The relevance of outcrossing for the dynamic management of genetic resources in predominantly selfing Triticum aestivum L. (bread wheat)

HAL Id: hal-00894241

https://hal.archives-ouvertes.fr/hal-00894241

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.

The relevance of outcrossing for the dynamic

management of genetic resources in predominantly

selfing Triticum aestivum L. (bread wheat)

Jérome Enjalbert, Isabelle Goldringer, Jacques David, Philippe Brabant

To cite this version:

Original

article

The relevance of

_outcrossing

for

the

dynamic

management

of

genetic

resources

in

predominantly

selfing

Triticum aestivum

L.

_{(bread wheat)}

Jérome

_Enjalbert

Isabelle

_Goldringer

Jacques

David

b

_Philippe

Brabant

a

Station de

_{génétique végétale,}

Institut national de recherche

_agronomique,

Inapg, Ups,

Ferme du

Moulon,

91190

Gif-sur-Yvette,

France

b

Station

de

_génétique

et amélioration des

_plantes,

Institut national de recherche

agronomique,

Domaine de

Melgueil,

34130

_Mauguio,

France c

Institut national

_{agronomique Paris-Grignon, 16,}

rue Claude

_Bernard,

75005

Paris,

France

Abstract - The

_mating

_system is of

_major

concern for the

dynamic

management of

_genetic

resources. We

_investigated

the

_outcrossing

rate of six

_experimental

wheat

populations,

derived from two

_{genetic pools}

_(PA

and

_PB)

which have been cultivated at four French sites. The

replicates

of the two

_segregating

populations

have

undergone

10 _years of

_{multiplication}

in

_isolation,

with no artificial selection. For each

_population,

78 individuals were

_{randomly sampled}

and

genotyped

for 25 codominant loci revealed

by

15 restriction

_{fragment length polymorphism}

_(RFLP)

_probes.

_Outcrossing

rates

were estimated on the basis of the multilocus

_{heterozygote deficiency.}

Estimates

ranged

from 2.4 to 10.1

_%,

_depending

on the

_population.

The

_outcrossing

rate

appeared

to be affected

_by

the cultivation

_site,

and also

by

the interaction between the

genetic pool

and the cultivation site. No

significant pool

effect was found over sites. The use of natural or modified

_outcrossing

rates is

_suggested

for the _management of gene flows between the

subpopulations

of a

_{metapopulation. Consequences}

of recombination events within and between

_populations

are discussed in the context of short-term

_adaptive

_response of

populations

and

_long-term

elimination of

_weakly

deleterious mutations.

_©

_{Inra/Elsevier,}

Paris

outcrossing rate _{/ gene flow / dynamic management}

_/

_genetic resources

_/

Triticum aestivum L.

*

Correspondence

and

_reprints

du taux

_{d’allogamie}

dans six

_{populations expérimentales}

de blé

tendre,

issues de deux

pools génétiques

différents

_(PA

et

_PB)

cultivés dans _quatre sites distincts.

_Chaque

population

a subi dix années de

_{multiplication}

en

_isolement,

sans sélection consciente.

Dans chacune

d’elles,

78 individus ont été aléatoirement échantillonnés et

_génotypés

pour

vingt-cinq

locus

codominants, grâce

à quinze sondes RFLP. Les taux

d’allogamie

ont été estimés à partir de

l’hétérozygotie

résiduelle multilocus. Un niveau

_{d’allogamie}

élevé _{pour cette}

_espèce

_cléistogame

a été observé. Les estimations varient de

_2,4

à 10,1 % suivant les

_populations.

Ces taux d’allofécondation

_dépendent

d’une part

du site de

_culture,

et d’autre _part d’une interaction entre

_{composition génétique}

initiale et site de culture. L’intérêt de

_{l’allogamie}

tant pour la

_génération

de nouvelles combinaisons

_génétiques

que pour la purge des mutations faiblement délétères est discuté. L’utilisation des taux naturels

_{d’allogamie}

pour instaurer des flux de

gènes

entre les

populations

est

proposée

dans

l’optique

d’une

_gestion

en

métapopulation.

©

Inra/Elsevier,

Paris

taux _{d’allogamie}

_/

flux de gènes

/

gestion dynamique

/

ressources _génétiques

_/

Triticum aestivum L.

1. INTRODUCTION

During

the

_past

30 years, national and international efforts have led to the construction of

_large

collections of

_genetic

resources. For the

_major

cereal _crops

(rice,

corn,

wheat),

these collections

mainly

comprise

sampled

seeds,

stored in

cold rooms of central _gene banks

(Frankel

et

al.,

1995).

Though

gene banks

are a safe and efficient _way to _preserve

_genetic

resources from

_extinction,

the

long-term

conservation of resources in isolation from the environment leads to

a decrease in their usefulness. Whereas

_genetic

resources are

_protected

in cold

rooms,

pathogens

are

_evolving

and the climate _may

_change.

Resources are no

longer exposed

to selective pressures, and

eventually

will prove

ill-adapted

to a new environment

(Simmonds,

_{1962; Henry}

et

_al.,

_1991).

_{Hence, complementary}

strategies,

qualified

as ’in situ or ex situ

dynamic

managements’

(DM)

are

presently developed

to circumvent this

_evolutionary

freeze. Their aim is to maintain or mimic natural _processes

_responsible

for the diversification and

conservation of

_{genetic diversity.}

On the basis of the

_{metapopulation}

_concept

_(Levins,

_1970;

Olivieri et

_al.,

1990),

a

pilot

_programme of

dynamic

_management

of

_genetic

resources of bread

wheat

_(TW!CMm

aestivum

_L.)

started in France in 1984. Its

_general

rules are to

let

_{genetically heterogeneous}

_populations

evolve in different environments and

to manage gene flows between

_populations.

This ex situ

_management

_develops

the

_concept

of ’mass reservoir’ described

_by

Simmonds

_(1962).

In a first

_10-year

period,

no _gene flows were realised in order to allow the

_study

of differentiation and local

_adaptation

with no interference from

_migration.

After a few

_generations

of cultivation in a French multilocal

network,

the

_populations

showed

_significant

differentiation for

_{agromorphological}

and biochemical traits. These

_{genetic changes}

are

_mostly

due to the three

_following

selective _pressures:

i)

environmental

_factors,

such as

climate,

which has induced a north-south

ii)

between-plant competition,

which led to the undesirable elimination of

dwarfing

genes

(rhtl

and

rht2)

segregating

in the initial

_populations

_(Pontis,

1992);

iii)

pathogens,

as shown

by

the differentiation of

_populations

for their

composition

in resistance _genes to

_powdery

mildew

_(Le

_{Boulc’h, 1994;}

Le Boulc’h et

_al.,

_1994).

Besides these selection _pressures, the

mating

system

is of

great

importance

for the

adaptive

response of each

population,

as it

_plays

a

_major

role in the

structuring

of within- and

_{between-population}

_diversity,

when _gene flows are

set _up between

_populations.

On a

_{within-population scale,}

a

_high

level of

_selfing

allows the

_rapid

fixation of the best

_genotypes

and thus a short-term

adaptation,

whereas

outcrossing

maintains

_genetic

_diversity

and the

_potential

for future

_adaptation,

by

producing

new recombinant

_genotypes

_{(Allard, 1979).}

David et al.

₍₁₉₉₃₎

showed

_that,

in a finite

population

under

selection,

the

_{optimal adaptive}

response

(accumulation

of

positive

alleles)

is obtained with an intermediate

outcrossing

rate,

whose value

_depends

on the size and fitness of the

population.

Outcrossing,

recombination and natural selection are also critical for

_purging

populations

of low effective sizes

_{(< 1 000)}

from the

genetic

load. Recent studies of natural

_populations

and mutation accumulation

_experiments

have shown that

_slightly

deleterious mutations

_(inducing

a loss of total fitness of 1 to 5

_%)

occur at a rate

_equivalent

to 0.5-1 new mutation _per

_diploid

_genome and _per

generation

(Mukai et

al., 1972; Lopez

and

_{Lopez-Fanjul, 1993;}

_Keightley,

_1996).

Such

_empirical

data combined with theoretical considerations

_{(Charlesworth}

et

_al.,

₁₉₉₃₎

shed new

light

on what should be the

_{long-term goals}

of

_dynamic

management

of

_genetic

_diversity.

Mutational load is

_expected

to build up

rapidly

in a

purely selfing

_population

of a few hundred individuals and should

lead such a

_population

to extinction due to the reduced

_{fertility/viability}

of individuals

_(phenomenon

_commonly

known as Muller’s ratchet

_{[Muller, 1964!).}

In this

context,

a mixed

_mating

_system

is

_optimal

because it allows both efficient

_purging

of the _genome and recombination

_(David

et

_{al., 1993;}

Ronfort and

_Couvet,

_1995).

On a

_{metapopulation scale,}

while selection over the

metapopulation

con-tributes to the conservation of

_genetic

_{diversity by}

the fixation of different combinations of

_adaptive

alleles in each

_site,

it also increases the

_{genetic drift,}

leading

to the random loss of alleles with a

slightly

_positive

effect on fitness.

Genetic drift also leads to the differential fixation of

_weakly

deleterious

alle-les

_{randomly appearing through}

mutation in the different

_{subpopulations.}

The

occurrence of

_{interpopulation}

recombination enables the reintroduction of lost

positive

alleles,

and to a lesser

_degree,

_{incorporation}

of new

_positive

alleles

appearing

through

mutation in other

_populations.

_Knowledge

of natural

out-crossing

rate is thus essential to

_optimise

the

_management

of _gene flows between

populations

and could facilitate its

_practical

realisation.

It is therefore

_strategic

to

_study

the

_breeding

_system

of the

_populations

cultivated in the wheat

_dynamic

_management

network. Triticnm aestivnm L.

is

_usually

considered as a

selfing species

because of its

_{cleistogamous}

flowers.

However, outcrossing

has been

_reported,

like for other

_{self-fertilising}

_species

of the Triticeae _genus

_(2-3

%

for T. aestivnm L. in

_Hayes,

1918 and

Martin, 1990;

This _paper

_gives

estimates of

outcrossing

rates of six

_genetically

heteroge-neous wheat

_populations

of the DM programme submitted to natural

selec-tion for 10 _years. We used molecular markers to derive

_outcrossing

rates from

the estimated fixation

parameters

f

(Wright’s statistics).

As these

_populations

originate

from two

_segregating

_populations

_(PA

and

_PB)

cultivated at three different

sites,

our second aim was to detect if

original genetic composition

and cultivation sites influence the

_outcrossing

rate of

_populations.

Lastly,

we

discuss the _consequences of

_outcrossing

rates for the

_adaptive

_response of the wheat

_populations

and consider the use of these natural

crossing

events for the

setting

of gene flows between

populations.

2. MATERIALS AND METHODS

2.1.

_History

of

_populations

Two

_segregating

_populations

_(PA

and

_PB)

were created each

_by

a manual

pyramidal

cross of two distinct sets of 16 inbred lines

_(for

_PA,

see Thomas

et

_al.,

_1991).

In

_1984,

after 3 _years of bulk

_{multiplication,}

the

_populations

were

_split

between,

_{respectively,}

seven and

_eight

sites of a French multilocal

network

_(either

Inra stations or

_agricultural

and

_agronomic

_high

_schools)

to

settle local

subpopulations.

The

_demographic

size of each

_founding

population

was about 10 000 individuals. At each

_site,

10 000 to 15 000

_plants

have been

grown each year in isolated

plots,

separately

harvested and

sampled

to

_generate

the next

_population.

As

_{previously mentioned,}

wheat is

_{usually cleistogamous,}

with a

_relatively

low

pollen/ovule

ratio

_{(2 000}

to

_{4 000;}

_Cruden,

_1977).

When

outcrossing

occurs, flowers are

wind-pollinated.

2.2. Plant material

Six

_populations

were studied after 10 _years of

multiplication

at the same

site: three PA

_populations

cultivated in Le Moulon

_(48°

9’N

_2°E),

Rennes

_(48°

1’N

_2°W)

and Toulouse

_(43°

6’N

_1°E),

and three PB

_populations

cultivated

in Le

_Moulon,

Rennes and Venours

_(45°

8’N

_1°W).

From the bulk harvest of the 1994 _season, 78 individuals were

_{randomly sampled}

and

_{analysed. Adding}

the 3 years of

multiplication prior

to the initial distribution of seeds over the

network to the

_{10-year multiplication period,}

these

_populations

resulted from

13

_cycles

of natural

_mating

after the last manual crosses. To estimate the initial

frequencies

of markers in the

_founding

_populations,

two random

samples

of inbred lines derived

_by

_single

seed descent

_(SSD)

from the initial

_populations

PAO and PBO were also studied.

2.3. Molecular

_study

Because of the low level of

_polymorphism

at the

_protein

level

_(Gale

and

Sharp,

1988),

we used restriction

_{fragment length}

_polymorphism

_(RFLP)

Total

_{deoxyribonucleic}

acid

_(DNA)

was extracted from

_lyophilised

_young

leaves

_following

a

_{rapid procedure adapted}

from

Dellaporta

et al.

_(1983).

Enzyme restriction, electrophoresis,

blotting

onto

_Hybond

N

+

membranes

(Amersham)

and nonradioactive

_{hybridisation}

_(DIG

_@

_{Boehringer-Mannheim)}

were

_performed

as described

_by

Lu et al.

_(1994).

P.

_{Leroy provided}

us with 35

_genomic

DNA

_probes,

obtained and

_mapped

by

Inra-Génoblé of

_{Clermont-Ferrand,}

France

_(Nelson

et

_al.,

_1995a,b).

These

probes

were chosen for their

_high

_polymorphism

information content

_(Nelson

et

_al.,

_1995a,b)

estimated over 14 lines. K. Devos

_provided

us with five

additional

_probes

obtained and

_mapped

_by

the John Innes

_{Centre, Norwich,}

UK

_(Gale

et

_al.,

_1997).

The RFLP

_diversity

of this set of

_probes

was screened on the 32

_genitors

of

the PA and PB

_pools,

and the 12 most

_polymorphic

_enzyme/probe

combina-tions were retained: EcoRl with

_{fba69, fba881}

and

_fbb09;

HindIII with

_fba65,

fbal27,

fba152, fba!4!

and

_fbbl!;

Dra I with

_fba!04

and

_fba251

for the Inra-Génoblé

_probes;

EcoRI with PSR

₁₄₄

and PSR 6!! for the John Innes Centre. Information on the

_probes

can be obtained from the GrainGenes database

(http://wheat.pw.usda.gov;

French mirror site:

_{http://grain.jouy.inra.fr).}

As some

_probes

exhibited

_polymorphism

for

_{nonmapped bands,}

the relations

of allelism between new

_segregating

bands were tested over the two PAO and PBO SSD and the DM

_populations.

2.4. Genetic

_analysis

To estimate the

_outcrossing

_rates,

we chose measurement of mean

heterozy-gosity

for two main reasons.

_First,

after 13 seasons of natural

_mating

we

ex-pected

the neutral markers to be very close to the

_inbreeding

_equilibrium,

so

that we could use the

_relationship

between t and

_{f. Second,}

whereas _progeny

analysis

(Ritland

and

_Jain,

₁₉₈₁₎

allows

_unambiguous

detection of

_outcrossing

events,

the estimated

_outcrossing

rate is

_only

relevant for one

_breeding

season.

Moreover,

we

_needed,

for other _purposes not

_{developed herein,}

accurate

esti-mates of allelic

_frequencies

more

_easily

achieved in

_{predominantly}

selfer

_plants

by

random

_sampling

than

_by

_progeny

_analysis.

Outcrossing

rates of the

_populations

were estimated from the observed

level of

_{heterozygosity deficiency f}

of the

_sampled

genotypes.

In a

_population

of infinite size without over- or underdominant

_selection,

with a constant

outcrossing

rate

_t,

the

_{heterozygosity}

reaches an

_equilibrium

value after a few

generations

(from

one

generation

for

complete outcrossing

to ten

_generations

for

a 99

%

selfing

population).

As the census size of

_populations

is

_large

_(>

10

₀₀₀₎

and because of the 13 _years of free

_pollination,

we assumed that

_f

was close to

the

_equilibrium

value.

With these

_assumptions,

the estimation of

_outcrossing

rate is

_given

_by

Ho is the observed

_proportion

of

_{heterozygotes}

in the

_sample,

the _gene

_diversity

as defined

_by

Nei

_(1958),

_n, the number of alleles for a

_given

locus and

_pi

the estimated

_frequency

of the allele i.

f

was estimated

_by

the maximum likelihood method

proposed

by

Li and Horovitz

₍₁₉₅₃₎

and Curie-Cohen

₍₁₉₈₂₎

for

_monolociis

data.

_Assuming

that there is no

_{gametic disequilibrium,}

multilocus

f

is the root of

(Rousset

and

_Raymond,

_1995),

where

_NH

_et

is the number of

_{heterozygotes}

at locus

_I,

n,zi the number of

homozygotes

for allele i at locus and

_pi

the

frequency

for allele i at the locus I observed in the

sample.

Estimate

_f

was

numerically

_computed

after estimation of the allelic

fre-quencies,

and used to calculate the

t value.

A

_{bootstrap resampling procedure}

over individuals was

performed

to determine the confidence interval of

_{t (Weir,}

1990).

For each

_population,

500

_{bootstrap samples}

were used to calculate a

distribution of values

_(b

=

1, ...,

500).

To test the

_significance

of differences in

_outcrossing

rates

_(t

_i

<

_t

_j

₎

between

populations

i and

_j,

_pairwise

subtraction between

_bootstrapped

values of each

_population,

_0,

_b

_-

_4,

_b

allowed us to

_perform

a one-tailed test

_(Efron

and

Tibshirani,

1982).

The

_percentage

of

_negative

differences

provided

the

probability

associated with the test

_{Ho: t}

_i

_{= t}

_j

_against

H1:

_t

_i

<

_t!.

To test the effects of the

_{genetic composition}

_(PA

versus

PB)

and of the

site

_(Le

Moulon versus

_Rennes),

the

_bootstrap

distributions of the

_following

contrasts were calculated:

3. RESULTS

3.1.

_Polymorphism

of RFLP markers

Most of the RFLP

_{genomic probes}

used for this

_study

revealed multiband

profiles,

due to the

_hexaploid

constitution of wheat. Over the 12

_{assayed probes,}

23 codominant loci were

examined,

some

probe/enzyme

combinations

_showing

up to five

_polymorphic

loci.

_Thus,

a

_single

_{hybridisation}

led to a mean of

revealed in

_comparison

with the

_{mapping populations}

_(Nelson

et

_al.,

_1995a,b).

Consequently,

the

_mapping

of these 23 loci on the

_genetic

_maps was not

_always

possible.

The Mendelian inheritance of the new

polymorphic

bands was verified

using

the

_segregation

data of the SSD

_populations

extracted from the initial

populations

in which a

_high

level of fixation

_{(96 %)}

allowed

_unambiguous

determination of allelism

_{relationships}

_(results

not

_shown).

Over the 23

loci,

the mean allelic

diversity

was

_{2.48, ranging}

from two to five alleles _per locus

(table 1).

The mean _gene

diversity

was He =

_{0.35, ranging}

from 0.09 _to 0.59.

3.2.

_Outcrossing

rates

The

_{heterozygosity}

observed after 13

_generations

was much

_higher

than

expected:

1 to 5

%

of mean

_{heterozygosity}

was measured

(data

not

_shown),

instead of 0.006

% (2!3 )

213

expected

for strict

self-pollination.

Values of

_single

locus estimates of

_outcrossing

rates

_ranged

from 0 to 30

_%,

most of them

_being

lower than 10

%

_{(figure 1).}

The

_highest

_outcrossing

rates were observed for loci

with a _{poor gene}

diversity,

and thus a

_high

standard error.

Analysis

of variance

_(ANOVA)

over the

_single-locus

estimates

_(data

not

shown)

did not reveal a

_significant

RFLP effect: no locus exhibited

consistently

high

or low levels of

_{heterozygosity}

across the four

_populations.

This indicated

that no RFLP locus was in

linkage

disequilibrium

with

polymorphic

_genes

controlling

the

_outcrossing

rate or submitted to overdominant selection in all

Multilocus estimates of

_outcrossing

rates of the six

_{populations ranged}

from 2.4 to 10.1

%

_{(table 77).}

This range is

higher

than the values

_reported

_by

Martin

(1990)

for 12 commercial lines of bread wheat: 0.1 to 5.6

%

measured over

1 year, with a maximum mean over 3 _years of 3.1

%.

The estimated

_outcrossing

rates are

_roughly

distributed in three _groups

rates

_(around

3

_%),

PA Moulon and PA Rennes with intermediate values

_(near

6

_%)

and PB Rennes and PA Toulouse with the

_highest

rates

_(around

10

_%).

According

to

_comparison

_tests,

PB Moulon differed

_{significantly}

from all the others with the

_exception

of PB

_Venours,

a

_{population which,}

in

_turn,

differs

significantly

from all the others

_except

PA

_Moulon,

all other

_comparisons

_being

not

_significant.

A

_significant

site effect was

_found,

with

_Pr[6site

<

_0]

=

0.004,

whereas no

pool

effect was detected

(Pr[bpool

<

_0]

=

0.4).

Nevertheless, site-pool

interac-tion was

_present,

as the PA and PB

_populations

had inverse classifications in

Rennes and Le Moulon.

4. DISCUSSION

Our results showed that the wheat

_populations

of the DM network cultivated

under natural

conditions,

though

cleistogamous,

can exhibit a

relatively

_high

outcrossing

rate

_(ranging

from 2.4 to 10.1

_%).

These estimations

_rely

upon the

assumption

of selective

_neutrality

and

_consequently

could be biased

_upward

due to the selective

_advantage

of

_{heterozygotes}

in

_populations

in which

_strong

competition

takes

_place

between individuals. In winter

_wheat,

low heterosis

(!

10

_%)

has been

_reported

for

_{yield surveyed}

on

plots

of

homogeneous

_genetic

composition

(Oury

et

_al.,

_1990),

but we

_might

_expect

heterosis for

individ-ual fitness in

_{heterogeneous}

_populations

to be

_stronger,

since

_hybrids

_are, on

average, taller and bloom earlier than their inbred

parents,

both traits

con-tributing

to a better

_competitive

_aptitude.

Even with 20

%

heterosis on fitness

explained

by

a

_{single-locus model, however,}

13

_cycles

of

_{complete selfing}

_only

raised the

_{heterozygote frequency}

from 0.006 to 0.08

%

_(data

not

_shown),

a

value

_{significantly}

lower than the rates observed in this

_study.

This theoretical

consideration,

associated with the observation that no locus exhibited

signifi-cant excess of

heterozygotes

across all studied

populations,

leads us to _argue

that the

_outcrossing

rate of our

_populations

is often as

_high

as several

_percent,

Such levels of

_outcrossing

are

_commonly

_reported

in other

species

classically

described as

selfing

(table

I in Schoen and

_Brown,

1991).

In the few

_highly

selfing

species

(outcrossing

rate <

1

_%),

such as

_Arabidopis

thaLiana or some

Avena

_{species, outcrossing}

events

_might

be due to accidents in the

_development

of some anthers or to the

_exceptional

infiltration of exogenous

pollen

and

_might

have low

_{evolutionary impact.}

On the other

_{hand, outcrossing}

_rate,

even as low as 5

_%,

should be

_{explained by}

different mechanisms.

4.1.

_Origin

and

_stability

of

_outcrossing

rates

in

_dynamically

_{managed populations}

In some _{grasses, environment} has a

_strong

effect on

_fertility

and

outcross-ing propensity.

Low

_luminosity

and low

_temperature

_during

meiosis have been shown to cause

pollen

_sterility

in rice

(Li

et

_al.,

₁₉₉₆₎

and winter wheat

(Demotes-Mainard

et

_al.,

_1995).

Demotes-Mainard et al.

₍₁₉₉₅₎

showed that this

_sensitivity

to low

_luminosity

has a

_{genetic basis,}

the two studied lines Per-nel and Moulin

_exhibiting

very contrasted responses to the stress. This

phys-iological

response should lead to

strong year-to-year

and site-to-site variation

in the rate of

_outcrossing.

Contrasted climatic conditions of the DM network

are most

_likely

to affect the amount of

_outcrossing

realised in the

_populations,

either

_{through partial}

male sterilities or

_{by shifting}

of male and female

phe-nologies.

Thus,

one

explanation

for the

_high

_outcrossing

rate of

_populations

grown in Rennes could be the

cloudy

weather in

_spring

and in the

_beginning

of the summer in the west of France. On the other

_hand,

_Henry

et al.

_(1991),

by studying

a locus

coding

for seed

_storage

_protein,

have

_previously

shown

that

_populations

_{grown in} the south of France

_{displayed significant}

excesses of

heterozygotes

as

_compared

with

_populations

_{grown in} the north. This was

ex-plained

by

a

_protogynous

_flowering

at

_high

_{temperatures.}

Our

_results,

in accord with this

_previous

_study,

underline the

importance

of environmental factors for

the

_mating

system.

Although

we did not detect an effect of the

_genetic

_background

(PA

or

_PB)

on the

_outcrossing

_ability,

the PA

_population

of Le Moulon showed a

signifi-cantly higher

rate of

_outcrossing

than PB Le

Moulon,

and the two PB popu-lations of Le Moulon and Venours were the most

_selfing

_populations.

A rather

high

level of

_outcrossing

has also been observed in a recurrent selection pro-gramme based on the same

_genetic

_background

as the PA

_{populations. Spikes}

or

_spikelets

have often been found with _open flowers

_receptive

to _exogenous

pollen,

indicating

that no viable

_pollen

was available for a

_{cleistogamous}

polli-nation,

a

phenomenon

well known in wheat. This trend to

_partial

male

_sterility

may be due to the _{presence in} the

_genitors

of PA of the Courtot

_cultivar,

a line

known for its

_{high degree}

of

pollen

sterility. Savy

(1991)

showed that within a

population

the individuals

_presented

different rates of

_selfing,

with the

_greatest

proportion

of

_{plants being mainly selfing,}

with some individuals

_showing

com-plete

outbreeding.

Thus, genetic

and environmental factors

_probably

interact to determine individual levels of

_outcrossing.

If site-to-site variation of

_outcrossing

rates could be

_estimated,

the

year-to-year

or

_{plant-to-plant}

variations need to be evaluated because

_they

will affect the

_efficiency

of _gene flow in schemes based on the

_{population’s}

mean

However,

which forces maintain the

outcrossing

ability

of these

_{populations ?}

Simple

theoretical models for the evolution of

_{selfing predict}

that a

selfing

variant should

_spread

due to its

_{segregational}

_advantage

as soon as

inbreeding

depression

(defined

as one minus the relative fitness of selfed versus outcrossed

progeny)

does not exceed one half

_{(Fisher, 1958).}

Other theoretical models

(assuming

population

of finite size or

_spatial

structuration)

show that even

with low heterosis for

fitness,

recombination is

_advantageous

and maintains

evolutionary

stable rates of

_outcrossing

at a few

_percent

(David

et

_{al., 1993;}

Ronfort and

Couvet,

1995).

If the

_optimal

multilocus

genotype

is not

_initially

present

in a

_population,

the selection for favourable recombinant

_types

slows

down the counterselection of

outcrossing

abilities within the

_population.

In the

present

study,

the low heterosis of wheat combined with

_significant

outcrossing

rates led us to the conclusion either that a

_large

_part

of the

population

must

exhibit a few

_percent

_outcrossing,

or that the

heritability

for

high

outcrossing

rates must be low. Otherwise

_{outcrossing plants}

should have been

quickly

eliminated and the

_outcrossing

rates lower than

_reported

here.

Beyond

the exact nature of the selective forces

_acting

on the

mating

_system,

outcrossing

is

_expected

to

_{play a}

_major

role in the local

adaptation

of the

populations

of the DM network.

_By

allowing

occasional recombination events

between

_individuals,

such levels of

_outcrossing

enable the

_production

of better

adapted

genotypes

(Allard,

1979)

and

_{simultaneously}

_prevent

rapid

loss of favourable alleles

_trapped

in inferior lines that would otherwise be

_wiped

out of the

_population.

Hence, comparing

the level of

_diversity

maintained on

polygenic

traits and the structure of

_genetic

correlations between

traits,

a

_population

with a mixed

_mating

_system

_involving

a few

_percent

_outcrossing

is

qualitatively

different from a _pure

selfing

one.

4.2.

_Consequences

for

_managing

the DM network

The

_diversity

of selective pressures

present

at the various sites of the

net-work is a warrant for the maintenance of

_polymorphism

on loci involved in

adaptation

(Henry

et

_al.,

_1991).

_However,

we _argue that _gene flows between

populations

are _necessary to

_compensate

for the

variability

lost within

popu-lations

_through

drift and

_linkage

drag

due to selection. A rule of thumb could be the

_preservation

of a certain level of

_{within-population}

polymorphism

for neutral

_traits,

_expecting

that in case of

_change

in selection

_gradients

these

previously

neutral traits become

adaptive.

Several

_examples

can be

_given

to

illustrate this

_possible

feature.

_First,

dramatic

_changes

in environmental

con-ditions can induce inversions in the

hierarchy

of the traits

determining

local

fitness.

_{Second, heterogeneous populations}

can

_experiment

different successive

phases

of selection. When the

_adaptation

for an

_important

trait is

finally

re-alised

_by

the exhaustion of the

variability

for that

trait,

natural selection will

act more

_strongly

on another trait which was of minor

importance

in the first

stage

of

adaptation.

In these cases of

_change

in selective

_gradients,

gene flows

would allow local

populations

to enrich their

variability

for

_secondary

_traits,

as

_they

restart selection on

_primary

traits when their

variability

has been

ex-hausted

_{by linkage drag}

or

_genetic

drift. On the other

hand,

it is

important

not to

_impede

local

_adaptation

of

_populations

to their site because of too

_high

evolution as a

_guideline,

the

_intensity

of _gene flow could be determined in order

to limit the level of

between-population

differentiation on neutral _genes, i.e. to

ensure a minimum rate of

_migration

_m, so that Ne*m =

1,

where Ne is the

local effective size of a

_population

_(Crow

and

_Kimura,

_1970).

_However,

a

_higher

migration

could be necessary to _preserve

_diversity

of chromosomal

_segments

in

the

_{neighbourhood}

of _genes under

_strong

selection.

To favour recombination between

_{individuals, pollen migration}

should be favoured instead of seed

_migration.

In

_{outcrossing species}

both modes of

migration

are

_expected

to

_produce

similar effects

_(except

for

_cytoplasmic

genomes),

but

_markedly

different _consequences are

_expected

in the case of

selfers. Seed

_migration

could

_{potentially split}

the

_{recipient population}

into two

compartments.

If

_migrant

_genotypes

are

_{poorly adapted}

to local

_conditions,

hybrid/outbreeding

depression

is

_expected

and the

_efficiency

of gene transfer via such schemes will be low. To circumvent such

_problems,

Hauser et al.

₍₁₉₉₄₎

suggested

to realise back crosses instead of

_{first-generation hybrids}

to enhance

genetic diversity

in

_endangered

_populations.

On the other

_hand,

when some

migrants

display

more

_{competitive ability,}

_they

could

_potentially

_outcompete

resident

_genotypes

and eliminate the initial

_variability

_present

in the

_population

(especially

if

_migrant

and resident

_genotypes

_hardly

_recombine).

_Migration

via

pollen

should avoid these

_phenomena

_{by enforcing}

recombinatioa of resident and

_migrant

_genotypes.

On the basis of the data

obtained,

natural

_outcrossing

rates are sufficient

to allow the

_{incorporation}

of

_{foreign migrants}

_by

the local

_population.

To achieve a level of _gene flow of one effective

_migrant

_per

_generation

(neutralist

guideline),

two

_outcrossings

_per

_{migrant pollen}

_per

_population

are

_required.

Assuming

a 5

%

rate of

_outcrossing

in a

_{population composed}

of 10 000

plants,

500 individuals will

_potentially

outcross. To ensure _{two crosses,}

_migrant

_pollen

should thus be

_provisioned

as to

_represent

_1/250

of the total

_{pollen pool.}

For

wheat,

this should be

_easily

realised

_{by surrounding populations}

with a belt of

migrant plants.

Estimation of rates of natural

_outcrossing

in

_populations

combined with the

_knowledge

of local effective sizes will make it

_possible

to rationalise the

management

of the network. Such

_optimisation

should be achieved

_by

real

experiments,

analytical approaches

and

_through

_modelling

based on realistic

parameters.

ACKNOWLEDGEMENTS

The authors are

_grateful

to T. Bataillon and D. Schoen for scientific comments and for

_help

in the translation of the paper, and

they

thank the anonymous reviewers for very

helpful

comments on the

_manuscript.

The wheat

_dynamic

network was initiated

_by

the ’Direction

générale

de

1’enseigne-ment et de la recherche’

_(DGER)

of the French

_{agriculture ministry}

and received scientific and financial _support from the

_DGER,

’Institut national de la recherche

agronomique’

and ’Bureau des Ressources

_{Génétiques’}

_(BRG).

This work is

_supported

by

a _grant from the BRG. J.

_Enjalbert

is the

_recipient

of a doctoral

_fellowship

from

REFERENCES

Allard

_R.W.,

The

mating

system and

microevolution,

Genetics 79

₍₁₉₇₉₎

115-126. Charlesworth

D., Morgan M.T.,

Charlesworth B., Mutation accumulation in finite

outbreeding

and

_{inbreeding populations,}

Genet. Res. 61

₍₁₉₉₃₎

39-56.

Crow

_J.F.,

Kimura

_M.,

An Introduction to

_Population

Genetics

_{Theory, Harper}

International Edition, New

_York,

1970.

Cruden

R.W.,

Pollen-ovule ratios: a conservative indicator of

breeding

_systems in

flowering plants,

Evolution 31

₍₁₉₇₇₎

32-46.

Curie-Cohen

M.,

Estimate of

inbreeding

in natural

populations:

a _comparison of

sampling properties,

Genetics 100

₍₁₉₈₂₎

339-358.

David J.L.,

Savy J.,

Trottet

_M.,

Pichon M., Méthode de

_{gestion dynamique}

de la variabilité

_génétique

en milieu naturel.

_Exemple

de

_{populations composites}

de

_ble,

in:

Colloque

international en

_hommage

à Jean

_Pernes,

8-10

_{janvier 1992, Paris, France,}

1992,

pp. 337-350.

David

J.L., Savy

Y., Brabant P.,

Outcrossing

and

selfing

evolution in

_populations

under directional

_{selection, Heredity}

71

₍₁₉₉₃₎

642-651.

Dellaporta S.,

Wood J., Hicks

_J.,

A

plant

DNA

minipreparation:

version II, Plant Mol. Biol.

Rep.

1

₍₁₉₈₃₎

19-21.

Demotes-Mainard

S.,

Doussinault

_{G., Meynard}

J.M., Effect of low radiation and low _temperature at meiosis on

_{pollen viability}

and

_grain

set in

_{wheat, Agronomie}

15

(1995)

357-365.

Devos _K., Gale M.D., The

_genetic

_maps of wheat and their

_potential

in

_plant

breeding,

Outlook

_Agric.

22

₍₁₉₉₃₎

93-99.

Efron

_B.,

Tibshirani

_R.J.,

An Introduction to the

Bootstraps, Chapman

and

_Hall,

New

York,

1982.

Fisher

_R.A.,

The Genetical

Theory

of Natural Selection, Oliver and

_{Boyd, London,}

1958.

Frankel

_O.H.,

Brown

_A.H.D.,

Burdon

_J.J.,

The Conservation of Plant

_{Biodiversity,}

Cambridge University Press, Cambridge,

1995.

Gale

_{M.D., Sharp P.J.,}

Genetic markers in

_{wheat-developments}

and prospects,

in: Seventh International Wheat Genetic

_{Symposium, Cambridge,}

13-19

July 1988,

Institute of Plant Science

Research, 1988,

pp. 469-473.

Gale

M.D.,

Atkinson

_{M.D., Chinoy C.N.,}

Harcourt

R.L.,

Jia

J.,

Li

Q.Y.,

Devos

K.M.,

Genetic maps of

hexaploid wheat,

in:

Eighth

International Wheat Genetics

Symposium, Beijing, China,

1997, pp. 29-40.

Harrison

_{S., Hasting A.,}

Genetic and

_evolutionary

consequences

of metapopulation

structure, Tree 11

₍₁₉₉₆₎

180-183.

Hauser

_{T.P., Damgaard}

C., Loeschcke V., Effects of

_Inbreeding

in Small Plant

Populations: Expectations

and

_Implications

for

Conservation,

Birkhauser

Verlag,

Basel,

1994.

Hayes H.K.,

Natural

_crossing

in wheat. A cause of _impurities in

breeding

plots-belief of some

_agronomists

that

hybrids frequently

revert to the

parental

type, J. Hered. 9

₍₁₉₁₈₎

326-330.

Henry J.P.,

Pontis

C.,

David

_J.L.,

_{Gouyon P.H.,}

An

_experiment

on

_dynamic

conservation of

_genetic

resources with

_{metapopulations,}

in: Seitz

_A.,

Loeschcke V.

(Eds.),

Species

Conservation: A

_{Population-Biological Approach,}

Birkhaiiser

_Verlag,

Basel,

1991.

Le Boulc’h

_V.,

Evolution

de la resistance à 1’6idium

_(Erysiphe

_graminis)

dans des

populations

de ble tendre

_{( Z’riticum}

aestivum

_L.)

menées en _gestion

dynamique, these,

Institut national

_{agronomique Paris-Grignon,}

Paris, 1994.

Le Boulc’h

V.,

David

J.L.,

Brabant

P., Vallavieille-Pope C., Dynamic

conservation of

_variability:

_response of wheat

populations

to different selective forces

_including

powdery mildew,

Genet. Sel. Evol. 26

₍₁₉₉₄₎

221-240.

Levins

R., Extinction,

in: Gerstenhaber M.

_(Ed.),

Some Mathematical

_Questions

in

_Biology,

American Mathematical

_{Society, Providence, RI, 1970,}

pp. 77-107.

Li

_C.C.,

Horovitz

_D.,

Some methods of

_estimating

the

_{inbreeding coefficient,}

Am. J. Hum. Genet. 5

₍₁₉₅₃₎

107-117.

Li H.B.,

Zhang (a.,

Liu

_A.M.,

Zou

_J.S.,

Chen

Z.M.,

A

genetic analysis

of

low-temperature-sensitive sterility

in

_{indica-japonica}

rice

hybrids,

Plant

_Breeding

115

(1996)

305-309.

Lopez M.A., Lopez-Fanjul C., Spontaneous

mutation for a

_quantitative

trait in

Drosophila melav,ogaster.

II. Distribution of mutant effects on trait and

fitness,

Genet.

Res. 61

₍₁₉₉₃₎

117-126.

Lu Y.H., Merlino

M.,

Isaac

_{P.G., Staccy}

_J., Bernard M.,

Leroy

P., A comparative

analysis

between 32p and

digoxigenin-labelled single-copy probes

for RFLP detection in

_{wheat, Agronomic}

14

₍₁₉₉₄₎

33-39.

Martin

T.J., Outcrossing

in twelve hard red winter wheat

_{cultivars, Crop}

Sci. 30

(1990)

59-62.

Mukai T.,

Chigusa S.I.,

Mettler L.E., Crow J.F., Mutation rate and dominance of genes

affecting viability,

Genetics 72

₍₁₉₇₂₎

335-355.

Muller

_H.J.,

The relation of recombination to mutational

_advance,

Mutat. Res. 1

(1964)

2-9.

Nei _M.,

_{Syakudo M.,}

The estimation of

_outcrossing

in natural

populations, Jpn.

J. Genet. 33

₍₁₉₅₈₎

46-51.

Nelson

J.C.,

Sorrells M.E., Van

_{Deynze A.E.,}

Lu

_Y.H.,

Atkinson

M.,

Bernard

M.,

Leroy P.,

Faris

J.D.,

Anderson

J.A.,

Molecular

_mapping

of wheat:

major

genes and rearrangements in

_homoeologous

group 4, 5 and 6, Genetics 141

_(1995a)

721-731.

Nelson

_J.C.,

Van

_{Deynze A.E., Autrique E.,}

Sorrells

_M.E.,

Lu

_{Y.H., Negre S.,}

Bernard

M., Leroy P.,

Molecular

_mapping

of wheat.

_Homoeologous

_{group 3,} Genome 38

_(1995b)

525-533.

Olivieri 1., Couvet D.,

Gouyon

P.-H., The

_genetic

of transient

_populations:

research at the

metapopulation level,

Tree 5

₍₁₉₉₀₎

207-210.

Oury F.-X.,

Brabant

_P.,

Pluchard

_P.,

B6rard

_P.,

Rousset

_M.,

Etude

multilocale de blés

_{hybrides :}

niveaux d’heterosis et elaboration du

_{rendement, Agronomie}

10

₍₁₉₉₀₎

735-748.

Pontis

_C.,

Utilisation de marqueurs

génétiques

pour le suivi de la variabilité de trois

_composites

de ble tendre d’hiver

_(T.

aestivam

_L.)

menées en

_{gestion dynamique,}

these,

Institut national

_{agronomique Paris-Grignon, Paris,}

1992.

Ritland

K.,

Jain

_S.,

A model for the estimation of

_outcrossing

rate and gene

frequencies using

n

_{independent loci, Heredity}

47

₍₁₉₈₁₎

35-52.

Ronfort

_J.,

Couvet

_D.,

A stochastic model of selection on

_selfing

rates in structured

populations,

Genet. Res. 65

₍₁₉₉₅₎

209-222.

Rousset F.,

Raymond

M.,

Testing

heterozygote

excess and

_deficiency,

Genetics 140

(1995) 1413-1419.

Savy Y.,

Evolution

de la variabilité

génétique

en selection naturelle en relation avec

le

_regime

de

reproduction :

étude

agronomique

de trois

_{pools géniques}

de ble

tendre,

étude

théorique

par simulations

informatiques, Rapport

de

DEA,

Institut national

agronomique Paris-Grignon,

Paris, 1991.

Simmonds

N.W., Variability

in _crop

_plants,

its use and _{conservation,} Biol. Rev. 37

(1962)

442-465.

Thomas

G.,

Rousset

_M.,

Pichon

_M.,

Trottet

M.,

Doussinault

G.,

Picard

E.,

Méthodologie

de I’amelioration du ble tendre

_(T.

aestivum

_L.).

I. Creation par croisements et

_analyse

d’une

_population

artificielle à 16 _parents, base de cette étude

méthodologique, Agronomie

11

₍₁₉₉₁₎

359-368.

Tsegaye S.,

Estimation of

_outcrossing

rate in landraces of

tetraploid

wheat

(Triticum

turgidum

L.),

Plant

_Breeding

115

₍₁₉₉₆₎

195-197.