overall survival of embryos was maximal
(56% and 62%, respectively). Thus, any
pressure from 6140-6840 psi may be used
to generate half-tetrad embryos, with maximal survival at the two lowest pressures,
6140 and 6240 psi.
An additional prediction of successful
suppression of the second meiotic disjunction by EP is the production of triploid zygotes when eggs are fertilized with
nonirradiated sperm and subjected to EP
(Figure 3). Eggs from a single clutch were
fertilized using nonirradiated sperm and
divided into two groups: one was subjected to EP and the other group was allowed
to complete the second meiotic division
and develop normally. Lane 1 in each
group shows the maternal genotype, and
the subsequent lanes show embryo genotypes. SSR genotypes show that embryos
exposed to EP were triploid (Figure 3A),
while the normal embryos were diploid
(Figure 3B). The lower panel also indirectly indicates that the pooled sperm DNA
contains all three alleles. The evidence
consistent with triploidy consists of the
presence of three alleles for SSR 15 in 6
out of the 18 triploids (third, fifth, seventh,
and last three embryo lanes). Furthermore, when only two alleles were present,
one was darker than the other, consistent
with the presence of two copies of the
darker allele. In contrast, untreated diploid embryos allowed to complete normal
meiosis and fertilization contained only
one or two of the parental alleles; when
two alleles were present, they were of
equivalent intensity.
Generation of triploids in zebrafish may
be used to study the effects of gene dosage on recessive and dominant mutations
(Ashburner 1989). Application of this pressure-based generation of triploids offers
an alternative to heat shock for generation
of triploids in other organisms in which
sterile animals may be desired to control
overpopulation, and in which triploids
may be associated with increased growth
rates, final size, or survival in mature fish
(Thorgaard 1983).
In order to determine whether the derived pressure of 6240 psi produced results consistent with previously published
data (Streisinger et al. 1986), we determined the frequency of golden half-tetrad
embryos produced by six heterozygous
golden mothers in a wild-type background.
Of 506 half-tetrad embryos, 27 (0.053 of total) were phenotypically golden. Assuming
reciprocal genetic exchange and the absence of recessive lethal mutations in the
region, the frequency of half-tetrads that
are homozygous wild type at the golden
locus is equal to the frequency of golden
homozygotes. Therefore, the frequency of
golden heterozygotes is 0.89, or l-2m,
where m is the frequency of golden homozygotes. This is identical to the value
derived by Streisinger et al. (1986) from
the examination of 1,151 half-tetrad progeny of golden heterozygotes in a wild-type
background.
In conclusion, SSR genotypes of embryos were determined to confirm the generation of gynogenic half-tetrads using specific pressures. Similar microsatellite analysis may be used to confirm the generation of candidate half-tetrad or triploid
embryos in any organism for which microsatellite markers are available. Utilizing
our improvements in the EP technique,
linkage data deriving from these experiments have been successfully used to confirm strong chiasma interference in the zebrafish, map centromeres, and confirm
linkage of nearby markers (Kauffman et al.
1995). Half-tetrad analysis was used to
map the remaining centromeres to the
current linkage map (Johnson et al. 1996)
and may increase our understanding of
chiasma interference. Finally, half-tetrad
gynogenesis has been used to screen for
recessive mutations in carrier females at
the University of Oregon (Johnson and
Weston 1995; Henion et al. 1996). The
methods described herein are successfully being used to find mutant screens in
progress (Moore JL, Gestl E, Tsao-Wu G,
Cheng KC, unpublished data).
From the Division of Experimental Pathology (Gestl,
Kauffman, Moore, and Cheng) and the Department of
Biochemistry and Molecular Biology (GesU and
Cheng), Milton S. Hershey Medical Center, Hershey, PA
17033. We thank M. Westerfield for his support and
hospitality, S. Johnson and C Walker (University of Oregon) for demonstrating EP at the U. of Oregon and
Cliff Tabln (Harvard) for providing the SSRs. This work
was supported by the Jake Glttlen Memorial Golf Tournament and grants from the American Cancer Society
(IRG-196 and JFRA-581), the Four Diamonds Fund, and
the National Science Foundation (MCB-9317817) toKX.
Address correspondence to K. C. Cheng, Division of Experimental Pathology C7804, 500 University Dr., Hershey, PA 17033.
The Journal of Heredity 1997*8(1)
Reference*
Ashburner M, 1989. Dmsophilcr. a laboratory handbook.
Cold Spring Harbor, New York; Cold Spring Harbor Laboratory Press.
Concordet J-P and Ingham P, 1994. Catch of the decade.
Nature 369:19-20.
Goff DJ, Gahin K, Katz H, Westerfield M, Lander ES, and
Tabln CJ, 1992. Identification of polymorphic simple sequence repeats In the genome of the zebrafish. Genomlcs 14200-202.
Henion PD, Ralble DW, BeatUe CE, Stoesser KL, Weston
JA, FJsen JS, 1996. Screen for mutations affecting development of zebrafish neural crest. Dev Genet 18:1117.
Johnson SL, Africa D, Home S, and Postlethwalt JH,
1995. Half-tetrad analysis In zebrafish: mapping the ros
mutation and the centromere of linkage group I. Genetics 139:1727-1735.
Johnson SL, Gates MA, Johnson M, Talbot WS, Home
S, Balk K, Rude S, Wong JR. and Postlethwait JH, 19%.
Centromere-linkage analysis and consolidation of the
zebrafish genetic map. Genetics 142:1277-1288.
Johnson SL and Weston JA, 1995. Temperature-sensitive mutations that cause stage-specific defects In zebrafish fin regeneration. Genetics 141:1583-1595.
Kahn P, 1994. Zebrafish hit the big time. Science 264:
904-905.
Kaulfman EJ, Gestl EE, Walker C, Hite JM, Yan G, Rogan
PK, Johnson SL, and Cheng KC, 1995. Mlcrosatellltecentromere mapping In the zebrafish (Damo rerio).
Genomics 30337-341.
Kavumpurath S and Pandian TJ, 1990. Induction of triploidy In the zebrafish, Bmchydanio rerio (Hamilton).
Aquacult Fish Manage 21 299-306.
Mulllns MC, Hammerschmidt M, Haffter P, and NOssleln-Volhard C, 1994. Large-scale mutagenesls In the
zebrafish: In search of genes controlling development
In a vertebrate. Curr Blol 4:189-202.
Solnlca-Krezel L, Schler AF, and Driever W, 1994. Efficient recovery of ENlHnduced mutations from the zebrafish germllne. Genetics 136.1401-1420.
StrShle U and Ingham PW, 1992. Flight of fancy or a
major new school? Curr Blol 2.135-138.
Streisinger G, Singer F, Walker C, Knauber D, and Dower
N, 1986. Segregation analyses and gene-centromere distances In zebrafish. Genetics 112:311-319.
Streisinger G, Walker C, Dower N, Knauber D, and Singer F, 1981. Production of clones of homozygous dlplold
zebra fish (Bmchydanio rerio) Nature 291:293-2%.
Thorgaard GH, 1983. Chromosome set manipulation
and sex control In fish. In: Fish physiology, vol. IX, Reproduction, part B, Behavior and fertility control (Hoar
WS, Randall DJ, Donaldson EM, eds). New York: Academic Press, 405-434
Westerfield M, 1995 The zebrafish book. Eugene, Oregon: University of Oregon Press.
Received December 29, 1995
Accepted May 20, 1996
Corresponding Editor Robert Wayne
Applications of Single Locus
Minisatellite DNA Probes to
the Study of Atlantic Salmon
(Salmo salar L.) Population
Genetics
J. P6rez, P. Mordn, A. M. Pendas,
and E. Garcfa-VAzquez
Five single-locus minisatellite probes
(SLPs) derived from Salmo salar have
been used to describe genetic variability
of Atlantic salmon in a Spanish river
(Esva). A total of 202 individuals (48 juveniles and 154 returning adults) were analyzed in 1992 and 1993. The five loci
were highly polymorphic, with the number
Brief Communicatjoris 7 9
of different alleles per locus ranging from
4 to 10. In adult samples, allele polymorphism was demonstrated to be independent of both sex and sea returning age.
Between-generation stability in frequencies for the different alleles was measured
comparing samples from consecutive
generations. Results indicate the utility of
SLPs for the study of Atlantic salmon population genetics.
During the past two decades, protein electrophoresis has been used as the main
tool to examine the genetic structure of
fish populations (Utter 1991). Yet the dependence on a small number of polymorphic loci and the existence of fitness differences among alleles of a particular locus have limited the understanding of
population dynamics of many fish species.
On this basis, the development of molecular fingerprinting technologies has
revolutionized studies of genetic variation.
In comparison to the complex patterns detected using minisatellite probes (Jeffreys
et al. 1985; Taggart and Ferguson 1990b),
single-locus probes (SLPs) from hypervariable noncoding regions of nuclear
DNA (VNTR) have simplified the study of
species like Atlantic salmon (Salmo salar
L.). Although SLPs were first used to identify individuals and trace parentages (Debenham 1992), the extensive allelic variability detected in many species may represent an important advantage over previous methods of population genetics
analysis such as isozyme and mitochondrial DNA studies (Bentzen et al. 1991).
The Atlantic salmon is one of the most Important sport fish species in the western
world and an important fish for commercial aquaculture. In this species, lack of appropriate allozyme and mitochondrial
DNA markers (Davidson et al. 1989) does
not allow evaluation of population enhancement programs or of the genetic effects of escaped fanned salmon on natural
populations. Atlantic salmon exhibit low
levels of allozyme variation. Polymorphism at five allozyme loci accounts for
more than 90% of the total allozyme variation (Stahl 1987), and genetic selection
has been implied for one of the most polymorphic ones, malic enzyme MEP-2* (Verspoor and Jordan 1989). The identification
and cloning of several specific Salmo salar
SLPs (Prodohl et al. 1994a; Taggart and
Ferguson 1990a) with a high number of alleles detected per locus, may allow a better understanding of population structure.
The SLP hypervariable loci are molecular markers, thus they could be consid-
8 0 The Journal of Heredity 1997:88(1)
ered to be selectively neutral; they present single-locus inheritance patterns not
sex-linked patterns (Taggart et al. 1995).
Therefore they offer a major advantage in
comparison to isozyme and mitochondrial
markers.
The Atlantic salmon shows considerable variation in life-history components.
For example, parental brood stocks show
a generation overlap. Fish can return to
the river after 1, 2, or more years in the
sea. On the other hand, juveniles can migrate to the sea after a variable number of
years spent in the river (in northern
Spain, typically one or two). These two
characteristics imply that fish hatched in
three or four different years can join together in the same reproductive population. Genetic stability in Atlantic salmon
populations depends on the existence of
genetic homogeneity between generations, which are composed of variable proportions of adults born in different years.
In this work we studied patterns of genetic variation using five Salmo salar single-locus probes in a Spanish Atlantic
salmon population. The objectives were to
determine the degree of between-generation genetic stability and to test the actual
neutrality of SLP allelic frequencies with
respect to sex and time spent at sea (sea
returning age).
Material and Methods
Fish Samples
Forty-eight Atlantic salmon juveniles of
the same year class were collected during
September 1993 by electrofishing throughout the whole spawning area of the Esva
River (northern Spain). Muscle samples of
58 and 96 returning adult salmon were collected in the same river in 1992 and 1993,
respectively, during the legal angling seasons (March-July). The proportion of the
fish analyzed from the total sport catch
(official records) was 35% in 1992 and 30%
in 1993. These values represent 10-15% of
the total river population estimated
through redd counts (Garda-Vazquez E et
al., unpublished data).
Sex and Age Determination
Ages were determined by scale analysis as
described by Bagliniere (1985). Age classes were assigned a two-digit number, with
the first indicating river age and the second sea age; that is, 1.1 refers to a salmon
that has spent 1 year in the river and 1
year in the sea. Sex determination was
based on the detection by immunoaglutination of the serum vitellogenin of females,
following the method of Le Bail and Breton
(1981).
DNA Analyses
Total DNA was isolated from muscle tissue
following the method of Taggart et al.
(1992) and digested (4 (ig of DNA/individual) with Pall (Pharmacia) following manufacturer's recommendations. Restriction
fragments were separated by electrophoresis through 0.7% agarose gels in 1X TAE
buffer (Sambrook et al. 1989), transferred
to nylon membrane (Hybond-N Amersham) by capillary action (Southern
1975), and immobilized on the filters by
cross-linking. Hybridization was carried
out at 65°C in 1.5x SSPE (1.5x SSPE is 0.27
M NaCl, 15 mM sodium phosphate pH 7.7,
1.5 mM EDTA), 0.5% dried milk, 1% SDS,
and 6% polyethyleneglycol. Probes were
^P-labeled according to Dalgleish (1987).
The five SLPs used in this work were pSsaA45/1, pSsaA45/2, pStr-A22/l, pStr-A9, and
pSsa-A60, kindly provided by Dr. J. Taggart, Dr. P. Prodohl, and Dr. A. Ferguson.
Nomenclatures employed followed Prodohl et al. (1994b). After 12 h of hybridization, blots were washed for 30 min in 2x
SSC 0.1% SDS at 65°C and 0.2X SSC, 0.1%
SDS at 65°C. The results were visualized
after exposure to Kodak X-OMAT-S film at
-80°C with two intensifying screens for 2
days.
Alleles for each of the loci were designated sequentially by the molecular
weight of the different DNA fragments,
with allele A representing the DNA fragment of the highest molecular weight.
Sizes were estimated by consecutive running and probing with different commercial DNA markers (Boehringer Mannheim). Analyses were performed using the
BIOSYS-1 computer package (Swofford
and Selander 1989). Chl-square analysis
was used to test heterogeneity of allele frequencies among all the samples. Levels of
statistical significance were taken according to the Bonferroni approach. Tests of
conformation to Hardy-Weinberg equilibrium were made by pooling genotypes for
rare alleles.
Results and Discussion
All the individuals had one or two bands
for each of the probes assayed. The variation fit a single-locus model of inheritance (Prodohl et al. 1994b; Taggart et al.
1995). The maximum numbers of alleles
obtained with pSsa-A45/l, pSsa-A45/2,
pStr-A22/l, pStr-A9, and pSsa-A60 were 6,
10, 8, 7 and 4, respectively (Table 1), and
Table 1. Allele frequencies of the five VNTR lod
for the three analyzed samples
Table 2. Comparison of the 1993 adult samples separated by sex and returning age
Males/Females
Locus
Allele
Ssa-A45/1
A
B
C
D
E
F
H
Str-A9
H
Ssa-A60
H
Ssa-A45/2
H
Str-A22/1
B
C
D
F
G
H
I
A
B
Z
F
B
D
G
H
0
Q
S
L
C
R
Q
M
L
K
H
E
B
A
Juveniles
Adult
1992
Adult
1993
0.402
0.185
0.402
0.011
0.000
0.000
0.642
0.073
0.427
0500
0.000
0000
0.000
0.000
0.562
0.426
0489
0.011
0.074
0.574
0.160
0.043
0.000
0.021
0.468
0.298
0.000
0.000
0.000
0.011
0.664
0.065
0.054
0.054
0.043
0.457
0.087
0.239
0.000
0.715
0.348
0.250
0.348
0.036
0.009
0.009
0.694
0.103
0.371
0.371
0.000
0.095
0.052
0.009
0.703
0.422
0.448
0.026
0.103
0.609
0.152
0.196
0.009
0.018
0.277
0 313
0.009
0.000
0.009
0 018
0.763
0 091
0.045
0.064
0 082
0409
0.082
0 218
0.009
0.757
0.397
0.139
0.361
0.062
0.026
0.015
0.688
0.076
0.386
0.408
0.016
0.071
0.043
0.000
0.672
0.588
0.335
0.041
0.036
0.539
0.149
0.211
0.010
0.031
0.268
0.284
0.005
0.015
0.026
0.000
0.779
0.067
0.046
0.129
0.093
0.371
0.124
0.170
0.000
0.782
H refers to heterozygoslty values of each locus.
the size ranged from 1.9 to 17 kb (Taggart
et al. 1995). High levels of heterozygosity
per locus were found in all the samples
analyzed. Mean values were 0.711 in the
Esva River (1993) adult sample, 0.628 in
1992, and 0.660 in the juvenile sample.
When the three samples were considered
together, it was found that one or two alleles accounted for nearly half of the variation for each particular locus. The presence of unique alleles at low frequencies
in one of the samples analyzed may be
due to sampling error. Any difference between samples reflects not the number or
size of alleles at a locus but their relative
frequencies. The extent of variability
found by this method represents a real advantage with respect to previous methods
employed to study this particular population, such as protein variation (Moran et
al. 1994a) and chromosomal markers (Garcfa-Vazquez et al. 1991). These methods
detect too little genetic variation for clear
definition of the population genetic structure, and not enough to compare it to oth-
Adult/grilse
Locus
Chi square
df
P
Chi square
Ssa-A45/1
Str-A9
Ssa-A60
Sso-A4S/2
SIT-A22/]
Global
8.589
12.108
5.276
9.674
4.781
40.429
5
5
4
8
6
28
.12662
.03333
.26011
.28866
.57218
.06053
2.386
4.951
2.846
4.805
3.592
18.580
df
5
5
4
8
6
28
P
.79361
.42191
58394
.77816
.73169
.91060
Chl-square values for each locus and their significances,
df: degrees of freedom; R statistical significance.
er adjacent or more distant European populations.
To examine the utility of single-locus
probes in population genetics analysis it
is necessary to demonstrate the independence of the variation observed with respect to ethological, physiological, and
morphological traits. This work focuses
on two aspects: sex and returning age. To
this end, 1993 adult salmon have been
grouped by sex, giving 47 males and 49 females. They were also classified by their
returning age as 64 grilse (returning after
1 year in the sea) and 32 multi sea winter
(MSW) fish.
Immunological sexing of salmon is a
very accurate method and allows the evaluation of possible differences between
sexes in population dynamics. Few studies
have been done where adult fish were
sexed by biochemical or immunological
criteria. Sex determination by morphological traits is confusing when performed
out of the breeding season because sexual
dimorphism appears only at time of
spawning. There were no differences observed between male and female groups
(X2 = 40.429, NS) even considering the five
probes independently (Table 2). Taggart
et al. (1995) demonstrated that the five
SLPs assayed were not sex linked. Our results support their finding and, moreover,
the similar distribution of alleles among
sexes indicates that the SLP loci are not
influenced by sex.
An association between age of return
and the different alleles (*100 and *125) of
the MEP* locus has been described by Jordan et al. (1990). Similar results were obtained in the Esva isozyme study (Moran
et al. 1994b). In that work, it was clear that
the allele *125 had a higher frequency in
adults returning after only one winter in
the sea, that is, the *125 allele was related
to early maturity. The overlapping of generations because of the irregular proportions of early maturing adults of each cohort and/or river (probably depending on
environmental factors during the juvenile
freshwater stage) (Nicieza and Brana
1993) makes it difficult to interpret the
variation found for this enzyme. This
problem can be easily overcome using the
approach presented in the present work.
The five SLPs assayed are independent of
returning age (Table 2), permitting a
meaningful Interpretation of population relationships that could then be related to
postglacial colonization of rivers (Stahl
1987).
For a reasonable study of population genetics, an assessment of the temporal stability of populations should be done before assessing the significance of interpopulation heterogeneity (or homogeneity).
In the present case, between-generation
stability was measured through the comparison of allelic frequencies of adult samples that returned to the river in 1992 and
1993. Feeling confident that the adults
caught during the angling season are a
good representation of the brood stock,
Table 3. Comparisons between 1992 and 1993 adult samples and between 1992 adults and Juveniles
1993 samples
Adult 1992/1993
Adult 1992/Juvenlles 1993
Locus
Chi square:
df
P
Chi square
df
P
Ssa-A45/1
Str-A9
Ssa-A60
Ssa-A45/2
Str-A22/\
Global
10.748
5.005
12.093
7.158
7.491
42.495
6
6
3
9
7
31
.0965
.5431
.0070*
.6206
.3796
.0818
4.648
15.486
1.303
17512
2.925
41.904
5
5
3
8
7
28
.4563
.0084
.7284
.0252
.8918
.0442
Chl-square values for each locus.
dh degrees of freedom; P: statistical significance; *: significant after Bonierroni correction.
Brief Communications 8 1
Table 4. Comparison between 1990-hatcbed
salmon returned to the river as grilse 1.1 in 1992
and as MSW 1.2 in 1993
Locus
Chi square
di
Ssa-A45/J
Str-A9
Ssa^60
Ssa-A45/2
Str-A22/1
Global
1.935
7.424
1.972
9.892
7544
28.767
5
5
3
8
7
28
.85812
.19097
.57815
.27268
.37454
.42443
From the Departamento de Blologla Funclonal (Genetlca) Unlversidad de CMedo, 33071 Oviedo, Spain. We
are grateful to Dr. J. Taggart, Dr. P. ProdOhl, and Dr. A.
Ferguson (Queen's University of Belfast) for IrJndly supplying SLPs and to Dr P. Y. Le Bail for providing vltellogenin antibody. P M received a research fellowship
from EC AIR-1-3003-92-0719. This study was supported
by the Spanish DGICYT UE94-0020 and by a Grant of
the Conse)eria de Medlo Ambiente, Princlpado de Asturias.
The Journal of Heredity 1997:88(1)
df: degrees of freedom; P: statistical significance.
we have also compared 1992 adults with
the salmon parr aged 0+ sampled In 1993,
as theoretically, the 1992 adults represent
the parental generation of the Juvenile
salmon. In both global cases (Table 3), between-sample differences were not statistically significant (x2 = 42.49, df = 31, P <
.081 and x 2 = 41.9, df = 28, P < .044; Table
3). Only one of the individual tests performed was statistically significant; SsaA60 when comparing 1992 and 1993 adult
samples. In both samples, alleles A and B
had a high frequency, whereas alleles E
and F were present in low frequency. In
general terms, the results obtained are indicative of a great degree of genetic stability in this population.
An additional comparison was made between adults of the same cohort (born in
1990) that returned to the river in consecutive years, as 1.1 in 1992 and as 1.2 in
1993. There were no differences between
these two groups (Table 4). Conformance
to Hardy-Weinberg expectations indicated
that the 1990 cohort was in equilibrium.
Homing behavior (returning to the natal
river) is a typical feature of Salmo salar
populations (Stabell 1984), probably giving the genetic stability observed between
samples and cohorts in the absence of
gene flow from neighboring populations.
From a genetic perspective, minisatellites constitute a new approach to the
study of genetic variability in Atlantic
salmon populations. They are useful indicators of population mixture, providing
also an alternative method of estimating
levels of heterozygosity. In the present
case, the use of minisatellite variation has
demonstrated the genetic stability of an
Atlantic salmon population. This procedure ought to have direct application to
monitoring the unpredictable genetic effects of large-scale introductions of exogenous populations on native fish (Hindar
et al. 1991), and can be used to study genetic changes at the populational level by
breakdown of native gene complexes
through interbreeding and genetic drift.
8 2 The Journal of Heredity 1997:88(1)
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