Hereditas 123: 191-195 (1995)
Brief report
Genetic variability at minisatellite and allozyme loci in brown trout
(Salmo trutta) -A comparison
LINDA LAIKRE',*, PAUL0 A. PRODOHL2.3, PER ERIK JORDE' and NILS RYMAN'
Division of Population Genetics, Stockholm University, S - 106 91 Stockholm, Sweden
School of Biology and Biochemistry, Queen's University of Belfast, Berfast BT7 INN, Northern Ireland,
UK
Present address: Department of Genetics, University of Georgia, Athens, GA 30602, USA
I
(Received June 9, 1995. Accepted October 25, 1995)
Most of our present knowledge regarding the
genetic structure of natural animal and plant
populations has been derived from protein electrophoretic studies. New molecular techniques are
evolving quickly, however, and increased attention
is focused on the analysis of mini- and microsatellite loci (e.g., PENAet al. 1993; QUELLERet al.
1993; AVISE1994). Techniques for analyzing these
types of loci are presumed to be of great interest
to population biologists because of their high
resolution power in relation to that of protein
electrophoresis (AMOSand HOELZEL1992; BRUFORD and WAYNE 1993a). It is important to
evaluate the extent to which allozyme and mini-/
microsatellite data provide approximately the
same overall picture of the population genetic
parameters of interest ( CHAKRABORTY
et al.
1992). To date, the number of studies comparing
population genetic data for the same set of samples using both types of loci is still limited (e.g.,
CHAKRABORTYet al. 1992; BRUFORD and
WAYNE1993b; SCRIBNER
et al. 1994).
We have analyzed brown trout (Salmo trutta)
samples using three minisatellite and 16 protein
electrophoretic loci. The results indicate that although the minisatellite loci show higher levels of
genetic variability, the two sets of markers are
equally efficient in delineating genetic differentiation as evaluated from contingency tests for allele
frequency homogeneity and gene diversity statistics (GST)in brown trout.
lakes in the province of Jamtland, central Sweden
(Table I). Populations A and B represent naturally
derived first generation offspring (F,) from stocks
with a hatchery background which had previously
been transplanted into one of the sampled lakes.
The C group of fish represents naturally produced
hybrid offspring between these two stocks. Discrimination between A, B, and C in the same lake
was possible through genetic marking-transplanted fish from the A and B stocks were homozygous for alternate alleles at the G3PDH-2*
allozyme locus, resulting in C being heterozygous
at this locus; all fish from A-C were from the F,
generation as determined by otolith ageing. The
fourth sampled population, D, represents a natural
brown trout population from a nearby lake that
lacks connection with the lake containing A, B,
and C. These four populations and additional ones
in the same area are subject to an ongoing,
longterm population genetics study (RYMANet al.
1986).
Allozyme loci were selected on the basis of the
prior information that they are polymorphic in
Scandinavian brown trout. Similarly, the minisatellite loci were selected based on the knowledge that
they were polymorphic in Irish and Icelandic
brown trout populations, although no information
regarding their variability in brown trout in Scandinavia was available prior to our present study
(PRODOHLet al. 1994a).
Allozyme analysis
Materials and methods
A total of 60 brown trout from four populations
(A-D) were collected in 1991 from two separate
* Corresponding author.
Electrophoretic procedures and genotype scoring
followed ALLENDORF
et al. (1977), AEBERSOLD
et
al. (1987), JORDEet al. (1991), and JORDE(1994).
The following 16 enzyme loci where scored (enzyme name and Enzyme Commission number in
192
Hereditas I23 (1995)
L. LAIKRE ET AL.
Table 1. Summary measures of genetic variability at allozyme and minisatellite loci in brown trout from Sweden. ( H =expected
heteroaygosity, P = percent polymorphic loci, A = average number of alleles per locus). X*-values refer to tests for allele frequency
homogeneity among all four populations, summed over all variable loci
Population
A
B
C
D
Total
Source
Hatchery
background
Hatchery
background
Hybrid
AxB
Natural
population
Number
of fish
Allozyme data (16 loci)
Minisatellite data (3 loci)
H
P
Total number
of alleles
A
20
0.24
69
27
20
0.16
56
10
0.19
Number of
unique
genotypes
H
P
1.7 20
0.40
100
25
1.6
17
0.52
62
26
1.6
9
10
0.23 75
28
1.8
60
0.24 88
30
A
Number of
unique
genotypes
8
2.7
13
100
8
2.7
14
0.44
100
7
2.3
7
10
0.66
100
14
4.1
10
1.9 55
0.59
100
16
5.3
39
G,, = 0.16 i0.04
x2 = 63.9, df = 9, P << 0.001
G, = 0.17 i- 0.05
x2 = 213.0, df = 39, P<<0.001
parentheses): sAA T-4* (aspartate aminotransferase, 2.6.1. l), CK-2* (creatine kinase, 2.7.3.2),
DIA* (diaphorase, 1.6.2.2), bCALA -2* (P-Nacetylgalactosaminidase, 3.2.1.53), bCL UA * (Nacetyl-P-glucosaminidase, 3.2.1.30), IDDH- 1 *
(L-iditol dehydrogenase, 1. I . 1.14), slDHP- 1 *
(isocitrate dehydrogenase (NADP +), 1.1.1.42),
LDH-l*,-5* (lactate dehydrogenase, I . 1.1.27),
aMAN* (a-mannosidase, 3.2.1.24), sMDH-2*,
-3,4* (malate dehydrogenase, 1.1.1.37), ME*
(malic enzyme (NAD+), 1.1.1.39), MPZ* (mannose-6-phosphate isomerase, 5.3.1A), PEPLT*
(leucyl-tyrosine peptidase, 3.4.-.-). In addition to
these 16 loci, G3PDH-2* (glycerol-3-phosphate dehydrogenase, 1.1.1.8) was analyzed on fish from
one of the lakes for discrimination between stocks
A and B and their hybrid C, but this locus was
excluded from subsequent statistical analyses.
Total number
of alleles
been confirmed at these loci, and their characteristics have been described in detail by PRODOHL
et
al. (1994a, b, in prep.).
Results and discussion
None of the allozyme loci segregated for more than
two alleles. Two of the 16 allozyme loci were
monomorphic in all of the present populations,
and the other 14 were polymorphic in one or more
of the populations. A total of 7, 4, and 5 alleles
were observed at the Str-AS, Str-A3, and SsaA45/ 1 minisatellite loci, respectively.
The total amount of genetic variability observed
at the allozyme loci is typical for that found at the
loci examined in brown trout in Scandinavia (RYMAN 1983). To our knowledge, no previous data
have been published on minisatellite variability in
Scandinavian brown trout. Comparisons can be
made, however, with the number of alleles found at
Minisatellite analysis
the same three minisatellite loci in Irish and IceTotal genomic DNA was isolated from muscle landic brown trout populations (PRODOHLet al.
tissue following the simplified methodology of 1994a). The number of alleles observed in our
TAGGARTet al. (1992). Standard protocols were present study is somewhat lower than that reported
followed for gel electrophoresis (3 pg Pal I di- by PRODOHLet al. (1994a), but not markedly so.
gested DNA per sample), Southern blotting (onto The difference is most likely due to the larger
Hybond N membrane), and isotopic labelling of sample sizes of that study (cf. EWENS1979) and to
probe (random priming). Prehybridization and hy- the hatchery background of populations A-C (see
bridization conditions as well as posthybridization below).
procedures followed those described by PRODOHL
There are highly significant allele frequency hetet al. (1994a).
erogeneities among populations at both sets of loci
Three minisatellite loci were studied, Str-A3, (2 x 4 x2 contingency tests with pooling of alleles
Str-AS, and Ssa-A45/1. Mendelian inheritance has into two classes at the minisatellite loci, accounting
Herediras 123 (1995)
for low expected values, and summing the x2 values yields P<<0.001 in both cases; Table 1).
The amount of genetic variability observed at
the two sets of loci is summarized in Table 1. The
(expected) average heterozygosity, the proportion
of polymorphic loci, and the average number of
alleles per locus are all conspicuously higher for
the minisatellite loci.
The D population exhibits the generally highest
level of genetic variation at both sets of loci (Table
I). The lower amount of genetic variation in populations A-C is most likely a reflection of their
recent hatchery backgrounds (hatchery propagation is frequently based on few parental fish). For
the minisatellite loci the number of alleles is significantly higher in population D as compared to A,
B, and C (Mann-Whitney rank sum statistics yields
P < 0.05), but the difference is not significant at the
allozyme loci. This observation is in accordance
with the expectation that loci with many alleles
lose more alleles during a population size bottleneck than loci with few alleles ( N E Iet al. 1975).
The proportion of the total gene diversity attributable to differences between populations
(G,,, NEI 1987) is almost identical for the two sets
of loci (Table 1). In contrast, pairwise genetic
identity values ( N E I 1972) range from 0.886 to
0.985 at isozyme loci and from 0.594 to 0.957 at
minisatellite loci, which results in larger genetic
distance values at minisatellite loci. These observations are consistent with a higher mutation rate at
minisatellite loci, which affects genetic identity,
whereas for selectively neutral alleles G,, is largely
independent of the mutation rate and primarily
represents a reflection of the population structure
(CHAKRABORTY
and LEIMAR1987; NEI 1987 and
references therein).
Our observations are somewhat different from
those of SCRIBNER
et al. (1994), who compared
variability patterns in three populations of the
common toad (Bufo hujb) for different sets of
genetic markers including six allozyme and three
minisatellite loci which were all polymorphic. For
example, both the GSTvalues and their variances
for the two sets of loci are very similar in our study
(Table I ) , whereas SCRIBNER
et al. (1994) report a
considerably higher average F,, value (and variance) for the allozyme loci. Further, we found
more unique multilocus genotypes at the allozyme
than at the minisatellite loci (55 vs. 39; Table 1)
whereas the opposite was observed by SCRIBNER
et
al. (1994). For the six most variable allozyme loci
in our present study, the total number of multilo-
BRIEF REPORT
193
cus genotypes is about the same as that observed at
the three minisatellite loci (40 vs. 39). It is
presently not clear if this discrepancy between the
brown trout and the toad is due to a larger number
of toads being analyzed (120 vs. 60 brown trout)
or the greater number of polymorphic allozyme
loci in our study (14 vs. 6). It must be emphasized,
however, that the present study, as well as that of
SCRIBNER
et al. (1994), were based on very few
minisatellite loci.
In the present context it is of interest to compare
the power of the two sets of loci for detection of
population structuring as reflected by deviations
from Hardy-Weinberg expectations. For this purpose we pooled the data from the 50 fish representing samples A-C, considering them a single
sample from a single lake (which they are, but we
have additional information about their background). On this pooled material we tested for
deviations from Hardy-Weinberg expectations, applying an extended version of VITHAYASAI’S
( 1973) exact method ( LAGERCRANTZ
and RYMAN
1990). We expect to find deviations from HardyWeinberg proportions (deficiency of heterozygotes)
because of the significant genetic heterogeneity
among these three stocks at both sets of loci (contingency x2 tests for allele frequency homogeneity
yields P << 0.001 in both cases). For the allozyme
loci, one of 1 1 tests showed a clearly significant
deficiency of heterozygotes ( P = 0.003), whereas
none of the three tests was significant for the
minisatellite loci.
In summary, it is obvious that the minisatellite
loci exhibit a larger degree of genetic variability
than the allozyme loci. However, in the present
study there is no clear difference between the two
sets of loci in the ability to detect and quantify
genetic population structuring as revealed by contingency tests and G,, statistics. Both the time
required to go from tissue sample to genotype and
the cost for consumables are considerably higher
for the minisatellites as compared to the allozymes.
Taking all of these factors into account our results
suggest that, for addressing questions relating to
population structure, allozymes may be advantageous as compared to minisatellites for a species
like the brown trout, for which a large number of
variable allozyme loci have been identified.
Mini- and microsatellites are currently fashionable within the field of population biology, but it
appears that protein electrophoresis may in many
cases still be a preferable tool (UTTER1991; AVISE
1994; PARKand MORAN1994; UTTER1994). It is
194
L LAIKRE ET AL.
Heredims 123 (1995)
obvious, however, that for species that require
non-destructive sampling or those exhibiting low
levels of protein electrophoretic variability, miniand microsatellite loci provide a valuable tool.
Likewise, studies warranting a large number of
alleles, such as problems relating to population
bottlenecks, may benefit from mini- or microsatellite
genotype data (MARTINSON
et al. 1993; MENOTTIRAYMONand OBRIEN 1993). Clearly, it is ideal to
analyze as many different types of genetic markers
as possible, and reports of discrepancies between, for
instance, allozyme and RFLP data (KARL and
AVISE1992; POGSON
et al. 1995) stress the need for
further studies including both allozyme and various
DNA marker data (ALLENDORF1994).
Of course, the present study is small and therefore
not generally conclusive. It is important, however,
that results are reported so that, in combination with
findings of other studies, they aid in providing
information on the relationship between genetic
markers reflecting different parts of the genome.
Acknowledgements. -We thank Andrew Ferguson and John
Taggart for valuable discussions and suggestions. The facilities
made available to L. Laikre during a visit to the School of
Biology' and Biochemistry, Queen's University of Belfast, Belfast,
are gratefully acknowledged. The study was supported by a grant
to N. Ryman from the Swedish Natural Science Research Council. The award of a Postgraduate Studentship to P. A. Prodohl by
the Brazilian Federal Agency of Postgraduate Education
(CAPES-No. 487/89-5) and a scholarship from Nilsson-Ehlefonden, Kungl. Fysiografiska Sallskapet i Lund to L. Laikre are
greatly appreciated.
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