Download Linkage Mapping of the ACE I Gene in Pig Vincent Nguyen

Linkage Mapping of the Angiotensin I Converting Enzyme Gene in Pig
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V.Q. Nguyen , K.L. Glenn , B.E. Mote , and M.F. Rothschild
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Department of Biological Science, University of California, Irvine, Irvine, CA 92697, USA
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Department of Animal Science and Center for Integrated Animal Genomics
Iowa State University, Ames, Iowa 50011, USA
ABSTRACT
Sow productive life plays an important role in the
economic efficiency of pork production. Several
genes have been isolated in model organisms and
humans that are associated with lifespan. Our
hypothesis is that these same genes or regulatory
pathways are also important for sow productive life.
Angiotensin I converting enzyme (ACE) has been
identified as being key to several diseases known to
shorten human lifespan. In human, the ACE gene is
located on chromosome 17, but it has not been
mapped in the pig. Three primer sets were designed
to amplify the pig ACE sequence and are anchored
in exons and spanned introns covering from exon 1exon 2, exon 9 - exon 10, and exon 12 - exon 13
based on pig sequence and human intronic sizes
based on Ensembl. Sequence results from the
primer set designed to amplify sequence from exon
12- exon 13 showed an exonic C/T single nucleotide
polymorphism located 95 bases from the start of the
amplified fragment. The restriction enzyme AluI was
used to perform PCR-RFLP test on this SNP in the
Berkshire x Yorkshire Resource Family. The
genotypes were used to linkage map this loci using
CRIMAP. The results showed that ACE is located
between microsatellite markers S0229 and SW874 on
pig chromosome 12 (SSC12), as expected by
comparative mapping with the human location.
Radiation Hybrid (RH) mapping confirmed that ACE
is located on pig chromosome 12. The association
studies of this SNP with sow productive life are
ongoing.
MATERIAL AND METHODS
 Primers were designed using pig sequence
accession number NM_001033015 from NCBI.
 Preparation for polymerase chain reactions are
summarized in Table 1.
Polymerase chain reactions (PCR) were set up
under the following protocol: Initial denaturation
temperature at 94˚C for 2 min with 36 cycles of 94˚C
for 30 sec, 53˚C for 45 sec, 72˚C for 50 sec, and final
extension at 72 ˚C for 5 min.
PCR products were sequenced and SNPs were
identified.
 A PCR-RFLP test was performed for the C/T SNP
in exon 12 using the ISU Berkshire x Yorkshire
Resource Family (Malek et al. 2001) under the above
conditions with the restriction enzyme AluI.
 ACE-I12F: 5’ tca tca tcc agt tcc agt tcc 3’
 ACE-I12R: 5’ gtt cgg cgt cca gtt gta ct 3’
TABLE 1. PCR PREPARATION
Ingredients
Amount
Concentration
Buffer
2 µl
n/a
dNTPs
0.5 µl
Forward
Primer
TABLE 2. PCR-RFLP TESTS
SNP
Location
Enzyme
10 mM
C/T
Exon 1
Cac8I
0.1 µl
25 pM
C/T
Exon 9
ApeKI
Reverse
Primer
0.1 µl
25 pM
C/T
Exon 12
EarI
GoTaq
0.05 µl
.35 µM
C/T
Exon 12
BseRI
Water
6.35 µl
n/a
C/T
Exon 12
AluI
DNA
1.0 µl
12.5 ng
FIGURE 1. CHROMATOGRAM
FIGURE 2. ACE LINKAGE MAP
Pig Chromosome 12
C/T
C
LOCI
T
S0229
0.00
ACE
16.0
SW874
38.5
S0090
52.8
S0147
65.4
SWC23
88.3
100.5
SW2180
cM
RESULTS AND DISCUSSION
REFERENCES
Sequencing of PCR products, using the
designated primers, revealed 17 single
nucleotide polymorphisms, of which five
were verified by PCR-RFLP tests
(Table 2).
Green, P., Falls, K. and Crooks, S.
(1990) Documentation for CRI-MAP,
version 2.4 (St. Louis, MO:
Washington University School of
Medicine).
An exonic C/T SNP in the amplified
fragment from exon 12 - exon 13 was
identified in the sequence chromatogram
(Figure 1).
ACE mapped to SSC12 between
microsatellites, S0029 and SW874,
(Figure 2) on pig chromosome 12, which
was expected when compared to human
map.
RH mapping confirmed the location of
ACE on pig chromosome 12 between
markers RH and SW957.
Malek, M., et al. 2001. A molecular
genome scan analysis to identify
chromosomal region influencing
economic traits in the pig. I. Growth
and body composition. Mammalian
Genome 12: 630-636.
Milan D, et al. IMpRH server: an RH
mapping server available on the
Web. Bioinformatics 16 (2000):
558-9.
 ACE-I1F: 5’ tca tcc tcc tcc tgc tct gc 3’
 ACE-I1R: 5’ gcc gtg atg ttg gtg ttg ta 3’
 ACE-I9F: 5’ ggg cca cat tca gta ttt ca 3’
ACKNOWLEDGEMENTS
 ACE-I9R: 5’ tcc acc act cct ggt tgt ag 3’
 Two point and multipoint linkage analysis of the
genotypes were completed using CRI-MAP software
(Green et al. 1990).
This study was supported by the
National Science Foundation Research
Experiences for Undergraduate
Program.
RH mapping was performed using the IMpRH panel
(Milan et al. 2000)
 ACE-RHF: 5’ gtc cac cct ctg gcc tac tt 3’
 ACE-RHR: 5’ tta ctg agg ccc agc ttc at 3’
We also thank members of Dr.
Rothschild’s lab for technical support.
F2 GENERATION PIGS