B R I E F N O T E S

Characterization of the genetic diversity on
natural populations of Chinese miniature pig
breeds

B. Fan, S.-L. Yang, B. Liu, M. Yu, S.-H. Zhao and K. Li

Laboratory of Molecular Biology & Animal Breeding, College of
Animal Science & Technology, Huazhong Agricultural University,
Wuhan 430070, China

Accepted for publication 9 August 2003

Source/description: There are four indigenous miniature pig
breeds in China: Xiang pig, Wuzhishan pig, Diannan Small-Ear
pig and Tibetan pig. These pigs are mainly found in the
mountain areas of south and south-west China, where com-
munication and transportation links are limited.

1

Although a

few studies have been published on genetic purity analysis of
inbred populations of these miniature pigs,

2,3

no report deals

with the natural populations.

Animals: Blood and ear tissue samples were collected from
conservation farms and the home areas of these breeds: Xiang
pig (XING; n

¼ 30, Guizhou Province livestock breeding farm),

Wuzhishan pig (WZSN; n

¼ 30, Hainan Province Wuzhishan

pig conservation farm of Academy of Agricultural Science),
Diannan Small-Ear pig (DNSE; n

¼ 30, Yunnan Province pig

breeding farm of Xishuangbanna Municipality), Tibetan pig
(TBTN; n

¼ 31, Linzhi of Tibet). Erhualian (EHLN; n ¼ 30)

and Duroc (n

¼ 32) pigs were used as outgroups in the study.

Genomic DNA was extracted according to a modified phenol
and chloroform method.

Microsatellite markers: Of the 37 microsatellites used, 20 were
from No. X set of fluorescent microsatellites distributed by the
US Pig Genome Coordinated Project and were donated kindly
by Prof. Max Rothschild; 17 were chosen from those recom-
mended by ISAG-FAO which were synthesized by Sangon
Biotechnology Company (Shanghai, China). DNA amplifica-
tions were implemented using PE 480 and Amp 9700 Ther-
mal

Cyclers

(Perkin-Elmer,

Norwalk,

CT,

USA),

and

polymerase chain reaction (PCR) conditions were optimized on
the basis of reference protocols. The PCR products of fluores-
cent microsatellites were scanned through an ABI PRISM 310
Genetic Analyzer (ABI System, Foster City, CA, USA) and
genotyping was performed using

GENESCAN

software. Genotyp-

ing of the other 17 microsatellites were carried out as reported
earlier.

4

Two control DNA samples of French9110010 and

French9110012 were used in order to ensure the precision of
allele calling.

5

Statistical analysis: Allele frequencies were accounted using the

GENEPOP

software package.

6

The genetic heterozygosity, gene

differential coefficients (Gst), D

A

distance and Nei’s standard

genetic distance, neighbour-joining (NJ) topology tree and
bootstrap value were estimated using

DISPAN

.

7

The allele shar-

ing measure (D

AS

) between individuals was estimated with

MICROSAT,

8

while the NJ topology tree was constructed with the

NEIGHBOR

program of the

PHYLIP

software package.

9

The effective

number of allele was calculated according to the methods
described by Kimura and Crow.

10

The polymorphism content

information (PIC) was obtained with

CERVUS

.

11

Comments: All loci were polymorphic except that Sw2435,
Sw860 and S0034 were monomorphic in DNSE and XING,
respectively. PIC values ranged from 0.488 (Sw1092) to 0.897
(S0227). The number of alleles per locus varied from three
(Sw860) to 12 (S0227), and the mean across all loci was 7.62.
The expected genetic heterozygosities of miniature pigs were
between 0.603 and 0.746 (Table 1), which were similar to
those of Spain Iberian pigs, Chinese Taihu pigs and European
pigs,

12–14

and were higher than those of inbred populations

used as experimental animals.

2,3

On the basis of these meas-

ures, the level of genetic variability within TBTN was the
highest, followed by WISN, DNSE and XING in descending
order. Duroc used for outgroups had the least genetic variab-
ility. TBTN mainly inhabit the high-altitude areas of Qing-Tibet
plateau, which are maintained outdoors all year around. Easy
mating and gene exchange among pig populations account for
the level of intrabreed genetic variability. State-owned WISN,
DNSE and XING pigs were subjected to artificial selection and
long-term systematic breeding, so they have a narrower genetic
base. The gene differential coefficient (Gst

¼ 0.21) indicated

that 21% of total genetic variation existed among breeds, and
genetic structures of these pigs were obviously distinctive.

D

A

genetic distances among breeds were between 0.255 and

0.516, and Nei’s standard distances were between 0.447 and
1.083. The NJ topology tree constructed from D

A

distances is

illustrated in Fig. 1. According to the classification results in
the book of Pig Breeds in China, XING, DNSE and WZSN are
classified into South China Type. TBTN is the only breed of
Plateau Type. EHLN is one of Low Yangtze River Basin Type.
Both XING and DNSE are distributed in the mountain areas of
south-west China, and they had closer genetic relationship
because of their similar geographical distribution and adapta-
bility to ecological conditions. TBTN was separated from the

Table 1 Genetic variability within miniature pig breeds, Erhualian and
Duroc.

Breed

Mean number of alleles

Mean heterozygosity

PIC

Observed

Effective

Observed

Expected

Duroc

3.676

2.596

0.329

0.493

0.439

EHLN

4.351

3.399

0.387

0.642

0.580

TBTN

6.108

4.982

0.515

0.746

0.696

XING

5.108

3.606

0.407

0.603

0.552

WZSN

5.703

4.293

0.498

0.707

0.653

DNSE

5.243

3.614

0.354

0.640

0.585

EHLN

WZSN

XING

DNSE

TBTN

Duroc

49

51

39

Figure 1 The NJ dendrogram of miniature pig breeds, Erhualian and
Duroc, and the numbers on the branches indicate the percentage
occurrence in 1000 bootstrap replicates.

2003 International Society for Animal Genetics, Animal Genetics, 34, 465–476

other Chinese indigenous breeds as a single branch, possibly for
its special appearance and living surroundings. In addition,
most of the individuals could be classified into their source
populations, based on a NJ topology tree constructed from allele-
sharing measures among individuals. The cluster relationship of
individuals was tight for XING, DNSE, WISN, EHLN and Duroc,
but TBTN was divided into three subpopulations, which reflec-
ted the high genetic variability within this population.

Acknowledgements: This study was funded by the International
Foundation for Science (B/3093-1) to Dr Bin Fan and partly
supported by National Key Projects for Basic Research and
Development Plans of China (G2000016103), National Out-
standing Youth Science Foundation (39925027) and National
High Science and Technology Project of China (863) to Dr Kui
Li. Comments made by Dr Alan L. Archilbald and other
anonymous reviewers are also gratefully acknowledged.

Supplementary material: The following material is available
from http://www.blackwellpublishing.com/products/journals/
suppmat/AGE/AGE1057/AGE1057sm.htm
Table S1 Allele frequencies for all populations by locus.

References

1 Zhang Z. G. (1986) Pig Breeds in China. Shanghai Scientific

& Technical Publishers, Shanghai.

2 Niu R. et al. (2001) Acta Genetica Sinica 28, 518–26.
3 Li K. et al. (2002) 7th World Congress on Genetics Applied to

Livestock Production 26, 26–36. Montpellier, France.

4 Fan B. et al. (2002) Animal Genetics 33, 422–7.
5 Archibald A. et al. (1995) Mammalian Genome 6, 157–75.
6 Raymond M. et al. (1995) Journal of Heredity 86, 248–9.
7 Ota T. (1993) DISPAN: Genetic Distance and Phylogenetic

Analysis. Pennsylvania State University. University Park.

8 Minch E. et al. (1995) MICROSAT. Version 1.4. A computer

program for calculating various statistics on microsatellite
allele data.

9 Felsenstein J. (1996) PHYLIP: Phylogeny Inference Pack-

age, Version 3.5c. Department of Genetics. University of
Washington, Seattle.

10 Kimura M. & Crow J. F. (1964) Genetics 49, 725–38.
11 Marshall T. C. et al. (1998) Molecular Ecology 7, 639–55.
12 Martı´nez A. M. et al. (2000) Animal Genetics 31, 295–301.
13 Laval G. N. et al. (2000) Genetics, Selection, Evolution 32,

187–203.

14 Li K. et al. (2000) Animal Genetics 31, 322–25.

Correspondence: K. Li ([e-mail])

Fine mapping of the bovine heart fatty
acid-binding protein gene (

FABP3

)

to BTA2q45 by fluorescence

in situ

hybridization and radiation hybrid mapping

R. Roy*, J. H. Calvo

†

, H. Hayes*, C. Rodellar

‡

and A. Eggen*

*Laboratoire de Ge´ne´tique Biochimique et de Cytoge´ne´tique,
INRA, Jouy-en-Josas, France.

†

Departamento de Mejora Gene´tica

Animal, INIA, Madrid, Spain.

‡

Laboratorio de Gene´tica Bioquimica,

Universidad de Zaragoza, Zaragoza, Spain

Accepted for publication 19 August 2003

Source/description: Fatty acid-binding proteins (FABPs) repre-
sent a group of evolutionarily conserved proteins that are
involved in cellular uptake and metabolism of long-c7hain fatty
acids.

1

They are small intracellular proteins involved in fatty

acid transport from the plasma membrane to the sites of
b-oxidation and triacylglycerol or phospholipid synthesis. Fur-
thermore, FABPs may modulate the intracellular fatty acid
concentration and in this manner regulate various cellular
processes and lipid metabolism in particular.

2

The heart type

FABP (FABP3) protein is present in several tissues with a high
demand of fatty acids such as cardiac and skeletal muscle and
lactating mammary gland.

PCR primers: In order to obtain a bovine FABP3 DNA frag-
ment, PCR amplification was performed using the follow-
ing set of primers: FABP3.F:5

¢

-GGTTTTGCTACCAGGCAGGT-3

¢

FABP3.R:5

¢

-TTCCCTATTCCCCTTCAGGG-3

¢

Primers were designed from the ovine FABP3 (AY157617)

sequence and amplified a 222-bp fragment.

PCR conditions: Polymerase chain reactions (PCR) were per-
formed in 15

ll using 20 ng of genomic DNA, standard PCR

buffer, 1.5 m

M

MgCl

2

, 200 m

M

each dNTP, 10 pmol each pri-

mer and 0.5 U Taq DNA polymerase (Promega, Madison, USA).
Cycling conditions were 94

C for 30 s, 55 C for 30 s and

72

C for 30 s for 35 cycles. PCR products were cloned and

sequenced. Homology searc4hes were performed with BLAST
programs at the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/BLAST/).

The isolated bovine DNA sequence showed a high level of

sequence identity with the ovine, swine, horse and human
FABP3 genes: 97, 93, 92 and 90%, respectively.

Screening of a bovine BAC library: Using the ovine primers, a
bovine Bacterial Artificial Chromosome (BAC) library

3

was

screened by PCR and a BAC clone containing the bovine
FABP3 gene was identified. Partial sequence analysis of the
BAC clone (500E02) confirmed the presence of FABP3
sequences (Accession number: AY327461).

Fluorescence in situ hybridization: Fluorescence in situ hybrid-
ization (FISH) was carried out on RBP-banded bovine, ovine
and caprine chromosome preparations (Fig. 1), as described
previously by Hayes et al.

4

The BAC clone, 500E02, with a

150-kb insert containing the bovine FABP3 gene was used as a
probe. FISH on cattle, ovine and caprine chromosomes was
performed using 500 ng of digoxigenin-labelled BAC DNA.

Radiation hybrid mapping: To confirm the cytogenetic localiza-
tion obtained by FISH, a bovine

· hamster radiation hybrid (RH)

panel was analysed.

5

Primers and PCR conditions for RH map-

ping were the same as those used for screening the BAC library.

The Carthagene software

6

was used to perform two-point

and multipoint analyses of the RH data and to provide a map of
the region where the FABP3 gene is located (Fig. 2). The dis-
tances between markers on the most likely map were calculated
with the RHMAP3.0 software

7

under the equal retention

probability model.

2003 International Society for Animal Genetics, Animal Genetics, 34, 465–476

Brief notes

466

Chromosomal location: The chromosomal locations of the
bovine FABP3 gene and its ovine and caprine homologues on
BTA2q45, OAR2q45 and CHI2q45 were determined by FISH
of the bovine BAC clone 500E2 to bovine, ovine and caprine
metaphase chromosomes (Fig. 1). This localization was con-
firmed by analysis of a bovine RH panel.

Comments: The assignment of the bovine FABP3 gene on BTA
2 agrees with comparative mapping data. Indeed, BTA2 is
conserved with chromosomal segments on HSA1, OAR2, CHI2,
MMU4 and RN05 (H. Hayes, personal communication).
However, this assignment is inconsistent with the bovine
FABP3-like sequence previously located on BTA6.

8

This bovine

FABP3-like sequence may code for the mammary derived

growth inhibitor (MDGI), which has a 95% protein identity
with FABP3.

9

Moreover, the full-length cDNA coding for mouse

MDGI displayed strong sequence similarity to mouse FABP3.

10

In conclusion, the sequence located in BTA6 may be the
mammary derived growth inhibitor gene.

Acknowledgements: This study was supported by a grant from
Institute National de Recherche Agronomique (INRA).

References

1 Hirsch D. et al. (1998) Proc Natl Acad Sci USA 95, 8625–9.
2 Veerkamp J. H. (1995) Prog Lipid Res 34, 17–52.
3 Eggen A. et al. (2001) Genet Sel Evol 33, 543–8.
4 Hayes H. et al. (2000) Cytogenet Cell Genet 90, 315–20.
5 Williams J. L. et al. (2002) Mamm Genome 13, 469–74.
6 Schiex T. (1997) Proc Int Conf Intell Syst Mol Biol 5,

258–67.

7 Lange K. et al. (1995) Genome Res 5, 136–50.
8 Billich S. et al. (1988) Eur J Biochem 175, 549–56.
9 Specht B. et al. (1996) J Biol Chem 271, 19943–9.

10 Binas B. et al. (1992) In Vitro Cell Dev Biol 28A, 625–634.

Correspondence: Rosa Roy ([e-mail])

Exclusion of

PISRT1

as a candidate locus for

canine

Sry

-negative XX sex reversal

K. S. D. Kothapalli*, E. Kirkness

‡

, L. J. Natale* and

V. N. Meyers-Wallen*

†

*J.A. Baker Institute for Animal Health, College of Veterinary
Medicine, Cornell University, Ithaca, NY 14853, USA.

†

Department

of Biomedical Sciences, College of Veterinary Medicine, Cornell
University, Ithaca, NY 14853, USA.

‡

The Institute for Genomic

Research (TIGR), Rockville, MD 20850, USA

Accepted for publication 22 August 2003

Source/description: Human and animal studies have shown that
testis induction can occur in the absence of the Sry gene.
Specifically, in individuals with a female karyotype (46,XX
humans and 78,XX dogs) testicular tissue develops in the
absence of the Y chromosome and Sry. This disorder is termed
Sry-negative XX sex reversal. The mechanism of testis induc-
tion in such individuals is unexplained, but indicates that

Figure 1 Chromosome mapping of FABP3 by fluorescence in situ hybridization (FISH). A specific signal is observed on chromosomes 2q45 proximal.
In cattle (a), goat (b) and sheep (c).

483.00

0

30

TGLA226

TNP1

ARO28
TEXAN4
SERPINE2

BMS1987

INRA135
TEXAN5

FABP3

LAPTM5
IDVGA64
PTP4A2
BM4117
ALP1
BMS356

BM2113

RSJW85

60

90

120

150

180

210

240

270

300

330

360

390

420

450

480

Figure 2 Radiation hybrid (RH) mapping of FABP3 gene to bovine
chromosome 2.

2003 International Society for Animal Genetics, Animal Genetics, 34, 465–476

Brief notes

467

undiscovered autosomal genes may be involved.

1,2

A region

(PIS, polled intersex) on goat chromosome 1 has been linked to
caprine Sry-negative XX sex reversal.

3

Subsequent studies

identified a deletion in this region that affects transcription of at
least two genes, FOXL2 and PISRT1.

4

The 48 kb genomic

sequence surrounding PISRT1 (GenBank AF404302) in nor-
mal and affected goats was examined, and a homologous
sequence (GA_X5028QUEVST) in the canine genome was
identified in the database maintained by The Institute for
Genomic Research (TIGR, Rockville, MD, USA). A simple
dinucleotide (CA) repeat of approximately 30 bp was identified
in this region (GenBank AY319655) and used to screen a
canine Sry-negative XX sex reversal pedigree.

Primer sequence: QUEVST: Forward: 5

¢-TTA GTG AGG AGA

AGA TGA CAG T-3

¢ Reverse: 5¢-TAG AAG AGA CCA GAG GCG

GA-3

¢

The forward primer was 5

¢ end labelled with 6-FAM (IDT,

Coralville, IA, USA).

PCR conditions: Polymerase chain reactions (30

ll) were per-

formed using genomic DNA (50 ng), 1

l

M

of each primer,

0.25 m

M

each of dNTPs, 3

ll of 10

· PCR buffer (Perkin-Elmer

Life Sciences, Foster City, CA, USA), 3.0 m

M

MgCl

2

and 1.5 U

Taq polymerase (Ampli Taq II; Perkin-Elmer Life Sciences).
Cycling conditions were: initial denaturation at 95

C for 5 min

followed by 35 cycles of denaturation at 95

C for 30 s,

annealing at 62

C for 1 min and extension at 72 C for 1 min,

with a final extension at 72

C for 2 min.

A portion (3

ll) of the reaction was mixed with TAMRA

standard (PRISM Genescan-500; Applied Biosystems, War-
rington, UK) and deionized formamide. Denatured samples
were assayed by capillary electrophoresis (310 Genetic Ana-
lyzer; Applied Biosystems) and product size determined by
software (

GENESCAN

version 2.1; Perkin-Elmer Life Sciences)

relative to the internal TAMRA standard.

Pedigree: In previous studies of the canine Sry-negative XX sex
reversal model,

5,6

a pedigree was generated from one sire, a

proven carrier American cocker spaniel, producing F1, F2 and
F1 backcross generations. Together with later molecular find-
ings,

7

these studies indicate that Sry-negative XX sex reversal in

this pedigree is inherited as an autosomal recessive trait with
expression limited to 78,XX individuals homozygous for the trait.

All affected dogs in the above pedigree inherited Sry-negative

XX sex reversal from the founder sire, the American cocker
spaniel (C1 in Fig. 1). Thus, the disease causing mutation
should be identical by descent in all affected dogs.

Polymorphism: Four alleles in the QUEVST microsatellite ran-
ging from 369 to 397 bp were identified in the pedigree. A total
of six different genotypes at the marker locus were found in
affected dogs, none of which were of the same genotype (1 2) as
the founder sire (Fig. 1). In addition, none of the allele in three
affected dogs (C10, C16 and C18) was inherited from the
founder sire. Our results indicate that the mutation causing
canine Sry-negative XX sex reversal in this pedigree is not
located within the region homologous to goat PISRT1.

Chromosomal

location: The

GA_X5028QUEVST

sequence

(1–740 nucleotides) falls within a larger canine contig (Gen-
Bank AACN010076059), which contains nucleotides 180–
1345 and appears to be orthologous to human chromosome 3
(nucleotides

139855971–139857365;

ENSEMBL

release

13.31.1, http://www.ensembl.org), downstream from

Ôtestes-

specific

Õ BPESC1 (nucleotides 139679588–139740027). This

region of the human genome is orthologous to canine chro-
mosome 23 (CFA23) between RH map markers 3472 and
4096 (in TSP units, as defined by Guyon et al.).

8

Acknowledgements: This study is supported by the NIH R01
HD40351. The authors wish to thank Anita Hesser for
manuscript preparation.

Figure 1 Pedigree of the Sry-negative XX sex reversal syndrome, genotyped with the QUEVST microsatellite marker. Marker genotypes are given
below the animal’s symbol. The Ô?Õ symbol represents an unavailable sample. C1 is the founder animal. B1–B4 are beagles.

2003 International Society for Animal Genetics, Animal Genetics, 34, 465–476

Brief notes

468

References
1 McElreavey K. et al. (1993) Proc Natl Acad Sci USA 90,

3368–72.

2 Koopman P. (1999) Cell Mol Life Sci 55, 839–56.
3 Schibler L. et al. (2000) Genome Res 10, 311–8.
4 Pailhoux E. et al. (2001) Nat Genet 29, 453–8.
5 Selden J. R. (1981) PhD Thesis. Graduate Group in Genetics,

University of Pennsylvania, Philadelphia, PA.

6 Meyers-Wallen V. N. & Patterson D. F. (1988) Hum Genet 80,

23–30.

7 Meyers-Wallen V. N. et al. (1995) Mol Reprod Dev 41, 300–5.
8 Guyon R. et al. (2003) Proc Natl Acad Sci USA 29, 5296–301.

Correspondence: V N Meyers-Wallen ([e-mail])

Assignment of the

porcine peptide YY

gene to

chromosome 12

R. H. Pita*, A. M. Ramos

†

, P. S. Lopes*,

S. E. F. Guimaraes* and M. F. Rothschild

†

*Departamento de Zootecnia, Universidade Federal de Vic¸osa,
Vic¸osa, MG 36570-000, Brazil.

†

Department of Animal Science,

Center for Integrated Animal Genomics, Iowa State University,
2255 Kildee Hall, Ames, IA 50011, USA

Accepted for publication 28 August 2003

Source/description: Peptide YY is a member of the neuropeptide
Y (NPY) protein family and it is secreted by endocrine cells of
both the intestine and the pancreas.

1

The PYY participates in

regulating a number of physiological actions of the digestive
organs, such as pancreatic exocrine secretion, gastric acid
secretion, gastrointestinal motility, contraction of gallbladder
and colonic absorption of water and electrolytes.

2,3

Consensus

primers (PYY1-F and PYY1-R) were designed across conserved
regions among the human, mouse and bovine PYY gene
sequences (GenBank accession nos. NM_004160, NM_145435,
and L37369, respectively). Polymerase chain reaction (PCR)
was used to amplify c. 900 bp of the corresponding porcine PYY
gene spanning exon 2 and the 3

¢ UTR. The resulting sequence

(AY344365) showed 86 and 76% identity to the corresponding
human and bovine exon PYY sequences, respectively. Using this
sequence, pig specific primers, PYY2-F and PYY2-R, were
designed to amplify a 175 bp product. Sequence analysis of the
PCR products from several individual pigs of different breeds
revealed a nucleotide substitution in the 3

ÕUTR at position

(nucleotide) 566 which affected a HinfI recognition site.

Primer sequences:
PYY1-F: 5

¢- AAC CGC TAC TAC GCC TCC CTG-3¢

PYY1-R: 5

¢-ACC ACA CAC AGC CCT CCA GCC -3¢

PYY2-F: 5

¢- GAG AGC TGG AAG AAT AGA AGC-3¢

PYY2-R: 5

¢- ACC ACA GCC CTC CAG CC-3¢

PCR conditions: The PCR reactions were performed using
12.5 ng of porcine DNA, 1

· PCR buffer, 0.75 m

M

MgCl

2

(PYY1-F and PYY1-R) or 1.0 m

M

MgCl

2

(PYY2-F and PYY2-R),

0.125 m

M

dNTPs, 0.3

l

M

of each primer, and 0.5 U Taq DNA

polymerase (Promega, Madison, WI, USA) in a 10

ll final

volume. The PCR profile included 4 min at 94

C; 35 cycles of

45 s at 94

C, 30 s at 58 C (PYY1-F and PYY1-R) or 54 C

(PYY2-F and PYY2-R), 1 min at 72

C (PYY1-F and PYY1-R)

or 30 s (PYY2-F and PYY2-R) at 72

C; and a final 7 min

extension at 72

C in a PTC200 thermalcycler (MJ Research,

Inc., Watertown, MA, USA).

Polymorphism and Mendelian inheritance: HinfI digestion of the
175 bp PCR product produced allelic fragments of 175 bp
(allele 1), or 105 and 70 bp (allele 2). Mendelian segregation of
the polymorphism was verified in the three-generation Iowa
State Berkshire

· Yorkshire resource population.

4

Allele fre-

quencies of the polymorphism were determined by genotyping
several commercial breeds. Allele 1 was the most common
allele, observed with a frequency of 0.78 in Landrace (n

¼ 23),

0.48 in Yorkshire (n

¼ 23), 1.0 in Berkshire (n ¼ 24), 0.89 in

Hampshire (n

¼ 52), 0.58 in Pietrain (n ¼ 30) and 1.0 in

Duroc (n

¼ 24).

Chromosomal location: The porcine PYY gene was assigned to
chromosome 12 (Prob.

¼ 1.00) and the p11–(2/3 p13) region

(Prob.

¼ 0.89) by PCR analysis of a pig-rodent somatic cell

hybrid panel.

5

This assignment was confirmed by two-point

and multipoint linkage analyses using CRIMAP 2.4 and geno-
types in the Iowa State Berkshire

· Yorkshire resource popu-

lation.

4

Linkage

was

confirmed

between

PYY

and

microsatellites

S0229

(recombination

fraction

¼ 0.19;

LOD

¼ 41.2) and SW874 (recombination fraction ¼ 0.13;

LOD

¼ 59.9) that had been previously mapped to chromosome

12. The best map order was (with distances between markers in
centimorgans): S0229 – 20.9 – PYY – 14.6 – SW874 – 12.3 –
S0090 – 14.7 – S0147 – 22.9 – SWC23 – 12.2 – SW2180.

Comments: PYY may affect feeding behaviour and hence may
be related to obesity in mammals.

Acknowledgements: The authors thank CAPES/Brazilian Minis-
try of Education, for its sponsorship of R.H. Pita during her stay
at Iowa State University. This work was supported in part by
funding from Sygen International, PIC USA and the Iowa
Agriculture and Home Economics Experiment Station, Ames,
Iowa and by Hatch Act and State of Iowa funds, project 3600.
The authors also thank Dr K.S. Kim for technical advice.

References
1 Schartz M. W. et al. (2002) Nature 418, 595–7.

2 Chen Z. et al. (2001) FEBS Letters 492, 119–22.
3 Imamura M. (2002) Peptides 23, 403–7.
4 Malek M. et al. (2001) Mamm Genome 12, 637–45.
5 Yerle M. et al. (1996) Cytogenet Cell Genetics 73, 194–202.

Correspondence: M. F. Rothschild ([e-mail])

Mapping of the porcine

peroxisome proliferator

activated receptor alpha

gene to chromosome 5

R. H. Pita*, A. M. Ramos

†

, P. S. Lopes*,

S. E. F. Guimaraes* and M. F. Rothschild

†

*Departamento de Zootecnia, Universidade Federal de Vic¸osa,
Vic¸osa, MG 36570-000, Brazil.

†

Department of Animal Science,

2003 International Society for Animal Genetics, Animal Genetics, 34, 465–476

Brief notes

469

Center for Integrated Animal Genomics, Iowa State University,
2255 Kildee Hall, Ames, IA 50011, USA

Accepted for publication 28 August 2003

Source/description: The peroxisome proliferator activated receptor
alpha gene (PPARA) is a ligand-activated nuclear hormone
receptor that mediates the effects of fatty acids and their
derivatives

at

the

transcriptional

level.

1

Primers

were

designed in exon 4 (PPARA1-F) and exon 5 (PPARA1-R)
from a published porcine partial cDNA sequence (GenBank
accession number AF228696). The 1700-bp product was
sequenced (GenBank accession number AY364466) with 85
and 87% nucleotide identity to the corresponding human and
mouse exon sequences, respectively. Sequence analysis of the
polymerase chain reaction (PCR) products from several indi-
vidual pigs of different breeds revealed a nucleotide substitu-
tion in intron 4 at nucleotide 251. Because a natural
restriction endonuclease recognition site did not exist, a
restriction site for enzyme BsrGI was created using a mis-
match primer (PPARA2-F and PPARA2-R). The PCR frag-
ment from these primers was 317 bp in length and spanned
intron 4 and exon 5.

Primer sequences:
PPARA1-F: 5

¢-TCT CCA GCC TCC AGC CCC TC-3¢

PPARA1-R: 5

¢-CAC AGG CTT CAT ACG CAG GA-3¢

PPARA2-F: 5

¢-CAT TCG GCT AAA GCT GGT CT-3¢

PPARA2-R: 5

¢-TGA CTA GTT CTA ATT ATT CCG AGG ATC

TGC TGT AC-3

¢

PCR conditions: Both PCR reactions were performed using
12.5 ng porcine DNA, 1

· PCR buffer, 1.25 m

M

MgCl

2

(PPARA1-F and PPARA1-R) or 1.0 m

M

MgCl

2

(PPARA2-F and

PPARA2-R), 0.125 m

M

dNTPs, 0.3

l

M

of each primer, and

0.5 U Taq DNA polymerase (Promega, Madison, WI, USA) in a
10

ll final volume. The PCR profile included 4 min at 94

C;

35 cycles of 45 s at 94

C, 45 s at 60 C (PPARA1-F and

PPARA1-R) or 57

C (PPARA2-F and PPARA2-R) and 30 s at

72

C; and a final 7 min extension at 72 C in a PTC200

thermocycler (MJ Research, Inc., Watertown, MA, USA).

Polymorphism and Mendelian inheritance: The BsrGI digestion of
the 317-bp PCR product produced allelic fragments of 317 bp
(allele 1), or 286 and 31 bp (allele 2) that were resolved on 4%
agarose gels. Mendelian segregation of the BsrGI PCR-RFLP
was observed in the three-generation Iowa State Berk-
shire

· Yorkshire resource population.

2

Allele frequencies were

determined by genotyping several commercial breeds. Allele 1
was the most common allele, with a frequency of 0.87 in
Landrace (n

¼ 16), 0.68 in Yorkshire/Large White (n ¼ 331),

0.75 in Berkshire (n

¼ 16) and 0.90 in Duroc (n ¼ 20).

Chromosomal location: Two-point and multipoint linkage ana-
lyses (CRIMAP

4

) were performed using the genotypes of the

Iowa State Berkshire

· Yorkshire resource population.

3

PPARA

was significantly linked with several markers on porcine
chromosome 5 (SSC5). Two-point linkage analysis determined
that the most closely linked markers (recombination fraction
and LOD score) were ACR (0.06, 21.8) and SW413 (0.10,
15.0). The best map order for this region of SSC5 produced by
multipoint linkage analysis (with distances in centimorgans

listed between markers) was: PPARA – 6.3 – ACR – 2.3 –
SW413 – 27.3 – SW1482.

Comments: Peroxisome proliferator activated receptor alpha
belongs to the superfamily of steroid/thyroid nuclear hormone
receptors. Its role in lipid metabolism makes it an interesting
candidate gene affecting intramuscular fat and other fat-related
meat quality traits.

4

PPARA expression is mainly detected in

tissues exhibiting high rates of

b-oxidation, i.e. liver, kidney,

heart and skeletal muscle, where it promotes cellular uptake,
activation and oxidation of fatty acids through activation of
target gene expression.

5

PPARA trancriptionally regulates the

production of enzymes such as acyl-coenzyme A (CoA) oxidase
(the key enzyme in the peroxisomal

b-oxidation pathway) and

carnitine palmitoyl transferase I (implicated in the transloca-
tion of fatty acids across the inner mitochondrial membrane) as
well

as

CYP4A6,

which

is

an

important

microsomal

x-hydroxylase.

1

Acknowledgements: The authors thank CAPES/Brazilian Minis-
try of Education for its sponsorship of R.H. Pita during her stay
at Iowa State University. This work was supported in part by
funding from Sygen International, PIC USA and the Iowa
Agriculture and Home Economics Experiment Station, Ames,
Iowa and by Hatch Act and State of Iowa funds, project 3600.
The authors also thank Dr K.S. Kim for technical advice.

References

1 Hihi A. K. et al. (2002) Cell Mol Life Sci 59, 790–8.
2 Malek M. et al. (2001) Mamm Genome 12, 637–45.
3 Green P. et al. (1990) Documentation for CRIMAP, Version

2.4. Washington University, School of Medicine, St Louis,
MO, USA.

4 Bocher V. et al. (2002) Ann NY Acad Sci 967, 7–18.
5 Sundvold H. et al. (2001) Gene 273, 105–13.

Correspondence: M. F. Rothschild ([e-mail])

Mapping of bovine

CEBPD

to

BTA14q15–17

N. Ihara*, H. Yamakuchi

†

, Y. Taniguchi

‡

, Y. Sasaki

‡

,

G. L. Bennett

§

, S. Kappes

§

and Y. Sugimoto*

*Shirakawa Institute of Animal Genetics, Nishigo, Fukushima 961-
8061, Japan.

†

Cattle Breeding Development Institute Kagoshima,

Kagoshima 899-8212, Japan.

‡

Laboratory of Animal Genetics and

Breeding, Graduate School of Agriculture, Kyoto University, Sak-
yoku, Kyoto 606-8502, Japan.

§

USDA-ARS, U.S. Meat Animal

Research Center, Clay Center, NE 68933, USA

Accepted for publication 28 August 2003

Source/description: CCAAT/enhancer-binding proteins (CEBPs)
and peroxisome proliferator activated receptor

c (PPARG) are

transcription factors that regulate the expression of fat-specific
genes, and have been reported to control differentiation of
mouse preadipocyte cell lines.

1

These bovine orthologs may

influence body fat composition and distribution, which are
economically important traits in beef cattle. We have mapped

2003 International Society for Animal Genetics, Animal Genetics, 34, 465–476

Brief notes

470

bovine

CEBPA

and

PPARG

genes

to

BTA18q24

and

BTA22q24, respectively.

2

Here we report the mapping of

bovine CEBPD gene to BTA14q15–17 by fluorescence in situ
hybridization (FISH) using a yeast artificial chromosome (YAC)
clone harbouring the gene, and by genetic linkage mapping of a
polymorphic microsatellite marker isolated from the YAC clone.
Polymerase chain reaction (PCR) primers (DS4; 5

¢-AGC TGC

CGC GTG GAC CCT AAG T-3

¢ and DA4; 5¢-CAT GCT CAG TCT

TTC CCT CGT ATC-3

¢) were designed from the bovine CEBPD

gene sequence

3

(GenBank accession no. D82986). Three YAC

clones, Y122D11, Y130E11 and Y175F8, were isolated by PCR
screening

4

and validated by sequencing. Clone Y122D11,

which contained a 800 kb non-chimeric insert was randomly
amplified with primers designed from consensus bovine short
interspersed nuclear element (SINE) regions with slight modi-
fications (retro1a: 5

¢-AAT ACT GGA GTG GGT TGC C-3¢ and

retro2a: 5

¢-AGG CTACAG TCC ATG GGA T-3¢),

5

(AMS1a:

5

¢-AAT CTT CTC CAA CAC CAC AG-3¢)

6

and YAC right/left

arm regions (YAC-right-R: 5

¢-AAC GCC CGA TCT CAA GAT

TA-3

¢ and YAC-left-S: 5¢-CAA GTT GGT TTA AGG CGC AA-3¢).

7

The PCR products containing microsatellites were verified by
Southern hybridization using a fluorescent-labelled (dC-dA/dG-
dT) probe (Pharmacia, Uppsala, Sweden) and subcloned into
pCR2.1 (TA Cloning Kit; Invitrogen, Carlsbad, CA, USA) fol-
lowed by colony hybridization. Positive clones were sequenced
using an ABI 377 automated DNA Analyzer (Applied Byosi-
stems, Foster City, CA, USA), and primers that amplified
microsatellite DIK121 were designed (5

¢-CTA CAG TCC ATG

GGA TCA CA-3

¢ and 5¢-GCA GCT TGC AGC AAG AAT TC-3¢;

GenBank accession nos. AB107878 and AB107879, respect-
ively).

PCR conditions: SINE PCR mixture contained 2 m

M

MgCl

2

,

0.2 m

M

each dNTPs, 2.5 U Ex Taq

TM

(Takara Biomedical, Otsu,

Japan), 50 p

M

primer, and 100 ng of Y122D11 DNA. The profile

for SINE PCR was 2 min at 94

C, 30 cycles of 30 s at 94 C,

2 min at 53

C, and 3 min at 74 C, and a final 10 min at 74 C

followed by cooling to 4

C. The PCR mixture for DIK121 con-

tained 1.7 m

M

MgCl

2

, 0.2 m

M

each dNTP, 0.75 U Taq DNA

polymerase (ABgene, Epsom, UK), 6.25 p

M

primer, and 20 ng of

genomic DNA. The profile for microsatellite PCR was 2 min at
94

C, 29 cycles of 1 min at 94 C, 2 min at 60 C, and 20 s at

72

C, and a final 10 min at 72 C followed by cooling to 4 C.

Polymorphism and chromosomal locations: Using FISH, Y122D11
DNA was hybridized to BTA14q15–17 (data not shown) cor-
responding to HSA8q11 where human CEBPD gene is located.

8

Heterozygosity of DIK121 was 0.62 across 28 parents in the
USDA-MARC mapping population,

9

with identification of seven

alleles and a product size of 189–201 bp. Using CRIMAP
version 2.4,

10

DIK121 was linked to RM180 (recombination

fraction

¼ 0.004, LOD ¼ 65.2) and RM011 (recombination

fraction

¼ 0.073, LOD ¼ 42.1) on BTA14 (Fig. 1). Thus there

is a good agreement between the physical assignment of CEBPD
gene by FISH and genetic localization by linkage analysis.

Acknowledgements: The authors thank H. Takeda for valuable
suggestions and Y. Kaneuchi for laboratory assistance. This
work was supported by a grant from the Japan Racing and
Livestock Promotion Foundation.

References

1 Lowell B. B. (1999) Cell 99, 239–42.
2 Ihara N. et al. (1998) Anim Genet 29, 398–400.
3 Taniguchi Y & Sasaki Y. (1997) J Anim Sci 75, 586.
4 Takeda H. et al. (1998) Anim Genet 29, 216–9.
5 Kaukinen J. & Varvio S. L. (1992) Nucleic Acids Res 20,

2955–8.

6 Lenstra J. A. et al. (1993) Anim Genet 24, 33–9.
7 Kuhn R. M. & Ludwig R. A. (1994) Gene 141, 125–7.
8 Cleutjens C. B. et al. (1993) Genomics 16, 520–3.
9 Bishop M. D. et al. (1994) Genetics 136, 619–39.

10 Green P. et al. (1990) Documentation for CRIMAP Version

2.4. Washington University School of Medicine, St Louis,
MO, USA.

Correspondence: Y. Sugimoto ([e-mail])

Assignment

**

of the porcine stearoyl-CoA

desaturase (

SCD

) gene to SSC14q27 by

fluorescence

in situ

hybridization and by hybrid

panel mapping

J. Ren*

,†

, C. Knorr*, F. Habermann

‡

, R. Fries

‡

,

L. S. Huang

†

and B. Brenig*

*Institute of Veterinary Medicine, Georg-August-University of
Go¨ttingen, Go¨ttingen, Germany.

†

Jiangxi Provincial Key Laboratory

for Animal Biotechnology, Jiangxi Agricultural University,
Nanchang, P.R. China.

‡

Institute of Animal Breeding and Molecular

Genetics, Technical University of Munich, Munich, Germany

Accepted for publication 29 August 2003

Figure 1 Genetic localization of bovine CEBPD gene on BTA14.

**This is a more precise localization of SCD previously mapped to
SSC14q15–16 by Wimmers et al. (2002).

2003 International Society for Animal Genetics, Animal Genetics, 34, 465–476

Brief notes

471

Source/description: Stearoyl-CoA desaturase (SCD) is a key
enzyme in the biosynthesis of monosaturated fatty acids, it
catalyses the introduction of the first double bond between
carbons 9 and 10 of palmitoyl-CoA and stearoyl-CoA to form
the monounsaturated fatty acyl-CoA esters, palmitoleoyl-CoA
and oleoyl-CoA, respectively.

1

Monosaturated fatty acids con-

trol triacylglycerol storage in adipose tissue, membrane fluidity,
signal transduction, apoptosis and senescence. The altered
function of the SCD gene has been implicated to be associated
with obesity, cancer and aging.

2

To date, several mammalian

SCD cDNAs have been isolated from rat, mice, human, sheep,
goat and cattle.

3–6

The genomic structure for the identified

human, ovine and bovine SCD gene are very similar, spanning
approximately 15–21 kb and consisting of six exons and
five introns with an unusually long 3

¢ UTR.

3

The human

SCD gene has been assigned to chromosome 10

4

and

more precisely to 10q23–q24 (http://www.ncbi.nlm.nih.gov/
LocusLink). Previously, 1003 bp of the porcine SCD mRNA was
identified in an EST clone and assigned to SSC14q15–16 by
somatic cell hybrid panel analysis.

7

Here, we report the precise

localization of the porcine SCD gene to SSC14q27 using the full
sequence information of the gene. The chromosomal localiza-
tion confirms the homology between the

ÔqÕ arms of chromo-

somes HSA10 and SSC14.

Isolation of a PAC clone containing the porcine SCD gene: The
porcine PAC library TAIGP714

8

was initially screened with two

primer pairs to isolate PAC clones harbouring the SCD gene.
Primers A (5

¢-ATC CGA GAG CCA AGA TGC C-3¢) and B

(5

¢-ATG GTA GTG GTG GTT GTG TAG-3¢) were designed based

on the consensus sequence after comparison of the human and
the caprine SCD gene. The amplicon encompassed parts of
exons 1 and 2, and the complete intron 1 (GenBank accession
no: AF097514 and AF325499). Primers K (5

¢-CAC AAC TAC

CAC CAC ACC-3

¢) and L (5¢-GTC ATA AGC CAG ACC CAG-3¢)

were adopted from exon 6 according to the porcine SCD mRNA
sequence (EMBL accession no: Z97186). Polymerase chain
reaction (PCR) amplification was performed in a total volume of
25

ll using 50 ng of porcine genomic DNA, 100 l

M

of each

dNTP, 10 pmol of each primer and 2.5 U Taq polymerase
including the supplemented 10

· PCR buffer (Qiagen, Hilden,

Germany). The DNA was pre-heated at 95

C for 2 min. The

following PCR profile was used: 35 cycles of 92

C for 30 s,

63

C for 30 s and 72 C for 30 s. The final cycle had an

extension time of 10 min at 72

C. The resulting fragments of

591 bp (primer combination A/B) and 114 bp (primer combi-
nation K/L) were cloned into the pGEM-T vector (Promega,
Madison, WI, USA) and bidirectionally sequenced. A similarity
of 86% between the 591-bp fragment and the human SCD
gene, and a similarity of 100% between the 114-bp fragment
and the porcine mRNA confirmed the sequence identity. Sub-
sequently, clone TAIGP714K14177 of approximately 100 kb
size harbouring the complete porcine SCD gene was isolated
by PCR screening of the library with the primers mentioned
above.

Fluorescence in situ hybridization: Fluorescence in situ hybrid-
ization (FISH) was performed as described previously

9,10

using

swine metaphase spreads (prepared from peripheral lympho-
cytes) obtained from a normal, healthy boar. Prior to FISH,

the QFQ-banded spreads were photographed using a cooled
CCD camera. Hybridization signals were detected and amplified
by incubation with Streptavidin-Cy3 (Rockland, Gilbertsville,
NY, USA). The chromosomes were then DAPI-counterstained
(Sigma, Deisenhofen, Germany). The relative positions of the
signals on the chromosomes were measured considering the
distance to the telomere and the length of the entire chro-
mosome enabling the calculation of the fractional length
(Flqter).

Hybrid panel analyses: A porcine rodent somatic cell hybrid
panel

11

and the INRA/University of Minnesota porcine radi-

ation hybrid panel (IMpRH)

12

were screened for porcine SCD

by PCR. The PCR primers C (5

¢-AGT TTG TTG GGA ATG TGG-3)

and D (5

¢-AAC TGT GCG GAG AAA TTG-3¢) originated from

intron 1 of the porcine SCD gene. The PCR amplifications
of the 211-bp fragment were performed in a total volume
of 25

ll with 25 ng of panel DNA as template. Polymerase

chain reaction conditions were 92

C for 30 s, 57 C for

30 s, and 72

C for 30 s for 35 cycles with an initial dena-

turation step at 95

C for 5 min plus a final extension at

72

C for 10 min. All the PCR products were analysed by

agarose gel electrophoresis and ethidium bromide staining.
Panels were scored by presence/absence of the amplified
product. Obtained PCR results were evaluated using the web-
pages http://imprh.toulouse.inra.fr (radiation hybrid panel)
and http://www.toulouse.inra.fr/lgc/pig/hybrid.htm (somatic
cell hybrid) at INRA.

Chromosomal location: The chromosomal localization of the
porcine SCD gene was carried out by FISH (most precise posi-
tion 14q27, 29 chromosomes measured, Flqter: 0.17 ± 0.04)
of the PAC clone TAIGP714K14177 to metaphase chromo-
somes (Fig. 1). Analysis of the somatic cell hybrid panel

Figure 1 Chromosomal assignment of the porcine SCD gene by FISH
analysis.

2003 International Society for Animal Genetics, Animal Genetics, 34, 465–476

Brief notes

472

revealed a significant correlation of 0.98 between the SCD gene
and SSC14 (error risk <0.5% and maximal correlation of 1.00).
Within SSC14, chromosomal region C14G indicated the highest
probability with 0.87 and a correlation of 1.00. Radiation
hybrid mapping was carried out with 118 clones. The retention
frequency of the SCD gene was 24%. Two-point analysis
revealed that the most significantly linked marker is S0007 on
chromosome 14 at a distance of 17cR (LOD score of 16.83).
Multipoint analysis led to linkage group SW2122-SW361-
SW328-SCD-S0007-SW1333-SW2057-SWR1042-SW886.

Comment: The SCD gene has previously been mapped to
SSC14q15-16 by the somatic cell hybrid panel with oligonu-
cleotides designed from the partial SCD mRNA

7

. Our inde-

pendent mapping of the SCD gene by somatic cell hybrid panel,
radiation hybrid panel and FISH confirms the chromosomal
assignment of SCD to SSC14 and provides the accurate local-
ization to q27.

Acknowledgements: The authors would like to thank Dr Martine
Yerle (INRA, Castanet-Tolosan, France) for kindly providing the
hybrid panels. The project was supported by budgetary funds
for the Institute of Veterinary Medicine.

References

1 Enoch H. G. et al. (1976) J Biol Chem 251, 5095–103.
2 Cohen P. et al. (2002) Science 297, 240–3.
3 Bernard L. et al. (2001) Gene 281, 53–61.
4 Zhang L. et al. (1999) Biochem J 340, 255–64.
5 Tabor D. E. et al. (1998) Mamm Genome 9, 341–2.
6 Campbell E. M. G. et al. (2001) J Anim Sci 79, 1954–5.
7 Wimmers K. et al. (2002) Anim Genet 33, 255–63.
8 AI-Bayati H. K. et al. (1999) Mamm Genome 10, 569–72.
9 Solinas-Toldo S. et al. (1995) Cytogenet Cell Genet 69, 1–6.

10 Toldo S. et al. (1993) Mamm Genome 4, 720–7.
11 Yerle M. et al. (1996) Cytogenet Cell Genet 73, 194–202.
12 Yerle M. et al. (1998) Cytogenet Cell Genet 82, 182–8.

Correspondence: Bertram Brenig ([e-mail])

Sequencing, tissue distribution and physical
mapping of the porcine homologue of
cardiomyopathy associated 3 (

CMYA3

)

P.-W. Pan*, K. Li*, C. K. Tuggle

†

, M. Yu*, B. Liu*

and S.-H. Zhao*

*Laboratory of Molecular biology and Animal Breeding, Huazhong
Agricultural University, Wuhan 430070, P R China.

†

Department of

Animal Science, Iowa State University, Ames, IA 50011, USA

Accepted for publication 29 August 2003

Source/description: Data obtained by sequencing the 5

¢ and 3¢ end

of M223, a clone from a porcine skeletal muscle cDNA library,
identified two expressed sequence tags (ESTs) (AA063663 and
AA063664) with no overlap (http://www.ans.iastate.edu/fac-
ulty/Muscle_E.html). The BLAST results indicated these two
ESTs matched several porcine, bovine and human skeletal mus-

cle ESTs but not any known gene. Transcriptional profiling
revealed that this clone was differentially expressed during dif-
ferent skeletal muscle developmental stages (Zhao and Tuggle,
unpublished data). To localize this interesting clone, detect its
tissue distribution and obtain longer sequences by rapid ampli-
fication of cDNA ends (RACE), primers GSP1 and GSP2 were
designed from AA063663. An in silico cloning method was used
to extend the sequence towards the 5

¢-end when no 5¢-RACE

product was obtained.

Primer sequences: GSP1: 5

¢-GGACCGAGGATACAAAGAGTAA

GAGG-3

¢

GSP2: 5

¢-CGTCTGCACCATCAGGTTCATTAG-3¢

GSP2F: 5

¢-CCTCTTCATCACATAGCTCAGAAG-3¢

WP1: 5

¢-TGGCATCGGAAGCAGGACTTAT-3¢

WP2: 5

¢-GCGGAAGATATGCTTGTGTCCT-3¢

Tissue distribution: Total RNAs were extracted from adult porcine
heart, skeletal muscle, liver, kidney, spleen, lung and bladder
with TRIzol reagent (GIBCO) and treated with DNase I (Promega).
One microgram RNA was reverse transcripted with 300 U
M-MLV (Promega). Polymerase chain reaction (PCR) was per-
formed in 25

ll reactions containing 1 ll of cDNA, 1

· PCR

buffer, 1.5 m

M

MgCl

2

, 100

l

M

dNTPs, 0.4

l

M

of GSP1 and GSP2,

and 2 U Taq DNA polymerase. Amplification profiles were 94

C

for 3 min, followed by 30 cycles of 94

C (40 s), 65 C (40 s),

72

C (50 s), with a final extension step of 10 min at 72 C. The

PCR product size was 117 bp. The glyceraldehyde-3-phosphate
dehydrogenase was used as control

1

. Expression was primarily

restricted to skeletal and cardiac muscle, although a weak signal
was observed for spleen RNA (Fig. 1A). Northern blot revealed
that the transcript of the pig cardiomyopathy associated 3
CMYA3 is around 10 kb and expresses strongly in 3 and 7 week
post-natal skeletal muscle (Fig. 1B).

RACE: SMART

TM

RACE cDNA Amplification Kit (Clontech)

was used with skeletal muscle total RNA as template. The
3

¢-RACE product obtained was sub-cloned into the pGEM-T

vector (Promega) and two independent clones were seq-
uenced. A 1275 bp sequence was obtained and the 5

¢- and

3

¢ of this sequence matched AA063663 and AA063664

completely.

In silico cloning: In silico cloning refers to obtaining larger or
full-length cDNA by sorting and assembling the large amount of
ESTs in GenBank. The 1275 bp sequence was used in BLAST
analyses to search for homologous human sequences. Over-
lapping human and pig sequences were assembled by DNAStar
software. This assembling allowed prediction of pig cDNA se-
quence 5

¢ to current pig cDNA sequence. Experimental verifi-

cation of this predicted pig cDNA was performed by using RT–
PCR and pig muscle RNA with primers GSP2 and GSP2F, as well
as WP1 and WP2. The PCR products were recovered and se-
quenced. The lengths of these two PCR fragments were as
expected (GSP2 and GSP2F generates 1120 bp, WP1 and WP2
generates 1592 bp and there was a 362 bp overlap between the
two sequences) and matched the human sequence with an
identity of 88.8%. The 3516 bp experimentally identified se-
quence was deposited in GenBank (AY246700).

Physical mapping: The chromosomal location of the pig gene
was detected by the pig

· rodent somatic cell hybrid panel

2003 International Society for Animal Genetics, Animal Genetics, 34, 465–476

Brief notes

473

(SCHP) with GSP1 and GSP2

2

and was assigned to SSC15

q15–22 by the SCHP analysis. To determine the precise
location, the INRA-University of Minnesota porcine radiation
hybrid (IMpRH) panel was typed by using the same primer pair.
Statistical analysis was performed as described

3

and it indicated

that this gene was linked most closely to microsatellite S0088,
situated on SSC15 with a LOD score of 12.01.

Comments: The AY246700 (3516 bp) was compared to
sequences available in GenBank and found to be similar to
mRNA of human hypothetical protein FLJ32020 (NM_152381),
now known as cardiomyopathy associated 3, CMYA3, and
which is localized on human chromosome 2q31.1. This gene
produces, by alternative splicing, four different transcripts a, b,
c, d, that altogether encode four different protein isoforms. The
pig CMYA3 cloned in our study has 82.9 % sequence identity
over 3516 bp to variant a (the longest cDNA, 11.51 kb) and
76.1% identity over 390 aa to the corresponding protein.
Localization of the porcine homologue of CMYA3 to SSC15 is
consistent with orthology to human CMYA3 as SSC15
sequences have been shown to map to HSA2 through
heterologous chromosome painting.

4

Acknowledgements: Provision of the SCHP and IMpRH panel by
Dr Martine Yerle is greatly appreciated. This research was
supported by National Natural Science Foundation of China
(30100131), Key Project of National Basic Research and

Developmental Plan (G2000016103) of China, National High
Science and Technology Foundation of China (2001AA222241)
and

National

Outstanding

Youth

Foundation

of

China

(39925027).

References
1 Janzen M. A. et al. (2000) J Anim Sci 78, 1475–84.
2 Yerle M. et al. (1996) Cytogenet Cell Genet 73, 194–202.
3 Milan D. et al. (2000) Bioinformatics 16, 558–9.
4 Rettenberger G. et al. (1995) Genomics 26, 372–78.

Correspondence: Shu-Hong Zhao ([e-mail])

Mapping of the goat

stearoyl coenzyme A

desaturase

gene to chromosome 26

M. H. Yahyaoui*, D. Vaiman

†

, A. Sa´nchez* and

J. M. Folch*

*Departament de Cie`ncia Animal i dels Aliments, Facultat de
Veterina`ria, Universitat Auto`noma de Barcelona, Bellaterra 08193,
Spain.

†

Laboratoire de Ge´ne´tique Biochimique et de Cytoge´ne´-

tique, INRA-CRJ, 78352 Jouy-en-Josas, France

Accepted for publication 12 September 2003

Source/description: Stearoyl Coenzyme A desaturase (SCD) is an
iron-containing enzyme that catalyses the

D 9-cis desaturation

of palmitoyl and stearoyl-CoA to produce palmitoleoyl and
oleoyl-CoA, respectively. Palmitoleic acid and oleic acid are the
major constituents of triglycerides that are stored in adipocytes
and membrane phsopholipids. A polymorphism was detected
within a 447 bp polymerase chain reaction (PCR) amplification
product of the goat SCD gene, that contained the complete exon
5 sequence (239 bp). The polymorphic site consists of a single
nucleotide substitution (G to T) in exon 5, at position 931 of the
cDNA sequence

1

(GenBank accession number AF339909) with

no amino acid change. This nucleotide substitution results in
the disappearance of a RsaI recognition site. Therefore, RsaI
digestion of the 447 bp PCR product from the G allele produces
three fragments of 98, 111, and 238 bp, whereas the T allele
generates only two fragments of 98 and 349 bp.

Primer sequences: Primers were located in the intron 4 (forward
primer) and in the intron 5 (reverse primer) of the SCD gene.
Forward primer: 5

¢-AGT GTA GAA GGG ACA GCC CAG C-3¢;

reverse primer: 5

¢-GTG GAA TGA CAC ATG GAG AGG G-3¢.

PCR conditions: The PCR reaction was performed in a 25-

ll

final volume, containing 0.625 units of Taq DNA polymerase
(Invitrogen, Barcelona, Spain) with 1

· PCR buffer, 2 m

M

MgCl

2

, 200

l

M

of each dNTP, 0.4

l

M

of each primer and

approximately 100 ng of goat genomic DNA. Thermal cycling
conditions were 95

C for 5 min, 10 cycles of 97 C for 15 s,

63

C for 1 min, and 72 C for 90 s, then 25 cycles of 95 C for

Figure 1 The RNA expression analysis of cardiomyopathy associated 3
(CMYA3) shows primarily post-natal muscle-specific expression. (a)
Tissue distribution of porcine CMYA3, M: 100 bp ladder. (b) Northern
blot hybridization results of porcine CMYA3. 45, 60, 75, and 105
represent day fetal hind-limb muscles; 1, 3 and 7 wk represent 1-, 3-
and 7-week old post-natal semitendinosus muscle.

Present address: D. Vaiman, U361 INSERM, 123 Boulevard de Port-
Royal, 75014 Paris, France.

2003 International Society for Animal Genetics, Animal Genetics, 34, 465–476

Brief notes

474

30 s, 63

C for 1 min, and 72 C for 90 s, followed by a final

extension at 72

C for 5 min.

Polymorphism and allele frequencies: Protocol for rapid genotyp-
ing of the polymorphic site was developed by PCR-RFLP proce-
dure, using RsaI (Roche Molecular Diagnosys, Indianapolis, IN,
USA). Ten microlitres of the PCR product were digested overnight
at 37

C and separated by electrophoresis in a 2% agarose gel.

A total of 99 genomic DNA samples from animals of different
Spanish (Canaria n

¼ 27, Malaguen˜a n ¼ 12, Payoya n ¼ 7,

and Murciano–Granadina n

¼ 24) and French (Saanen n ¼ 29)

breeds were analysed. The frequencies of the G allele were 1.0,
1.0, 1.0, 0.92, and 0.69, respectively, in the five breeds.

Chromosomal

location

and

comparative

mapping

informa-

tion: Chromosomal location was obtained by genetic analysis of
the genotypes of goat reference half-sib families.

2

Three of 12

progenitors were informative, for a total of 55 informative
meioses. Two-point analysis was carried out using the CRI-
MAP software in order to calculate distances and LOD scores
and resulted in significant linkage of SCD with five markers
previously mapped on caprine chromosome 26 (CHI26):
INRA081 (

h

¼ 0.09; LOD ¼ 4.36), INRABERN172 (h ¼ 0.13;

LOD

¼ 3.34), BM4505 (h ¼ 0.11; LOD ¼ 3.66), LSCV046

(

h

¼ 0.13; LOD ¼ 4.18), and LSCV052 (h ¼ 0.11; LOD ¼

5.46). Primer sequences for the amplification of markers are
available at the GoatMap database (http://locus.jouy.inra.fr/
cgi-bin/bovmap/livestock.pl). Haplotype analysis was carried

out by ordering the markers such that the number of double-
recombinants was minimized. This analysis suggested that the
most probable order is Centromere – HEL11 – BM1314 –
INRA081 – INRABERN172 – LSCV052 – SCD – BM4505 –
LSCV046 – Telomere. However, CRI-MAP analysis with the
ÔBuildÕ option indicated that LSCV046 could also be in the
centromeric position, with a likelihood ratio <10 between the
two maps. The physical mapping of LSCV046 to 26q13 and
LSCV052 to 26q21

3

suggests that the true order is Centromere

– LSCV046 – HEL11 – BM1314 – INRA081 – INRABERN172
– LSCV052 – SCD – BM4505 – Telomere. Using this order and
removing double recombinants allowed us to calculate the
distances presented in Fig. 1. There is one notable difference
between our map and that of Schibler et al.

3

in that we have

located INRABERN172 in the middle of the chromosome
whereas it was previously located near the telomere.

We found that LSCV052 (isolated from a BAC clone that was

identified with PAX2 primers

3

) and SCD were separated by

1 cM, suggesting that PAX2 and SCD are physically close in the
goat species. In humans, PAX2 and SCD are separated by
<500 000 bp (NCBI mapview site http://www.ncbi.nlm.nih.
gov/mapview/), which is consistent with our mapping data.
Furthermore, linkage mapping of goat SCD is in accordance
with the previously reported physical mapping using fluores-
cence in situ hybridization.

4

Acknowledgements: M.H. Yahyaoui was supported by a fellow-
ship from AECI (Agencia Espan

˜ ola de Cooperacio´n Internac-

ional), Spain.

References
1 Yahyaoui M. H. et al. (2002) J Anim Sci 80, 886–7.
2 Vaiman D. et al. (1996) Genetics 144, 279–305.
3 Schibler L. et al. (1998) Genome Res 8, 901–15.
4 Bernard L. et al. (2001) Gene 281, 53–60.

Correspondence: J. M. Folch ([e-mail])

The chicken

bone morphogenetic protein

receptor type II (BMPR2)

gene maps to

chromosome 7

1

C. R. Cisar*, J. M. Balog*, R. Okimoto

†

,

N. B. Anthony

†

and A. M. Donoghue*

*Poultry Production and Product Safety Research Unit, Agricultural
Research Service, United States Department of Agriculture, Fay-
etteville, AR, USA.

†

Poultry Science Department, University of

Arkansas, Fayetteville, AR, USA

Accepted for publication 26 September 2003

Source/description: Bone morphogenetic protein receptor type II
is a member of the transforming growth factor (TGF) beta
superfamily of receptors.

1

The TGF beta cytokines and their

Figure 1 Partial genetic map of goat chromosome 26.

1

This article is the material of the US Government and can be

produced by the public at will.

2003 International Society for Animal Genetics, Animal Genetics, 34, 465–476

Brief notes

475

receptors are important cell signalling proteins involved in
regulation of growth and differentiation of many tissues and
organs. A chicken bone morphogenetic protein receptor type II
(BMPR2) cDNA has been sequenced (GenBank accession no.
L77660).

2

In addition, several single nucleotide polymorphisms

(SNPs) have been reported for the chicken BMPR2 gene (dbSNP
accession nos. 12709541-54).

3

Primer sequences: Polymerase chain reaction (PCR) primers
were designed to amplify a 419 bp product from putative exon
12 of the chicken BMPR2 gene (nts 2015–2433) using Mac-
Vector 7.0 (Genetics Computer Group, Madison, WI, USA).
BMPR2/201-5for: 5

¢-CACCAGTTTGTCCACCAATACTACTACC-3¢

BMPR2/243-3rev: 5

¢-AAGGATAGACCTGAGCAGATGGG-3¢

PCR conditions: Reactions contained 100 ng genomic DNA,
10 pmol of each primer, 200

l

M

of each dNTP, 1X buffer

(60 m

M

Tris-HCl, pH 9.25, 1.5 m

M

MgSO

4

, 30 m

M

NaCl,

10

lg/ml BSA, 0.01% Triton

 X-100), and 2 units Thermal-

Ace

TM

DNA polymerase (Invitrogen, Carlsbad, CA, USA) in a

total volume of 50

ll. After an initial denaturation step of

95

C for 3 min, samples were subjected to 30 cycles of

amplification consisting of 95

C for 30 s, 55 C for 30 s, and

74

C for 45 s followed by a final incubation step of 74 C for

10 min.

SNP analysis: Amplicons from Jungle Fowl and White Leghorn,
parents of the East Lansing reference population,

4

were purified

(MinElute PCR Purification Kit, Qiagen, Valencia, CA, USA),
sequenced (DNA Resource Center, University of Arkansas,
Fayetteville, AR, USA) and compared (AssemblyLIGN, Genetics
Computer Group). Sequence variation between the parents was
identified at nt 2228 (T in the Jungle Fowl parent, C in the
White Leghorn parent). This SNP creates a polymorphic HinfI
restriction site (HinfI digest, allele T : 210, 185, and 24 bp;
allele C: 234 and 185 bp).

Linkage analysis: The East Lansing reference population was
used for linkage analysis (52 birds from a backcross).

4

Two

microlitres of each purified PCR product were digested with
5 units Hinf I (New England Biolabs, Beverly, MA, USA) at
37

C for 1 h. The restriction fragments were separated on 3%

agarose gels (Agarose 1000, Invitrogen) and stained with
ethidium bromide. Linkage analysis was performed using Map
Manager.

5

Chromosomal location: Chicken BMPR2 was mapped by two-
point analysis to chromosome 7 at position 14.6 between
markers MSU0339/MSU0381 and MSU0481 (LOD 13.5)

(Fig. 1). BMPR2 is closely linked to CD28 in the chicken gen-
ome (LOD 10.7). These genes are also closely linked in the
human and mouse genomes (http://www.ncbi.nlm.nih.gov/
Homology). These and other data indicate that synteny is
conserved between human chromosome 2q, mouse chromo-
some 1 and chicken chromosome 7.

6

Acknowledgements: This work was supported in part by funds
from the Poultry & Egg Association (Project #285) and Cobb-
Vantress, Inc. Mention of trade names or commercial products
in this article is solely for the purpose of providing specific
information and does not imply recommendation or endorse-
ment by the USDA.

References
1 Massague J. (1998) Annu Rev Biochem 67, 753–91.
2 Kawakami Y. et al. (1996) Development 122, 3557–66.
3 Cisar C. R. et al. (2003) Poult Sci 82, 1494–9.
4 Crittenden L. B. et al. (1993) Poult Sci 72, 334–48.
5 Manly K. F. (1993) Mamm Genome 4, 303–13.
6 Groenen M. A. M. (2000) Genome Res 10, 137–47.

Correspondence: C. R. Cisar ([e-mail])

MSU0339/MSU0381

EEF1B

ABR0326

ROS0331

MSU0481

CD28

MSU0410

TUS0026

BMPR2

10.7

12.6
13.6
14.6
15.5
16.5

19.4

22.2

25.1

Figure 1. Consensus linkage map of section of chicken chromosome 7
showing position of BMPR2.

2003 International Society for Animal Genetics, Animal Genetics, 34, 465–476

Brief notes

476