Organization of Microsatellites Differs between Mammals

and Cold-water Teleost Fishes

Amanda L. Brooker, Doug Cook, Paul Bentzen,1 Jonathan M. Wright,2 and Roger W. Doyle

Marine Gene Probe Laboratory, Department of Biology, Dalhousie University, Halifax, NS B3H 4J1, Canada

Brooker, A.L., D. Cook, P. Bentzen, J.M. Wright, and R.W. Doyle. 1994. Organization of microsatellites differsbetween mammals and cold-water teleost fishes. Can. J. Fish. Aquat. Sci. 51:1959-1966.

Microsatellites, in particular (dG-dT)n and (dG-dA)n dinucleotide repeats, are abundant and display a high

degree of length polymorphism and heterozygosity in eukaryotic genomes. Here, we report the cloning andcharacterization of 64 microsatellite sequences from Atlantic cod, Gadus morhua. The microsatelliteswere classified as perfect, imperfect, and compound repeats. The length and integrity of these repeatswere compared with microsatellites characterized from two other teleosts, rainbow trout (Oncorhynchusmykiss) and Atlantic salmon (Salmo salar), and from three mammalian genomes, human, porcine, andcanine. Differences were found in the proportions of the repeat classes; however, the most significantdifference between microsatellites from teleost fishes and mammals was the propensity of the former to beof greater length: some cod and rainbow trout microsatellites were more than twice the size of the longestmicrosatellite repeats reported for any mammalian genome. Primers for PCR amplification were constructed for seven of the cod microsatellites. Allele frequencies, degree of polymorphism, and heterozygositywere estimated for a sample population. Amplification with these cod primers was also carried out ona number of related gadids. These polymorphic microsatellite loci have enormous potential utility asgenetic markers for use in population, breeding, and evolutionary studies.

Les chaines d'ADN microsatellite, et plus particulierement les sequences r£petitives des dinucleotides(dG-dT)net (dG-dA),,, sont abondantes et leurs genomes eucaryotes presentent un taux eleve de poly-morphisme et d'heterozygosite". Dans ce rapport, nous pre*sentons les n§sultats du clonage et de la carac-terisation de 60 sequences d'ADN microsatellite de la morue franche, Gadus morhua. Les ADN microsatellites etaient classees en trois categories : parfaites, imparfaites et a sequence de repetition composee. Lalongueur et I'integrite de ces sequences de repetition ont et<§ comparees a celles d'ADN microsatellitecaracterisee chez deux autres tel£osteens, la truite arc-en-ciel {Oncorhynchus mykiss) et le saumon deI'Atlantique {Salmo salar), et chez trois genomes de mammiferes, (humain, porcin et canin). On a trouvedes differences dans les proportions des categories de sequences de repetition; toutefois, la difference laplus significative entre ies ADN microsatellites de te§l<§osteens et celle de mammiferes etait une tendancea une longueur supe>ieure chez ces derniers : les ADN microsatellites de certaines morues et truites arc-en-ciel etaient deux fois plus longues que les sequences rep<§t<§es d'ADN microsatellite observees pourtous les genomes de mammiferes. Des amorces pour I'amplification par la PCR ont et§ creees pour sept desADN microsatellites de morue. Les frequences d'alleles, le degre de polymorphisme et I'heterozygositeont ete estime*s pour une population e*chantillon. On a egalement fait I'amplification de ces amorces de

morue pour un certain number de gadides connexes. Ces locus d'ADN microsatellites polymorphespeuvent etre extremement utiles comme marqueurs genetiques dans les etudes de population, de reproductionet devolution.

Received November 24, 1993

Accepted March 16, 1994

(J12177)

Re$u le 24 novembre 1993

Accepte le 16 mars 1994

Atlantic cod, Gadus morhua, is one of the most com

mercially important fish in the North Atlantic. Highly

polymorphic genetic markers assayable via the poly-

merase chain reaction (PCR) hold the potential for consid

erable utility in selective breeding, fisheries management,

and genome mapping of cod (Bentzen et al. 1991; Wright

1993; Wright and Bentzen 1994). To this end, we have

cloned and sequenced microsatellite repeats from cod.

Microsatellites consist of tandem repeats of simple

sequence DNA, where the varying number of repeats at a

particular locus creates a polymorphism (Litt and Luty 1989;

'Current address: School of Fisheries HF-10, MAR Building,Room 201, University of Washington, Seattle, WA 98195, USA.

2Author to whom correspondence should be addressed.

Can. J. Fish. Aquat. Sci., Vol. 51, 1994

Tautz 1989; Weber and May 1989). These arrays of differing

lengths can be amplified by PCR and size fractionated on

acrylamide gels. The most abundant and widely used of these

simple repeats are the dinucleotide sequences (dG-dT)n and

(dG-dA)^. Cloning of these microsatellites has been reported

for a variety of species, although mostly mammals (Stallings

et al. 1991). At present, extensive microsatellite data for

human (Weber 1990), mouse (Love et al. 1990), rat (Beckmann

and Weber 1992), dog (Ostrander et al. 1993), pig (Wintero

et al. 1992), horse (Ellegren et al. 1992), bees (Estoup et al.

1993), Atlantic salmon {Salmo salar) (Slettan et al. 1993),

brown trout {Salmo trutta) (Estoup et al. 1993), and zebrafish

(Goff et al. 1992) have been published.

Highly polymorphic microsatellite markers have great

potential utility as genetic tags for use in aquaculture and

1959

Table 1. Repeat sequence type and size of microsatellites from mammalian and

teleost fish species. Figures in parentheses are the second highest size class due to abimodal distribution.

Atlantic Rainbow Atlantic

cod trout salmon Human Canine Porcine

n

% perfect

% imperfect

% compound

Most common

size class3

Largest

size classa

64

48.4

45.3

6.3

6-11

(30-35)

>60

51

56.9

31.4

11.7

24-29

>60

45

80

20

0

6-9

(21-24)

>33

114

64

25

11

12-15

27-30

101

80

8

12

14-16

23-25

105

71

19

10

16-18

28-30

"Number of dinucleotide repeats.

□ Perfect

E3 Imperfect

Compound

Table 2. Nucleotide sequence of Atlantic cod microsatellites

and primers for amplification of loci by PCR.

6-11 12r17 18-23 24-29 30-35 36-41 42-47 48-53 54-59 >60

Number of repeat units (longest continuous run)

Fig. 1. Frequency of different size classes of the longest uninter

rupted microsatellite arrays from Atlantic cod. Size classes are rep

resented as number of repeat units.

fisheries biology. They may prove particularly valuable for

stock discrimination and population genetics due to the high

levels of polymorphism compared with conventional allozyme

markers (Bentzen et al. 1991; Wright and Bentzen 1994).

The utility of the PCR approach also makes possible the pro

cessing of large sample numbers required for population sur

veys and large-scale breeding programs. Moreover, the minute

amount of tissue required to genotype individuals allows

larvae as well as retrospective studies of archival samples

such as otoliths to be examined (Wright and Bentzen 1994).

Recently, microsatellites from Atlantic salmon were reported

to differ significantly in length and composition from mam

malian microsatellites (Slettan et al. 1993). In order to deter

mine if this finding is a general trend for microsatellites iso

lated from teleost fishes, we have investigated the organization

of Atlantic cod and, to a lesser extent, rainbow trout

(Oncorhynchus mykiss) microsatellites and compared them

with microsatellites isolated from other species. We also

report the allele distributions and heterozygosities of seven

microsatellite loci that exhibit a wide range of polymorphism

in a sample of 127 cod from the western North Atlantic

Ocean and investigate whether or not these microsatellite

loci are conserved in related gadid species.

i960

Primer Sequence 5' -> 3'

Gmo 1 (GA)4N(GA)2N,(GA)7N2(GA)4N2(GA)5Fa AAA AAC AAA ACG AGA GCG GTRb GGA GTT GCT TGT GGT GGC AT

57

Gmo2

Rb

Gmo9

F

R

Gmo 10

F

R

Gmol23

F

R

Gmol32

F

R

Gmol45

F

R

(GT)I4

CCC TCA GAT TCA AAT GAA GGA

GTG TGA GAT GAC TGT GTC G

(GT)6N2GTN2GTN2(GT)46

GCA GAT TTG AAT CAG CGT GT

CTC TGT GGG TAT GTG CAA AG

(GT)5N2(GT)3N,(GT)2N2(GT)44

GGA GTT TGA CTC ATC AGA TGC

ACA GCG TAT GAC CAT GAT TCG

(GT)gN2(GT)IIN(GT)2N3(GT)43N3(GT)8GAG GGA CAT AAA GAC ACT T

AAA CAT GCA TGC AGG CAA C

(GT)19

GGA ACC CAT TGG ATT CAG GC

CGA AAG GAC GAG CCA ATA AC

GI2(GA)29

GCA TTG TAG GAA CAA CAA TTA AC

GTG CAT GTG CTC ATT ATA GC

51

50

53

56

52

50

aF = forward primer.

bR = reverse primercAnnealing temperature.

Methods and Materials

Isolation of Microsatellites from Cod

Two cod genomic libraries were constructed. The first

employed the vector M13mp9 and was constructed from

purified, size-selected fragments of 200-500 bp. These frag

ments were generated by digestion with enzymes A/wI,

Haelll, Hindi, and Rsal (Pharmacia) according to the man

ufacturer's instructions and cloned into the Smal site of the

vector. Construction of a second library was perfqrmed by

cleaving cod genomic DNA with Alul, Hindi, Pall, and

Rsal, followed by ligation of size-selected fragments of

300-800 bp into the Smal site of the pUC18 plasmid.

Can. J. Fish. Aquat. ScL, Vol. 51, 1994

Table 3. Pedigree analysis of three two-generation families of Atlantic cod using

six polymorphic microsatellite loci.

Fish

AI,AI2

All,

AII2

AII3

AII4

AII5

AII6

AII7

AII8AII9

AII.o

BI,

BI2

BII,

BII2

BII3

BII4

BII5

BH6

CI,

CI2

CII,

CII2

CII3

CII4

CII5

CII6

CII7

Size of

alleles

(bp)

Gmol Gmo2

1»

1»

'»

'»

l »

» ■

» •

» •

» ■

» i

» J

» J

» J

1 3,4

1 2,5*

1 2,4

1 3,5

1 2,3

1 2,4

1 3,5

1 2,4

1 2,4

1 3,5

1 2,3

I 4,5

1 2,3

1 1,5

I 3,5

I 1,3

1 3,5

1 1,2

1 2,5

1 1,3

2,5

1 2,5

2,5

2,2

2,5

2,2

2,5

5,5

5,5

1 = 104 1 = 116

2= 114

3 = 112

4= 110

5= 108

Gmo9

4,7

8,9

4,8

4,9

7,9

4,9

4,9

4,9

4,8

4,9

7,8

7,9

2,3

6,6

3,6

3,6

3,6

2,6

3,6

3,6

1,8

5,7

5,8

1,7

7,8

7,8

1,5

1,7

5,8

1 =236

2 = 220

3 = 216

4 = 212

5 = 208

6 = 206

7 = 202

8 = 194

9= 190

Gmol 23

1,4

2,5

4,5

1,5

4,5

1,2

2,4

2,4

4,5

4,5

1,5

2,4

6, 10

2,4

2, 10

2, 10

2, 10

2,6

4, 10

4, 10

7,9

3,8

3,7

8,9

3,7

3,9

3,9

8,9

7,8

1 =221

2 = 219

3 = 213

4 = 203

5 = 201

6= 199

7= 195

8= 191

9= 187

10= 141

Gmol 32

4,5

4,5

4,4

5,5

4,5

4,4

5,5

4,4

4,4

5,5

5,5

4,5

1,2

3,5

1,5

1,3

2,5

1,5

2,3

1,5

5,5

3,5

3,5

5,5

5,5

5,5

5,5

5,5

5,5

1 = 135

2= 123

3= 119

4= 113

5= 111

Gmol45

2,8

5, 10

2, 10

2, 10

8, 10

8, 10

5,8

2,5

2, 10

8, 10

2, 10

5,8

4,7

3,9

4,9

7,9

4,9

4,9

4,9

3,7

1, 10

5,6

1,5

1,5

1,5

1,6

1,6

5, 10

6, 10

1 = 183

2= 181

3= 179

4= 177

5 = 175

6= 173

7= 171

8= 169

9= 167

10 = 165

Following transformation, bacterial colonies or plaques

were transferred to nylon filters (Amersham) according to the

manufacturer's instructions and the immobilized DNA was

hybridized to (GT)15 synthetic oligonucleotide, radiolabeled

with [7 32P]ATP and T4 polynucleotide kinase. Filters wereincubated in prehybridization solution of Westneat et al.

(1988) at 65°C for 1 h before addition of the radiolabeled

probe. The hybridization reaction was allowed to proceed

overnight. Filters were washed once with 2 X SSC/0.2%

SDS at room temperature for 15 min and once with 2 X

SSC/0.2% SDS at 65°C for 15 min. Dideoxy-chain termi

nation sequencing of DNA from purified positive clones

was accomplished using T7 DNA polymerase (Pharmacia).

PCR Analysis

PCR primers complementary to the sequence flanking the

microsatellite were synthesized. For all PCR analyses, except

those that amplify the locus Gmo9, the reverse primer was

5' end-labeled with [7 32P]ATP (37 kBq/10 pmol primer)using T4 polynucleotide kinase (Pharmacia). PCR was carried

Can. J. Fish. Aquat. Sci., Vol. 51, 1994

out in a Perkin Elmer thermal cycler using Taq Polymerase

(Perkin Elmer). The standard reaction contained 10 ng of

template DNA, 0.6 \xM labeled reverse primer, 0.6 \M for

ward primer, 10 mM Tris-HCl (pH 8.3), 1 mM MgCl2,

50 mM KC1, 0.01% gelatin, 200 \iM dNTPs, and 0.25 unit

of Taq Polymerase in a 10-jjlL volume. For Gmo9, the reverse

primer was not labeled; instead, [a 32P]dATP was includedin the PCR mix along with 20 mM MgCl2. Amplifications

were achieved by running 30 cycles of 1 min at 95°C, 2 min

at the appropriate annealing temperature (see Table 2), and

20 s at 72°C. Stop dye (10 jjiL, Pharmacia) was added to

each reaction and the products were denatured at 80°C for

5 min. Two microlitres of the reaction was subjected to gel

electrophoresis on a 6% denaturing polyacrylamide gel.

Results and Discussion

Characterization of Cod-derived (dG-dA)n and (dG-dT)wSequences

As a result of the two cloning efforts, over 120 clones

1961

A C G T bp

13 112 111112 2

243444443434 ':

Fig. 2. Allelic products generated by PCR of the Gmo9 locus from a two-generation Norwegian

pedigree of Atlantic cod. Offspring demonstrate stable inheritance of parental alleles which

are numbered and shown at the bottom of the figure. Size of alleles are estimated using an

M13 sequence ladder.

that hybridized to the dinucleotide probe were isolated and

sequenced from Atlantic cod, approximately half from each

library. A crude estimate of the number of times (dG-dT),,

arrays occur in the genome and the distance between these

arrays was calculated. A plasmid library of approximately

7000 clones containing an average insert size of 400 bp

was constructed, representing 2.8 X 106 bp of cod genomicDNA. We make the assumption that (GT)rt sequences are

distributed evenly throughout the cod genome and also

that the 54 positive clones that we sequenced were repre

sentative of all the positive clones (about 10%). Thirty-one

of 54 clones, or 57%, contained a (GT)n array. Based on a

"c" value of 0.9 pg, the haploid genome of cod is 821 Mbp

(Hinegardner and Rosen 1972); we estimate that there are

approximately 2.34 X 105 copies of (GT),, microsatellitesequence present in the cod genome at intervals of 7 kbp.

These estimates are comparable with those suggested for

mammalian genomes (Tautz 1989) and approximately three

times more frequent than in brown trout (Estoup et al. 1993).

Forty-nine clones harbored repeats bracketed by nonrepet-

itive sequences. These were classified as perfect, imperfect,

and compound according to Weber (1990). Briefly, perfect

1962

repeats are uninterrupted stretches of the repeat unit, while

imperfect repeats have one to three intervening bases with

repeat sequence on either side. Compound repeats consist

of neighboring blocks of different types of repeat sequence.

Sixty-four independent microsatellite arrays were identi

fied from the 49 clones. The longest perfect stretch of each

of these arrays was plotted according to class and number of

uninterrupted repeat units (Fig. 1). The percentage of each

class of repeat, the most common size class, and the longest

size class for cod were compared with microsatellites from

other species: human (Weber 1990), pig (Wintero et al.

1992), dog (Ostrander et al. 1993), Atlantic salmon (Slettan

et al. 1993), and rainbow trout (unpublished data) (Table 1).

Slightly different procedures have been used to isolate

microsatellites from different species. It is possible that we

may have biased our screening towards longer arrays with

the use of a (GT)I5 oligonucleotide and higher hybridization and

washing temperatures (65°C); however, we feel that this is

unlikely. Less stringent screening efforts for mammals, as

used by Weber (1990), Beckmann and Weber (1992),

Ellegren et al. (1992), Wintero et al. (1992), and Ostrander

et al. (1993) have failed to reveal microsatellites over 30 repeats

Can. J. Fish. Aquat. ScL, Vol. 51, 1994

ACGT ACGT bp

-140

hi30

-120

-110

Fig. 3. PCR amplified alleles from 48 Atlantic cod individuals at microsatellite locus Gmo2. The sizes of the PCR products were esti

mated by co-migration with those of the M13 sequence fragments shown at either side of the cod samples.

in length. Screening at a lower stringency should enhance

detection of long, short, and more degenerate dinucleotide

arrays, but there is no evidence of this in the literature.

In all animal species, (dG-dT),, microsatellites predomi

nate with the next abundant type being (dG-dA)w. For this

reason, we chose to focus our search on (dG-dT)n. We only

succeeded in isolating two (dG-dA),, microsatellites, detected

by a (GT)W probe; these were found in clones containing a

(dG-dT),, array. The proportions of the different classes of

repeat types differ between species, with the highest per

centage of imperfect repeats found in cod and rainbow trout.

Proportions of perfect and imperfect microsatellites isolated

from Atlantic salmon (Slettan et al. 1993) more closely

resemble the mammalian genomes, except for the absence

of compound repeats, than do microsatellites from Atlantic cod

and rainbow trout (Table 1). An even more striking difference

between mammalian and cold-water fish microsatellites is

the most common size class of the microsatellite arrays.

Sixty-four percent of the smallest class of arrays (contain

ing 6-11 repeats) in cod were separated by four or five bases

adjacent to a larger repeat in the same clone. They were

classified as separate repeats because of the arbitrary defin

ition of Weber (1990). If these shorter microsatellites are

viewed as part of a larger degenerate microsatellite array,

and the criteria which define an imperfect repeat are altered

to incorporate four or five intervening bases rather than three,

then the most common size class for cod microsatellites

would be at least 30-35 repeats, approximately twice the

size of those reported in mammalian genomes. Regardless

of this, the next abundant size classes for cod and Atlantic

salmon are 30-35 and 21-24 dinucleotide repeats, respec

tively, significantly larger than the most common size classes

reported for mammals. This conclusion is consistent with

the size of microsatellites found in rainbow trout (unpub

lished data), where smaller adjacent repeats were not found.

The absolute lengths of the largest microsatellite arrays

in cod, rainbow trout, and Atlantic salmon (Slettan et al.

1993) are also greater than those reported in mammalian

genomes. In a study of microsatellite arrays from zebrafish,

although not providing data on all repeat arrays, three of 16

sequences were over 30 repeat units in length, the longest

consisting of 38 dinucleotide units (Goff et al. 1992). Data

were statistically analyzed to see if this difference in

Can. J. Fish. Aquat. Sci, Vol. 51, 1994

Table 4. Allele size and heterozygosity at Atlantic cod

microsatellite loci detected by PCR amplification.

Locus

Gmol

Gmo2

Gmo9

GmolO

Gmol23

Gmol 32

Gmol45

No. of

alleles

8

15

38

1

46

15

26

Size

(bp)a

94-114

106-140

116-246

214

137-239

109-155

123-217

% heterozygosity

14

82

92

0

89

85

92

aEstimated size of the PCR fragment when compared

with M13 sequence fragments of known length.

microsatellite length was significant for fishes and mam

mals. A chi-squared test was inappropriate because of the

varying size classes used by the different authors to group

data from the different species. A new data set was con

structed by taking the midpoint of the microsatellite length

of each size class and then weighting the length data by

frequency of the class. Bartlett's test for homogeneity of

variance showed that the fish data were more variable than

the mammal data (P < 0.001). A Kruskal-Wallis nonpara-

metric analysis on the pooled mammal and fish data demon

strated that the maximum lengths of fish and mammalian

microsatellites are highly different (P < 0.001).

Also noteworthy, the longest microsatellite arrays in the

cod genome consisted of perfect dinucleotide repeats. In

rainbow trout, six of seven microsatellites longer than

47 repeat units were perfect motifs. Cod, rainbow trout, and

possibly Atlantic salmon and zebrafish microsatellites are

longer and more degenerate than similar mammalian

sequences. Cod and rainbow trout microsatellites are similar

in length, but there is a higher proportion of imperfect

repeats in the cod, often with smaller degenerate repeats

adjacent to larger ones.

Inheritance and Allele Frequencies at Seven Microsatellite

Loci f

PCR primers were designed from flanking regions for a

number of microsatellites and conditions such as temperature,

1963

0 I ™l " " I H 1 1 ™ I

94 98 102 106 110 114

l—I—P"H—Fl-l* I

106 114 122 130 138

0.06

D0.4

w 0.2

.III ill ,in-l*l i-i-i-iI109 117 125 133 141 149

Fig. 4. Allele frequency distribution of alleles at microsatellite loci from Atlantic cod from the Northwest Atlantic Ocean (n127). Microsatellite loci: (A) Gmol, (B) Gmo2, (C) Gmo9, (D) Gmol23, (E) Gmol32, and (F) Gmol45.

radiolabeling (end labeling versus incorporation), and mag

nesium concentration were optimized (Table 2). Seven primer

pairs produced a product readily scored. All seven loci exhib

ited stable inheritance of alleles by PCR assay in three unre

lated, two-generation pedigrees of Atlantic cod (Table 3).Figure 2 demonstrates that even the most complex fragments

produced by PCR of the Gmo9 locus can be readily scored.

Genomic DNA samples from 127 fish caught off the coast

of Nova Scotia in the western North Atlantic were used to

1964

amplify seven microsatellites with the primer pairs described

in Table 2. Alleles for each individual were scored by comparing their migration with sequence fragments from M13 of

known length (Fig. 3). The seven microsatellites varied

widely in the degree of polymorphism exhibited (Table 4).Of the seven microsatellites, six were polymorphic, with 8-46

alleles and heterozygosities of 14-92%. Five of th'e micro-satellites exhibited heterozygous arrays in excess of 80%. It is

interesting that the only locus which was monomorphic

Can. J. Fish. Aquat. ScL, Vol. 51, 1994

(GmolO; (GT)5N2(GT)3N2(GT)2N2(GT)44 consists of a long

imperfect repeat, while all of the short repeats such as Gmo2,

Gmol32, and Gmol45 were highly polymorphic. This result

suggests that factors other than overall length of a microsatel

lite array, as suggested by Weber (1990), may determine

whether a microsatellite will exhibit allelic variation (see

Wright 1993).

The allele frequency distributions varied markedly for

the seven microsatellite loci assayed (Fig. 4; Table 4). Some

microsatellites showed wide variation in allele size (e.g.,

Gmo9 and Gmol23). Most allele distributions were uni-

modal, but those of Gmo9 and Gmol32 appeared weakly

and strongly bimodal), respectively (Fig. 4; Table 4). Similar

allele frequency distributions, reported for human microsatel

lites (Valdes et al. 1993), have been attributed to a stepwise

mechanism for the generation of new alleles. Presumably,

a similar mechanism is probably responsible for the gener

ation of allelic variation at microsatellite loci in cod.

In order to determine the evolutionary conservation of

microsatellite loci in related gadids, PCR amplification was

attempted with the seven primer pairs for the Atlantic cod

microsatellite loci on genomic DNA from the following

species: haddock (Melanogrammus aeglefinus), pollock

(Pollachius virens), and white hake {Urophycis tennis). PCR

products were detected in all of the gadid samples for the loci

Gmol, Gmo2, and Gmo9, while GmolO, Gmol32, and

Gmol45 produced fragments in only haddock and pollock.

No products were detected after PCR with the Gmol23

primers. Although only three individuals for each species

were assayed, all of the loci except GmolO and Gmol23

appeared to be polymorphic for at least one of the other

gadids (data not shown).

General Discussion

We report here the characterization of a group of micro-

satellites from Gadus morhua. It appears that the microsatel

lites in teleost fishes differ significantly in length and com

position from those of mammals. We can only speculate as

to why the microsatellites found in Atlantic cod, rainbow

trout, and Atlantic salmon are larger than microsatellites

from mammals. It remains to be seen, with subsequent iso

lation of microsatellites from other species, whether poikilo-

therms consistently have longer arrays than homiothermic

species. The longer, degenerate microsatellite arrays found

in teleost fishes may be caused by a predisposition to slip

page owing to fluctuations in temperature. The DNA poly-

merase may not function efficiently when subjected to

changes in temperature of several degrees, or temperatures

close to freezing, such as occur in the Atlantic cod envi

ronment and that of the salmonid species.

Disparities in genomic composition between warm- and

cold-blooded vertebrates have already been established by

Bernardi and Bernardi (1990). They found that fish iso-

chores were much more homogeneous and lower in GC con

tent than those of warm-blooded vertebrates. A subsequent

study by Cross et al. (1991) makes a further distinction

between fish genomes and other warm- and cold-blooded

vertebrates with regard to the GC-poor CpG islands that

are associated with some genes. It is possible that different

evolutionary forces are acting on the fish genome, gener

ating longer repeat arrays at microsatellite loci, or con

versely, shorter arrays in the genomes of mammals.

Can. J. Fish. Aquat. Sci, Vol. 51, 1994

Using PCR and primers specific to the nonrepetitive

flanks, we have successfully amplified from cod genomic

DNA seven of the cod microsatellites isolated in this study.

Five of these microsatellites exhibit heterozygosities greatly

in excess of those reported for allozyme or mitochondrial

studies. The average heterozygosity of these microsatellite

markers is H = 0.85 compared with H = 0.071 for allozymes

(Mork et al. 1985) and an equivalent measure for nucleon

diversity, h = 0.36, for mitochondrial genotypes (Carr and

Marshall 1991).

Breeding and population studies of Atlantic cod utilizing

these microsatellites as genetic markers are in progress in our

laboratory. The family Gadidae to which Atlantic cod belong

includes many other economically important species. Our

results have shown that the microsatellites reported here

may prove useful as genetic markers in other gadid species,

similar to a study previously carried out on domestic mam

mals (Moore et al. 1991). Furthermore, a comparative study

of the conservation and polymorphism of these microsatellite

loci in related gadids may elucidate the mode and tempo

of mutation at these fascinating sequences (Bentzen and

Wright 1993; Wright 1993, 1994).

Acknowledgements

Technical assistance was provided by Michiko Fllipak, Linda

Aitkinson, Barbara Edgar, and Harvey Domoslai. Rainbow trout

microsatellite data were provided by Dianne Morris prior to

publication. DNA from cod pedigrees was a generous gift of

Geir Dahle, Institute of Marine Research, Bergen, Norway. This

work was supported by funds from the Ocean Production

Enhancement Network (OPEN), the Nova Scotia government,

Department of Industry Trade and Technology, to the Marine

Gene Probe Laboratory, and the Natural Sciences and Engineer

ing Research Council of Canada to J.M.W. OPEN fellowship

support to A.L.B. is also acknowledged.

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1966Can.J.Fish.Aquat.Sci.,Vol.51,1994

Organization of Microsatellites Differs between Mammals

and Cold-water Teleost Fishes

Amanda L. Brooker, Doug Cook, Paul Bentzen,1 Jonathan M. Wright,2 and Roger W. Doyle

Marine Gene Probe Laboratory, Department of Biology, Dalhousie University, Halifax, NS B3H 4J1, Canada

Brooker, A.L, D. Cook, P. Bentzen, J.M. Wright, and R.W. Doyle. 1994. Organization of microsatellites differs

between mammals and cold-water teleost fishes. Can. J. Fish. Aquat. Sci. 51:1959-1966.

Microsatellites, in particular (dG-dT)n and (dG-dA),, dinucleotide repeats, are abundant and display a high

degree of length polymorphism and heterozygosity in eukaryotic genomes. Here, we report the cloning andcharacterization of 64 microsatellite sequences from Atlantic cod, Cadus morhua. The microsatelliteswere classified as perfect, imperfect, and compound repeats. The length and integrity of these repeats

were compared with microsatellites characterized from two other teleosts, rainbow trout (Oncorhynchusmykiss) and Atlantic salmon {Salmo salar), and from three mammalian genomes, human, porcine, andcanine. Differences were found in the proportions of the repeat classes; however, the most significantdifference between microsatellites from teleost fishes and mammals was the propensity of the former to beof greater length: some cod and rainbow trout microsatellites were more than twice the size of the longestmicrosatellite repeats reported for any mammalian genome. Primers for PCR amplification were constructed for seven of the cod microsatellites. Allele frequencies, degree of polymorphism, and heterozygositywere estimated for a sample population. Amplification with these cod primers was also carried out on

a number of related gadids. These polymorphic microsatellite loci have enormous potential utility asgenetic markers for use in population, breeding, and evolutionary studies.

Les chatnes d'ADN microsatellite, et plus particulierement les sequences rep£titives des dinucleotides(dG-dT)net (dG-dA),,, sont abondantes et leurs genomes eucaryotes pr£sentent un taux eleve de poly-morphisme et d'heterozygosite\ Dans ce rapport, nous pr£sentons les r£sultats du clonage et de la carac-terisation de 60 sequences d'ADN microsatellite de la morue franche, Gadus morhua. Les ADN microsatellites etaient classees en trois categories : parfaites, imparfaites et a sequence de r£p£tition composed. Lalongueur et l'integrit£ de ces sequences de r£p£tition ont £t£ comparers a celles d'ADN microsatellitecaracterisee chez deux autres teleost£ens, la truite arc-en-ciel (Oncorhynchus mykiss) et le saumon deI'Atlantique (Salmo salar), et chez trois genomes de mammiferes, (humain, porcin et canin). On a trouvedes differences dans les proportions des categories de sequences de repetition; toutefois, la difference laplus significative entre les ADN microsatellites de teleosteens et celle de mammiferes etait une tendancea une longueur supe>ieure chez ces derniers : les ADN microsatellites de certaines morues et truites arc-en-ciel Etaient deux fois plus longues que les sequences re>§t§es d'ADN microsatellite observees pourtous les genomes de mammiferes. Des amorces pour I'amplification par la PCR ont £te cr£ees pour sept desADN microsatellites de morue. Les frequences d'alleles, le degre de polymorphisme et I'heterozygosityont £t£ estim£s pour une population £chantillon. On a Sgalement fait I'amplification de ces amorces de

morue pour un certain number de gadid£s connexes. Ces locus d'ADN microsatellites polymorphespeuvent etre extremement utiles comme marqueurs gen&iques dans les eludes de population, de reproductionet devolution.

Received November 24, 1993

Accepted March 16, 1994

(J12177)

Regu le 24 novembre 1993

Accepte le 16 mars 1994

Atlantic cod, Gadus morhua, is one of the most com

mercially important fish in the North Atlantic. Highly

polymorphic genetic markers assayable via the poly-

merase chain reaction (PCR) hold the potential for consid

erable utility in selective breeding, fisheries management,

and genome mapping of cod (Bentzen et al. 1991; Wright

1993; Wright and Bentzen 1994). To this end, we have

cloned and sequenced microsatellite repeats from cod.

Microsatellites consist of tandem repeats of simple

sequence DNA, where the varying number of repeats at a

particular locus creates a polymorphism (Litt and Luty 1989;

'Current address: School of Fisheries HF-10, MAR Building,Room 201, University of Washington, Seattle, WA 98195, USA.

2Author to whom correspondence should be addressed.

Can. J. Fish. Aquat. Sci.. Vol. 51, 1994

Tautz 1989; Weber and May 1989). These arrays of differing

lengths can be amplified by PCR and size fractionated on

acrylamide gels. The most abundant and widely used of these

simple repeats are the dinucleotide sequences (dG-dT)w and

(dG-dA),,. Cloning of these microsatellites has been reported

for a variety of species, although mostly mammals (Stallings

et al. 1991). At present, extensive microsatellite data for

human (Weber 1990), mouse (Love et al. 1990), rat (Beckmann

and Weber 1992), dog (Ostrander et al. 1993), pig (Wintero

et al. 1992), horse (Ellegren et al. 1992), bees (Estoup et al.

1993), Atlantic salmon (Salmo salar) (Slettan et al. 1993),

brown trout (Salmo trutta) (Estoup et al. 1993), and z^rafish

(Goff et al. 1992) have been published.

Highly polymorphic microsatellite markers have great

potential utility as genetic tags for use in aquaculture and

1959

Table 1. Repeat sequence type and size of microsatellites from mammalian and

teleost fish species. Figures in parentheses are the second highest size class due to abimodal distribution.

n

% perfect

% imperfect

% compound

Most common

size classa

Largest

size class3

Atlantic

cod

64

48.4

45.3

6.3

6-11

(30-35)

>60

Rainbow

trout

51

56.9

31.4

11.7

24-29

>60

Atlantic

salmon

45

80

20

0

6-9

(21-24)

>33

Human

114

64

25

11

12-15

27-30

Canine

101

80

8

12

14-16

23-25

Porcine

105

71

19

10

16-18

28-30

aNumber of dinucleotide repeats.

□ Perfect

0 Imperfect

El Compound

Table 2. Nucleotide sequence of Atlantic cod microsatellitesand primers for amplification of loci by PCR.

6-11 12r17 18-23 24-29 30-35 36-41 42-47 48-53 54-59 >60

Number of repeat units (longest continuous run)

Fig. 1. Frequency of different size classes of the longest uninter

rupted microsatellite arrays from Atlantic cod. Size classes are rep

resented as number of repeat units.

fisheries biology. They may prove particularly valuable for

stock discrimination and population genetics due to the high

levels of polymorphism compared with conventional allozyme

markers (Bentzen et al. 1991; Wright and Bentzen 1994).

The utility of the PCR approach also makes possible the pro

cessing of large sample numbers required for population sur

veys and large-scale breeding programs. Moreover, the minute

amount of tissue required to genotype individuals allows

larvae as well as retrospective studies of archival samples

such as otoliths to be examined (Wright and Bentzen 1994).

Recently, microsatellites from Atlantic salmon were reported

to differ significantly in length and composition from mam

malian microsatellites (Slettan et al. 1993). In order to deter

mine if this finding is a general trend for microsatellites iso

lated from teleost fishes, we have investigated the organization

of Atlantic cod and, to a lesser extent, rainbow trout

(Oncorhynchus mykiss) microsatellites and compared them

with microsatellites isolated from other species. We also

report the allele distributions and heterozygosities of seven

microsatellite loci that exhibit a wide range of polymorphism

in a sample of 127 cod from the western North Atlantic

Ocean and investigate whether or not these microsatellite

loci are conserved in related gadid species.

I960

Primer Sequence 5' -> 3' 7-ann <QC

Gmo 1 (GA)4N(GA)2N,(GA)7N2(GA)4N2(GA)5

AAA AAC AAA ACG AGA GCG GT

57

Rb

Gmo2

Rb

Gmo9

F

R

Gmo 10

F

R

Gmol23

F

R

Gmol32

F

R

Gmo 145

F

R

GGA GTT GCT TGT GGT GGC AT

(GT)I4

CCC TCA GAT TCA AAT GAA GGA

GTG TGA GAT GAC TGT GTC G

(GT)6N2GTN2GTN2(GT)46

GCA GAT TTG AAT CAG CGT GT

CTC TGT GGG TAT GTG CAA AG

(GT)5N2(GT)3N2(GT)2N2(GT)44

GGA GTT TGA CTC ATC AGA TGC

ACA GCG TAT GAC CAT GAT TCG

(GT)8N2(GT)nN(GT)2N3(GT)43N3(GT)8GAG GGA CAT AAA GAC ACT T

AAA CAT GCA TGC AGG CAA C

(GT)I9

GGA ACC CAT TGG ATT CAG GC

CGA AAG GAC GAG CCA ATA AC

G12(GA)29

GCA TTG TAG GAA CAA CAA TTA AC

GTG CAT GTG CTC ATT ATA GC

51

50

53

56

52

50

ZF = forward primer.

bR = reverse primercAnnealing temperature.

Methods and Materials

Isolation of Microsatellites from Cod

Two cod genomic libraries were constructed. The first

employed the vector M13mp9 and was constructed from

purified, size-selected fragments of 200-500 bp. These frag

ments were generated by digestion with enzymes Alul,

Haelll, Hindi, and Rsal (Pharmacia) according to the man

ufacturer's instructions and cloned into the Smal site of the

vector. Construction of a second library was perfqrmed by

cleaving cod genomic DNA with Alul, Hindi, Pall, and

Rsal, followed by ligation of size-selected fragments of

300-800 bp into the Smal site of the pUC18 plasmid.

Can. J. Fish. Aquat. ScL, Vol. 51, 1994

Table 3. Pedigree analysis of three two-generation families of Atlantic cod using

six polymorphic microsatellite loci.

Fish

AI,AI2

All,

AII2

AII3

AII4

AII5

AII6

AII7

AH8AII9

AH,o

BI,BI2

BII,

BII2

BII3

BII4

BII5

BH6

CI,

CI2

CII,

CII2

CII3

CII4

CII5

CII6

CII7

Size of

alleles

(bp)

Gmol Gmo2

[ y

[ ?

[ ^

[ ^

L ?

»

»

»

»

, ■

I 3,4

1 2, 5V

1 2,4

1 3,5

1 2,3

1 2,4

I 3,5

I 2,4

I 2,4

I 3,5

1 2,3

I 4,5

I 2,3

I 1,5

I 3,5

I 1,3

I 3,5

I 1,2

I 2,5

I 1, 3

2,5

I 2,5

I 2,5

2,2

2,5

i 2,2

2,5

5,5

5,5

1 = 104 1 = 116

2 = 114

3=112

4= 110

5 = 108

Gmo9

4, 7

8,9

4, 8

4,9

7,9

4,9

4,9

4,9

4,8

4,9

7,8

7,9

2,3

6,6

3,6

3,6

3,6

2,6

3,6

3,6

1,8

5,7

5,8

1,7

7, 8

7,8

1, 5

1,7

5,8

1 =236

2 = 220

3 = 216

4 = 212

5 = 208

6 = 206

7 = 202

8= 194

9= 190

Gmol 23

1,4

2,5

4,5

1, 5

4, 5

1, 2

2,4

2,4

4,5

4,5

1,5

2,4

6, 10

2,4

2, 10

2, 10

2, 10

2,6

4, 10

4, 10

7,9

3,8

3,7

8,9

3,7

3,9

3,9

8,9

7, 8

1 =221

2 = 219

3 = 213

4 = 203

5 = 201

6= 199

7= 195

8= 191

9= 187

10= 141

Gmol32

4, 5

4, 5

4, 4

5,5

4, 5

4,4

5,5

4,4

4,4

5,5

5,5

4, 5

1,2

3,5

1,5

1,3

2,5

1,5

2,3

1,5

5,5

3,5

3,5

5,5

5, 5

5, 5

5,5

5, 5

5,5

1 = 135

2= 123

3 = 119

4= 113

5 = 111

Gmol45

2,8

5, 10

2, 10

2, 10

8, 10

8, 10

5, 8

2,5

2, 10

8, 10

2, 10

5,8

4,7

3,9

4,9

7,9

4,9

4,9

4,9

3,7

1, 10

5,6

1,5

1,5

1,5

1,6

1,6

5, 10

6, 10

1 = 183

2= 181

3 = 179

4= 177

5 = 175

6= 173

7= 171

8= 169

9= 167

10= 165

Following transformation, bacterial colonies or plaques

were transferred to nylon filters (Amersham) according to the

manufacturer's instructions and the immobilized DNA was

hybridized to (GT)15 synthetic oligonucleotide, radiolabeled

with [7 32P]ATP and T4 polynucleotide kinase. Filters wereincubated in prehybridization solution of Westneat et al.

(1988) at 65°C for 1 h before addition of the radiolabeled

probe. The hybridization reaction was allowed to proceed

overnight. Filters were washed once with 2 X SSC/0.2%

SDS at room temperature for 15 min and once with 2 X

SSC/0.2% SDS at 65°C for 15 min. Dideoxy-chain termi

nation sequencing of DNA from purified positive clones

was accomplished using T7 DNA polymerase (Pharmacia).

PCR Analysis

PCR primers complementary to the sequence flanking the

microsatellite were synthesized. For all PCR analyses, except

those that amplify the locus Gmo9, the reverse primer was

5' end-labeled with [7 32P]ATP (37 kBq/10 pmol primer)using T4 polynucleotide kinase (Pharmacia). PCR was carried

Can. J. Fish. Aquat. ScL, Vol. 51, 1994

out in a Perkin Elmer thermal cycler using Taq Polymerase

(Perkin Elmer). The standard reaction contained 10 ng of

template DNA, 0.6 pM labeled reverse primer, 0.6 |nM for

ward primer, 10 mM Tris-HCl (pH 8.3), 1 mM MgCl2,

50 mM KC1, 0.01% gelatin, 200 \xM dNTPs, and 0.25 unit

of Taq Polymerase in a 10-jiL volume. For Gmo9, the reverse

primer was not labeled; instead, [a 32P]dATP was includedin the PCR mix along with 20 mM MgCl2. Amplifications

were achieved by running 30 cycles of 1 min at 95°C, 2 min

at the appropriate annealing temperature (see Table 2), and

20 s at 72°C. Stop dye (10 jxL, Pharmacia) was added to

each reaction and the products were denatured at 80°C for

5 min. Two microlitres of the reaction was subjected to gel

electrophoresis on a 6% denaturing polyacrylamide gel.

Results and Discussion

Characterization of Cod-derived (dG-dA)w and (dG-dT)n

Sequences

As a result of the two cloning efforts, over 120 clones

1961

OtD

13 112 111112 2

243444443434

Fig. 2. Allelic products generated by PCR of the Gmo9 locus from a two-generation Norwegian

pedigree of Atlantic cod. Offspring demonstrate stable inheritance of parental alleles which

are numbered and shown at the bottom of the figure. Size of alleles are estimated using an

M13 sequence ladder.

that hybridized to the dinucleotide probe were isolated and

sequenced from Atlantic cod, approximately half from each

library. A crude estimate of the number of times (dG-dT),,

arrays occur in the genome and the distance between these

arrays was calculated. A plasmid library of approximately

7000 clones containing an average insert size of 400 bp

was constructed, representing 2.8 X 106 bp of cod genomicDNA. We make the assumption that (GT)n sequences are

distributed evenly throughout the cod genome and also

that the 54 positive clones that we sequenced were repre

sentative of all the positive clones (about 10%). Thirty-one

of 54 clones, or 57%, contained a (GT)n array. Based on a

"c" value of 0.9 pg, the haploid genome of cod is 821 Mbp

(Hinegardner and Rosen 1972); we estimate that there are

approximately 2.34 X 105 copies of (GT)n microsatellitesequence present in the cod genome at intervals of 7 kbp.

These estimates are comparable with those suggested for

mammalian genomes (Tautz 1989) and approximately three

times more frequent than in brown trout (Estoup et al. 1993).

Forty-nine clones harbored repeats bracketed by nonrepet-

itive sequences. These were classified as perfect, imperfect,

and compound according to Weber (1990). Briefly, perfect

1962

repeats are uninterrupted stretches of the repeat unit, while

imperfect repeats have one to three intervening bases with

repeat sequence on either side. Compound repeats consist

of neighboring blocks of different types of repeat sequence.

Sixty-four independent microsatellite arrays were identi

fied from the 49 clones. The longest perfect stretch of each

of these arrays was plotted according to class and number of

uninterrupted repeat units (Fig. 1). The percentage of each

class of repeat, the most common size class, and the longest

size class for cod were compared with microsatellites from

other species: human (Weber 1990), pig (Wintero et al.

1992), dog (Ostrander et al. 1993), Atlantic salmon (Slettan

et al. 1993), and rainbow trout (unpublished data) (Table 1).

Slightly different procedures have been used to isolate

microsatellites from different species. It is possible that we

may have biased our screening towards longer arrays with

the use of a (GT)I5 oligonucleotide and higher hybridization and

washing temperatures (65°C); however, we feel that this is

unlikely. Less stringent screening efforts for mammals, as

used by Weber (1990), Beckmann and Weber (1992),

Ellegren et al. (1992), Wintero et al. (1992), and Ostrander

et al. (1993) have failed to reveal microsatellites over 30 repeats

Can. J. Fish. Aquat. Set., Vol. 51. 1994

ACGT

Fig. 3. PCR amplified alleles from 48 Atlantic cod individuals at microsatellite locus Gmo2. The sizes of the PCR products were esti

mated by co-migration with those of the M13 sequence fragments shown at either side of the cod samples.

in length. Screening at a lower stringency should enhance

detection of long, short, and more degenerate dinucleotide

arrays, but there is no evidence of this in the literature.

In all animal species, (dG-dT),, microsatellites predomi

nate with the next abundant type being (dG-dA),,. For this

reason, we chose to focus our search on (dG:dT)w. We only

succeeded in isolating two (dG-dA),, microsatellites, detected

by a (GT)n probe; these were found in clones containing a

(dG-dT)w array. The proportions of the different classes of

repeat types differ between species, with the highest per

centage of imperfect repeats found in cod and rainbow trout.

Proportions of perfect and imperfect microsatellites isolated

from Atlantic salmon (Slettan et al. 1993) more closely

resemble the mammalian genomes, except for the absence

of compound repeats, than do microsatellites from Atlantic cod

and rainbow trout (Table 1). An even more striking difference

between mammalian and cold-water fish microsatellites is

the most common size class of the microsatellite arrays.

Sixty-four percent of the smallest class of arrays (contain

ing 6-11 repeats) in cod were separated by four or five bases

adjacent to a larger repeat in the same clone. They were

classified as separate repeats because of the arbitrary defin

ition of Weber (1990). If these shorter microsatellites are

viewed as part of a larger degenerate microsatellite array,

and the criteria which define an imperfect repeat are altered

to incorporate four or five intervening bases rather than three,

then the most common size class for cod microsatellites

would be at least 30-35 repeats, approximately twice the

size of those reported in mammalian genomes. Regardless

of this, the next abundant size classes for cod and Atlantic

salmon are 30-35 and 21-24 dinucleotide repeats, respec

tively, significantly larger than the most common size classes

reported for mammals. This conclusion is consistent with

the size of microsatellites found in rainbow trout (unpub

lished data), where smaller adjacent repeats were not found.

The absolute lengths of the largest microsatellite arrays

in cod, rainbow trout, and Atlantic salmon (Slettan et al.

1993) are also greater than those reported in mammalian

genomes. In a study of microsatellite arrays from zebrafish,

although not providing data on all repeat arrays, three of 16

sequences were over 30 repeat units in length, the longest

consisting of 38 dinucleotide units (Goff et al. 1992). Data

were statistically analyzed to see if this difference in

Can. J. Fish. Aquat. ScL. Vol. 51, 1994

Table 4. Allele size and heterozygosity at Atlantic cod

microsatellite loci detected by PCR amplification.

Locus

Gmol

Gmo2

Gmo9

GmolO

Gmol 23

Gmol 32

Gmol45

No. of

alleles

8

15

38

1

46

15

26

Size

(bp)a

94-114

106-140

116-246

214

137-239

109-155

123-217

% heterozygosity

14

82

92

0

89

85

92

Estimated size of the PCR fragment when compared

with M13 sequence fragments of known length.

microsatellite length was significant for fishes and mam

mals. A chi-squared test was inappropriate because of the

varying size classes used by the different authors to group

data from the different species. A new data set was con

structed by taking the midpoint of the microsatellite length

of each size class and then weighting the length data by

frequency of the class. Bartlett's test for homogeneity of

variance showed that the fish data were more variable than

the mammal data (P < 0.001). A Kruskal-Wallis nonpara-

metric analysis on the pooled mammal and fish data demon

strated that the maximum lengths of fish and mammalian

microsatellites are highly different (P < 0.001).

Also noteworthy, the longest microsatellite arrays in the

cod genome consisted of perfect dinucleotide repeats. In

rainbow trout, six of seven microsatellites longer than

47 repeat units were perfect motifs. Cod, rainbow trout, and

possibly Atlantic salmon and zebrafish microsatellites are

longer and more degenerate than similar mammalian

sequences. Cod and rainbow trout microsatellites are similar

in length, but there is a higher proportion of imperfect

repeats in the cod, often with smaller degenerate repeats

adjacent to larger ones.

Inheritance and Allele Frequencies at Seven Microsatellite

Loci ,

PCR primers were designed from flanking regions for a

number of microsatellites and conditions such as temperature,

1963

©4 98 102 106 110 114

106

B

Fig. 4. Allele frequency distribution of alleles at microsatellite loci from Atlantic cod from the Northwest Atlantic Ocean (n127). Microsatellite loci: (A) Gmol, (B) Gmo2, (C) Gmo9, (D) Gmol23, (E) Gmol32, and (F) Gmol45.

radiolabeling (end labeling versus incorporation), and mag

nesium concentration were optimized (Table 2). Seven primer

pairs produced a product readily scored. All seven loci exhib

ited stable inheritance of alleles by PCR assay in three unre

lated, two-generation pedigrees of Atlantic cod (Table 3).

Figure 2 demonstrates that even the most complex fragments

produced by PCR of the Gmo9 locus can be readily scored.

Genomic DNA samples from 127 fish caught off the coast

of Nova Scotia in the western North Atlantic were used to

1964

amplify seven microsatellites with the primer pairs described

in Table 2. Alleles for each individual were scored by com

paring their migration with sequence fragments from M13 of

known length (Fig. 3). The seven microsatellites varied

widely in the degree of polymorphism exhibited (Table 4).

Of the seven microsatellites, six were polymorphic, with 8-46

alleles and heterozygosities of 14-92%. Five of th'e micro-

satellites exhibited heterozygous arrays in excess of 80%. It is

interesting that the only locus which was monomorphic

Can. J. Fish. Aquat. ScL, Vol. 51, 1994

(GmolO; (GT)5N2(GT)3N2(GT)2N2(GT)44 consists of a long

imperfect repeat, while all of the short repeats such as Gmo2,

Gmol 32, and Gmol45 were highly polymorphic. This result

suggests that factors other than overall length of a microsatel

lite array, as suggested by Weber (1990), may determine

whether a microsatellite will exhibit allelic variation (see

Wright 1993).

The allele frequency distributions varied markedly for

the seven microsatellite loci assayed (Fig. 4; Table 4). Some

microsatellites showed wide variation in allele size (e.g.,

Gmo9 and Gmol23). Most allele distributions were uni-

modal, but those of Gmo9 and Gmol32 appeared weakly

and strongly bimodai), respectively (Fig. 4; Table 4). Similar

allele frequency distributions, reported for human microsatel

lites (Valdes et al. 1993), have been attributed to a stepwise

mechanism for the generation of new alleles. Presumably,

a similar mechanism is probably responsible for the gener

ation of allelic variation at microsatellite loci in cod.

In order to determine the evolutionary conservation of

microsatellite loci in related gadids, PCR amplification was

attempted with the seven primer pairs for the Atlantic cod

microsatellite loci on genomic DNA from the following

species: haddock (Melanogrammus aeglefinus), pollock

(Pollachius virens), and white hake (Urophycis tenuis). PCR

products were detected in all of the gadid samples for the loci

Gmol, Gmo2, and Gmo9, while GmolO, Gmol32, and

Gmol45 produced fragments in only haddock and pollock.

No products were detected after PCR with the Gmol23

primers. Although only three individuals for each species

were assayed, all of the loci except GmolO and Gmol23

appeared to be polymorphic for at least one of the other

gadids (data not shown).

General Discussion

We report here the characterization of a group of micro-

satellites from Gadus morhua. It appears that the microsatel

lites in teleost fishes differ significantly in length and com

position from those of mammals. We can only speculate as

to why the microsatellites found in Atlantic cod, rainbow

trout, and Atlantic salmon are larger than microsatellites

from mammals. It remains to be seen, with subsequent iso

lation of microsatellites from other species, whether poikilo-

therms consistently have longer arrays than homiothermic

species. The longer, degenerate microsatellite arrays found

in teleost fishes may be caused by a predisposition to slip

page owing to fluctuations in temperature. The DNA poly-

merase may not function efficiently when subjected to

changes in temperature of several degrees, or temperatures

close to freezing, such as occur in the Atlantic cod envi

ronment and that of the salmonid species.

Disparities in genomic composition between warm- and

cold-blooded vertebrates have already been established by

Bernardi and Bernardi (1990). They found that fish iso-

chores were much more homogeneous and lower in GC con

tent than those of warm-blooded vertebrates. A subsequent

study by Cross et al. (1991) makes a further distinction

between fish genomes and other warm- and cold-blooded

vertebrates with regard to the GC-poor CpG islands that

are associated with some genes. It is possible that different

evolutionary forces are acting on the fish genome, gener

ating longer repeat arrays at microsatellite loci, or con

versely, shorter arrays in the genomes of mammals.

Can. J. Fish. Aquat. Sci, Vol. 51. 1994

Using PCR and primers specific to the nonrepetitive

flanks, we have successfully amplified from cod genomic

DNA seven of the cod microsatellites isolated in this study.

Five of these microsatellites exhibit heterozygosities greatly

in excess of those reported for allozyme or mitochondrial

studies. The average heterozygosity of these microsatellite

markers is H = 0.85 compared with H = 0.071 for allozymes

(Mork et al. 1985) and an equivalent measure for nucleon

diversity, h = 0.36, for mitochondrial genotypes (Carr and

Marshall 1991).

Breeding and population studies of Atlantic cod utilizing

these microsatellites as genetic markers are in progress in our

laboratory. The family Gadidae to which Atlantic cod belong

includes many other economically important species. Our

results have shown that the microsatellites reported here

may prove useful as genetic markers in other gadid species,

similar to a study previously carried out on domestic mam

mals (Moore et al. 1991). Furthermore, a comparative study

of the conservation and polymorphism of these microsatellite

loci in related gadids may elucidate the mode and tempo

of mutation at these fascinating sequences (Bentzen and

Wright 1993; Wright 1993, 1994).

Acknowledgements

Technical assistance was provided by Michiko Fllipak, Linda

Aitkinson, Barbara Edgar, and Harvey Domoslai. Rainbow trout

microsatellite data were provided by Dianne Morris prior to

publication. DNA from cod pedigrees was a generous gift of

Geir Dahle, Institute of Marine Research, Bergen, Norway. This

work was supported by funds from the Ocean Production

Enhancement Network (OPEN), the Nova Scotia government,

Department of Industry Trade and Technology, to the Marine

Gene Probe Laboratory, and the Natural Sciences and Engineer

ing Research Council of Canada to J.M.W. OPEN fellowship

support to A.L.B. is also acknowledged.

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