Hb content of cathodal components from the juvenile to the adult (Fyhn and Withler 1991~). In the present study the adult Hb phenotypes were well established in 2- and 3- year-old fish. A certain development in phenotype I occurred in captive 2-year-old immature and wild 3-year-old mature salmon, but the Hb phenotypes were definitely manifested in all individ-

uals tested. The Hb phenotype did not depend on the length or sex of the salmon, and no variation in Hb patterns was found to correspond to environmental variability. All three phenotypes were observed in both wild and captive chinook salmon. An Hb pattern intermediate between phenotypes I1 and 111, similar to the transitional pattern of younger, post-smolt chinook salmon (Fyhn and Withler 1991a), was observed in a single thin, heavily infected 3-year-old fish not included in the data. Manifestation of the adult Hb phenotype may therefore be delayed by develop- mental or conditional factors, as for Atlantic salmon retained in freshwater (Koch 1982) or affected by disease or injury (Koch et al. 1966).

The electrophoretic mobility of Hbs from fishes and higher vertebrates may be influenced by the oxidation state of the iron atoms and the formation of dimers or aggregates larger than tetramers (Fyhn and Sullivan 1975; Reischl 1976; Borgese et al. 1988). Methodological studies (Fyhn and Withler 199 1 b) have shown that the Hb phenotypes in the anodal components of chinook salmon hemolysates are stable and not a result of experimental or storage artifacts. No polymers are formed as a result of disulphide-bridge formation, and the oxy- and carboxy- hemoglobins show the same gel pattern. The phenotypes are evident in patterns of fully oxidized Hbs, although the distribu- tion of Hb among the anodal bands differs somewhat from the patterns of nonoxidized samples.

Phenotypes I1 and I11 are distinguished by a difference in the quantitative distribution of Hb among the anodal bands, but this difference can be masked by overloading of the gels (Fyhn and Withler 199 1 b). A true lack of specimens with phenotype I in the material and heavy loading of the gels may be the reason why the Hb polymorphism has not previously been reported in electrophoretic studies of chinook salmon hemolysates (Tsuyuki and Gadd 1963; Tsuyuki et al. 1965; Tsuyuki and Ronald 197 1 ; Harrington 1986).

An interpretation of the globin patterns produced by the three main anodal Hbs of chinook salmon is given in Fig. 4. The terminology regarding a- and P-globins follows that of Wilkins (1970, 1985), Ronald and Tsuyuki (1971), and Tsuyuki and Ronald (197 1) for other salmonids. The terminology for chinook salmon globins, as well as a conclusion as to which of the globin

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zones contains more than one globin species, must await further study. The model assumes the presence of one type of a-chain, aF, in the slow-migrating zone and two types of P-chains, PFf and pF" in the fast migrating zone. This is similar to the pattern of globins of other salmonids with three main anodal components (Ronald and Tsuyuki 197 1 ; Tsuyuki and Ronald 197 1 ; Southard et al. 1986 (in the last study the a and P designations are reversed).

The presence of all three anodal bands (1 A-3A) in phenotypes I1 and I11 requires a minimum of one structural genetic locus for the a-chain(s) and two loci for the P-chains. The Hb polymor- phism may result from a dimorphism in one or both of the P- chain loci. The D allele encodes the P-chain PFf and the H allele encodes the P-chain pF" Because the Hb band containing only pFf chains was present in all phenotypes, it seems likely that one of the p loci is polymorphic and the other is monomorphic for the D allele. The two P-chain loci may be the two copies of a duplicated gene which diverged into two functionally distinct genes similar to that suggested for many other salmonid struc- tural loci (Ohno 1970). The a-chain gene may also be duplicated into two isoloci encoding structurally identical (i.e., the same electrophoretic mobility) a-chains, as in various human popula- tions (Weatherall and Clegg 1976).

If the high-pH oxy gels are assumed to be a true expression of the native Hbs (i.e., each of the three main Hb bands contains only one species of Hb), and band 4A is derived from band 3A, the Hb distribution values in Table 2 may be used for calculating the proportions of the pFs- and PFf-chains contributing to each phenotype. In spawning fish the values of phenotype 111, which is homozygous at both P-chain loci, are 64% PF%nd 36% pFf, giving a pF7pFf ratio of 1.78. Apparently the protein product of one P-chain locus occurs about 1.8 times as frequently as the protein encoded by the second locus. In mature fish of phenotype 11, the percentage of PFVs 40.5% and the pFS:pFf ratio is 40.559.5 (= 0.68). If the protein product of the first locus occurs 1.8 times as frequently as the product of the second locus, the percentage of ~ ~ " h o u l d be 32%, and a pFS:pFf ratio of 32/68 (= 0.47) would be expected in phenotype 11. The discrepancy between the observed percentage of PFs (0.68) and that expected on the basis of phenotype I11 (0.47) may result from individual variation in the Hb distribution within phenotypes and (or) the occurrence of more than one tetramer species in each band. The results suggest the existence of different regulatory genes for the two P-chain loci. Differences in gene expression in Hb subunits encoded by different loci have also been reported for Atlantic salmon (Wilkins 1983, 1985).

The present data do not exclude another model for the Hb polymorphism, in which individuals of all three phenotypes are homozygous in all three globin loci with the genotype aFaF, pFspFs, pFfpFf, but the regulator gene for the pFvFs locus is polymorphic (or one of the P-chain loci is polymorphic for a null allele). The regulator gene with both alleles HH (phenotype 111) is expressed by full amount of the pFyFs gene product (PFs) (which is still 1.8 times the amount of the pFfpFf gene product), DH is expressed by a reduced amount (phenotype 11), and DD results in no detectable product (phenotype I). This model is as compatible with the observations as the model with the diallelic locus pFyFs, and cannot be distinguished from the latter without detailed quantitative measurements of the Hbs. Such a regulator gene polymorphism may be similar to polymorphisms in man and other vertebrates (e.g., various types of P-thalassemia (Weatherall and Clegg 1976)).

The lack of common globins for the Hb A and Hb C of

chinook salmon agrees with findings for other salmonids (Wilkins 1985). For the main cathodal bands (2C4C), which are present in all individuals, a minimum of three or four structural loci is required. None of these loci seem to be polymorphic in chinook salmon. Allelic Hb polymorphisms have been reported in cathodal components of Arctic charr, Salvelinus alpinus (Giles and Rystephanuk 1989), and probably also in cutthroat trout, Salmo clarkii (Braman et al. 1980). Further studies are necessary to reveal the genetic basis and biological significance of the polymorphisms in salmonid Hbs and their value in breeding experiments and for stock identification.

Acknowledgments The staff of the hatcheries at Big Qualicum, Conuma, and

Quesnel rivers kindly made mature chinook salmon available for blood sampling. Henrik Kreiberg and Alvin Solmie assisted in the sampling of captive salmon. Debra Tuck was helpful in the laboratory. Jan Whyte kindly offered us access to necessary equipment. Maria S. Evjen prepared the figures. Unni E. H. Fyhn acknowledges support from the Norwegian Fisheries Research Council.

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