Length variation in mtDNA control region in hatchery stocks of European sea bass subjected to acclimation experiments

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Length variation in mtDNA control region in hatchery

stocks of European sea bass subjected to acclimation

experiments

Giuliana Allegrucci, Donatella Cesaroni, Federica Venanzetti, Stefano

Cataudella, Valerio Sbordoni

To cite this version:

Original

article

Length

variation

in

mtDNA

control

_region

in

_hatchery

stocks

of

_European

sea

bass

subjected

to

acclimation

_experiments

Giuliana

_Allegrucci*

Donatella

_Cesaroni,

Federica

Venanzetti

Stefano Cataudella Valerio Sbordoni

Department

of

Biology,

Tor

_{Vergata University,}

via della Ricerca

Scientifica,

00133

_{Rome, Italy}

(Received

29

_May

_1997;

_accepted

17 March

₁₉₉₈₎

Abstract -

_{Length polymorphism}

of the mtDNA control

_region

was

_analysed

in

samples

from the sea bass Dicentrarchus labrax before and after acclimation to

freshwater. Acclimation trials were

_repeated

twice for two

_{samples originating}

from the same broodstock. DNA

_{amplification}

of 221 individuals made it

_possible

to detect 40 different

_{D-loop length}

variants and a mean level of _gene

_diversity

(/c!)

of 0.850.

Out of 52 scored distinct _genotypes, 18 were

_homoplasmic

and the

_remaining

ones were

heteroplasmic

for _{up to} four variants. Shifts in

frequencies

between

_starting

and acclimated

_samples

in the same direction of the same _genotypes in both _years were not observed. Patterns of mtDNA control

_region

variation were

_compared

to

previous

allozymic

and RAPD studies carried out on the same

samples.

While

variation found in the

_allozyme

alleles and the RAPD markers in

_{replicate experiments}

revealed differential survival of the genotypes, stochastic processes, such as

_genetic

drift, explained

the observed variation of mtDNA control

region.

Very high

levels of _genotype

_diversity

were observed within each

_{sample, ranging}

from 0.77 to 0.97. This

_{high diversity}

associated with the maternal inheritance of mtDNA makes this molecular marker

_especially

suitable for studies in

_{aquaculture genetics,}

when the

allozyme

studies fail to reveal

_genetic

variation or when the identification of _progeny

based on the mother’s contribution is relevant.

_©

_{Inra/Elsevier,}

Paris

fish mtDNA

_/

D-loop region

/

European sea bass

_/

hatchery stock

_/

acclimation trials

Résumé - Polymorphisme de la longueur de la région de contrôle de l’ADN mitochondrial dans des souches de bar _européen soumis à des _expériences d’acclimatation à l’eau douce. Le

_{polymorphisme}

de

longueur

de la

région

de contrôle de l’ADN mitochondrial

_(ADNmt)

a été

_analysé

dans des échantillons de

bar

_{(D. labra!)}

avant et

_après

des

_expériences

d’acclimatation à l’eau douce. Les

expériences

d’acclimatation ont été

répétées

sur deux échantillons de même

_origine.

L’amplification

de l’ADNmt de 221 individus a _permis de détecter 40 variantes de

longueur

de la boucle D et une diversité

_génique

(K

c

)

de

0,850

en _moyenne. Sur

52

_{génotypes analysés,}

18 ont été

homoplasmiques

et le reste

_{hétéroplasmiques,}

avec

jusqu’à

quatre variants chez le même individu. La variation de la

_région

de contrôle

de l’ADNmt a été

_comparée

à celle observée lors d’études

précédentes

sur les mêmes

échantillons pour les

allozymes

et aux _marqueurs RAPD. Alors _que la variation liée aux

_allozymes

et aux _marqueurs RAPD trouvés a

_indiqué

des différences de survie

des

génotypes après acclimatation,

des processus

stochastiques,

comme la dérive

génétique,

peuvent

expliquer

la variation observée.

A

l’intérieur de

_chaque

échantillon,

on a observé de hauts niveaux de diversité

génétique

avec une variation

_comprise

entre l’indice 0,77 et l’indice

0,97.

Cette diversité associée à l’hérédité maternelle de

l’ADNmt semble rendre ce _marqueur moléculaire très utile pour les études

génétiques

appliquées

à la

_{pisciculture.}

_{© Inra/Elsevier,}

Paris

poisson

/

bar

_/

ADN mitochrondrial / D loop

/

acclimatation

1. INTRODUCTION

The

_European

sea

_bass,

Dicentrarchus

labrax,

is an

_{Euryhaline species}

found in the northeastern Atlantic Ocean and in the Mediterranean sea. It occurs in coastal

_waters,

_lagoons

and estuaries. This

_species

_{is very}

_important

in the

_{fishery industry}

and is also

_widely

used in

_aquaculture

_{[7, 26].}

The

genetic

identification and discrimination of

_aquaculture

stocks is a fundamental

requirement

in culture _programmes

_especially

for those directed at

_using

genetically

characterized breeders. The

_knowledge

of

_genetic

_{heterogeneity}

within and _{among stocks} makes it

_possible

to minimize the deleterious effects of

_inbreeding

_and/or

_specific

selective

_agents

_by

_planning

_{appropriate crossing}

schedules.

For the

_past

few _years we have been

analysing

the distribution of

genetic

variability

in wild and cultivated

populations

of sea bass D.

labrax, using

differ-ent

_{genetic markers,}

such as

_allozymes

[1, 3!,

RAPDs

[2, 13]

and mitochondrial DNA

_(mtDNA;

_{[14-16, 29].}

Over two

subsequent

years

(1989

and

1990)

we followed the

_changes

in

genetic

variation in reared

_samples,

before and after acclimation to freshwater. Both

_allozyme

and RAPD markers showed that survival to acclimation to freshwater was not random.

_Variability

estimates based on 28

_allozyme

loci decreased between the

_starting

and acclimated

_{samples by}

about 50

%.

Survival rates and relative fitnesses per

genotype

per locus were estimated and in both

years the same

_genotype

at certain loci

_(i.e.

_Ck-4,

_Est-2,

_G6pd,

_Me,

_Np)

showed the

_{highest probability}

of survival

_[1].

Similar shifts were also observed in RAPD markers. Even

_{though only}

one band out of the 126 markers scored showed concordant shifts in both _years, which were

_{statistically}

_significant,

several other markers also varied

concordantly

!2!.

These results

_suggested

the

within an

_{heterogeneous}

_array of

_genotypes,

_probably

reflect the different

geographic

origins

of broodstock

_{[1, 2J.}

We followed the behaviour of both

_allozymes

and RAPD markers in wild

populations

from the Mediterranean sea,

sampled

either in open sea or in coastal

_lagoons.

_{Interestingly,}

the

_allozyme

_genotypes

and RAPD

_bands,

which increased in

_frequency

in the

_samples

acclimated to

_freshwater,

were also more

frequently

found in wild

_populations

from brackish water rather than from the open sea

[3, 13J.

Wild

_populations

were also

_investigated

_using

a third

_marker,

the

_D-loop

or control

_region

of

_mtDNA,

which revealed the existence of

_length

polymorphism

!16J.

In this _paper we

_analyse

the same cultivated

_samples

_previously

studied

by allozymes

and

_{RAPDs, studying}

the

_{length polymorphism}

of the mtDNA control

_region,

before and after acclimation to freshwater. This

_region

has proven to be

particularly

suitable for

population genetic

studies

!9J,

because it has the

_highest

nucleotide substitution rate of all the mitochondrial genome,

with an

_evolutionary

_{rate up to} five times

_higher

than the

_coding

_portion

[20, 30J.

The control

_region

is

_{primarily responsible}

for the observed variation in the total

_length

of the vertebrate mitochondrial _genome

_(see

review in

_Meyer

[21, 22]).

In

_vertebrates,

the

D-loop region

often varies in

_length

because of tandem

_duplications

_{[23, 28J.}

In

_fish,

_length

variation is not a rare

_phenomenon

and in most cases it is due to variation in the _copy number of tandem

_repeats

in the control

_region

_[5,

_{8, 11, 12, 22, 24,}

_31J.

In D.

_labrax,

the control

_region

has proven to be

_{only responsible}

for the observed individual variation in total

length

of the mtDNA molecule

_[29].

The

_sequencing

of

_length

variants from three distinct D. labrax individuals revealed the existence of

_a)

a variable number of

_copies

of two

_strictly

related

_17-bp

_tandemly

_repeated

sequences;

b)

a variable number of

copies

of two

_strictly

related

48-bp tandemly repeated

sequences; and

c)

an

_{’imperfect’ 17-bp}

_repeat

_{occurring only}

once in the

D-loop region,

which

_separates

the _array of

_17-bp

_repeats

from the _array of the

_48-bp

repeats

(figure

1;

(15J).

A survey of 209 individuals collected in

eight

Mediterranean localities confirmed that mtDNA

_length

variation in D. labrax is the outcome of the

contemporary

variation in

_repeat

_copy number of these two

repeat

arrays.

Moreover,

it revealed

_high

levels of

_heteroplasmy

_{(33.3-70 %),}

and

_{genetic diversity, especially}

between individuals within

_populations

_!16J.

The aim of the

_present

_study

was to characterize and

_{quantify length}

vari-ation in a

_{large sample}

of reared

individuals,

in order to evaluate differences after acclimation to freshwater.

_Comparisons

with

_allozyme

and RAPD vari-ation were also carried

_out,

in order to examine the usefulness of the three molecular markers as a source of

_genetic

markers in sea bass

_aquaculture.

2. MATERIAL AND METHODS

Samples

were taken from the ENEL fish

farms, ’Torrevaldaliga’

(Civitavec-chia,

Latium)

and ’La Casella’

_{(Piacenza, Lombardy).}

_Samples

from

’Torreval-daliga’

were obtained from artificial

reproduction

in 1989 and 1990. In both

years, the same

_broodstock,

made _up of 190 individuals from different local-ities

_along

the Italian coasts

_(i.e.

50

%

from

_Thyrrenian

sea and 50

%

from northern Adriatic

_sea),

contributed to artificial

_{reproduction,}

the sex ratio

seawater conditions _up to a size of 70 _{g in}

weight.

Average

_age was 10 months

(1989

sample)

and _{1 year}

_{(1990 sample).}

_{Yearly samples}

_(ca.

7000 sea basses

in 1989 and 2 600 in

₁₉₉₀₎

were taken from this stock and transferred to the ENEL fish farm ’La

_Casella’,

where

_they

were acclimated to freshwater

_(for

fur-ther details see

_Allegrucci

et al.

_!1!).

At the

_sampling

time

_(i.e.

after 15 months for 1989

_sample

and 7 months for 1990

_one)

_mortality

rates were 94.4

%

for 1989

_sample

and 74.7

%

for 1990 one.

Starting

samples

from

Torrevaldaliga

collected before freshwater acclimation were named S89 for 1989 and S90 for 1990.

_Samples

from La Casella collected after acclimation trials were named A89 for 1989 and A90 for 1990.

DNA was extracted from individual

_{fishes, following}

a standard

phenol-chloroform

_procedure

detailed in

_Allegrucci

et al.

_(2!.

We

_amplified

the variable

portion

of the

_{D-loop region by}

PCR. Primer sequences were derived from Cecconi et al.

_!15!.

_They

are

complementary

to

_unique

_sequences

_flanking

the tandem arrays located at

_positions

284-302

_{(CCCGAGTGCCACAAACGCG)}

and

_positions

1509-1490

_{(TTGTCCCTGAACTAACAGCC)}

of the D. labrax

D-loop

sequence

(EMBL

Data

Library

accession no.

X81472).

Amplifications

were

_performed

with a Perkin-Elmer Cetus thermal

_cycler

in

50

_R

_L

of a solution

_containing

10 mM

_’I!is-HCI,

50 mM

KCI,

1.5 mM

_MgCl

₂

_,

each dNTP at 2.5

_mM,

about 20 _ng of

_template

_DNA,

1U of

_Amplitaq,

15 _ng for each of the two

_primers

used.

_{Amplification}

conditions included a total of

30

_cycles

of 1 min at

_{94 °C,}

1 min at 55 °C and 2 min and 30 s at

_{72 °C, using}

the fastest available transition between each

_temperature.

Individual PCR

amplification products

were

_separated

_{by electrophoresis through}

ethidium-bromide stained 1-1.5

%

_agarose

_gels

in Tris-borate buffer and their size was scored in

_comparison

to

_appropriate

molecular

_weight

standards.

Each individual

_fragment

was double

_digested

with the restriction endonucle-ases Nde

_{1, recognizing}

a site within all the _sequences of R48

_repeats,

and Xba

_I,

which cleaves the

_amplified

_region

at an

_unique

site 143

_bp

_away from 3’ end

(figure

1).

After

_digestion,

the

_products

were visualized on 2

%

TBE _agarose

in the

_figure)

and a residual

_fragment

whose variable

_length

_depends

on the number of R17

repeats.

The

_{143-bp fragment}

derives from the

_cleavage

site of Xba I _away from 3’ end of the

_{amplified region;}

the

_163-bp

is

_comprised

on the

D-loop

map between the Xba I restriction site and the first Nde I site away from

3’

end. Nde

_I,

_cleaving

once in each R48

_repeat,

_{produces n 48-bp fragments.}

In

_figure

_2,

the three

_fragments

of

290,

307 and 324

bp

(belonging

to different

individuals)

illustrate the

_length

_variability

of the

_D-loop

_{fragment including}

the R17

_repeats.

Each one of these variable

_fragments

includes the

_primer

(at

the

5’

_end),

26

_bp

of the first R48 element and 194

_bp

_{(non-repeated}

_sequence between the

_primer

and the first R17

_element)

for a total of 239

_{bp plus}

n R17. The number of R48

_repeats

was inferred

_{by comparing}

the

_fragment

sizes before and after

_digestion.

In

_fact,

the difference in molecular

weight

among D. labrax

D-loops

is due

_only

to the different

_composition

in number of R17

_and/or

R48. Once we calculated the

_repeat

combination for each

_{amplified fragment,}

the molecular

_weight

of each

D-loop

examined was also

indirectly

inferred.

The statistical data

_analysis,

i.e.

_frequency

distribution and multivariate

analysis

were

_performed

_by

_using

standard

_procedures

included in the STA-TISTICA

_package

_(ver.

_5.1,

_StatSOFT,

Inc.

_!27!).

Estimates of

_{genetic diversity}

were calculated within each

_sample

for both

length

variants and

_genotypes,

_according

to the

_general

formula K = 1 -

Ex2,

where xi is the

frequency

of the ith size

length

variant or

_composite

_genotype

within the

_sample

_(10!.

Estimates of _gene

_diversity

_(K!)

were obtained when

frequencies

of

_length

variants were used and estimates of

_genotype

_diversity

(G)

were obtained when

frequencies

of

_composite

_genotypes

were used. Standard errors were estimated

_using

a

_jackknife

procedure

within each

sample.

Each

estimates. ‘1’he

_jackknife

mean is the _average of the n estimates and the

jackknife

variance is their variance.

Likelihood ratio test

_analyses

were used to determine if the

_genotype

frequencies

were

_{significantly}

different _among

_samples.

The exact test was used because the _sparseness of data

sets,

using

SPSS

_package

_(vers.

_6.0,

Metha and

Patel/SPSS

Inc.,

1993).

Multivariate ordination of individual fish based on the

D-loop

_genotypes

was studied

_by

means of

_{correspondence analysis, using}

individual

_profiles.

Each individual was characterized for each variable

(length variant)

as 1 in the presence of the

length

variant and as 0 in its absence. This

_analysis

was carried

out

_separately

on the two

_yearly

_samples

_(S89-A89

and

_S90-A90,

_{respectively).}

3. RESULTS

Each individual

_{amplification produced}

a maximum of four

_fragments

of different

_lengths,

which were

_interpreted

as

_{heteroplasmic}

variants if

_they

were

present

in the same individual. In table

_I,

the total number of

_fragments,

number of

_length

variants and number of distinct

_genotypes

for each

_sample

(S89,

A89,

S90 and

_A90)

are

_reported.

Estimates of

_heteroplasmy

within each

sample

are also

_reported.

The

_percentage

of

_heteroplasmy

was about 30

%

in

S89,

S90 and A90 and reached 70

%

in A89.

Among

the 221

_specimens

of D. labra!

_analysed,

40 different

_D-loop

_length

variants were detected. For each

_variant,

the overall size of the

_amplified

product,

the number of

_tandemly

_repeated

R17 and R48 sequences, and the relative

_frequency

in each

sample

are

_reported

(table

77).

The minimum and maximum

_length

of the

_{amplified portions}

were 884 and 1 463

_bp,

_{respectively.}

The total

_length

variation was due to the combined variation of the number R17 and R48

_repeats,

as described in Cesaroni et al.

_!16!.

More than 50

%

of the scored

_{D-loop length}

variants

₍₂₂

out of

₄₀₎

were

singletons,

and occurred

only

in one of the four

_samples.

Between the two

_{yearly samples only}

15 out of 40

_length

variants

_{(35 %)}

were in common.

_Starting

and acclimated

_samples

within each year shared 21 and 44

%

(in

1989 and

_1990,

_{respectively)}

of the

D-loop length

variants

_(table

_II).

Estimates of gene

diversity,

K!,

for this mtDNA

region

ranged

from 0.750 to 0.899 and are

_reported

in table IIL

Out of 221

_{assayed specimens}

in the two

_{yearly samples,}

a total of 52 distinct

homoplasmic. Heteroplasmic

genotypes

with two bands were named

LP18-LP39. Eleven

_genotypes

were made _up of three

_fragments

(LP40-LP50)

and

only

two

_genotypes

_(LP51

and

_LP52)

were

_{heteroplasmic}

for four

_fragments.

Heteroplasmic

individuals with three or four bands were rare, and

they

were

Mean

genotype

diversity

estimates,

G,

calculated for each

_sample,

indi-cated a reduction between

_starting

and acclimated

_samples

in 1989

(G

S

g

9

=

0.865 t

_{0.0004, G}

_A

_g

₉

= 0.772 !

0.001)

and a

_slight

increase in the 1990

_sample

(Gs9o

= 0.876 t

0.0006, G

A90

= 0.911 !

0.0004).

Although

_genotype

diversity

estimates were rather similar in both _years, the

_pattern

of distribution of the

D-loop

genotypes

was

remarkably

different

(figure 3).

In

1989, only

six out of 35

_genotypes

were shared

_by

the two

_samples,

S89 and

A89,

before and after acclimation to freshwater. The other

_genotypes

either in the

_starting

_sample

or

in the acclimated one were

mostly singletons.

In

1990,

9 out of 30

_genotypes

were in common between the S90 and A90

samples,

before and after acclimation to freshwater. The other

_genotypes

occurred in one or at least two individu-als either in the

_{starting sample}

or in the acclimated one. Between the two

yearly samples only

13 out of 52

_genotypes

were in common,

although

_diversity

estimates were

similarly high

(table

111).

In both _years,

only

a few

_genotypes

had a

_{high frequency}

of occurrence

(>

10

_%).

In

_1989,

four

_genotypes

_(LP1,

LP8,

LP10 and

_LP19)

were the most common. In

_1990,

three

_genotypes

(LP6,

LP8 and

_LP10)

had

_frequencies

_higher

than 10

%.

_Only

the LP8 and LP10

genotypes

were

frequent

in both _years

(figure 3).

Likelihood ratio tests were carried out to evaluate differences in

_genotype

fre-quencies

between

_starting

and acclimated

_samples

and between the two

_yearly

samples.

Statistically significant

differences

_(exact

probability,

P « 0.01 were observed between all

_samples,

_except

for the

_S90/A90

_comparison

which showed

an exact

_{probability equal}

to 0.087.

Results from the multivariate

correspondence

analysis,

carried out on indi-vidual

_D-loop

_genotype

_profiles,

did not reveal _any shift in

_genotype

_frequencies

between the

_starting

and acclimated

_samples

in both _years.

4. DISCUSSION

4.1. Levels of

genetic

variation

The results of this

_study

confirmed the occurrence of

_high

levels of

_length

polymorphism

in the mtDNA

_D-loop

_region

of D.

labrax,

as

already

observed

in wild

_populations

_!16!.

The

_high

_{variation in copy} number of both the R17 and R48

_repeats,

associated with the

_high

_degree

of

_heteroplasmy

found in D. labra!

_populations,

were

probably responsible

for the

high

number of distinct

genotypes

found either in the wild

_populations

or in the cultivated

_samples.

Statistically significant

differences were found between the

_genotype

fre-quency distribution of the two

_{yearly samples}

_(exact

_probability

«

0.01,

figure

3).

This

_suggested

that these

_samples,

since mtDNA is

_maternally

in-herited,

originated

from

_genetically

different maternal lines within the same broodstock. This result is in

_agreement

with those obtained from

_allozyme

and RAPD markers

_analyses,

carried out on the same individuals

[1, 2!.

As with

allozymes

and RAPD

_markers,

the

_{D-loop length}

variation

_suggested

that the two

_starting

_populations

were

_{genetically heterogeneous. Indeed,}

the

_original

broodstock was

_{heterogeneous,}

as it was

_composed

of individuals from different

geographic populations

from Italian coastal waters.

When the levels of

diversity

of the

hatchery

stock

_samples

were

compared

length

variation could be observed. The

_study

of wild

_{populations coming}

from

_eight

Mediterranean

_localities,

_ranging

from France

_(Gulf

of

_Lyons)

to

Egypt

(Bardawill lagoon),

revealed a total of 104

_{D-loop length}

variants and an _average level of _gene

_diversity,

K!,

_equal

to 0.937 f 0.013

_!16!.

As

_previously

pointed

out,

the cultivated

_samples

of sea bass

_originated

from a broodstock

composed

of individuals

_belonging

to different Italian

populations

both from Adriatic and

_Thyrrenian

seas.

Unfortunately,

we did not

_analyse

the mtDNA

length

variation in any other Adriatic

_populations.

We could

_only

_compare the

_genetic

_diversity

between

_hatchery

stock

samples

and wild

populations,

by

using

the wild

_populations

_coming

from

_Thyrrenian

sea. The four

_Thyrrenian

populations

showed 76

_length

variants across 121

assayed

individuals and an

average level of gene

diversity,

K!, equal

to 0.930 f 0.022. The cultured stock showed a

_significant

reduction in mtDNA

_length

variation with

_respect

to the wild

_populations,

_having

_only

40

_D-loop

_length

variants over 221

_specimens,

with a

_K

_c

of 0.850 t 0.033.

The reduction in

_genetic

_variability

in cultivated stocks of

_{aquatic organisms}

is a known

_phenomenon.

This reduction can be due to the limited number of

founding

parents

relative to wild

_populations,

where

_genetic

drift mediated

by

reductions in effective

_population

size can cause lower levels of observed

genetic

variation

_[4].

Studies carried out on wild and cultured stocks of salmonids

_by

restriction

_{fragment length polymorphisms}

_(RFLPs)

in mtDNA demonstrated the existence of lower variation in cultured stocks. For

_example,

Ontario

aquaculture

stocks of rainbow trout

_{(Onchorhynchus mykiss)}

showed

an _average nucleon

diversity

less than one-half of that of the wild

populations

!17!.

Swedish stocks of brown trout have

_only

25

%

of the mtDNA

_variability

of the wild

populations

!19!.

The loss of

diversity

in D. labrax is

_probably

due to domestication.

_However,

we do not know the level of variation in the Adriatic

populations.

If

_they

have less variation than the

_Thyrrenian

_ones, this could also

_explain

the reduced

_polymorphism

levels of the cultured stocks.

When

_{analysing allozyme polymorphisms}

of wild and

_hatchery

stocks of D.

_labrax,

we did not observe a loss of

_genetic

_variation,

in fact the _average

allozyme heterozygosity

was of the same order of

_magnitude

in both the hatch-ery and the wild stocks of sea bass

(average

_{heterozygosity}

was of about 10

%;

A

_{possible explanation}

of these data can be found in the

_properties

of the mtDNA. It is

_{predominantly}

inherited

_maternally

and no recombination between mitochondrial _genomes is known

_!6!.

These factors contribute to the reduction of the effective

_population

size for mtDNA with

respect

to that for the nuclear genes of the same

_organisms

(generally,

in case of a 1:1 sex

_ratio,

the effective

_population

size is one-fourth _{of that for nuclear genes;}

_[25])

and make it more

susceptible

to

_genetic

drift and founder effect.

4.2.

_D-loop

_length

variation versus

_rapid

and

_allozyme

polymorphisms

As

_{previously pointed}

_out,

the same individuals were studied for

_allozymic

and RAPD variation

_{[1, 2].}

Multivariate ordination

_analysis

_{(correspondence}

analysis)

was carried out for each of the three data sets.

_Using

the first

_principal

axis score as a

_synthetic

_descriptor

of variation

expressed by

each

genetic

marker,

we were able to _compare the results from the three different molecular

techniques.

Using

the three

_synthetic

_descriptors

as dimensional axes of a 3D _space, we obtained a schematic

_{representation}

of the cumulative

_genetic

variation in sea bass

(figure !,).

_Using

the axis relative to

_D-loop

_genotype

variation,

it was not

_possible

to

_distinguish

between

_starting

and acclimated

samples

in both _years. This outcome contrasts with those from RAPDs

and,

especially,

allozymes,

where a clear

_separation

can be observed in both _years

(,figure

4).

RAPD markers and

_allozyme

loci revealed a differential survival rate for the different

_genotypes

to acclimation to

_freshwater,

because some of the RAPD bands and most of the

_allozyme

alleles

_changed

in the same direction

in both _years. On the basis of these

results,

we have

_suggested

that selection was

responsible

for the observed shifts in

frequencies

in these markers

[1, 2].

In the mtDNA control

_region,

we also

_only

observed

_{statistically significant}

differences in

_genotype

_frequencies

between

_starting

and acclimated

_samples

in 1989

_(exact

P «

_0.01,

_figure

_3).

Differences between

_starting

and acclimated

samples

were also found in

1990,

although they

were not

_significant

_(exact

P =

_0.08,

figure

3).

_However,

_we did _not observe shifts in

frequencies

in the

same direction of the same

_genotypes

in both _years.

This result is not

_surprising

if one considers that these three

_genetic

markers

are

_marking

different

parts

of the genome.

Allozymes

are

_parts

of the functional genome and therefore

they

are more

_{probably subjected}

to selective _pressures. RAPD markers are,

by

definition,

randomly amplified

and most of them may mark less functional

_parts

of the genome and thus are not

_expected

to trace selective effects. The control

_region

of mtDNA

_(D-loop)

is a

_non-coding

_region

of the mitochondrial genome and is more

likely

to be neutral than

_allozymes.

On the other

_hand,

_specific

_D-loop

_genotypes

could in

_principle

be linked to

genes

subjected

to selection.

_However,

selection would have to be _very

_strong

in order to overcome

_genetic

drift because the effective

_population

size of mtDNA is much lower than that of nuclear _genes.

mother contribution may be relevant. Wild

populations

show

genotype

diversity

ranging

from 0.855 to

_0.967,

_comparable

to that obtained in the

_present

paper for

starting samples

(table III).

Such

_high

_diversity

within

_populations

ensures a

_probability

of 73-93.5

%

for

_identifying

_any individual

_by

a

_given

genotype.

Moreover,

this level of

_polymorphism

is

_comparable

to that observed in a

_single

microsatellite locus

!18!.

When

_studying

reared and wild stock of

species

of economic relevance such as D.

_labrax,

the

_opportunity

of individual

characterization could be beneficial.

The combined

_study

of

_highly

variable

_markers,

such as mtDNA control

re-gion

and microsatellites would be

_highly

desirable in

_aquaculture

_programmes, for three

_major

reasons:

_a)

both these molecular

_techniques

use a PCR

me-diated

_approach

which has the

_advantage

of

_{characterizing}

samples

by taking

tissue

_biopsies

from reared

_specimens

without

_sacrificing

_them;

_b)

the

analy-sis of nuclear

loci,

such as

microsatellites,

could be used as a

complement

to avoid the bias of

_{using only}

one

_maternally

inherited

_{genetic marker;}

and

c)

it could

_provide

information on both maternal and

_{paternal genetic}

contribution

in reared stocks. Such information could be useful for

_{reconstructing pedigrees}

of cultured

_stocks,

which are essential for

_identifying

founder

_individuals,

avoid-ing

deleterious

_inbreeding

and

_{maintaining variability by maximizing}

founder contributions.

ACKNOWLEDGEMENTS

The authors

_{gratefully acknowledge}

Dr P. Bronzi for

_providing

the sea bass

_samples.

to A. Caccone for useful hints on this work and to G. Carchini who

_suggested

the

use of the Exact test. C. Di Russo

_helped

us to write the summary in French. This

research was

_{supported by}

a National Research Council _grant to V.

Sbordoni, Special

Project, RAISA, Sub-project

n.3.

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