Rat gene mapping using PCR-analyzed microsatellites.

(1)

Rat Gene Mapping Using PCR-Analyzed Microsatellites

Tadao Serikawa,* Takashi Kuramoto,* Pascale Hilbert:* Masayuki Mori,* Junzo Yamada,*

Christopher

J. Dubay,t Klaus Lindpainter,g Detlev Ganten! Jean-Louis Guhnet,**

G. Mark Lathrop’ and Jacques S. Beckmannt

*Institute

of Laboratory Animals, Faculty of Medicine, Kyoto University, Kyoto 606, Japan, ?Centre d’Etude du Polymorphisme

Humain, 75010 Paris, France, $IRIBNH, Campus Erasme, Brussels I070, Belgium, “German Institute

for

High Blood Pressure and

Department of Pharmacology, University of Heiderberg,

0-6900

Heiderberg, Germany, and Institut Pasteur de Paris, 75724 Paris**

Cedex 15, France

Manuscript received February

6 ,

1992

Accepted for publication March 20, 1992

ABSTRACT

One hundred and

seventy-four rat loci which contain short tandem repeat sequences were extracted

from the GenBank

or

EMBL data bases and used to define primers for amplification by the polymerase

chain reaction (PCR) of the microsatellite regions, creating PCR-formatted sequence-tagged micro-

satellite sites (STMSs).

One hundred and thirty-four STMSs for 1 18 loci, including 6 randomly cloned

STMSs, were characterized: (i) PCR-analyzed loci were assigned to specific chromosomes using a

panel of rat

X

mouse somatic cell hybrid clones. (ii) Length variation of the STMSs among

8 inbred

rat strains could be visualized at

85 of 107 loci examined (79.4%). (iii) A genetic map, integrating

biochemical, coat color, mutant and restriction fragment length polymorphism loci, was constructed

based on the segregation of

125

polymorphic markers in seven rat backcrosses and in two F2 crosses.

Twenty four linkage groups were identified, all of which were assigned to a defined chromosome. As

a reflection of the bias for coding sequences in the public data bases, the STMSs described herein are

often associated with genes. Hence, the genetic map we report coincides with a gene map. The

corresponding map locations of the homologous mouse and human genes are also listed for compar-

ative mapping purposes.

T

HE construction of a detailed rat genetic map is

important to exploit the potential of the rat as

a n experimental animal (GILL

et

al.

1989). Until now,

progress in rat gene mapping

has relied largely on the

use of somatic cell-hybrid clone panels (YOSHIDA

1978, SZPIRER

et

al.

1984; YASUE, SERIKAWA

and

YAMADA

1991

;

YASUE

et

al.

1992), and precise chro-

mosomal localizations have been determined by

in

situ

hybridization

(KANO, MAEDA

and SUCIYAMA

1976;

MORI

et

al.

1989; TAKAHASHI

et

al.

1986;

ZHANC

et

al.

1988). In contrast, linkage information has been

very limited. A large collection of informative genetic

markers has not been available for linkage analysis

and mapping until recently. Consequently, only 13

linkage groups were known today in the rat, of which

only 6 have been assigned to a specific chromosome

(LEVAN

et

al.

1991; HEDRICH

1990).

A new class of polymorphic markers, broadly re-

ferred to as microsatellites, has recently been applied

to human and mouse genetic studies. These sequence

tagged microsatellite sites (STMSs) consist of simple,

tandemly repeated mono-

to

tetranucleotide sequence

motifs flanked

by

unique sequences, that serve as

primers for polymerase chain reaction (PCR) amplifi-

cation. Variability at these sites is mostly due to a

variation in the number of repeat units in the motif.

As

a result of the very small size differences (as few as

Genetics 131: 701-721 (July, 1992)

a single nucleotide base pair), visualization

of

the

length polymorphism can often be

achieved on

4%

agarose gels. Although for smaller size differences

resolution may require electrophoretic separation of

allelic fragments on denaturing sequencing gels.

STMSs found in human, mouse and cattle have

been shown to be highly polymorphic and randomly

distributed throughout the genomes (HAMADA and

KAKUNACA 1982;

HEARNE

et

al.

1991

;

LITT and LUTY

1989; LOVE

et

al.

1990; MONTACUTELLI,

SERIKAWA

a n d G U ~ N E T

1991

;

M O Y Z I ~

et

al.

1989;

TAUTZ

1989;

WEBER 1990; WEBER and

MAY

1989). There are

numerous STMSs in the eukaryote genome; for the

(dT-dG),A(dC-dA), motif alone it is estimated that the

number of these sequences

in the mammalian genome

exceeds

IO5

copies per haploid genome (HAMADA,

PETRINO

and

KAKUNACA

1982).

A search

within the public data bases of human

sequences for the

presence of microsatellite motifs

yielded some 368 potentially polymorphic loci (WIL-

(2)

702 T. Serikawa

et

al.

genes

(or

in

their

flanking sequences). Primers

for

PCR amplification were chosen

to define each

of

these

as STMSs (PCR-formatting). These were character-

ized

for

their potential variability among different rat

strains and used to construct

a

genetic

map of

t h e

rat.

MATERIALS AND METHODS

Screening of rat microsatellite loci and design

of

primer

sequences:

GenBank (version 63) and EMBL (version 22+)

were screened for rat sequences which contain microsatel-

lites, following the same procedure as described in BECK-

MANN

and WEBER

(1 992). Searches were performed using

the FIND protocol of the “Genetics Computer Group Se-

quence Analyses Software Package” of the University of

Wisconsin, version 6.1 (DEVEREUX,

HAEBERLI

and SMITHIES

1984) for all simple

tandemly repeated rat sequences, having

a repeat unit of 4 bases or less, with total length for the

uninterrupted stretch of repeats of at least 20 base pairs

(WEBER

1990).

Independent entries from different non-overlapping

parts of the same gene were clustered as a single “locus.”

All redundant synonymous entries were removed. In several

instances, upstream and downstream sequence comparisons

had to be performed to ascertain identity. Satellite DNA

(e.g.,

RATSIMPA) and artefactual microsatellites (such as

mRNA poly(A) tails or those resulting from tailing or clon-

ing experiments) were also eliminated. Moreover, members

of large multigene families such as the MHC, rDNA and

immunoglobulin gene families were combined into a single

entry. (These loci are included even though they are unlikely

to yield an easily interpretable locus-specific polymorphism.)

This yielded 174 “unique” loci (excluding thus repetitive

DNA and large multigene families), each containing at least

one microsatellite.

Random cloned STMSs were derived from a rat total

genomic library, by screening for AC dinucleotide repeats,

and sequencing of positive clones

to determine PCR

primers.

Primers 16-26 nucleotides long were selected using the

“OLIGO” computer program (RYCHLIK

and RHOADS

1989)

and were custom synthesized by GENSET (France).

PCR and gel electrophoresis:

PCR was performed using

a

standard tube type PCR apparatus (DNA thermocyler

PJ

1000, Perkin-Elmer Cetus) and microtiter plate type PCR

apparatus (LEP PREM 111, Techne or

MJ research program-

mable thermal cycler). The reaction volume was 25

111. Final

concentrations were: 10 mM Tris-HC1, pH 8.4; 50 mM KCI;

0.1% Tween 20; 125

PM

dATP, dCTP, dGTP, dTTP

each.

MgCln concentration was adjusted to 1-3 mM according to

a preliminary titration (LOVE

et

al.

1990). One hundred

nanograms of genomic DNA and 0.6 units of Taq DNA

polymerase (Promega) were used. Programming of temper-

ature and time cycles was as follows: 3 min at 94”, 35 cycles

of 1

min at 94”,

1

min

at

50”, 55” or

60”

and

30

sec at

72”, followed by a final elongation step of 3 min at

7 2 ” .

PCR products were usually resolved

on 4%

agarose gel

electrophoresis (NuSieve 3:

1 agarose, FMC bioproduct) and

stained by ethidium bromide.

In the analyses of the F2 cross, in cases where polymor-

phism could not be detected on agarose gels, radioactively

labeled PCR amplification products were separated by elec-

trophoresis on standard denaturing sequencing gels, a

method enabling the visualization of small size differences.

Polymorphism was visualized after autoradiography.

Length polymorphisms and genetic linkage study:

The

rat inbred strains ACI/N, BN/N, F344/N, IS/Kyo, SHR/

Kyo, TM/Kyo, WTC/Kyo, ZI/Kyo were raised at the Insti-

tute of Laboratory Animals at Kyoto University. The prog-

eny from seven backcross matings (ACI

X

WTC)

X

WTC,

(ACI

X

F344)

X

F344, (BN

X

WTC)

X

WTC, (SHR

X

WTC)

X

WTC, (SHR

X

BN)

X

BN, (ZI

X

BN)

X

ZI and

(TM

X

ZI)

X

ZI were obtained from the same facility. The

FS animals from SHRSP and WKY are the same as those

described in HILBERT

et al.

(1 99 1). High molecular weight

DNAs were prepared from liver or spleens from all

10

inbred rat strains (including the parental strains of the FP

progeny), and backcross and FP progeny, following standard

protocols. All DNA samples were dialyzed and stored in a

10 mM Tris-HC1, 0.1 mM EDTA buffer (pH 8.0).

Table 1 lists 134 PCR-formatted markers corresponding

to 1 18 loci, which were used for chromosomal assignment

using somatic cell

hybrid clones and/or linkage analyses.

Twenty nine allelic forms of 16 biochemical loci, 9 restric-

tion fragment length polymorphism (RFLP) loci, three coat

color loci and one mutant locus (Table 2) were also typed

in the seven backcross sets.

Three loci AGT, REN and

SCN2A were typed with both RFLPs and STMSs. The

segregation patterns in the backcross progeny of the micro-

satellite loci and other markers were first analyzed with

“GENE-LINK”

(MONTAGUTELLI

1990). More detailed two

point and multipoint analyses were subsequently performed

using the “LINKAGE” and “GMS” programs (LATHROP

and

LALOUEL

1984; LATHROP

et aE.

1988) with genotypes from

all nine crosses. The GMS programs permits testing of a

large number of alternative orders, including all permuta-

tions of four adjacent loci and higher order permutations

involving closely linked groups of loci, during map construc-

tion. Anchor markers were chosen based on the criterion

that the

relative placement of these loci was

unchanged

within 1OO:l odds of the best supported order.

Chromosomal assignment

of

genes:

In this study, 18 rat

X

mouse somatic cell hybrid clones, segregating all individ-

ual rat chromosomes were used. The clones were developed

by fusing Sp2/0-Ag14 myeloma of BALB/c mice

origin

with lymphocytes of a male ACI/N rat. The panel of seg-

regation of rat chromosomes and assignment of biochemical

genes using the clones has already been reported (YASUE,

SERIKAWA and

YAMADA

1991). DNA samples were

prepared

from all somatic cell hybrid clones and from the parental

Sp2/0-Ag14 myeloma cells. The rat-specific amplification

products for the microsatellite regions were examined in

each hybrid clone, and compared to the known segregation

pattern of individual rat chromosomes in this panel.

In

this

manner,

it

was possible to assign most microsatellites to a

particular chromosome according to the most concordant

segregation pattern.

RESULTS

Development

of

rat microsatellites:

One

hundred

and seventy-four “unique”

loci, each

of

which contains

at

least

20 uninterrupted bases

of a repeat

of mono-

to

tetranucleotides were extracted from

the

GenBank

and

EMBL

data

bases.

Of 364

STMSs, 196 (53.8%)

were dinucleotides repeats, 42 were tri- and

91 were

tetranucleotide repeats (1 1.5% and 25.0%,

respec-

tively;

for a

more detailed description of

the

results

of this search, see

BECKMANN

a n d

WEBER

1992).

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