A genetic analysis of variable number of tandem repeats (VNTR) polymorphism in the horse

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A genetic analysis of variable number of tandem repeats

(VNTR) polymorphism in the horse

G Guérin, M Bertaud, B Billoud, Jc Mériaux

To cite this version:

Original

article

A

_{genetic analysis}

of variable number

of

tandem

_repeats

_(VNTR)

polymorphism

in the horse

G Guérin

M

Bertaud

B

Billoud

JC

Mériaux

2

1

INRA, Laboratoire de

_{Génétique Biochimique;}

2 INRA,

Laboratoire des

_{Groupes Sanguins,}

78352

Jouy-en-Josas Cedex,

France

(Received

3

_December

_1992;

_accepted

1

_July

₁₉₉₃₎

Summary - Restriction

_{fragment length polymorphism}

detected with the 33.6

minisatel-lite

_{probe (Jeffreys}

et al,

1985)

was

_analysed

in 3 horse families of

paternal

half-sibs and in

a

_sample

of stallions from 4 breeds.

_Among

the bands detected on Hae III

_genomic

DNA

digests,

it was found that

_{strongly hybridizing fragments}

behaved as alleles at 2 different

loci. These 2

_{loci, showing}

7 and 3 detectable

alleles,

were not

_closely

linked to each other

nor to informative blood groups and protein markers. No neo-mutation in allele

length

was observed at these 2 loci ip the 3 families. In the stallion

_sample,

8 alleles were detected at the first locus and no differences were found between their

frequencies

in the 4 breeds.

Heterozygosity

and

_polymorphism

information content

_(PIC)

values estimated in the 4 breeds show that the

_Thoroughbred

is the least variable breed and the Arab the most. In the whole

_population

the PIC values were 0.73 and 0.70

_{respectively.}

horse

_/

variable number of tandem repeats / linkage / genetic marker

_/

polymorphism

Résumé - Analyse génétique du polymorphisme du nombre variable de _{répétitions} en tandem

_(VNTR)

chez le cheval. Le

_{polymorphisme}

de

_longueur

de

_fragment

de

restriction détecté par la sonde minisatellite .!,!.6

(Jeffreys

et

al, 1985)

a été

_analysé

chez le cheval dans trois

_familles

de

_{demi-germains paternels}

et dans un échantillon d’étalons de _quatre races. Parmi toutes les bandes révélées à partir d’ADN

_génomique

digéré

par

HaeIII,

les

_fragments

donnant un

_signal

intense se _comportent comme des allèles

ségrégeant

à deux locus

_{différents. Ceux-ci,}

qui

possèdent

respectivement sept et trois allèles

détectables, ne sont pas étroitement liés entre eux ni avec les locus

informatifs

des _groupes

sanguins et des

protéines

du sang. Aucune néo-mutation de

longueur

des allèles n’a été détectée dans les trois

_familles.

Dans l’échantillon des

étalons,

huit allèles ont été observés

au _premier locus et aucune

différence

de

fréquence n’apparaît

entre les quatre races.

Les valeurs

d’hétérozygotie

et les valeurs

informatives

des

polymorphismes

(PICJ

estimées

la race Arabe. Les valeurs estimées sur la

population

totale sont respectivement de 0,73 et

de

0, 70.

cheval / nombre

variable de répétitions en tandem / liaison

_/

marqueur génétique

/

polymorphisme

INTRODUCTION

It is now well established that the _genome of a number of animal and

plant

species

contains

_regions

that are

_highly

variable,

_among which are the minisatellites.

These

_regions

are detected

by probes

from a different

_{origin containing}

_sequences several

_{base-pairs long, tandemly repeated. Initially, highly polymorphic}

_patterns

displaying

a

_large

number of

_fragments

on Southern blots

_resulting

from variation in the number of tandem

_repeats

and called

_fingerprints

were obtained in man

by

Jeffreys

et al

_(1985).

These authors used a

_repeat

found in the second intron of the

_myoglobin

_gene to

_design

a

_probe

that revealed related human _sequences, some

of which were themselves later used as

probes.

This was the case for the 33.6 and 33.15

_regions

that revealed extensive

_polymorphism

in a number of

_organisms.

Other

_{probes containing}

_repeated

_sequences such as a _sequence

_from

the M13

bacteriophage

(Vassart

et

al,

1987),

a _sequence

homologous

to the

_Drosophila

Per gene

(Shin

et

al,

1985),

in the 3’

_region

of the a

_globin

_gene

(Jarman

et

_al,

₁₉₈₆₎

and a number of

_others,

cloned from the human _genome

_(see

for

_example

Nakamura

et

_al,

_1987),

were found to detect _very

_high

levels of

_{polymorphism.}

In

_pets

and domestic farm

animals, polymorphisms

were found in

dogs

and cats

(Jeffreys

and Morton

_1987),

in cattle

_(Georges

et

al,

1988),

in

_poultry

_(Hillel

et

al,

1989),

in

_sheep

_(Drinkwater

et

_al,

₁₉₉₀₎

and in

_pigs

_(Coppieters

et

al,

1990)

using

either

_human,

viral or

_{homologous probes.}

In

_horses,

restriction

_{fragment length polymorphisms}

_(RFLP)

have been detected

using

different

_probes

such as

M13, Jeffreys’

minisatellites or core _sequence,

Per,

human

_VNTR,

_(TG)n,

a

homologous

horse

probe

and

synthetic

tandem

_repeats

(Georges

et

_{al, 1988;}

Massina et

_al,

_1989;

_Troyer

et

al,

1989;

Bernoco and

_Byrns,

1991;

Broad et

_{al, 1991; Hopkins}

et

_{al, 1991;}

_Ellegren

et

_al,

_1992;

Giulotto et

_al,

1992;

Mariat et

_al,

_1992).

_However,

_very little or no information about

_segregation

of the

fragments

and

_linkage

analysis

has been

reported

as limited families were

typed.

In this

_report,

we show the

_segregation

of DNA

_{fragments hybridized}

with

a human minisatellite

probe

in 3

large

horse

pedigrees

and

_give

some

_genetic

characteristics of the

_regions

detected in 4 horse breeds.

MATERIALS AND METHODS

Animals

Three horse families

_consisting

of a stallion and half or full

_{paternal sibs,}

_along

combinations were

_30,

27 and 30 for the 3

_stallions,

Iris

_Landais,

Artichaut and Matador du

Bois,

respectively.

All animals were of Selle

_Fran!ais

_origin.

A

sample

of stallions from 4

_breeds,

Arabs

_(AR,

N =

47),

Thoroughbred

(TB,

N =

43),

Selle

Fran!ais

(SF,

N =

40)

and Trotteur

Fran!ais

(TF,

N =

43)

_were also obtained

from different state stud-farms. All animals were blood

_{typed by}

the Laboratoire des

_{Groupes Sanguins}

des

_Chevaux,

INRA-CRJ,

Jouy-en-Josas,

for the blood group

systems

(A,

C, D,

K,

P, Q,

U)

and the

_protein

_systems

_(Alb,

_{Tf, A1B,}

_Es,

_Pi,

_Gpi,

Pgd,

Pgm)

and all

_{offspring qualified.}

Purification of DNA

Blood

_samples

were drawn in tubes

_{containing tripotassium}

EDTA

_(Vacutainer)

and DNA was extracted

according

to the method of

_Jeanpierre

_(1987).

Blood was

treated twice in a solution of 10 mM

NaCI,

10 mM

_EDTA,

_pH

_8,

for 30 min at

+4°C.

Pellets

_containing

_leucocytes

were

_homogenized

in a solution of 5 M

guanidine

HCI,

0.5 M ammonium acetate,

1.25%

_{N-lauroylsarcosine containing}

0.1

_mg/ml

proteinase

K for 1 h at 60°C. DNA was then

_precipitated

with 2 vol ethanol and washed twice with

70%

ethanol.

Restriction endonuclease

_digestion

and

electrophoresis

Seven pg

samples

of horse DNA were

digested

for 20 h at 37°C

_by

restriction endonucleases HaeIII or HinfI

(BRL

or

Boehringer)

_according

to the manufacturer’s recommendations.

_Complete

_digestion

was achieved with a total addition of 5 units per Ag DNA. Restriction endonuclease DNA

fragments

were

subjected

to horizontal

electrophoresis

in

1%

agarose

gels

25 cm in

_length

in Tris-acetate buffer

(40

mM

Tris,

20 mM

AcOH,

2 mM

_EDTA;

_pH

_8.3).

_{Electrophoresis}

was

_performed

with

circulating

buffer at 55 V

_(HaeIII)

or 60 V

_(Hinfl)

for 18 h. Calibration was achieved

by

adding

a

_sample

of

_{BstEII-KpnI-SmaI digested}

lambda

phage

DNA.

Transfer

Transfer was

_performed

on

_Biodyne

B membrane

_(Pall

_Industry,

_SA).

The DNA

digested fragments

in the

_gels

were

_depurinated

in 0.15 N HCI for 10

min,

denatured in a 0.4 N NaOH solution for 30 min and transferred

_using

the method of Southern

(1975)

at room

_temperature

for 18 h with 0.4 N NaOH.

Hybridization probes

The 2 human VNTR

_probes,

33.6 and 33.15 were

_{kindly provided by}

AJ

_Jeffreys.

They

were labelled

by

the

_primer

extension method

according

to

_Jeffreys

_(1985),

except

that the radiolabelled insert was not extracted from the

plasmid.

Prehybridization

and

_{hybridization}

except

that

0.05%

sodium

_{pyrophosphate}

was added and sufficient radiolabelled

probe

to

_{provide !!}

1 x

10

6

_cpm/ml.

Membranes were then washed twice for 15 min

at room

_temperature

in 2 x

_SSC,

0.1%

SDS then in the same solution for 30 min at

65°C

_(in

more

_stringent

solution at the same

_temperature

if

necessary)

and

finally

rinsed in 2 x SSC at room

_temperature.

The restriction endonuclease

_fragments

were vizualized

_{by autoradiography}

in a cassette with a

Kodak

XAR

film. and

Cronex LI-PLUS

_intensifying

screens at -70°C.

Dehybridization

This was

_{performed by washing}

the blot for 1 h at 37°C with 0.4 N

_NaOH,

followed

by

washing

for 1 h in a 0.1 x

_SSPE,

0.5%

_SDS,

0.2 M Tris-HCI

_pH

7.5 solution

(1

x SSPE = 0.18 M

NaCI,

0.01 M

NaH2P0!,

1 mM

EDTA).

Linkage

analysas

Calculations were

perforined according

to the Lod score method of Morton

(1955).

RESULTS

Probes and enzymes

DNA extracted from our _group of horses was

digested

with HaeIII and with Hinfl.

Whatever the _enzyme

_used,

the 33.15

_probe

_gave a

_large

number of

_fragments

with

rather

_homogeneous

intensities and no clear

polymorphism

(results

not

_shown).

Conversely,

the 33.6

probe

detected fewer bands with different

_{hybridization signal}

intensities. With

Hinfl,

all

_{strongly hybridizing}

bands were detected in the _range

of small

_{fragment lengths}

_(less

than 3.5

_{kb) (fig la). HaellI}

gave more

_evenly

distributed bands

_spread

over the

_{autoradiograms}

_{(fig lb).}

Since,

with DNA

digested

with

_Hinfl,

the different levels of band

_migration

_appeared

to be more

heterogeneous

than with

HaeIII,

it was decided to take into account

_only

results

obtained with the 33.6-HaeIII

_probe-enzyme

combination.

Fragment

identification and

_polymorphism

Blots

_usually

consisted of DNA

_digests

from 1 sire and its

_{offspring along}

with DNA

digests

from their mothers. The 3 sires were also run on the same

gel

to _compare

the

_respective

mobilities of their bands

_{(fig 2).}

Two

_major

_groups of bands with a

strong

radioactive

_signal

_appeared

in the range of medium

(2.5

to 5

_kb)

and small

fragment

length

inferior to 2 kb

_{(fig 2)}

on HaeIII blots

_probed

with 33.6. A total

of 15

_fragments

were

_given

a letter as an identification code

(A

to

₀₎

_according

to

their

mobility

and

_intensity

while the rest of the bands

_giving

low

_signals

were not

Segregation

of the

_fragments

Transmission of

_{single paternal}

_fragments

was followed in the 3 sire

_progenies.

_Only

informative

_{dam(s)-offspring}

_{combinations,}

that is dam

_negative

for each

_fragment,

were taken into account. In this way, the presence or absence of a band in the

offspring

could be

_{unambiguously}

_interpreted.

It was observed that a

_given

_fragment

present

in the sire was transmitted to half of his _progeny

_{(table I)}

_suggesting

a Mendelian inheritance of the

_{corresponding}

allele.

Locus identification

Two

_approaches

were

_{consecutively}

used to determine the number of loci

_controlling

the

_{fragment polymorphism.}

One is based on the

analysis

of the relative

_segregation

of

paternal

fragments

in their progeny, the

other

considers

that,

in the same

animal,

any

fragment

should not

triplicate

with others

already

assigned

to a locus. In the

medium _{size range} of

_fragments

_(2.5

to 5

_kb),

sires exhibited 2 bands that were

found to behave as alleles at one locus. As no dam or

_offspring

_possesses more

than 2 of these

bands,

it was concluded that these

fragments

were alleles at a

single

locus. Six alleles

_present

in the sires were thus identified

(B,

C, D, E, F,

I).

In

_addition,

one

_fragment

_(H)

which was

_only

_present

in some mares and

part

of their

_offspring,

but did not

_triplicate

with the 6

_previously

defined

_alleles,

was

also

_assigned

to this first locus. The same

_arguments

were

employed

for the smaller

but the existence of

_negative

animals in this band

_{category suggests}

that there are

other

fragments

generated

by

this locus not detected in our conditions. Some other

fragments

were not allocated to _any

_specific

other loci

_mainly

because insufficient

genetic

information could be obtained

_(bands

_{A, G, J,}

K and

_L).

Mutation rate in allele

_length

Each

fragment

belonging

to 1 of 2 loci

_present

in an

_offspring

was also

_present

in

one of its

_parents

which

strongly

_suggests

that no mutation or mutation-like event

had occurred in these 3 families. The upper limit of the

5%

confidence interval

of the mutation rate based on the Poisson distribution is thus

1.8%

at the first locus and

4.4%

at the second locus

_according

to the number of observed

_fragments

transmitted to the progeny

(168

and 66

respectively).

Linkage analysis

No close

_linkage

was found between these 2 loci and between them and _any of the

other markers that were

_heterozygous

in at least 1 of the 3 sires

_(A,

_{D, P,}

_Q

and

U blood groups and

Al, Tf, Es, A1B, Pi,

Pgd

and

Pgm

for the

proteins).

No Lod scores exceeded 1.0 for recombination values

_ranging

between 0.01 and 0.5.

Polymorphism

in the 4 breeds

As for the

_families,

all

_{strongly hybridizing fragments}

were

_polymorphic.

The F

fragment,

detected in

_families,

was

split

into Fl and F2

fragments

that were

considered to be alleles.

_Conversely,

H and I were difficult to

_distinguish

as these 2

_fragments

are not

_frequent

and

_migrate

_very close to each other and thus were

on the albumin locus

(mean

value

X

2

=

_1.28,

df

=

3).

Allelic

frequencies

for the

first

minisatellite locus are

_given

in table II and no differences were detected between the

populations

(

X

2

=

_0.94,

df

=

21).

Heterozygosities

and

polymorphism

information

content

_(PIC)

values

_(Botstein

et

_al,

₁₉₈₀₎

are

_given

in table III.

DISCUSSION

In an initial

_screening,

horse

_genomic

DNA was

_digested

with HaeIII or Hinfl and

Southern blots were

_subjected

to

_{hybridization}

with either the 33.6 or 33.15

_probe.

It

_appeared

that the 33.15

_probe

_gave about 25

_hybridizing

bands per individual with a low

_degree

of

polymorphism

and some

_{background signal.}

On the

_contrary,

33.6,

in the same

_experimental

conditions allowed the detection of a smaller number

observable on Hinfl DNA

_digests).

This observation confirms the

_prediction

that the

polymorphism

detected with VNTR

_originates

from the number of tandem

_repeats

and not from mutation in the DNA restriction site of the enzyme.

The

_banding

_pattern

of the 33.6-HaeIII

_probe-enzyme

combination resembles

more a

_single-locus

VNTR situation than a

_fingerprint

and shows that at least 2 loci are

_{predominantly recognized}

due to a

_great

similarity

between the horse

minisatellite core _sequence and the human

_probe

_sequence. On the

_contrary,

the 33.15

_probe

detects a

_family

of DNA

_fragments,

the sequences of which are more similar to each other and are most

_{probably generated by}

a

_greater

number of loci. The 33.6-HaeIII combination allowed an

_analysis

of the

_segregation

of

_strongly

hybridizing fragments

in sire

_progenies,

an

approach

which has

proved

to be very tentative or even

_impossible

with true

_{fingerprints.}

Two loci were identified with 7 and 3 detectable alleles in these 3 families. No close

_linkage

was detected between any of the red cell

antigen,

protein

and DNA loci detected in this

_study

and no

mutation event could be observed which indicates that these loci are rather stable. In the stallion

_populations,

8 alleles were detected at the first locus whose

fre-quencies

did not

_{significantly}

differ between the 4 breeds.

However,

this observation has to be confirmed on a

larger

number of animals. On the other

hand,

some bands

at this locus

_migrated

very close to each other and we have

_certainly

underesti-mated the real level of

_polymorphism

of this

_system.

It can be observed that the lowest PIC value is found in the

_{Thoroughbred,}

which confirms the well-known

homogeneity

of this breed

_(Bowling

and Clark

_1985;

Gu6rin and

_{M6riaux, 1986;}

Ellegren

et

_al,

_1992).

_Conversely,

the Arab

_sample

is the most

_{heterogeneous}

of the 4 breeds. At the second locus in the

population

sample,

identification of alleles was rendered somewhat hazardous due to the low _power of resolution in this

_part

of the

_gel.

Bands were found to be less distinct with variable intensities and levels

of

_{migration heterogeneous.}

These characteristics were much less

important

in the families

_probably

because animals were

interrelated,

thus

reducing

the

variability

observed in unrelated animals of the same breed.

In

_spite

of some limits

_{imposed by}

the resolution of the

technique,

the

_high

polymorphism

detected with the 33.6

probe

makes it a _very useful marker for horse

identification and

_{linkage analysis}

in this

_species.

ACKNOWLEDGMENTS

We are _very

grateful

to the Haras Nationaux for

_providing

blood

samples, pedigree

in-formation as well as financial _support, to the breeders of the horse families and to AJ

Jeffreys

for

_providing

the minisatellite _probes. The referees suggestions are

_greatly

appre-ciated. This

study

was

_{supported by}

the Minist6re de la Recherche et de

_{l’Enseignement}

Sup6rieur,

contract No 88R0703.

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