Journal of Fish Biology (1991) 39,45 1 4 5 4
BRIEF COMMUNICATION
New biochemical genetic markers in the brown trout (Salmo
trutta L.)
P. E. JORDE, A. GITTAND N. RYMAN
Division of Population Genetics, Stockholm University, S-106 91 Stockholm. Sweden
(Received 29 March I99 I , Accepted 5 May I99 I )
Three ' new ' polymorphic loci in the brown trout (Sulmo t r w u L.) was detected using starch gel
electrophoresis. Evidence for simple Mendelian inheritance at all three loci are presented.
Key words: brown trout; genetic markers; isozymes.
Polymorphic isozyme loci have proven to be valuable markers for use in studying the
genetic population structure of brown trout (Salmo trutta L.) and other salmonids (e.g.
Allendorf & Utter, 1979; Ryman, 1983; Ryman & Utter, 1987; Ferguson, 1989). This note
extends the knowledge of such loci in brown trout. We describe details of variation at
three previously unreported polymorphic loci (bCLUA*,a M A N * and P E P L P ) , including
electrophoretic conditions, methods for visualization, and the cumulative evidence for their
Mendelian inheritance.
Sampling and preparation of skeletal muscle, liver, and eye tissues and electrophoretic
techniques generally followed those of Allendorf et af. (1976, 1977) and Aebersold et al.
(1987). The nomenclature for loci and alleles follows the recommendations given by
Shaklee et al. (1990). Two continuous buffer systems were used for electrophoretic separations, viz. ( I ) AC6.5 (modified from Clayton & Tretiak, 1972):0.04 M citric acid adjusted to
pH 6.5 with N-(3-aminopropyl)-morpholine,and (2) TP8 (modified from Guyomard &
Krieg, 1983): 0.1 M Tris adjusted to pH 8.0 with monobasic sodium phosphate. Gels were
prepared from 1 1.5% hydrolysed starch and 1 :20 (AC6.5) or 1 : 10 (TP8) dilutions of these
buffers, which were used undiluted for the electrode trays. The agar overlay technique
(Harris & Hopkinson, 1976; Aebersold et al., 1987) was used for all enzyme stainings.
N-acetyl-P-glucosaminidase (bGLUA: EC 3.2.1.30) was visualized using liver extracts
separated in the TP8 buffer. Gels were stained with naphthol-AS-BI-N-acetyl-P-Dglucoseaminide (40 mg) and Fast Garnet GBC (20 mg) in 30 ml 0.1 M citrate/phosphate
buffer at pH 4.5 (Harris & Hopkinson, 1976). A single anodally migrating zone, exhibiting
three distinct phenotypes, was presumed to represent the expression of a single locus
(bGLUA*)segregating for the two alleles bGLUA*100 and *I50(Fig. 1). The three-banded
phenotype of the alleged heterozygous individuals corresponds with the pattern expected
for this dimeric protein (Aebersold et al., 1987). The variation observed for bGLUA* in
brown trout appears similar to that recently reported for rainbow trout [Oncorhynchus
mvkiss (Walbaum), formerly Salmo gairdneri Richardson; Reisenbichler & Phelps, 19891.
a-Mannosidase (aMAN: 3.2.1.24) was also detected in liver extracts, using electrophoretic conditions identical to those used for bGLUA. Isozymes were visualized using the
fluorogenic substrate 4-methylumbelliferyl-a-~-mannopyranoside
(10 mg) in 30 ml 0.1 M
citrate/phosphate buffer at pH 9.5 (Aebersold et al., 1987). Under ultraviolet light, we
detected one anodally migrating zone of fluorescence with three different phenotypes
(Fig. 1). The two single-banded phenotypes were interpreted to be the homozygous genotypes of the alleles aMAN*100and *70, respectively, segregating at a singlelocus (aMAN*).
The third, presumed heterozygous, phenotype was not resolved into distinct bands,
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0022-1 112/91/090451+04 $03.00/0
0 1991 The Fisheries Society of the British Isles
452
P. E. JORDE E T A L .
FIG.1. Zyrnograrn patterns of bGLUA, a M A N and PEPLT in brown trout. Electrophoretic phenotypes
were interpreted as (a) I j l , (b) 1/2 and (c) 2j2, where I denotes the common (*100) and 2 denotes
the variant allele at each particular locus. The relative positions of the various gene products are
indicated to the right.
denotes the anodal part of gel.
+
N E W GENETIC MARKERS I N B R O W N TROUT
453
suggesting the presence of one or more heteromeric isozymes. The quaternary structure of
aMAN appears to be polymeric in mammals (Cheng et al., 1986), but the actual number of
subunits is uncertain (Aebersold et al., 1987; Morizot & Schmidt, 1990).
Leucyl-tyrosine peptidase (PEPLT: EC 3.4.-.-), the present designation is tentative as we
have been unable to establish a clear homology between this enzyme and any of the major
peptidases on the basis of substrate specificity, quaternary structure, or tissue distribution
(cf Frick, 1983). PEPLT was detected in all tissues examined. Electrophoretic separation
was carried out in the AC6.5 buffer and the enzyme was visualized using L-leucyl-L-tyrosine
(20 mg), snake venom (10 mg, from Western diamondback rattlesnake), peroxidase (5 mg,
from horseradish), and 3-amino-9-ethyl carbazole (10 mg dissolved in I ml DMSO) in 30 ml
0.1 M sodium phosphate buffer at pH 7 (Harris & Hopkinson, 1976). Two major zones of
peptidase activity are apparent in the zymograms (Fig. 1). The less anodal zone displays
three different phenotypes, presumed to be coded for by a single locus ( P E P L P ) segregating for the two alleles PEPLPIOO and *70. The quaternary structure varies among
peptidases (Harris & Hopkinson, 1976; Frick, 1983); the electrophoretic expression of the
present one is consistent with that of a monomeric enzyme. A genetic basis of the variation
is strongly suggested by the consistently parallel expression of the same phenotypic pattern
in all three tissues routinely examined. The more anodal zone represents a peptidase with a
higher pH optimum and a somewhat different substrate affinity; it was assumed to reflect
the expression of an additional peptidase-coding locus. No variation was detected in this
poorly resolved zone.
Information bearing upon the inheritance of the observed variation was obtained from
comparisons of allele frequencies among the offspring from spontaneous matings between
and within two stocks of brown trout characterized by different allele frequencies at all
three loci. These two stocks were genetically tagged (by differential homozygosity at the
G3PDH-2* locus (formerly Agp-2);Stihl& Ryman, 1982)and had been transplanted into a
common water system previously devoid of this species. Original transplants (19 fish) and
their first generation descendants (186 fish) were sampled 10 years later and analysed by
electrophoresis and otolith ageing.
It was found that despite significant (at the 5% level) differences in allele frequency
between the stocks at each of the three loci, no such differences were detected between
parents and offspring within either stock. Furthermore, for each locus we found that (1)
there was no significant difference in allele frequency between sexes, (2) the hybrid group
(heterozygotes at the G3PDH-2* locus) had an allele frequency intermediate between the
two parental stocks, and (3) there was no significant deviation from Hardy-Weinberg
proportions, except among the hybrids, where a significant excess of heterozygotes was
found at one locus (aMAN*).
While not as conclusive as techniques such as family analysis, our observations are
consistent with Mendelian inheritance at all loci, justifying their inclusion in studies
of brown trout population genetics. In the natural and hatchery populations from
Scandinavia and Spain that have been studied to date, the frequency of the variant allele
ranges from M . 8 4 , from C0.56, and from W . 4 7 at bGLUA*, aMAN* and P E P L P ,
respectively.
We thank Dr F. Utter for comments on an earlier version of the manuscript. Financial support was
obtained from the Swedish Natural Science Research Council.
References
Aebersold, P. B., Winans, G. A., Teel, D. J., Milner, G. B. & Utter, F. M. (1987). Munualfor Starch Gel
Electrophoresis: A Method,for the Detection of Genetic Variation. NOAA Technical Report NMFS
61.
Allendorf, F. W., Mitchell, N., Ryman, N. & Stihl, G . (1977). Isozyme loci in brown trout (Sulmo truttu L.):
detection and interpretation from population data. Hereditas 86, 179-190.
Allendorf. F., Ryman, N., Stennek, A. & Stlhl, G . (1976). Genetic variation in Scandinavian brown trout
(Salmo truttu L,): evidence of distinct sympatric populations. Hereditas 8 3 , 7 3 4 2 .
Allendorf, F. W. & Utter, F. M. (1979). Population genetics. In Fish Physiology, Vol. 8 , (Hoar, W. S.,
Randall, D. J. & Brett, J. R., eds), pp. 407-454. New York: Academic Press.
454
P. E. JORDE E T A L .
Cheng, S. H., Malcolm, S., Pemble, S. & Winchester, B. (1986). Purification and comparison of the
structures of human liver acidic a-D-mannosidases A and B. Biochemical Journal 233,65-72.
Clayton, J. W. & Tretiak, D. N. (1972). Amine-citrate buffers for pH control in starch gel electrophoresis.
Journal of the Fisheries Research Board of Canada 29,1169-1 172.
Ferguson, A. (1989). Genetic differences among brown trout, Salmo trutta, stocks and their importance for
the conservation and management of the species. Freshwater Biology 21.3S46.
Frick, L. (1983). An electrophoretic investigation of the cytosolic di- and tripeptidases of fish: molecular
weights, substrate specificities, and tissue and phylogenetic distributions. Biochemical Genetics 21,
309-322.
Guyornard, R. & Krieg, F. (1983). Electrophoretic variation in six populations of brown trout (Salmo trutta
L.). Canadian Journal of Genetics and Cytology 25,403413.
Harris, H. & Hopkinson, D. A. (1976). Handbook of Enzyme Electrophoresis in Human Genelics.
Amsterdam: North-Holland.
Morizot, D. C. & Schmidt, M. E. (1990). Starch gel electrophoresis and histochemical visualization of
proteins. In Electrophoretic and Isoelectric Focusing Techniquesin Fisheries Management (Whitmore,
D. H., ed.),pp. 23-80. Ann Arbor: CRC Press.
Reisenbichler, R. R. & Phelps, S. R. (1989). Genetic variation in steelhead (Salmogairdneri)from the north
coast of Washington. Canadian Journal of Fisheries and Aquatic Sciences 46,6673.
Ryman, N. (1983). Patterns of distribution of biochemical genetic variation in sdlmonids: differences
between species. Aquaculture 33,l-21.
Ryman, N. & Utter, F. (1987). Population Genetics and Fishery Management. Seattle, WA: University of
Washington Press.
Shaklee, J. B., Allendorf, F. W., Morizot, D. C. & Whitt, G. S. (1990). Gene nomenclature for proteincoding loci in fish. Transactions of the American Fisheries Society 119,2-15.
Stihl, G. & Ryman, N. (1982). Simple Mendelian inheritance at a locus coding for a-glycerophosphate
dehydrogenase in brown trout (Salmo trutta). Hereditas 96,313-315.