Genetic variability organisation and gene flow in natural populations of Medicago polymorpha L. prospected in Tunisia

HAL Id: hal-00894236

https://hal.archives-ouvertes.fr/hal-00894236

Submitted on 1 Jan 1998

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of

sci-entific research documents, whether they are

pub-lished or not. The documents may come from

teaching and research institutions in France or

abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est

destinée au dépôt et à la diffusion de documents

scientifiques de niveau recherche, publiés ou non,

émanant des établissements d’enseignement et de

recherche français ou étrangers, des laboratoires

publics ou privés.

Genetic variability organisation and gene flow in natural

populations of Medicago polymorpha L. prospected in

Tunisia

Amel Salhi Hannachi, Mohamed Boussaid, Mohamed Marrakchi

To cite this version:

Original

article

Genetic

_{variability organisation}

and

_gene

flow in natural

_populations

of

_{Medicago polymorpha}

L.

prospected

in Tunisia

Amel Salhi

Hannachi

Mohamed Boussaid

Mohamed Marrakchi

!

Laboratoire de

_génétique

et de

_{biologie moléculaire,}

faculté des sciences de

Tunis,

campus universitaire 1060,

Tunis,

Tunisia

Institut

national des sciences

_appliquées

et de

_technologie,

Tunis, Tunisia

Abstract - Sixteen accessions of Tunisian

germplasm

of

Medicago polymorpha

L. were

characterised

_{using enzymatic}

markers to

_identify

useful

_phytogenetic

resources for

their

_integration

in programmes of

_safeguard

and conservation. Protein extracts from

populations

of this

_species

were

_{analysed by electrophoresis}

to estimate

isoenzyme

variation. Two _{enzymatic systems}

_providing

six alleles were chosen from six _systems tested. From statistical _treatments, it _appears that a considerable variation was found

between accessions,

indicating

an

_{interesting genetic potential}

for selection. Such a

result

₍₅₀

% of the total variation due to

_population

_{differentiation)}

is in accordance

with the

strictly

autogamous system of

_reproduction

in this taxon.

_Isozyme

data

suggest that the level of gene flow between

populations

of M.

polymorpha

is very

low.

_{Additionally, genetic}

drift may be

responsible

for strong

among-population

differentiation. It seems that the

_genotypic

structure of natural

_populations,

as seen

from

isoenzymatic markers,

is not affected

by

the

geographic

parameters

(distance

and

_altitude).

Thus, these markers are of

practical

interest in the

background

selection

and

_improvement

_{programmes. ©}

_{Inra/Elsevier,}

Paris

Medicago

/

genetic variability

/

isoenzyme / gene flow

/

germplasm conservation Résumé - _Organisation de la variabilité génétique dans les _populations naturelles de Medicago polymorpha L. Pour identifier les ressources

_{phytogénétiques}

utiles

et leur

_intégration

dans des programmes de

sauvegarde

et de conservation, seize accessions de

_germoplasm

Tunisien de

_{Medicago polymorpha}

_L., sont caractérisées

pour leur diversité enzymatique. Les extraits

_protéiques

des populations de cette

espèce

sont

_analysés

_par

_{électrophorèse}

_pour estimer la variation

isoenzymatique.

Deux

_{systèmes enzymatiques}

révélant six allèles ont été _retenus,

_parmi

les six

*

systèmes

testés par

électrophorèse

sur

_{gel d’amidon,}

_pour leur

profil électrophorétique

polymorphe.

Les

analyses statistiques

ont montré une _importante variabilité entre les

populations

étudiées

_indiquant

un

_{potentiel génétique}

intéressant _pour la sélection.

L’hétérogénéité interpopulation

est _élevée, un tel résultat est en accord avec le

_système

de

_reproduction

strictement _autogame de ce taxon. Les données

_{isoenzymatiques}

suggèrent

que le flux

génique

entre les

_populations

de cette

espèce

est faible. La dérive

génétique

est sans doute

_responsable

de la différenciation entre les

_populations.

Par

ailleurs,

aucun

_{paramètre éco-géographique}

_(distance

et

_altitude)

ne semble influencer leur structuration

génotypique.

Les marqueurs

isoenzymatiques

ainsi révélés sont très

utiles dans des schémas de sélection _pour une meilleure efficacité d’amélioration.

©

Inra/Elsevier,

Paris

Medicago

/

variabilité _génétique

_/

isoenzyme

/

flux _génique

_/

_{germoplasm /} conservation

1. INTRODUCTION

In

_Tunisia,

in the last 10 _years, the

_preservation

of

_{plant genetic}

resources has been of

_prime

concern for wild

_forage

and

_{pastoral legumes}

because

_they

have

been

_subject

to severe

_genetic

erosion.

Indeed,

the local

_genetic

resources have been

_{damaged by}

the reduction of _range land and

_{overgrazing; pastoral}

_spaces

are

relegated

to the

dry

areas of the

_country

dominated

_{by irregular}

rainfall. The results of the erosion factors have been the

_degradation

and rarefaction of the native

_flora,

and the

_disruption

of

_equilibrium

between

_{ever-increasing}

livestock

_requirements

and limited resources.

Species

of

Medicago

and

especially

the annual

_species

can be used to valorise and enhance resources. The annual

species

of

_Medicago

constitute an

extremely

diversified

phytogenetic

_patrimony.

By

their

_integration

into

_{ley-farming, they}

can

play

an

_important

role in crop

production

increase.

_They

are also able to

_improve

_{pastoral production}

in semi-arid areas.

Medicago

is

_recognised

as one of the most

_important

_genera of

pasture

plants

in the world. The Mediterranean basin is the centre of the

_origin

of

Medicago species

and includes the world’s

greatest

variability

(Heyn, 1963).

Conscious of the value of these

plants,

we initiated a

large

research

pro-gramme in order to introduce them to fallow and

_{marginal pastoral}

zones. The aim of this work is:

₁₎

to build an

_inventory

of local

_Medicago

_species

and to

acquire

great

genetic diversity

through

a collection of accessions of

_Medicago

species

and

₂₎

to evaluate

_genetically

the

_phytogenetic

resources

_using

mor-phological

and

_{physiological}

traits as well as biomolecular tools. Selection and

improvement

programmes are based on the search for

_genetic

markers. Iso-enzymes

electrophoresis

is a useful tool for this _purpose. The

_genetic

resources assessed

by

molecular

techniques

must be considered in conservation

_strategies.

We used

_enzymatic

markers to characterise

_{Medicago polymorpha.}

This

approach

has been

_widely

used in

_elucidating

the

_genetic

structure of

_Medicago

species

(Brunel,

1979;

McCoy

et

al., 1991;

Kiss et

_{al., 1993;}

Bullita et

_{al., 1994;}

Fayed-Lameche

et

_{al., 1996;}

Salhi Hannachi et

_al.,

_1997).

Medicago polymorpha

L. _syn.

_{Medicago hispida Gaertn.,}

the annual

_species,

belongs

to the section

_Spirocarpos

of the

_Medicago

_genus. It is

_autogamous

variety

of habitats. The

ubiquitous

and

_polyvalent

nature of M.

_polymorpha

should be noted

_(Prosperi

et

_al.,

_1996).

Their

_spiny

_pods

are one of the characteristics

_favouring

their invasion

ability;

such

pods

hook onto

_sheep

wool and

_hay

_(Coks

et

_al.,

_1980).

According

to

_Heyn

_(1963),

three botanical varieties should be

_recognised:

brevispina, polymorpha

and

_vulgaris.

Several cultivars of this

_species

are com-mercialised in Australia. The most common are ‘circle

_valley’,

’serena’ and

’santiago’.

These Australian selected varieties are not well

_adapted

to the en-vironmental conditions of North Africa and

_Europe

_(Olea

et

_al.,

_1989;

Bullita

et

_al.,

_1994).

Observations of M.

polymorpha

at natural sites show that it is well

_adapted

to neutral and

_slightly

acid soils

_(Reid

et

_al.,

_1989).

Its

_geographical

distribution would indicate a

_preference

for the upper semi-arid bioclimatic

stage

(Abdelkefi

et

_al.,

_{1990, 1992,}

_1996).

M.

_polymorpha

is

_{equally adapted}

to areas in which the altitude _ranges from 10 to 900 m and rainfall from 100 to 800 mm.

In the

present

study,

we

investigated

the

_genetic

diversity by

means of

isozyme

markers.

2. MATERIALS AND METHODS

2.1. Plant material

Isoenzymatic

variation was

analysed

in 16

_spontaneous

populations

of M.

_polymorpha,

collected from a wide _range of climatic and

_edaphic

condi-tions in 1990

_(Abdelkefi

et

_al.,

_1990,

_1992).

The distribution map

(figure 1)

shows that this

_species

covers a _range of bioclimatic

_stages

_ranging

from the humid to the upper arid. Seeds

randomly

selected from each

population

were

germinated,

and the

_plants

were _grown under uniform conditions

(25

°C and 12-h

_day

_length).

2.2. Protein extraction

Electrophoresis

procedures

have been described

_by

Salanoubat

₍₁₉₈₃₎

and Baatout et al.

_(1990).

For _every

_sample,

50 _mg of _young leaves were

_ground

in

200

_vL

of extraction sodium ascorbate buffer

_{(pH 8.4)}

_comprising

_{4.15 mg of} sodium

ascorbate,

8.35 mg of D sucrose and 35

_wL

of

2-beta-mercaptoethanol

at 4 ° C. The

_homogenate

was

_centrifuged

at 13 000 rpm for 2 min.

2.3.

_{Electrophoretic}

_procedures

The six

_enzymatic

_systems

_{(table !}

were

_{electrophoresed}

in 13

%

horizontal starch

_gels.

_Twenty-four

wicks of Whatman 3 MM

_{chromatography}

paper were moistened with the

_protein

extracts and inserted into slits in the

_gel.

Migration

was anodic and conducted under 50 mA for 6 h at 4 °C in the extraction buffer and at _{constant power}

_{(16 W).}

Procedures for

_staining

_enzyme

activity

were as in Brunel

_(1979),

Stuber and Goodman

_(1980),

_Cardy

et al.

2.4. Data

_analysis

The bands of each zymogram were

interpreted

in terms of loci and alleles. Allelic

_frequencies

were used to describe the distribution of

_genetic

variation within and between

_populations

and to estimate the _gene flow _among popu-lations. These

_parameters

were estimated from

Wright’s

F-statistics

(Wright,

Fst measures

_degree

of

_{between-population}

_{differentiation;}

Fis measures

within-population variability.

A

_positive

value of Fis indicates a deficit in

het-erozygotes

in

_comparison

with the

_{Hardy-Weinberg}

_{equilibrium expectations.}

Fst is

_widely

used to estimate the

_degree

of subdivision between

populations.

Moreover,

the

_strengths

of _gene flow and random drift can be assessed from the

formula Nm =

1/4 (1/Fst - 1),

where N _is the effective _size of _a

_population

and m is the rate of

_migration

_{(Wright, 1969).}

Geographical

distances between

_populations

have been measured on a _map,

except

when a barrier to

_migration

was

_present

_(e.g.

Mediterranean

_Sea).

Isolation

_by

distance was estimated

_according

to Slatkin

_(1993).

The

_{linkage disequilibrium}

between

_pairs

of loci was estimated for each

pop-ulation

_using

the correlation coefficient

_{(Weir, 1990).}

Either selection _pressures

or

_genetic

drift

_acting

on

_pairs

of loci can

_produce

a

_{linkage disequilibrium}

among two alleles. To

distinguish

between the two _cases,

_gametic

associations of the whole data set Dst were

decomposed

into four coefficients which show the

_parts

created within

_{populations concerning}

Dis and D’is indices and

be-tween

_populations

with Dst and D’st coefficients.

_According

to Ohta

_(1982),

the

_comparison

of Dis and Dst

_values,

on the one

_hand,

and of D’is and D’st

values,

on the

_other,

allows the two situations to be differentiated.

The F-statistics were calculated

_using

GENEPOP

(version

_1.2)

software

(Raymond

and

_Rousset,

_1995).

The overall

_significance

of tests for each locus

was estimated

_by

Fisher’s combined

_probability

test

_{(Fisher, 1970).}

3. RESULTS

3.1. Genetic

_{interpretation}

Allelic

_frequencies

at the three

_polymorphic

loci

_analysed

are

reported

in table IL A total of six alleles were identified in all loci. Four _{enzymes appear}

3.1.1. Glutamate oxaloacetate transaminase

_(GOT)

Two

_{staining regions}

are

_apparent

for this _enzyme. Each

_region

exhibits

three

patterns:

a

_{triple-banded}

_pattern

_{corresponding}

to

_{heterozygotes}

and

two

_{single-banded}

_homozygotes

for diallelic loci

GOT2A,

GO T2B and

GOTI C,

GOT1D.

_Enzymes

GOTI and GOT2 are thus dimeric. Interlocus interaction is

not

_apparent

_{(figure !) .}

3.1.2. Isocitrate

_{dehydrogenase}

_(ICD)

Only

one _gene

_coding

for this _enzyme has been detected in M.

_polymorpha.

This gene appears to be

polymorphic

and biallelic

(ICDIA, ICD1B).

The ICD

isozymes

(figure 2)

produced

zymograms with one or three

_{bands, suggesting}

that those with one band were

_{homozygote phenotypes,}

and those with three bands

heterozygotes.

Only

one

monomorphic

zone of _enzyme

_activity

with a

_single

band for LAP

(monomeric

enzyme)

and PGI

_{(dimeric enzyme)}

was stained. Two

monomor-phic

bands were detected in all

_populations

for PGM

_{(monomeric system)}

and 6-PGD

_{(dimeric protein) (figure 2).}

The rare

heterozygote

individuals detected

by electrophoresis prompted

us to use allelic

_frequency

to test the

_autogamous

_reproduction

_system

of M.

_polymorpha

_using

the Fis

parameter

(table 111).

This test showed that the

populations

studied were not

_panmictic.

The observed

_{heterozygosity deficiency}

confirms the

_autogamous

nature of the

_system

of

_reproduction

but with the occurrence of a residual

_allogamy.

The average

_{heterozygosity}

was 0 to 0.055

%

3.2.

_Population

differentiation

The distribution of

_genetic

variation within and among the 16

populations,

using F-statistics,

showed

_high

_{genetic interpopulational}

differentiation. The overall differentiation _among

_populations

was

_{highly significant}

_(P

<

10-

5

)

and

corresponded

to an Fst value of 0.49

(table Il!,

_{indicating significant}

_genetic

heterogeneity

between the

_populations

studied.

GOTI,

GOT! and ICDl loci contributed to the distinction between

popu-lations

_by

FST values of

_0.68,

0.34 and

_0.56,

_respectively

_{(table T!.}

3.3. Statistical

_{linkage disequilibrium}

_among loci

Genotypic

association among

pairs

of loci was estimated from correlation

coefficients. Estimate of

_{linkage disequilibrium}

was

_{only significant}

for the association of

_{GOT! ICD1,}

in the

_(TAI)

Tabarka

_population

_(P

<

10-‘’)

(table VI).

Linkage disequilibrium

between loci for

_{pooled populations}

(table VII)

indicated

_preferential

association or

_linkage

between G’OT’!-7C*737

(P

<

_0.05)

and

_independence

between GOTI-GOT2 and GOTI-ICD1

associ-ations

_{(table VI!.}

The

_analysis

of the Ohta indices

_(table

_VII!

_supports

the

_hypothesis

that the

_gametic

associations were

_only

due to the action of

_drift,

_except

for combination of the

_pair

GOT2-ICDI. Ohta indices are

presented

for both

populations

in table VIII.

_Comparison

of

_D

_IS

and

_D

_ST

_,

on the one

_hand,

and

of

_D’

_s

and

_DS

_T

_,

_on the

_other,

shows that in M.

_pol!!norpha

the

_{disequilibrium}

between the loci

_pairs

was not

_systematic

and created

_by

_genetic

drift in each

3.4.

_{Population dynamics}

3.4.1. Gene flow

Wright

(1951)

showed that Fst is related to the level of _gene flow. The Fst of 0.498 leads to a low value of Nm = 0.26

(Nm

<

_1),

_suggesting

a low level

of _gene flow _among

_populations.

3.4.2. Isolation

_by

distance

The distinct

_{ecogeographic}

origins

of studied accessions of M.

_polymorpha

were used to

_identify

whether environmental factors are

_responsible

for the

allozyme

variation.

_{Additionally,}

_gene flow was

_analysed

in relation to

geo-graphical

distances

_according

to the Slatkin method founded on

_{slope sign}

of

regression

between Nm and distance of a

_log.

base

(Slatkin,

_{1985, 1993;}

Chevil-lon et

_al.,

1995 a,

b).

Computing

of correlation between Nm and distance shows that the

_slope

of

_regression

was near zero

(figure 3).

Another correlation coefficient was calculated between Fst and

_geographical

distance

_(Pasteur

et

_al.,

_1995),

and

again slope

was weak

_(0.069)

and

regression

not

_significant

_(P

= 0.313 _>

0.05)

Structuration of

_{genetic diversity}

was

_analysed

in relation to the altitude of sites colonised

_by

M.

_polymorpha.

Three _groups of

_populations

were defined: group I:

populations

from low altitude

(from

10 to 80

_m);

group II:

populations

colonising

stations from 100 to 500 _m; and group III:

populations

native at

_high

altitude

_(from

650 to 900

_m).

Table IX shows a differentiation _among

_populations,

estimated

_by

Fst. The differentiation within each group was

_stronger

than the differentiation between groups

(Fst

within

groups > Fst

among

groups).

Altitude did not affect the

structuration of the

_genotypes

studied.

4. DISCUSSION AND CONCLUSION

Previous work showed that in M.

_polymorpha

natural accessions a

high

level of

_{phenotypic diversity}

exists _among

_populations.

The

_variability

range seemed

to be continuous over the

prospected

area. Parameters of

_{precocity, vegetative}

development

and seed

_production

contribute to the distribution of

_phenotypic

of

_populations

and the level of _gene flow _among them. In this

_study,

we

investigated

genetic

variability

using

several

_enzymatic

systems.

Among

the six

systems

tested,

two

_(GOT

and

_ICD)

were retained. These two

_systems

delivered

six alleles and were used to estimate the _gene flow between

_populations,

to

_give

information on the

_organisation

of

_{genetic diversity.}

The

_genetic

variation

_occurring

within M.

_polymorpha

is

_organised

with

_high

levels of differentiation between

_populations.

_According

to Hamrick and Godt

(1990),

species

that have

_selfing

or mixed

_mating

_systems

have lower levels of

genetic variability

than

_{predominantly}

outcrossed

_species,

and 51

%

of their total

_genetic

_diversity

is

_apportioned

between

_populations

in

_comparison

to

10

%

for outcrossed

species.

Our results on M.

_polymorpha

_agree with these

generalisations

(50

%

of the total

_divergence

is attributable to the differenti-ation among

populations).

Indeed,

the level of

_variability

between altitudinal

groups is

significantly

lower than that between

populations

within groups. The

high

levels of

_autogamy

in this

_species

result in a

_{strong pattern}

of

among-population genetic

differentiation. Distribution of

_{genetic variability}

and the

extent of gene flow between

populations

are

_important

for the

understanding

of

_genetic

evolution. The

_degree

of

_{among-population}

differentiation is affected

by

the rate at which genes are carried between

_{populations by}

the

_migration

of

pollen

or seeds. The Fst of 0.498 leads to Nm =

_{0.26, suggesting}

_a low

gene flow among

populations.

This could be due to the

high

levels of auto-gamy in M.

polymorpha.

Genetic drift and selection could also result in

strong

among-population

differentiation

_(Nm

<

_1).

That selection occurs is

_suggested

with a

_{high dispersal}

of seeds

(Hamrick

and

Godt,

1990).

In M.

polymorpha,

easy seed

dispersal

does not

_prevent

isolation of

_populations.

This is confirmed

by

the noncorrelation between Nm and

_geographic

distances

separating

pop-ulations.

_Genotypic

structure is not affected

_by

environmental factors such as distance and altitude. M.

_polymorpha

is

_interesting

because of its

_ability

to

adapt

to difficult soils and climates. The survey and collection of the

genetic

resources of this

_species

must be

incorporated

into conservation

_strategies.

To establish a _gene bank we need to know how the

_{genetic variability}

is

_organised

in the

_species.

In-situ _gene banks have been used in

_Syria

and in Iran to select the M.

_{polymorpha variety}

_(Cocks

et

_al.,

_1986;

Nazari-Dashlibrown and

Fran-cis,

1988).

The conservation

_strategies

were fundamental to the

_development

of successful mixtures of M.

_polymorpha

for

_ley-farming

_systems

in Tunisia.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Nicole Pasteur from

Montpellier

II

University

for

helpful

discussions and statistical assistance. The authors are also

_grateful

to

Dr. Hamouda Hemissi from the

Faculty

of Sciences of Tunis for

_correcting

the

_English

version of this

_manuscript.

Support

for this research was

provided by

_grants from the ’Minist6re de

I’Enseigne-ment

Superieur’,

the ’Secretariat

d’Etat

a la Recherche

Scientifique

et a la

Technolo-gie’,

the

CNRS,

France and the French

Embassy

in Tunisia.

REFERENCES

Abdelkefi

_A.,

Boussaid M., Marrakchi M.,

Erosion

génétique

et aridite. Cas de

trois

_{légumineuses pastorales, susceptibles}

d’intervenir dans l’amélioration des

par-cours en zones

_{arides, Biologia}

1

(1990)

91-98.

Abdelkefi

_A.,

Boussaid

M.,

Marrakchi

M., Prospection

et inventaire des

esp6ces

spontanées

du genre

Medicago

L. en _Tunisie, in : Actes Coll. Inter.

_Complexes

d’esp6ces,

flux de

_genes

et Ressources

_génétiques

des

_plantes,

Paris, 8-10

_janvier

1992.

Abdelkefi

A.,

Boussaid M., Biborchi

A.,

Haddioui

A.,

Salhi Hannachi

A.,

Mar-rakchi

_M.,

Genetic

_diversity

inventory and valuation of spontaneous

species belonging

to

Medicago

L. genus in Tunisia, Cah.

Options

méditerranéennes 18

₍₁₉₉₆₎

143-150.

Baatout

_H.,

Marrakchi

_M.,

Pernes

_{J., Electrophoretic}

studies of

_genetic

varia-tion in natural

_populations

of

_{allogamous Hedysarum}

capitatum and _autogamous

Hedysarum

euspinosissimum, Plant Sci. 69

₍₁₉₉₀₎

49-64.

Brunel D., Recherche du déterminisme

_génétique

de quelques

systèmes

enzyma-tiques

chez la luzerne

_(Medicago

sativa

_L.)

di- et

_{tetraploides, these,}

Université Paris

XI, Orsay, France,

1979.

Bullita

S.,

Floris

_R.,

Hayward M.D.,

Loi

_{A., Porqueddu C.,}

Veronesi _F.,

Mor-phological and biochemical variation in Sardinian populations of

Medicago

polymor-pha

L. suitable for Mediterranean

_{conditions, Euphytica}

77

₍₁₉₉₄₎

263-268.

Cardy B.H., Stuber C.W., Goodman M.M., Technique

for starch

_gel

electrophore-sis of enzymes from maize

(Zea mays),

Inst.

Statistics,

Mimeo N°

_1317,

North Carolina State

University, Raleigh, NC,

1980.

Chevillon

_C.,

Addis

_{G., Raymond M.,}

Marchi

_{A., Population}

structure in

Medi-terranean islands and of

_genetic

invasion in Cule! pipiens L.

_{(Diptera: Culicidae),}

Chevillon

_C.,

Pasteur

_{N., Marquine M., Heyse D., Raymond M., Population}

structure and

_dynamics

of selected genes in the

mosquito

Culex pipiens, Evolution 49

(1995b)

997-1007.

Coks

P.S.,

Mathison

M.J.,

Crawford

_E.J.,

From wild

_plants

to pasture cultivars:

annual medics and subterranean clover in southern

_Australia,

in: Summerfield

R.J.,

Bunting

A.H.

_(Eds.),

Advances in

_{Legume Science,}

Kew

_Royal

Botanic

Gardens, UK,

1980, pp.

569-596.

Coks

_{P.S., Sawmy H.,}

Swedan

Y.,

Mawlawi

_B.,

Medics on farmer’s fields -

adap-tation of

_farming

to northern

_{Syria. Pasture, Forage}

and Livestock

_Program,

Ann.

Rep. ICARDA, Aleppo, Syria,

1986.

Fayed-Lameche F.Z.,

Bellatar

_S.,

Bouabdallah

S.,

Yahia

_N.,

Between and within

species variation in annual

Medicago species,

Cah.

Options

Méditerranéennes 18

(1996)

161-170.

Fisher

_R.A.,

Statistical Methods for Research

_Workers,

14th

_{ed., Olivier, Boyd,}

Edinburgh,

1970.

Goodman

_{M.M., Stuber C.W., Lee C.N., Johnson F.M.,}

Genetic control of malate

dehydrogenase isozyme

in

_maize,

Genetics 94

₍₁₉₈₀₎

153-158.

Hamrick

J.L.,

Godt

_{N.J., Allozyme diversity}

in

_{plant species,}

in: Brown

_A.H.D.,

Clegg M.T.,

Kahler

_A.L.,

Weir B.S.

_(Eds.),

Plant

_{Population Genetics, Breeding}

and Genetic

Resources,

Sinaner

_{Associates, Sunderland, MA, 43-63;}

Evol. Biol. 7

₍₁₉₉₀₎

1-144.

Heyn C.C.,

The Annual

Species

of

_{Medicago. Scripta hierosolymitana,}

Hebrew

University Press, Jerusalem,

1963.

Kiss

_G.B.,

Csanadi

G.,

Kalman _K., Kalo _P., Okresz L., Construction of basic

genetic

map for

alfalfa using RFLP, RAPD, isozyme

and

_{morphological markers,}

Mol. Gen. Genet. 238

₍₁₉₉₃₎

129-137.

Lesins

_K.A.,

Lesins

_1.,

Genus

_Medicago

_{(Leguminosae).}

A

_Taxogenetic

_Study,

W.

Junk,

The

_{Hague, Boston, London,}

1979.

McCoy

T.J., Echt

C.S., Mancino L.C., Segregation

of molecular markers _supports

an

_{allotetraploid}

structure for

_Medicago

sativa x

_{Medicago papillosa interspecific}

hybrid,

Genome 34

₍₁₉₉₁₎

574-578.

Nazari-Dashlibrown P., Francis

C.M.,

Value and

_potential

of medic based

_ley

farming

in

_Iran,

technical _report,

_Forestry

and

_{Range Organization, Shamarin, Iran,}

1988.

Ohta

T., Linkage disequilibrium

with the island

_model,

Genetics 101

₍₁₉₈₂₎

139-155.

Olea

L.,

Parades

_J.,

Verdasco

_P.,

Caracteristicas

_productivas

de los pastos de la dehesa de S. O. DE la

_{peninsula Iberica,}

SEEP XXIX

_{Elvas, Portugal, 1989,}

pp. 147-172.

Pasteur N.,

Marquine M.,

Rousset F., Failloux

_A.,

Chevillon

_{C., Raymond M.,}

The role of

_{passive migration}

in the

dispersal

of resistance genes in Culex pipiens

quinquefasciatus

with French

_Polynesia,

Genet. Res. 66

₍₁₉₉₅₎

139-146.

Prosperi J.M.,

Angevain M.,

Bonnin L, Chaulet

E.,

Genier

G.,

Jenczewski

_E.,

Olivieri

L,

Ronfort

_J.,

Genetic

_diversity,

preservation and use of

_genetic

resources

of mediterranean

_{legumes: Alfalfa}

and

_Medics,

Cah.

_Options

Méditerranéennes 18

(1996)

71-90.

Raymond M.,

Rousset

_{F., GENEPOP,}

version

_1.2,

a

_{population genetics}

software for exact tests and ecumenicism, J. Hered. 86

₍₁₉₉₅₎

248-249.

Reid

_{R., Konopka}

_J., Rihana

_J.R.,

Needs and _priorities collection of annual

medic

_germoplasm,

in: Christian

S.,

Materon

_L.,

Falcinelli

_M.,

Cocks P.

_(Eds.),

Salanoubat

_{M., Polymorphisme enzymatique}

et evaluation des ressources

g6n6ti-ques :

application

a 1’etude d’une collection de mafs

_(Zea

_mays

_L.),

_these,

Univ.

Paris-sud, Orsay, France,

1983.

Salhi Hannachi

_{A., Analyse}

de la diversité

_génétique

des

_populations

spontanées

de

_{Medicago polymorpha L., these,}

Faculte des Sciences de

_{Tunis, Tunisia,}

1996.

Salhi Hannachi

_A.,

Boussaid

_M.,

Marrakchi

_M.,

Diversité

génétique

de

_quelques

populations spontanées

de

_{Medicago polymorpha}

L.: variabilité

morphologique

et

polymorphisme isoenzymatique,

in : Rencontre internationale Gestion des ressources

génétiques

des

plantes

en

Afrique

des _savanes,

_Bamako,

24-28 février

_1997,

Bureau des ressources

_{génétiques, Paris,}

1997.

Slatkin

_M.,

Rare alleles as indicators of gene

flow,

Evolution 39

(1985)

53-65.

Slatkin

M.,

Isolation

_by

distance in

_equilibrium

and

_{non-equilibrium populations,}

Evolution 47

₍₁₉₉₃₎

264-279.

Stuber

C.W.,

Goodman

_M.M.,

Genetics of 6-PGD in corn

_Maize,

Genet.

Cooperation

Newsletter 54

₍₁₉₈₀₎

99.

Weir

_{B.S., Intraspecific differentiation,}

in: Hillis

D.M.,

Moritz C.

_(Eds.),

Molecular

Systematics, Sunderland, MA,

Sinaner

_Associates,

_{1990, pp.} 373-410.

Weir

B.S.,

Cockerham

_{C.C., Estimating}

F-statistics for the

_analysis

of

population

structure, Evolution 38

₍₁₉₈₄₎

1358-1370.

Wright S.,

The

_genetical

structure of

_populations,

Ann.

_Eugen.

15

₍₁₉₅₁₎

323-354.

Wright S.,

The

_Theory

of Gene

_Frequencies,

vol. 2,

University

of

_{Chicago Press,}