U.S. patent application number 11/177498 was filed with the patent office on 2006-02-16 for selecting animals for desired genotypic or potential phenotypic properties.
This patent application is currently assigned to University of Liege. Invention is credited to Goran Andersson, Leif Andersson, Nadine Buys, Michel Georges.
Application Number | 20060037090 11/177498 |
Document ID | / |
Family ID | 32479929 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060037090 |
Kind Code |
A1 |
Andersson; Leif ; et
al. |
February 16, 2006 |
Selecting animals for desired genotypic or potential phenotypic
properties
Abstract
The invention relates to methods to select animals, such as
mammals, in particular, domestic animals such as breeding animals
or animals destined for slaughter, for having desired genotypic or
potential phenotypic properties, in particular, related to muscle
mass and/or fat deposition or, in the case of mammals, to teat
number. The invention provides a method for selecting an animal for
having desired genotypic or potential phenotypic properties
comprising testing the animal, a parent of the animal or its
progeny for the presence of a nucleic acid modification affecting
the activity of an evolutionary conserved CpG island, located in
intron 3 of an IGF2 gene and/or for the presence of a nucleic acid
modification affecting binding of a nuclear factor to an IGF2
gene.
Inventors: |
Andersson; Leif; (Uppsala,
SE) ; Andersson; Goran; (Uppsala, SE) ;
Georges; Michel; (Villers-aux-Tours, BE) ; Buys;
Nadine; (Leuven, BE) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Assignee: |
University of Liege
Liege
BE
Melica HB
Uppsala
SE
Gentec B.V.
Buggenhout
BE
|
Family ID: |
32479929 |
Appl. No.: |
11/177498 |
Filed: |
July 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/EP04/00149 |
Jan 9, 2004 |
|
|
|
11177498 |
Jul 8, 2005 |
|
|
|
Current U.S.
Class: |
800/17 ; 435/6.1;
435/6.12; 514/44R |
Current CPC
Class: |
C12Q 2600/124 20130101;
A61P 21/00 20180101; C12Q 2600/158 20130101; C12Q 2600/172
20130101; A61P 3/04 20180101; C12Q 2600/156 20130101; C12Q 2600/154
20130101; C12Q 1/6876 20130101 |
Class at
Publication: |
800/017 ;
435/006; 514/044 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12Q 1/68 20060101 C12Q001/68; A61K 48/00 20060101
A61K048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 10, 2003 |
EP |
03075091.3 |
Claims
1. A method for selecting an animal for having desired genotypic or
potential phenotypic properties comprising testing said animal, a
parent of said animal or its progeny for the presence of a nucleic
acid modification affecting the activity of an evolutionary
conserved CpG island, located in intron 3 of an IGF2 gene (SEQ ID
NO: 1).
2. A method for selecting an animal for having desired genotypic or
potential phenotypic properties comprising testing said animal, a
parent of said animal or its progeny for the presence of a nucleic
acid modification affecting binding of a nuclear factor to an IGF2
gene.
3. A method according to claim 2 wherein said nuclear factor is
capable of binding to a stretch of nucleotides which in the
wild-type pig, mouse or human IGF2 gene is part of an evolutionary
conserved CpG island, located in intron 3 of said IGF2 gene (SEQ ID
NO:113, SEQ ID NO:114, SEQ ID NO:115 and SEQ ID NO:116).
4. A method according to claim 3 wherein said stretch is
functionally equivalent to the sequence
5'-GATCCTTCGCCTAGGCTC(A/G)CAGCGCGGGAGCGA-3'(SEQ ID NO:1).
5. A method according to claim 1 wherein said nucleic acid
modification comprises a nucleotide substitution.
6. A method according to claim 5 wherein said substitution in the
pig comprises a G to A transition at IGF2-intron3-nt3072.
7. A method according to claim 2 wherein inhibiting binding of said
nuclear factor to said IGF2 gene allows for modulating IGF2 mRNA
transcription in a cell provided with said gene.
8. A method according to claim 1 wherein said desired genotypic or
potential phenotypic properties comprise muscle mass, fat
deposition or teat numbers.
9. A method for modulating mRNA transcription of an IGF2 gene in a
cell or organism provided with said gene comprising modulating the
activity of an evolutionary conserved CpG island (SEQ ID NO:1),
located in intron 3 of an IGF2 gene and/or modulating binding of a
nuclear factor to an IGF2 gene.
10. A method according to claim 9 wherein said nuclear factor is
capable of binding to a stretch of nucleotides which in the
wild-type pig, mouse or human IGF2 gene is part of an evolutionary
conserved CpG island, located in intron 3 of said IGF2 gene (SEQ ID
NO:113, SEQ ID NO:114, SEQ ID NO:115 and SEQ ID NO:116).
11. A method according to claim 10 wherein said stretch is
functionally equivalent to the sequence
5'-GATCCTTCGCCTAGGCTC(A/G)CAGCGCGGGAGCGA-3'(SEQ ID NO: 1).
12. A method for identifying a compound capable of modulating mRNA
transcription of an IGF2 gene in a cell or organism provided with
said gene comprising: providing a first cell or organism having a
nucleic acid modification affecting the activity of an evolutionary
conserved CpG island, located in intron 3 of an IGF2 gene and/or
affecting binding of a nuclear factor to an IGF2 gene and a second
cell or organism not having said modification; further comprising
providing said first or said second cell or organism with a test
compound and determining IGF2 mRNA transcription in said first and
second cell or organism.
13. A method according to claim 12 wherein said nuclear factor is
capable of binding to a stretch of nucleotides which in the
wild-type pig, mouse or human IGF2 gene is part of an evolutionary
conserved CpG island, located in intron 3 of said IGF2 gene (SEQ ID
NO: 113, SEQ ID NO: 114, SEQ ID NO: 115 and SEQ ID NO: 116).
14. A method according to claim 13 wherein said stretch is
functionally equivalent to the sequence
5'-GATCCTTCGCCTAGGCTC(A/G)CAGCGCGGGAGCGA-3'(SEQ ID NO: 1).
15. A method according to claim 12 wherein said nucleic acid
modification comprises a nucleotide substitution.
16. A method according to claim 15 wherein said substitution
comprises a G to A transition which in the pig is located at
IGF2-intron3-nt3072.
17. A method for identifying a compound capable of modulating
binding of a nuclear factor to an IFG2 gene: comprising providing a
stretch of nucleotides which in the wild-type pig, mouse or human
IGF2 gene is part of an evolutionary conserved CpG island, located
in intron 3 of said IGF2 gene; further comprising providing a
mixture of DNA-binding proteins derived from a nuclear extract of a
cell; further comprising providing a test compound; and determining
competition of binding of said mixture of DNA-binding proteins to
said stretch of nucleotides in the presence or absence of said test
compound.
18. A method according to claim 17 wherein said stretch is
functionally equivalent to the sequence
5'-GATCCTTCGCCTAGGCTC(A/G)CAGCGCGGGAGCGA-3'(SEQ ID NO:1).
19. A compound identifiable with a method according to claim
13.
20. A compound according to claim 19 comprising an oligonucleotide
or analogue thereof functionally equivalent to the sequence
5'-GATCCTTCGCCTAGGCTC(A/G)CAGCGCGGGAGCGA-3' (SEQ ID NO:1).
21. A pharmaceutical composition comprising a compound according to
claim 19.
22. (canceled)
23. (canceled)
24. A method for modulating mRNA transcription of an IGF2 gene in a
cell or organism provided with said gene comprising providing said
cell or organism with a compound of claim 19.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of PCT International
Patent Application No. PCT/EP04/000149, filed on Jan. 9, 2004,
designating the United States of America, and published, in
English, as PCT International Publication No. WO 2004/063386 A2 on
Jul. 29, 2004, which application claims priority to European Patent
Application Serial No. 03075091.3 filed on Jan. 10, 2003, the
contents of the entirety of each are incorporated herein by this
reference.
TECHNICAL FIELD
[0002] The invention relates to methods to select animals, such as
mammals, in particular, domestic animals such as breeding animals
or animals destined for slaughter for having desired genotypic or
potential phenotypic properties, in particular, related to muscle
mass and/or fat deposition or, in the case of mammals, to teat
number. Herein, a domestic animal is defined as an animal being
purposely selected or having been derived from an animal having
been purposely selected for having desired genotypic or potential
phenotypic properties.
BACKGROUND
[0003] Domestic animals provide a rich resource of genetic and
phenotypic variation. Traditionally, domestication involves
selecting an animal or its offspring for having desired genotypic
or potential phenotypic properties. This selection process has in
the past century been facilitated by growing understanding and
utilization of the laws of Mendelian inheritance. One of the major
problems in breeding programs of domestic animals is the negative
genetic correlation between reproductive capacity and production
traits. This is, for example, the case in cattle (a high milk
production generally results in slim cows and bulls), poultry
(broiler lines have a low level of egg production and layers have
generally very low muscle growth), pigs (very prolific sows are in
general fat and have comparatively less meat), or sheep (high
prolific breeds have low carcass quality and vice versa). WO
00/36143 provides a method for selecting an animal for having
desired genotypic or potential phenotypic properties comprising
testing the animal for the presence of a parentally imprinted
qualitative or quantitative trait locus (QTL). Knowledge of the
parental imprinting character of various traits allows selection
of, for example, sire lines homozygous for a paternally imprinted
QTL, for example, linked with muscle production or growth; the
selection for such traits can thus be less stringent in dam lines
in favor of the reproductive quality. The phenomenon of genetic or
parental imprinting has never been earlier utilized in selecting
domestic animals, nor was it ever considered feasible to employ
this elusive genetic characteristic in practical breeding programs.
A breeding program, wherein knowledge of the parental imprinting
character of a desired trait as demonstrated herein is utilized,
increases the accuracy of the breeding value estimation and speeds
up selection compared to conventional breeding programs. For
example, selecting genes characterized by paternal imprinting is
provided to help increase uniformity; a (terminal) parent
homozygous for the "good or wanted" alleles will pass them to all
offspring, regardless of the other parent's alleles, and the
offspring will all express the desired parent's alleles. This
results in more uniform offspring.
[0004] Alleles that are interesting or favorable from the maternal
side are often the ones that have opposite effects to alleles from
the paternal side. For example, in meat animals, such as pigs,
alleles linked with meat or carcass quality traits, such
as--intramuscular fat or muscle mass, could be fixed in the dam
lines while alleles linked with reduced back fat could be fixed in
the sire lines. Other desirable combinations are, for example,
fertility, teat number and/or milk yield in the female line with
increased growth rates, reduced back fat and/or increased muscle
mass in the male lines. The purpose of breeding programs in
livestock is to enhance the performances of animals by improving
their genetic composition.
[0005] In essence, this improvement accrues by increasing the
frequency of the most favorable alleles for the genes influencing
the performance characteristics of interest. These genes are
referred to as QTL. Until the beginning of the nineties, genetic
improvement was achieved via the use of biometrical methods, but
without molecular knowledge of the underlying QTL. Now, the
identification of causative mutations for Quantitative Trait Loci
(QTLs) is a major hurdle in genetic studies of multifactorial
traits and disorders. The imprinted IGF2-linked QTL is one of the
major porcine QTLs for body composition. It was first identified in
intercrosses between the European Wild Boar and Large White
domestic pigs and between Pietrain and Large White pigs (1, 2). The
data showed that alleles from the Large White and Pietrain breeds,
respectively, were associated with increased muscle mass and
reduced back-fat thickness, consistent with the existing
breed-differences in the two crosses. A paternally expressed
IGF2-linked QTL was subsequently documented in intercrosses between
Chinese Meishan and Large White/Landrace pigs (3) and between
Berkshire and Large White pigs (4). In both cases, the allele for
high muscle mass was inherited from the lean Large White/Landrace
breed. However, there is a large number of potentially important
elements that may influence IGF2 function. Recent sequence analysis
(Amarger et al. 2002) provided a partial sequence of the
INS-IGF2-H19 region and revealed as many as 97 conserved elements
between human and pig.
SUMMARY OF THE INVENTION
[0006] The invention provides a method for selecting an animal for
having desired genotypic or potential phenotypic properties
comprising testing the animal for the presence of a qualitative or
quantitative trait locus (QTL). Here, it is shown that a paternally
expressed QTL affecting muscle mass, fat deposition and teat number
is caused by a single nucleotide substitution in intron 3 of IGF2.
The mutation occurs in an evolutionary conserved CpG island that is
hypomethylated in skeletal muscle (SEQ ID NO: 1). The function of
the conserved CpG island was not known before. IGF2-intron3-nt3072
is part of the evolutionary conserved CpG island with a regulatory
function, located between Differentially Methylated Region 1 (DMR1)
and a matrix attachment region previously defined in mice (11-13).
The 94 bp sequence around the mutation shows about 85% sequence
identity to both human and mouse and the wild-type nucleotide at
IGF2-intron3-nt3072 is conserved among the three species (FIG. 4A).
A qualitative trait nucleotide (QTN) occurs three bp downstream of
an eight bp palindrome also conserved between the three species.
The methylation status of the 300 bp fragment centered on
IGF2-intron3-nt3072 and containing 50 CpG dinucleotides was
examined by bisulphite sequencing in four month old
Q.sup.pat/q.sup.mat and q.sup.pat/Q.sup.mat animals. In skeletal
muscle, paternal and maternal chromosomes were shown to be
essentially unmethylated (including the IGF2-intron3-nt3071 C
residue) irrespective of the QTL genotype of the individual (3.4%
of CpGs methylated on average; FIG. 5A). The CpG island was more
heavily and also differentially methylated in liver; 33% of the
CpGs were methylated on the maternal alleles versus 19% on the
paternal allele. Unexpectedly, therefore, this CpG island behaves
as a previously unidentified DMR in liver, the repressed maternal
allele being more heavily methylated than the paternal allele,
which is the opposite of what is documented for the adjacent DMR1
in the mouse. To further uncover a function for this element,
electrophoretic mobility shift analyses (EMSA) were performed using
27 bp oligonucleotides spanning the QTN and corresponding to the
wild-type (q) and mutant (O) sequences. Nuclear extracts from
murine C2C12 myoblast cells, human HEK293 cells, and human HepG2
cells were incubated with radioactively labeled q or Q
oligonucleotides.
[0007] One specific band shift (complex C1 in FIG. 5B) was obtained
with the wild-type (q) but not the mutant (O) probe using extracts
from C2C12 myoblasts; similar results were obtained using both
methylated and unmethylated probes. A band shift with approximately
the same but weaker migration was also detected in extracts from
HEK293 and HepG2 cells. The specificity of the complex was
confirmed since competition was obtained with ten-fold molar excess
of unlabeled q probe, whereas a 50-fold excess of unlabeled Q probe
did not achieve competition (FIG. 5B). Thus, the wild-type sequence
binds a nuclear factor and this interaction is abrogated by the
mutation. This also means that there could be other mutations in
this region that are important in pigs or other species.
[0008] Furthermore, the data show that the CpG island contains both
Enhancer and Silencer functions so that there may be several
nuclear factors binding to this CpG island except for the one
already shown here. The results provide a method for isolating such
nuclear factors and a stretch of oligonucleotides that can be used
to fish out such proteins. Pigs carrying the mutation have a
three-fold increase in IGF2 mRNA expression in postnatal muscle.
The mutation abrogates in vitro interaction with a nuclear factor,
most likely a repressor. The mutation has experienced a selective
sweep in several pig breeds.
[0009] As described in the detailed description herein, a haplotype
sharing approach was used to refine the map position of the QTL
(5). It was assumed that a new allele (O) promoting muscle
development occurred g generations ago on a chromosome carrying the
wild-type allele (q). It was also assumed that the favorable allele
has gone through a selective sweep due to the strong selection for
lean growth in commercial pig populations. Twenty-eight chromosomes
with known QTL status were identified by marker-assisted
segregation analysis using cross-bred Pietrain and Large White
boars. All 19 Q-bearing chromosomes shared a haplotype in the 90
kilobase pairs (kb) interval between the microsatellites PULGE1 and
SWC9 (IGF2 3'-UTR), which was not present among the q chromosomes
and was, therefore, predicted to contain the QTL. In contrast, the
nine q chromosomes exhibited six distinct marker haplotypes in the
same interval. This region is part of the CDKN1C-H19 imprinted
domain and contains INS and IGF2 as the only known paternally
expressed genes. With this insight, the invention provides a method
for selecting an animal for having desired genotypic or potential
phenotypic properties comprising testing the animal, a parent of
the animal or its progeny for the presence of a nucleic acid
modification affecting the activity of an evolutionary conserved
CpG island, located in intron 3 of an IGF2 gene and/or affecting
binding of a nuclear factor to an IGF2 gene.
[0010] In a preferred embodiment, the invention provides a method
for selecting an animal for having desired genotypic or potential
phenotypic properties comprising testing a nucleic acid sample from
the animal for the presence of a single nucleotide substitution. A
nucleic acid sample can, in general, be obtained from various parts
of the animal's body by methods known in the art. Traditional
samples for the purpose of nucleic acid testing are blood samples
or skin or mucosal surface samples, but samples from other tissues
can be used as well, in particular, sperm samples, oocyte or embryo
samples can be used. In such a sample, the presence and/or sequence
of a specific nucleic acid, be it DNA or RNA, can be determined
with methods known in the art, such as hybridization or nucleic
acid amplification or sequencing techniques known in the art. The
invention also provides testing such a sample for the presence of
nucleic acid, wherein the QTN or allele associated therewith is
associated with the phenomenon of parental imprinting, for example,
where it is determined whether a paternal or maternal allele
comprising the QTN is capable of being predominantly expressed in
the animal.
[0011] In a preferred embodiment, the invention provides a method
wherein the nuclear factor is capable of binding to a stretch of
nucleotides, which, in the wild-type pig, mouse or human IGF2 gene,
is part of an evolutionary conserved CpG island, located in intron
3 of the IGF2 gene. Binding should preferably be located at a
stretch of nucleotides spanning a QTN (qualitative trait
nucleotide) that comprises a nucleotide (preferably a G to A)
transition, which, in the pig, is located at IGF2-intron3-nt3072.
It is preferred that the stretch is functionally equivalent to the
sequence as shown in FIG. 4 that comprises the sequence
5'-GATCCTTCGCCTAGGCTC(A/G)CAGCGCGGGAGCGA-3' (SEQ ID NO:1)
identifying the overlap with the QTN, whereby functional
equivalence preferably entails that the stretch is spanning the
QTN, and preferably overlaps with at least two or three nucleotides
at or on both sides of the QTN, although the overlaps may be
longer.
[0012] Functional equivalence also entails a sequence homology of
at least 50%, preferably at least 60%, more preferably at least
70%, even more preferably at least 80%, most preferred at least 90%
of the stretch overlapping the QTN. The stretch is preferably from
at least 5 to about 94 nucleotides long, more preferably from about
10 to 50, most preferably from about 15 to 35 nucleotides, and it
is preferred that it comprises a palindromic octamer sequence as
identified in FIG. 4.
[0013] In a preferred embodiment, the invention provides a method
wherein the nucleic acid modification comprises a nucleotide
substitution, whereby in the pig, the substitution comprises a G to
A transition at IGF2-intron3-nt3072 (SEQ ID NO:6 and SEQ ID NO:5).
Abrogating or reducing binding of the nuclear factor to the IGF2
gene allows for modulating IGF2 mRNA transcription in a cell
provided (naturally or by recombinant means) with the gene.
[0014] To further characterize the functional significance of the
IGF2 Q mutation, its effect on transcription was studied by
employing a transient transfection assay in mouse C2C12 myoblasts.
Q and q constructs were made containing a 578 bp fragment from the
actual region inserted in front of a Luciferase reporter gene
driven by the herpes thymidine kinase (TK) minimal promoter. The
two constructs differed only by the IGF2-intron3-nt3072G.fwdarw.A
transition. The ability of the IGF2 fragments to activate
transcription from the heterologous promoter was compared with the
activity of the TK-promoter alone.
[0015] The presence of the q-construct caused a two-fold increase
of transcription, whereas the Q-construct caused a significantly
higher, seven-fold, increase (FIG. 5C). The interpretation of this
result, in light of the results from the EMSA experiment, is that
the Q mutation abrogates the interaction with a repressor protein
that modulates the activity of a putative IGF2 enhancer present in
this CpG island. This view is consistent with our in silico
identification of potential binding sites for both activators and
repressors in this intronic DNA fragment (14).
[0016] The in vivo effect of the mutation on IGF2 expression was
studied in a purpose-built Q/q.times.Q/q intercross counting 73
offspring. As a deletion encompassing DMR0, DMR1, and the
associated CpG island derepresses the maternal IGF2 allele in
mesodermal tissues in the mouse (12), the effect of the
intron3-nt3072 mutation on IGF2 imprinting in the pig was tested.
This was achieved by monitoring transcription from the paternal and
maternal IGF2 alleles in tissues of q/q, Q.sup.pat/q.sup.mat, and
q.sup.pat/Q.sup.mat animals that were heterozygous for the SWC9
microsatellite located in the IGF2 3'UTR. Imprinting could not be
studied in Q/Q animals that were all homozygous for SWC9. Before
birth, IGF2 was shown to be expressed exclusively from the paternal
allele in skeletal muscle and kidney, irrespective of the QTL
genotype of the fetuses. At four months of age, weak expression
from the maternal allele was observed in skeletal muscle, however,
at comparable rates for all three QTL genotypes (FIG. 6A). Only the
paternal allele could be detected in four-month-old kidney (data
not shown). Consequently, the mutation does not seem to affect the
imprinting status of IGF2. The partial derepression of the maternal
allele in skeletal muscle of all QTL genotypes may, however,
explain why in a previous study, muscular development was found to
be slightly superior in q.sup.pat/Q.sup.mat versus q/q animals, and
in Q/Q versus Q.sup.pat/q.sup.mat animals (2).
[0017] The Q allele was expected to be associated with an increased
IGF2 expression since IGF2 stimulates myogenesis (6). To test this,
the relative mRNA expression of IGF2 was monitored at different
ages in the Q/q.times.Q/q intercross using both Northern blot
analysis and real-time PCR (FIGS. 6B and C). The expression levels
in fetal muscle and postnatal liver was about ten-fold higher than
in postnatal muscle. No significant difference was observed in
fetal samples or in postnatal liver samples, but a significant
three-fold increase of postnatal IGF2 mRNA expression in skeletal
muscle was observed in (Q/Q or Q.sup.pat/q.sup.mat) versus
(q.sup.pat/Q.sup.mat or q/q) progeny. Herewith, the invention
provides a method for modulating mRNA transcription of an IGF2 gene
in a cell or organism provided with the gene comprising modulating
binding of a nuclear factor to an IGF2 gene, in particular, wherein
the nuclear factor is capable of binding to a stretch of
nucleotides (as identified above) that in the wild-type pig, mouse
or human IGF2 gene is part of an evolutionary conserved CpG island,
located in intron 3 of the IGF2 gene. The significant difference in
IGF2 expression revealed by real-time PCR was confirmed using two
different internal controls, GAPDH (FIG. 6C) and HPRT (15). An
increase of all detected transcripts originating from the three
promoters (P2-P4) located downstream of the mutated site was found.
Combined, these results provide strong evidence for IGF2 being the
causative gene. The lack of significant differences in IGF2 mRNA
expression in fetal muscle and postnatal liver are consistent with
the previous QTL study showing no effect of the IGF2 locus on birth
weight and weight of liver (2).
[0018] Accordingly, a method according to the invention is herein
provided allowing testing for, and modulation of, desired genotypic
or potential phenotypic properties comprising muscle mass, fat
deposition or teat numbers (of mammals). Such testing is applicable
in man and animals alike (animals herein defined as including
humans). In humans, it is, for example, worthwhile to test for the
presence for the presence of a nucleic acid modification affecting
the activity of an evolutionary conserved CpG island, located in
intron 3 of an IGF2 gene or affecting binding of a nuclear factor
to an IGF2 gene, as provided herein, to test, for example, the
propensity or genetic predisposition or likelihood of muscle growth
or muscularity in humans versus propensity or genetic
predisposition or the likelihood of obesity. In domestic animals,
such testing may be undertaken to select the best or most suitable
animals for breeding, or to preselect domestic animals destined for
slaughter. An additional trait to be selected for concerns teat
number, a quality highly valued in sow lines to allow for suckling
large litters. A desirable breeding combination as provided herein
comprises, for example, increased teat number in the female line
with increased growth rates, reduced back fat and/or increased
muscle mass in the male lines. It is herein also shown that the
mutation influences teat number. The Q allele that is favorable
with respect to muscle mass and reduced back fat is the unfavorable
allele for teat number. This strengthens the possibility of using
the paternal imprinting character of this QTL in breeding programs.
Selecting maternal lines for the q allele will enhance teat number,
a characteristic that is favorable for the maternal side. On the
other hand, paternal lines can be selected for the Q allele that
will increase muscle mass and reduce back fat, characteristics that
are of more importance in the paternal lines. Terminal sires that
are homozygous QQ will pass the full effect of increased muscle
mass and reduced back fat to the slaughter pigs, while selection of
parental sows that express the q allele will allow for the
selection of sows that have more teats and suckle more piglets
without affecting slaughter quality.
[0019] The invention also provides a method for identifying a
compound capable of modulating mRNA transcription of an IGF2 gene
in a cell or organism provided with the gene comprising providing a
first cell or organism having a nucleic acid modification affecting
the activity of an evolutionary conserved CpG island, located in
intron 3 of an IGF2 gene and/or affecting binding of a nuclear
factor to an IGF2 gene and a second cell or organism not having the
modification further comprising providing the first or second cell
or organism with a test compound and determining IGF2 mRNA
transcription in the first and second cell or organism and
selecting a compound capable of modulating IGF2 mRNA transcription.
An example of such a compound as identifiable herewith comprises a
stretch of oligonucleotides spanning a QTN (qualitative trait
nucleotide) that comprises a nucleotide (preferably a G to A)
transition, which in the pig, is located at IGF2-intron3-nt3072. It
is preferred that the stretch is functionally equivalent to the
sequence as shown in FIG. 4 that comprises the sequence
5'-GATCCTTCGCCTAGGCTC(A/G)CAGCGCGGGAGCGA-3' (SEQ ID NO:1)
identifying the overlap with the QTN, whereby functional
equivalence preferably entails that the stretch is spanning the QTN
and preferably overlaps with at least two or three nucleotides at
or on both sides of the QTN, although the overlaps may be longer.
Functional equivalence also entails a sequence homology of at least
50%, preferably at least 60%, more preferably at least 70%, even
more preferably at least 80%, most preferred at least 90%, of the
stretch overlapping the QTN. The stretch is preferably from at
least 5 to at about 94 nucleotides long, more preferably from about
10 to 50, most preferably from about 15 to 35 nucleotides, and it
is preferred that it comprises a palindromic octamer sequence as
identified in FIG. 4. An alternative compound as provided herein
comprises a functional analogue of the stretch, the alternative
compound or oligonucleotide analogue functionally at least capable
of modulating the activity of an evolutionary conserved CpG island,
located in intron 3 of an IGF2 gene and/or modulating binding of a
nuclear factor to an IGF2 gene, preferably effecting modulation at
the site of the QTN. In electrophoretic mobility shift analyses
(EMSA), such compounds, e.g., the identified nuclear factor in FIG.
2, or compounds competing with the binding of the factor to the
IGF2 gene, can be further identified and selected. A typical
example of such an EMSA is given in the detailed description. It is
preferred that the nuclear factor is capable of binding to a
stretch of nucleotides that in the wild-type pig, mouse or human
IGF2 gene, is part of an evolutionary conserved CpG island, located
in intron 3 of the IGF2 gene. Oligonucleotide compounds or probes
spanning the QTN are herein provided that have the desired effect.
Such compounds or probes are preferably functionally equivalent to
the sequence 5'-GATCCTTCGCCTAGGCTC(A/G)CAGCGCGGGAGCGA-3'(SEQ ID NO:
1).
[0020] The invention also provides a method for identifying a
compound capable of affecting the activity of an evolutionary
conserved CpG island, located in intron 3 of an IGF2 gene and/or
modulating binding of a nuclear factor to an IFG2 gene comprising
providing a stretch of nucleotides that in the wild-type pig, mouse
or human IGF2 gene, is part of an evolutionary conserved CpG
island, located in intron 3 of the IGF2 gene. Such testing may be
done with single oligonucleotides or analogues thereof, or with a
multitude of such oligonucleotides or analogues in an array
fashion, and may further comprise providing a mixture of
DNA-binding proteins derived from a nuclear extract of a cell and
testing these with the array or analogue or oligonucleotide under
study. Testing may be done as well with test compounds provided
either singularly or in an array fashion and optionally further
comprises providing a test compound and determining competition of
binding of the mixture of DNA-binding proteins to the stretch of
nucleotides in the presence or absence of test compound(s). To find
active compounds for further study or, eventually, for
pharmaceutical use, it suffices to select a compound capable of
inhibiting binding of the mixture to the stretch, wherein the
stretch is functionally equivalent to the sequence
5'-GATCCTTCGCCTAGGCTC(A/G)CAGCGCGGGAGCGA-3'(SEQ ID NO: 1).
[0021] The invention thus provides a compound identifiable with a
method as described herein. Such a compound is, for example,
derived from a stretch of oligonucleotides spanning a QTN
(qualitative trait nucleotide) that comprises a nucleotide
(preferably a G to A) (SEQ ID NO:6 and SEQ ID NO:5) transition,
which in the pig, is located at IGF2-intron3-nt3072. It is
preferred that the stretch is functionally equivalent to the
sequence as shown in FIG. 4 that comprises the sequence
5'-GATCCTTCGCCTAGGCTC(A/G)CAGCGCGGGAGCGA-3' (SEQ ID NO:1)
identifying the overlap with the QTN, whereby functional
equivalence preferably entails that the stretch is spanning the
QTN, and preferably overlaps with at least two or three nucleotides
at or on both sides of the QTN, although the overlaps may be
longer.
[0022] Also, functional equivalence entails a sequence homology of
at least 50%, preferably at least 60%, more preferably at least
70%, even more preferably at least 80%, most preferred at least 90%
of the stretch overlapping the QTN. The oligonucleotide compound is
preferably from at least 5 to at about 94 nucleotides long, more
preferably from about 10 to 50, most preferably from about 10 to 35
nucleotides, and it is preferred that it comprises a palindromic
octamer sequence as identified in FIG. 4. An alternative compound
or functional analogue as provided herein comprises a functional
analogue of the oligonucleotide compound, the alternative compound
or oligonucleotide analogue functionally at least capable of
modulating the activity of an evolutionary conserved CpG island,
located in intron 3 of an IGF2 gene and/or modulating binding of a
nuclear factor to an IGF2 gene, preferably effecting the modulation
at the site of the QTN. For example, in electrophoretic mobility
shift analyses (EMSA), such compounds, e.g., the identified nuclear
factor in FIG. 2, or compounds competing with the binding of the
factor to the IGF2 gene, can be further identified and selected. A
typical example of such an EMSA is given in the detailed
description. The invention also provides a pharmaceutical
composition comprising a compound as provided herein, and use of
such a compound, for the production of a pharmaceutical composition
for the treatment of obesity or for the treatment of muscle
deficiencies. Furthermore, the invention provides a method for
modulating mRNA transcription of an IGF2 gene in a cell or organism
provided with the gene comprising treating or providing the cell or
organism with a compound as provided herein.
[0023] There has been a strong selection for lean growth (high
muscle mass and low fat content) in commercial pig populations in
Europe and North America during the last 50 years. Therefore, how
this selection pressure has affected the allele frequency
distribution of the IGF2 QTL was investigated. The causative
mutation was absent in a small sample of European and Asian Wild
Boars and in several breeds that have not been strongly selected
for lean growth (Table 1). In contrast, the causative mutation was
found at high frequencies in breeds that have been subjected to
strong selection for lean growth. The only exceptions were the
experimental Large White population at the Roslin Institute that
was founded from commercial breeding stocks in the UK around 1980
(16) as well as the experimental Large White populations used for
the Pietrain/Large White intercross (1). These two populations thus
reflect the status in some commercial populations about 20 years
ago and it is possible that the IGF2*Q allele is even more
predominant in contemporary populations. The results demonstrate
that IGF2*Q has experienced a selective sweep in several major
commercial pig populations and it has apparently been spread
between breeds by cross-breeding.
[0024] The results have important practical implications. The
IGF2*Q mutation increases the amount of meat produced, at the
expense of fat, by 3-4 kg for an animal slaughtered at the usual
weight of about 100 kg. The high frequency of IGF2*Q among major
pig breeds implies that this mutation affects the productivity of
many millions of pigs in the Western world. The development of a
simple diagnostic DNA test now facilitates the introgression of
this mutation to additional breeds. This could be an attractive way
to improve productivity in local breeds as a measure to maintain
biological diversity. The diagnostic test will also make it
possible to investigate if the IGF2*Q mutation is associated with
any unfavorable effects on meat quality or any other trait. It has
been previously demonstrated that European and Asian pigs were
domesticated from different subspecies of the Wild Boar, and that
Asian germplasm has been introgressed into European pig breeds
(17). The IGF2*Q mutation apparently occurred on an Asian
chromosome as it showed a very close relationship to the haplotype
carried by Chinese Meishan pigs. This explains the large genetic
distance observed between Q- and q-haplotypes (FIG. 4). However, it
is an open question whether the Q mutation occurred before after
the Asian chromosome was introduced into European pigs.
[0025] This study provides new insights in IGF2 biology. The role
of IGF2 on prenatal development is well documented (18, 19). The
observation demonstrates that the Q mutation does not upregulate
IGF2 expression in fetal tissue but, after birth, demonstrates that
IGF2 has an important role for regulating postnatal myogenesis. The
finding that the sequence around the mutation does not match any
known DNA-binding site shows that this sequence binds an earlier
unknown nuclear factor (14). The results also imply that
pharmacological intervention of the interaction between this DNA
segment and the corresponding nuclear factor opens up new
strategies for promoting muscle growth in humans such as patients
with muscle deficiencies or for stimulating muscle development at
the cost of adipose tissue in obese patients.
[0026] Applications of these insights are manifold. Applications in
animals typically include diagnostic tests of the specific
causative mutation in the pig and diagnostic tests of these and
possible other mutations in this CpG island in humans, pigs or
other meat-producing animals.
[0027] It is now also possible to provide for transgenic animals
with modified constitution of this CpG island or with modified
expression of nuclear factors interacting with this sequence, and
the invention provides the use of pharmaceutical compounds
(including oligonucleotides) or vaccination to modulate IGF2
expression by interfering with the interaction between nuclear
factors and the CpG island provided herein. Thus, instead of
selecting animals, one may treat the animals with a drug, if not
for producing meat, then at least in experimental animals for
studying the therapeutic effects of the compounds.
[0028] In humans, diagnostic tests of mutations predisposing to
diabetes, obesity or muscle deficiency are particularly provided
and pharmaceutical intervention to treat diabetes, obesity or
muscle deficiency by modulating IGF2 expression based on
interfering with the interaction between nuclear factors and the
CpG island as provided herein is typically achievable with
compounds, such as the above-identified nucleotide stretches or
functional analogues thereof as provided herein.
DESCRIPTION OF THE FIGURES
[0029] FIG. 1: QTL genotyping by marker-assisted segregation
analysis. The graphs show, for 14 paternal half-sib pedigrees (P1,
P2, . . . P14), the phenotypic mean.+-.2 standard errors of the
offspring sorted into two groups according to the homologue
inherited from the sire. The number of offspring in each group is
given above and below the error bars, respectively. The upper graph
corresponds to the boars that were shown to be heterozygous "Qq"
for the QTL, the lower graph to the boars that were shown to be
homozygous at the QTL. Pedigrees for which the percentage of lean
meat was measured as "% lean cuts" (NEZER et al. 2002) are marked
by L(eft axis), those for which "Piglog" was used (see M&M) are
marked by R(ight axis). The graph reports a Z-score for each
pedigree, i.e., the log10 of the H.sub.1/H.sub.0 likelihood ratio
where H.sub.1 assumes that the boar is heterozygous "Qq" for the
QTL, while H.sub.0 assumes that the boar is homozygous "QQ" or
"qq." "Q" alleles associated with a positive allele substitution
effect on the percentage of lean meat are marked by a diamond, "q"
alleles by a circle. The number within the symbols differentiates
the "Q" and "q" alleles according to the associated marker
haplotype (see results of Example 1 and FIG. 2).
[0030] FIG. 2: A. Schematic representation of the human 11p15
imprinted domain according to Onyango et al. (2000). B. BAC contig
spanning the porcine orthologue of the 11p15 imprinted domain,
assembled by STS content mapping. The length of the horizontal bars
does not reflect the actual physical size of the corresponding
BACs. C. Marker haplotypes of the five "Q" chromosomes (diamonds)
and six "q" chromosomes (circles). Closely linked SNPs (<5 kb)
were merged into poly-allelic multisite haplotypes (cfr. Table 2).
The chromosome segments highlighted in green correspond to the
haplotype shared by all "Q" chromosomes and, therefore, assumed to
contain the QTL. The chromosome segment highlighted in red
corresponds to a haplotype shared by chromosome "q.sup.4" and
chromosomes "Q.sup.1-4" that, therefore, excludes the QTL out of
this region. The resulting most likely position of the QTL is
indicated by the arrow.
[0031] FIG. 3: Average probability for two chromosomes to be
identical-by-descent at a given map position conditional on
flanking marker data (p(IBD|MG)), along the chromosome segment
encompassing the p57-H19 imprinted domain, computed according to
MEUWISSEN and GODDARD (2001). The positions of the markers defined
according to Tables 1 and 2 are shown by the vertical dotted
lines.
[0032] FIG. 4: (A) DNA sequence polymorphisms identified in a 28.6
kb segment spanning the porcine TH (exon 14), INS and IGF2 genes.
The average (C+G) content of a moving 100-bp window is shown on a
gray scale (black 100%, white 0%). The positions of evolutionary
conserved regions (7) including the DMR1 and associated CpG island
in IGF2 intron 3 are marked by horizontal cylinders. The Viewgene
program (25) was used to highlight the 258 differences between the
reference Q P208 sequence, four Q, and ten q chromosomes. The
position of the causative intron3-nt3072G.fwdarw.A mutation is
marked by an asterisk. The sequence context of the conserved
footprint surrounding intron3-nt3072 is shown for pig, human, and
mouse. The conserved palindromic octamer sequence is underlined.
P=Pietrain; LW=Large White; LR=Landrace; H=Hampshire; M=Meishan;
EWB=European Wild Boar; JWB=Japanese Wild Boar. (B)
Neighbor-Joining tree of 18,560 bp of the porcine IGF2 gene based
on 15 sequences classified as representing q and Q alleles. The
analysis was restricted to the region from IGF2 intron 1 to SWC9 in
the 3'UTR to avoid problems with the presence of recombinant
haplotypes. The tree was constructed using MEGA version 2.1 (26)
and positions with insertions/deletions were excluded. Bootstrap
values (after 1000 replicates) are reported on the nodes.
[0033] FIG. 5: (A) Percentage methylation determined by bisulphite
sequencing for the 300 bp fragment centered around intron3-nt3072,
containing 50 CpG dinucleotides, in liver and skeletal muscle of
four-month-old Q.sup.pat/q.sup.mat and q.sup.pat/Q.sup.mat
individuals. The number of analyzed chromosomes as well as the
standard errors of the estimated means are given. Pat and Mat refer
to the paternal and maternal alleles, respectively, determined
based on the intron3-nt3072G.fwdarw.A mutation. (B) Electrophoretic
mobility shift analyses (EMSA) using 10 .mu.g nuclear extracts
(N.E.) from mouse C2C12 myoblast cells, human HEK293 embryonic
kidney cells, and human HepG2 hepatocytes. The q and Q
oligonucleotide probes corresponded to the wild-type and mutant
sequences, respectively. Competition was carried out with a 50-fold
excess of cold nucleotide. Complex 1 (C1) was specific and
exclusively detected with the q probe. C2 was also specific and
stronger in q but probably present also with the Q probe. C3 was
unspecific. (C) Luciferase assays of reporter constructs using the
TK promoter. The pig IGF2 fragments (Q and q) contained 578 bp from
intron 3 (nucleotide 2868 to 3446) including the causative G to A
transition at nucleotide 3072. The relative activities compared
with the basic TK-LUC vector are reported as means.+-.standard
errors based on triplicate experiments. A student's t-test revealed
highly significant differences for all pairwise comparisons;
***=P<0.001.
[0034] FIG. 6: Analysis of IGF2 mRNA expression. (A) Imprinting
analysis of IGF2 in skeletal muscle of qq, Q.sup.Pat/q.sup.Mat, and
q.sup.Pat/Q.sup.Mat animals before birth and at four months of age.
The QTL and SWC9 genotypes of the analyzed animals are given. In
these, the first allele is paternal, the second maternal. The lanes
corresponding to PCR product obtained from genomic DNA are marked
by continuous lines, those corresponding to RT-PCR products by the
dotted lines. The three SWC9 alleles segregating in the pedigree
(1, 2, and 8) are marked by arrows. The RT-PCR controls without
reverse transcriptase were negative (not shown). (B) Northern blot
analysis of skeletal muscle poly(A).sup.+ RNA from three-week-old
piglets using IGF2 and GAPDH probes. Animals 1-4 and 5-8 carried a
paternal IGF2*Q or *q allele, respectively. P3 and P4 indicate the
IGF2 promoter usage and the superscripts a and b indicate the
alternative polyadenylation signal used. All four IGF2 transcripts
showed a significantly higher relative expression (standardized
using GAPDH expression) in the *Q group (P<0.05, Kruskal-Wallis
rank sum test, two sided). (C) Results of real-time PCR analysis of
IGF2 mRNA expression in skeletal muscle and liver at different
developmental phases of pigs carrying paternal IGF2*Q (gray
staples) or *q (white staples) alleles. The expression levels were
normalized using GAPDH as internal control. Means.+-.SE are given,
n=3-11.*=P<0.05, **=P<0.01, Kruskal-Wallis rank sum test, two
sided. No significant differences in IGF2 expression levels between
genotypes were found in fetal (80 days of gestation) or liver
tissues (three weeks). w, week; mo, month.
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLE 1
[0035] Haplotype sharing refines the location of an imprinted QTL
with major effect on muscle mass to a 90 Kb chromosome segment
containing the porcine IGF2 gene.
[0036] Herein described is the fine-mapping of an imprinted QTL
with major effect on muscle mass that was previously assigned to
proximal SSC2 in the pig. The proposed approach exploits linkage
disequilibrium in combination with QTL genotyping by
marker-assisted segregation analysis. By identifying a haplotype
shared by all "Q" chromosomes and absent amongst "q" chromosomes,
the QTL to a .apprxeq.90 Kb chromosome segment containing INS and
IGF2 was mapped as the only known paternally expressed genes. QTL
mapping has become a preferred approach towards the molecular
dissection of quantitative traits, whether of fundamental, medical
or agronomic importance. A multitude of chromosomal locations
predicted to harbor genes influencing traits of interest have been
identified using this strategy (e.g. MACKAY 2001; ANDERSSON 2001;
FLINT and MOTT 2001; MAURICIO 2001). In most cases, however, the
mapping resolution is in the order of the tens of centimorgans,
which is insufficient for positional cloning of the underlying
genes. High-resolution mapping of QTL, therefore, remains one of
the major challenges in the genetic analysis of complex traits.
[0037] Three factors limit the achievable mapping resolution:
marker density, cross-over density, and the ability to deduce QTL
genotype from phenotype.
[0038] Increasing marker density may still be time consuming in
most organisms but is conceptually the simplest bottleneck to
resolve. Two options are available to increase the local cross-over
density: breed recombinants de novo or exploit historical
recombination events, i.e., use linkage disequilibrium (LD). The
former approach is generally used with model organisms that have a
short generation time (e.g., DARVASI 1998), while the latter is the
only practical alternative when working with human or large
livestock species. Optimal use of LD to fine-map QTL in outbred
populations is presently an area of very active research (e.g.,
ARDLIE et al. 2002). The ability to deduce QTL genotype from
phenotype can be improved by using "clones" (e.g., recombinant
inbred lines) (e.g., DARVASI 1998), by means of progeny-testing
(e.g., GEORGES et al. 1995), or by marker-assisted segregation
analysis (e.g., RIQUET et al. 1999).
[0039] Recently, a QTL with major effect on muscle mass and fat
deposition was mapped to the centromeric end of porcine chromosome
SSC2 (NEZER et al. 1999; JEON et al. 1999). The most likely
position of the QTL was shown to coincide with a chromosome region
that is orthologous to HSA11p15 in the human, which is known to
harbor an imprinted domain. The demonstration that the QTL was
characterized by a clear parent-of-origin effect, strongly
suggested that the underlying gene was imprinted and expressed only
from the paternal allele. The human 11p15 imprinted domain is known
to contain at least nine imprinted transcripts. Three of these are
paternally expressed: LIT-1 (KVLQT1-AS), IGF2 and IGF2-AS (e.g.,
REIK and WALTER 2001). Fifteen imprinted transcripts are known to
map to the orthologous domain on distal mouse chromosome MMU7, of
which four are paternally expressed: Lit-1 (Kvlqt1-as), Ins2, Igf2
and Igf2-as (e.g.,
http://www.mgu.har.mrc.ac.uk/imprinting/imprinting.html; ONYANGO et
al. 2000). Because of its known function in myogenesis (FLORINI et
al. 1996), IGF2 stood out as a prime positional candidate.
[0040] To refine the map position of this QTL and to verify whether
its position remained compatible with a direct role of the INS
and/or IGF2 genes, an approach was applied targeting the three
factors limiting the mapping resolution of QTL: (i) a
higher-density map of the corresponding chromosome region was
generated; (ii) the QTL genotype of a number of individuals by
marker-assisted segregation analysis was determined; and (iii) an
LD-based haplotype sharing approach to determine the most likely
position of the QTL was applied. This approach is analogous to the
one that was previously applied by RIQUET et al. (1999) to refine
the map position of a QTL influencing milk production in dairy
cattle. It makes the assumption that the observed QTL effect is due
to the segregation of a QTL allele with major substitution effect
("Q") that appeared by mutation or migration g generations ago, and
swept through the populations as a result of artificial selection.
As a consequence, at the present generation, n chromosomes carrying
the "Q" allele are expected to share a haplotype of size
.apprxeq.2/ng (in Morgan) containing the QTL (DUNNER et al.
1997).
[0041] By doing so, a shared haplotype spanning less than 90 Kb
that is predicted to contain the Quantitative Trait Nucleotide
(QTN: MACKAY 2001) was identified. The corresponding chromosome
segment contains INS and IGF2 as the only known paternally
expressed genes. This considerably enforces the candidacy of these
two genes and demonstrates that LD can be exploited to map QTL in
outbred populations to chromosome intervals containing no more than
a handful of genes.
Materials and Methods
Pedigree Material and Phenotypic Data
[0042] The pedigree material used for this work comprised a subset
of previously described Pietrain.times.Large White F2 pedigrees
(NEZER et al. 2000; HANSET et al. 1995), as well as a series of
paternal half-sib pedigrees sampled in commercial lines derived
from the Pietrain and Large White breeds (Buys, personal
communication). In the F2 animals, "% lean cuts" was measured as
previously described (HANSET et al. 1995), while in the commercial
lines, the percentage of lean meat was measured as "Piglog"
corresponding to (63.6882-0.4465 a-0.5096 b+0.1281 c) where a=mm
backfat measured between the third and fourth lumbar vertebra at 7
cm from the spine, b=mm backfat measured between the third and
fourth last rib at 7 cm from the spine, and c=mm loin thickness,
measured at the same position as b.
Marker-Assisted Segregation Analysis
[0043] The QTL genotype of each sire was determined from the
Z-score, corresponding to the log10 of the likelihood ratio
L.sub.H.sub.1/L.sub.H.sub.0, where L.sub.H1 corresponds to the
likelihood of the pedigree data assuming that the boar is of "Qq"
genotype, and L.sub.H.sub.0 corresponds to the likelihood of the
pedigree data assuming that the boar is of "QQ" or "qq" genotype.
The corresponding likelihoods were computed as: L = i = 1 n .times.
1 2 .times. .pi. .times. .sigma. .times. e - ( y i - ( y _ .+-. a )
) 2 2 .times. .sigma. 2 ##EQU1## In this, n is the number of
informative offspring in the corresponding pedigree, y.sub.i is the
phenotype of offspring i, {overscore (y)} is the average phenotype
of the corresponding pedigree computed over all (informative and
non-informative) offspring, .sigma. is the residual standard
deviation maximizing L, and a is the Q to q allele substitution
effect. a was set at zero when computing L.sub.H.sub.0, and at +1%
for "R" offspring and -1% for "L" offspring when computing L.sub.H1
(NEZER et al. 1999).
[0044] Boars were considered to be "Qq" when Z>2, "QQ" or "qq"
when Z<-2 and of undetermined genotype if 2>Z>-2.
Linkage Disequilibrium Analysis
[0045] Probabilities for two chromosomes to be identical-by-descent
(IBD) at a given map position conditional on flanking marker data
were computed according to MEUWISSEN and GODDARD (2001). The
effective population size (N.sub.e) was set at 200 based on
estimates of N.sub.e determined from LD data (Harmegnies,
unpublished observations), and the number of generations to the
base population at 20. A multipoint test for association was
performed using the DISMULT program described in TERWILLIGER
1995.
Results
[0046] QTL genotyping by marker-assisted segregation analysis: A
series of paternal half-sib families was genotyped counting at
least 20 offspring for two microsatellite markers located on the
distal end of chromosome SSC2 and spanning the most likely position
of the imprinted QTL: SWR2516 and SWC9 (NEZER et al. 1999; JEON et
al. 1999). These families originated either from a previously
described Pietrain.times.Large White F2 pedigree (NEZER et al.
2002) or from two composite pig lines derived from Large White and
Pietrain founder animals (Nadine Buys, personal communication).
[0047] The pedigrees from sires which were heterozygous for one or
both of these markers were kept for further analysis. Twenty such
pedigrees could be identified for a total of 941 animals. Offspring
were sorted in three classes based on their marker genotype: "L"
(left homologue inherited from the sire), "R" (right homologue
inherited from the sire), or "?" (not informative or recombinant in
the SWR2516-SWC9 interval).
[0048] Offspring were slaughtered at a constant weight of
approximately 105 Kgs, and a series of phenotypes collected on the
carcasses including "% lean meat," measured either as lean cuts"
(experimental cross) or as "Piglog" (composite lines) (see
Materials & Methods).
[0049] The likelihood of each sire family was then computed under
two hypotheses: H.sub.0, postulating that the corresponding boar
was homozygous at the QTL and H.sub.1, postulating that the boar
was heterozygous at the QTL. Assuming a bi-allelic QTL, H.sub.0
corresponds to QTL genotypes "QQ" or "qq," and H.sub.1 to genotype
"Qq." Likelihoods were computed using "% lean meat" as phenotype
(as the effect of the QTL was shown to be most pronounced on this
trait in previous analyses) and assuming a Q to q allele
substitution effect of 2.0% (NEZER et al. 1999). If the odds in
favor of one of the hypotheses were superior or equal to 100:1, the
most likely hypothesis was considered to be true. Results are
expressed as lod scores (z): the log.sub.10 of the likelihood ratio
H.sub.1/H.sub.0. Boars were considered to be of heterozygous "Qq"
genotype if z was superior to 2, of homozygous "QQ" or "qq"
genotype if z was inferior to -2, and of undetermined QTL genotype
if -2<z<2. Using these rules, we could determine the QTL
genotype for fourteen of the twenty boars. Seven of these proved to
be heterozygous "Qq," the other seven to be homozygous and, thus,
either of "QQ" or "qq" genotype (FIG. 1).
[0050] Constructing a physical and genetic map of the porcine
orthologue of the human 11p15 imprinted domain: The SWC9 marker was
known from previous studies to correspond to a (CA).sub.n
microsatellite located in the 3'UTR of the porcine IGF2 gene (NEZER
et al. 1999; JEON et al. 1999; AMARGER et al. 2002). A BLAST search
was performed with the sequence of the porcine SWR2516 marker
(gi|7643973|) against the sequence contigs spanning the human 11p15
imprinted domain (http://www.ensembl.org/Homo.sub.--sapiens/). A
highly significant similarity (expected value of 6.times.10.sup.-5
calculated based on the size of the NCBI "nr" database) was found
between SWR2516 and sequence contig AC001228 (gi|1935053|) at 3.3
Kb of the p57 gene. This suggested that the SWR2516-SWC9 marker
interval in the pig might correspond to the p57-IGF2 interval of
the human 11p15 imprinted domain.
[0051] Porcine sequence tagged sites (STS) were then developed
across the orthologous region of the human 11p15 imprinted domain.
Sixteen of these were developed in genes (TSSC5, CD81, KVLQT1
(3.times.), TH (2.times.), INS (3.times.), IGF2 (3.times.), H19
(3.times.)), and five in intergenic regions (IG.sub.IGF2-H19,
IG.sub.H19-RL23mep(4.times.)). The corresponding primer sequences
were derived from the porcine genomic sequence, when available
(AMARGER et al. 2002), or from porcine-expressed sequence tags
(EST) that were identified by BLAST searches using the human
orthologues as query sequences (Table 1).
[0052] A porcine BAC library (FAHRENKRUG et al. 2001) was screened
by filter hybridization using (i) human cDNA clones corresponding
to genes known to map to 11p15, as well as (ii) some of the 21
previously described porcine STS. Seven of the identified BACs were
shown by PCR to contain at least one of the porcine STS available
in the region and were kept for further analysis, together with two
BACs that were previously shown to span the TH-H19 region (AMARGER
et al. 2002). Three additional STS were developed from BAC end
sequences (389B2T7, 370C17T7, 370SP6). 370SP6 revealed a highly
significant BLAST hit (expected value 10.sup.-7) downstream from
the ASCL2 gene providing an additional anchor point between the
human and porcine sequence.
[0053] Using STS content mapping, the BAC contig shown in FIG. 2
was assembled. It confirms the overall conservation of gene order
between human and pigs in this chromosome region and indicates that
the gap remaining in the human sequence between the INS and ASCL2
genes may not be larger than 55 Kb.
[0054] All available STS were then amplified from genomic DNA of
the fourteen QTL genotyped boars (see above) and cycle-sequenced in
order to identify DNA sequence polymorphisms. A total of 43 SNPs
were identified: two in TSSC5, fifteen in KVLQT1, three in
389B2-T7, four in TH, seven in INS, four in IGF2, one in
IG.sub.(IGF2-H19), three in H19 and four in IG.sub.(H19-RL23MRP)
(Table 1).
[0055] Three microsatellites were added to this marker list: one
(KVLQT1-SSR) isolated from BAC 956B11 and two (PULGE1 and PULGE3)
isolated from BAC 370.
[0056] Assembling pools of "Q" versus "q" bearing chromosomes: To
reconstruct the marker linkage phase of the fourteen QTL genotyped
sires, for each boar, offspring were selected that were homozygous
for the alternate paternal SWR2516-SWC9 haplotypes. These were
genotyped for all SNPs and microsatellites available in the region
and from these genotypes, the linkage phase of the boars was
determined.
[0057] For six of the seven boars, shown by marker-assisted
segregation analysis to be of "Qq" genotype (FIG. 1), the "Q"
chromosomes associated with an increase in the percentage of lean
meat proved to be identical-by-state (IBS) over their entire
length. This haplotype was, therefore, referred to as "Q.sup.1."
The haplotype corresponding to the seventh "Q" chromosome (P7 in
FIG. 2) was different and referred to as "Q.sup.2." For three of
these sires, the haplotypes associated with a decrease in the
percentage of lean meat proved to be completely IBS as well and
were thus referred to as "q.sup.1." The other four "q" chromosomes
carried distinct haplotypes and were referred to as "q.sup.2,"
"q.sup.3," "q.sup.4," and "q.sup.5" (FIG. 2).
[0058] The first boar that proved to be homozygous for the QTL by
marker-assisted segregation analysis (P8) carried the "q.sup.4"
haplotype on one if its chromosomes. Its other haplotype,
therefore, had to be of "q" genotype as well and was referred to as
"q.sup.6."
[0059] Boar P9 appeared to be heterozygous "Q.sup.1/Q.sup.2." Boars
P10 and P11 carried the "Q.sup.1" haplotype shared by six of the
"Qq" boars. As a consequence, the other chromosomes of boars P10
and P11, which were IBS as well, were placed in the "Q" pool and
referred to as "Q.sup.3." Homozygous boar P12 carried haplotype
"Q.sup.2." As a consequence, its homologue was referred to as
"Q.sup.4." Following the same recursive procedure, boars P13 and
P14 were identified as being, respectively, "Q.sup.3Q.sup.4" and
"Q.sup.2Q.sup.5."
[0060] The marker genotypes of the resulting five "Q" and five "q"
chromosomes are shown in FIG. 2. In this figure, closely linked
(<5 Kb) SNPs were merged into a series of polyallelic multisite
haplotypes. The correspondence between SNP genotype and haplotype
number is given in Table 2.
[0061] All "Q" chromosomes share a .apprxeq.90 Kb common haplotype
encompassing the INS and IGF2 genes not present in the "q"
chromosomes: Visual examination of the "Q" and "q" pools
immediately reveals that all five chromosomes in the "Q" pool
indeed share an IBS haplotype spanning the 389B2T7-IGF2 interval
(FIG. 2). Four of the five "Q" chromosomes ("Q.sup.1," "Q.sup.2,"
"Q.sup.3" and "Q.sup.4") also carry a common haplotype in the
proximal KVLQT1(I12)-(I7) interval, while the fifth one ("Q.sup.5")
carries a completely different KVLQT1(I12)-(I7) haplotype. This
strongly suggests an ancestral recombination between KVLQT1(I7) and
389B2T7. Likewise, three of the five "Q" chromosomes ("Q.sup.1,"
"Q.sup.3," "Q.sup.4") carry the same haplotype distal from IGF2,
while the two remaining ones ("Q.sup.2," "Q.sup.5") are sharing a
completely distinct one. Again, this is best explained by assuming
an ancestral recombination event just proximal from the SWC9
microsatellite marker. These observations, therefore, strongly
suggest that the hypothesized "Q" allele associated with an
increase in "% lean meat" appeared by mutation or migration on a
founder chromosome carrying the haplotype highlighted in FIG. 2,
and that the QTL is located in the KVLQT1(I7)-SWC9 interval. At
present, our best estimate of the size of this interval is of the
order of 500 Kb (FIG. 2).
[0062] No such shared haplotype could be identified in the "q"
pool. As expected under our model, the "q" pool exhibited a higher
level of genetic diversity. The "q"-bearing chromosomes would
indeed be older, having had ample opportunity to recombine, thereby
increasing haplotype diversity. This can be quantified more
accurately by computing the average pair-wise probability for "Q"
and "q" chromosome to be IBD-conditional on flanking marker data,
using the coalescent model developed by MEUWISSEN and GODDARD
(2001). As shown in FIG. 3, the average pair-wise IBD probability
amongst the five "Q" chromosomes is superior to 0.4 over the entire
KVLQT1(SSR)-IG.sub.(H19-RL23mrp) interval and exceeds 0.9 in the
PULGE3-IGF2 interval. For the "q" chromosomes, the equivalent
parameter averages 0.25 in the same region. It is worthwhile
noting, however, that even amongst "q" chromosomes, the average
pair-wise IBD probability peaks just above 0.4 between TH and INS,
which is thought to reflect a "q"-specific haplotype signature.
[0063] It is noteworthy that chromosome "q.sup.4" carries a
KVLQT1(I12)-PULGE3 haplotype that is IBS with the ancestral "Q"
haplotype in the KVLQTI(I12)-PULGE3 interval. The probability that
this IBS status reflects IBD was estimated at 0.50 using the
coalescent model of MEUWISSEN and GODDARD (2001). Assuming IBD,
this would position the QTL in the PULGE3-SWC9 interval, measuring
less than 90 Kb and containing TH, INS and IGF2 as the only known
genes.
[0064] One could argue that the probability to identify a shared
haplotype amongst five chromosomes by chance alone is high and does
not support the location of the QTL within this region. To more
quantitatively estimate the significance of the haplotype sharing
amongst "Q" chromosomes, accounting for the distance between
adjacent markers, as well as allelic frequencies, a multipoint LD
analysis was performed using the DISMULT program (TERWILLIGER
1995). To test the significance of the haplotype sharing observed
amongst the five "Q" chromosomes, the same DISMULT analysis was
performed on all 462 possible combinations of the 11 chromosomes
taken five at a time. For each of these analyses, the highest
likelihood obtained anywhere along the analyzed chromosome segment
was stored. The likelihood obtained using the real five "Q"
chromosomes at the position of marker PULGE3 was the highest one
obtained across all chromosome permutations (data not shown),
clearly indicating that the observed haplotype sharing is very
unlikely to be fortuitous.
[0065] When we previously demonstrated that only the paternal SSC2
QTL allele influenced muscle mass and that the most likely QTL
position coincided with IGF2, this gene obviously stood out as the
prime candidate (NEZER et al. 1999; JEON et al. 1999). On the basis
of the initial linkage analysis, however, the confidence interval
for the QTL covered approximately 4cM, which were bound to contain
a multitude of genes other than IGF2. It was, therefore, useful to
corroborate these papers by refining the map position of the QTL by
exploiting both LD- and marker-assisted segregation analysis.
Because of the observed parent-of-origin effect, analysis was
focused on a chromosome region that is the ortholog of the human
11p15 imprinted domain. Strong evidence is provided herein that the
QTL indeed maps to the p57-H19 imprinted gene cluster and within
this region, to a chromosome segment of .apprxeq.90 Kb known to
contain the TH, INS and IGF2 genes. These findings, therefore,
considerably strengthen the candidacy of IGF2 and justify a
detailed analysis of this gene.
[0066] Success in refining the map position of this QTL down to the
subcentimorgan level supports its simple molecular architecture.
Together with recent successes in positional cloning and
identification of the mutations that underlie QTL (e.g. GROBET et
al. 1997; MILAN et al. 2001; GRISART et al. 2002; BLOTT et al.
2002), this clearly indicates that at least part of the genetic
variation of production traits in livestock is due to single
mutations with large effects on the traits of interest.
[0067] The success of haplotype-sharing approaches in fine-mapping
QTL in livestock also suggests that QTL may be mapped in these
populations by virtue of the haplotype signature resulting from
intense selection on "Q" alleles, i.e., haplotypes of unusual
length given their population frequency. The feasibility of this
approach has recently been examined in human populations for loci
involved in resistance to malaria (SABETI et al. 2002). QTL could
thus be identified in livestock in the absence of phenotypic
data.
EXAMPLE 2
[0068] Positional identification of a regulatory mutation in IGF2
causing a major QTL effect on muscle development in the pig.
[0069] The identification of causative mutations for Quantitative
Trait Loci (QTLs) is a major hurdle in genetic studies of
multifactorial traits and disorders. Here, it is shown that a
paternally expressed QTL-affecting muscle mass and fat deposition
in pigs is caused by a single nucleotide substitution in intron 3
of IGF2. The mutation occurs in an evolutionary conserved CpG
island that is hypomethylated in skeletal muscle. Pigs carrying the
mutation have a three-fold increase in IGF2 mRNA expression in
postnatal muscle. The mutation abrogates in vitro interaction with
a nuclear factor, most likely a repressor. The mutation has
experienced a selective sweep in several pig breeds. The study
provides an outstanding example where the causal relationship
between a regulatory mutation and a QTL effect has been
established.
[0070] The imprinted IGF2-linked QTL is one of the major porcine
QTLs for body composition. It was first identified in intercrosses
between the European Wild Boar and Large White domestic pigs and
between Pietrain and Large White pigs (1, 2). The data showed that
alleles from the Large White and Pietrain breeds, respectively,
were associated with increased muscle mass and reduced back-fat
thickness, consistent with the existing breed differences in the
two crosses. A paternally expressed IGF2-linked QTL was
subsequently documented in intercrosses between Chinese Meishan and
Large White/Landrace pigs (3) and between Berkshire and Large White
pigs (4). In both cases the allele for high muscle mass was
inherited from the lean Large White/Landrace breed.
[0071] Recently, a haplotype-sharing approach to refine the map
position of the QTL was used (5). It was assumed that a new allele
(O) promoting muscle development occurred g generations ago on a
chromosome carrying the wild-type allele (q). It was also assumed
that the favorable allele had gone through a selective sweep due to
the strong selection for lean growth in commercial pig populations.
Twenty-eight chromosomes with known QTL status were identified by
marker-assisted segregation analysis using cross-bred Pietrain and
Large White boars. All 19 Q-bearing chromosomes shared a haplotype
in the 90 kilobase pairs (kb) interval between the microsatellites
PULGE1 and SWC9 (IGF2 3'-UTR), which was not present among the q
chromosomes and was, therefore, predicted to contain the QTL. In
contrast, the nine q chromosomes exhibited six distinct marker
haplotypes in the same interval. This region is part of the
CDKN1C-H19 imprinted domain and contains INS and IGF2 as the only
known paternally expressed genes. Given the known functions of
these genes and especially the role of IGF2 in myogenesis (6), they
stood out as prime positional candidates. A comparative sequence
analysis of the porcine INS-IGF2 region revealed as many as 59
conserved elements (outside known exons) between pig and human, all
being candidate regions for harboring the causative mutation
(7).
[0072] In order to identify the causative mutation, one of the 19
Q-chromosomes (P208) and six q-chromosomes (each corresponding to
one of the six distinct marker haplotypes) were re-sequenced for a
28.6 kb segment containing IGF2, INS, and the 3' end of TH. This
chromosome collection was expanded by including Q- and
q-chromosomes from (i) a Wild Boar/Large White intercross
segregating for the QTL (2), (ii) a Swedish Landrace boar showing
no evidence for QTL segregation in a previous study (8), (iii)
F.sub.1 sires from a Hampshire/Landrace cross showing no indication
for QTL segregation (9), and (iv) an F.sub.1 sire from a
Meishan/Large White intercross. A Japanese Wild Boar was included
as a reference for the phylogenetic analysis; the QTL status of
this animal is unknown, but it is assumed that it is homozygous
wild-type (q/q). A total of 258 DNA sequence polymorphisms were
identified corresponding to a staggering one polymorphic nucleotide
per 111 base pairs (bp) (FIG. 4A). The sequences formed three major
and quite divergent clusters (FIG. 4B). The only exception to this
pattern was one Hampshire haplotype (H254) that was apparently
recombinant.
[0073] The two established Q haplotypes from Pietrain and Large
White animals (P208 and LW3) were identical to each other and to
the chromosomes from the Landrace (LRJ) and Hampshire/Landrace
(H205) animals for almost the entire region, showing that the
latter two must be of Q-type as well. The absence of QTL
segregation in the offspring of the F.sub.1
Hampshire.times.Landrace boar carrying the H205 and H254
chromosomes implies that the latter recombinant chromosome is also
of Q-type. This places the causative mutation downstream from IGF2
intron 1, the region for which H254 is identical to the other Q
chromosomes. The Large White chromosome (LW197) from the
Meishan/Large White pedigree clearly clustered with q chromosomes,
implying that the F.sub.1 sire used for sequencing was homozygous
q/q as a previous QTL study showed that the Meishan pigs carried an
IGF2 allele associated with low muscle mass (3). Surprisingly, the
Meishan allele (M220) was nearly identical to the Q chromosomes but
with one notable exception, it shared a G nucleotide with all q
chromosomes at a position (IGF2-intron3-nt3072) where all Q
chromosomes have an A nucleotide (FIG. 4A). Under a bi-allelic QTL
model, the causative mutation would correspond to a DNA
polymorphism for which the two alleles segregate perfectly between
Q- and q-chromosomes. The G to A transition at IGF2-intron3-nt3072
is the only polymorphism fulfilling this criterion, implying that
it is the causative Quantitative Trait Nucleotide (QTN) (10). So
far, 12 large sire families were tested where the sire is
heterozygous A/G at this position and all have showed evidence for
QTL segregation. In contrast, more than 40 sires were tested,
representing several different breeds, genotyped as homozygous A/A
or G/G at this position without obtaining any significant evidence
for the segregation of a paternally expressed QTL at the IGF2
locus. The results provide conclusive genetic evidence that
IGF2-intron3-nt3072G.fwdarw.A is the causative mutation.
[0074] IGF2-intron3-nt3072 is part of an evolutionary conserved CpG
island of unknown function (7), located between Differentially
Methylated Region 1 (DMR1) and a matrix attachment region
previously defined in mice (11-13). The 94 bp sequence around the
mutation shows about 85% sequence identity to both human and mouse,
and the wild-type nucleotide at IGF2-intron3-nt3072 is conserved
among the three species (FIG. 4A). The QTN occurs three bp
downstream of an eight bp palindrome also conserved between the
three species. The methylation status of the 300 bp fragment
centered on IGF2-intron3-nt3072 and containing 50 CpG dinucleotides
was examined by bisulphite sequencing in four-month-old
Q.sup.pat/q.sup.mat and q.sup.pat/Q.sup.mat animals. In skeletal
muscle, paternal and maternal chromosomes were shown to be
essentially unmethylated (including the IGF2-intron3-nt3071 C
residue), irrespective of the QTL genotype of the individual (3.4%
of CpGs methylated on average; FIG. 5A). The CpG island was more
heavily, and also differentially, methylated in liver. 33% of the
CpGs were methylated on the maternal alleles versus 19% on the
paternal allele. Unexpectedly, therefore, this CpG island behaves
as a previously unidentified DMR in liver, the repressed maternal
allele being more heavily methylated than the paternal allele,
which is the opposite of what is documented for the adjacent DMR1
in the mouse.
[0075] To uncover a possible function for this element,
electrophoretic mobility shift analyses (EMSA) were performed using
oligonucleotide probes spanning the QTN and corresponding to the
wild-type (q) and mutant (O) sequences. Nuclear extracts from
murine C2C12 myoblast cells, human HEK293 cells, and human HepG2
cells were incubated with radioactively labeled q or Q
oligonucleotides. One specific band shift (complex C1 in FIG. 5B)
was obtained with the wild-type (q) but not the mutant (O) probe
using extracts from C2C12 myoblasts; similar results were obtained
using both methylated and unmethylated probes. A band shift with
approximately the same migration, but weaker, was also detected in
extracts from HEK293 and HepG2 cells. The specificity of the
complex was confirmed since competition was obtained with ten-fold
molar excess of unlabeled q probe, whereas a 50-fold excess of
unlabeled Q probe did not achieve competition (FIG. 5B). Thus, the
wild-type sequence binds a nuclear factor and this interaction is
abrogated by the mutation.
[0076] To further characterize the functional significance of the
IGF2 Q mutation, its effect on transcription was studied by
employing a transient transfection assay in mouse C2C12 myoblasts.
Q and q constructs were made containing a 578 bp fragment from the
actual region inserted in front of a Luciferase reporter gene
driven by the herpes thymidine kinase (TK) minimal promoter. The
two constructs differed only by the IGF2-intron3-nt3072G.fwdarw.A
transition. The ability of the IGF2 fragments to activate
transcription from the heterologous promoter was compared with the
activity of the TK-promoter alone. The presence of the q-construct
caused a two-fold increase of transcription, whereas the
Q-construct caused a significantly higher, seven-fold, increase
(FIG. 5C). Interpreting this result, in the light of the results
from the EMSA experiment, the Q mutation abrogates the interaction
with a repressor protein that modulates the activity of a putative
IGF2 enhancer present in this CpG island. This view is consistent
with in silico identification of potential binding sites for both
activators and repressors in this intronic DNA fragment (14).
[0077] The in vivo effect of the mutation on IGF2 expression was
studied in a purpose-built Q/q.times.Q/q intercross counting 73
offspring. As a deletion encompassing DMR0, DMR1, and the
associated CpG island derepresses the maternal IGF2 allele in
mesodermal tissues in the mouse (12), the effect of the
intron3-nt3072 mutation on IGF2 imprinting in the pig was tested.
This was achieved by monitoring transcription from the paternal and
maternal IGF2 alleles in tissues of q/q, Q.sup.pat/q.sup.mat, and
q.sup.pat/Q.sup.mat animals that were heterozygous for the SWC9
microsatellite located in the IGF2 3'UTR. Imprinting could not be
studied in Q/Q animals that were all homozygous for SWC9. Before
birth, IGF2 was shown to be expressed exclusively from the paternal
allele in skeletal muscle and kidney, irrespective of the QTL
genotype of the fetuses. At four months of age, weak expression
from the maternal allele was observed in skeletal muscle, however,
at comparable rates for all three QTL genotypes (FIG. 6A). Only the
paternal allele could be detected in four-month-old kidney (data
not shown). Consequently, the mutation does not seem to affect the
imprinting status of IGF2. The partial derepression of the maternal
allele in skeletal muscle of all QTL genotypes may, however,
explain why in a previous study, muscular development was found to
be slightly superior in q.sup.pat/Q.sup.mat versus q/q animals and
in Q/Q versus Q.sup.pat/q.sup.mat animals (2).
[0078] The Q allele was expected to be associated with an increased
IGF2 expression since IGF2 stimulates myogenesis (6). To test this,
the relative mRNA expression of IGF2 was monitored at different
ages in the Q/q.times.Q/q intercross, using both Northern blot
analysis and real-time PCR (FIGS. 6B and 6C). The expression levels
in fetal muscle and postnatal liver was about ten-fold higher than
in postnatal muscle. No significant difference was observed in
fetal samples or in postnatal liver samples, but a significant
three-fold increase of postnatal IGF2 mRNA expression in skeletal
muscle was observed in (Q/Q or Q.sup.pat/q.sup.mat) versus
(q.sup.pat/Q.sup.mat or q/q) progeny. The significant difference in
IGF2 expression revealed by real-time PCR was confirmed using two
different internal controls, GAPDH (FIG. 6C) and HPRT (15). An
increase of all detected transcripts originating from the three
promoters (P2-P4) located downstream of the mutated site was found.
Combined, these results provide strong support for IGF2 being the
causative gene. The lack of significant differences in IGF2 mRNA
expression in fetal muscle and postnatal liver are consistent with
the previous QTL study, showing no effect of the IGF2 locus on
birth weight and weight of liver (2).
[0079] There has been a strong selection for lean growth (high
muscle mass and low fat content) in commercial pig populations in
Europe and North America during the last 50 years. Therefore, how
this selection pressure has affected the allele frequency
distribution of the IGF2 QTL was investigated. The causative
mutation was absent in a small sample of European and Asian Wild
Boars and in several breeds that have not been strongly selected
for lean growth (Table 1). In contrast, the causative mutation was
found at high frequencies in breeds that have been subjected to
strong selection for lean growth. The only exceptions were the
experimental Large White population at the Roslin Institute that
was founded from commercial breeding stocks in the UK around 1980
(16), as well as the experimental Large White populations used for
the Pietrain/Large White intercross (1). These two populations thus
reflect the status in some commercial populations about 20 years
ago and it is possible that the IGF2*Q allele is even more
predominant in contemporary populations. The results demonstrate
that IGF2*Q has experienced a selective sweep in several major
commercial pig populations and it has apparently been spread
between breeds by cross-breeding.
[0080] The results have important practical implications. The
IGF2*Q mutation increases the amount of meat produced, at the
expense of fat, by 3-4 kg for an animal slaughtered at the usual
weight of about 100 kg. The high frequency of IGF2*Q among major
pig breeds implies that this mutation affects the productivity of
many millions of pigs in the Western world. The development of a
simple diagnostic DNA test now facilitates the introgression of
this mutation to additional breeds. This could be an attractive way
to improve productivity in local breeds as a measure to maintain
biological diversity. The diagnostic test will also make it
possible to investigate whether the IGF2*Q mutation is associated
with any unfavorable effects on meat quality or any other trait. It
has been previously demonstrated that European and Asian pigs were
domesticated from different subspecies of the Wild Boar and that
Asian germplasm has been introgressed into European pig breeds
(17). The IGF2*Q mutation apparently occurred on an Asian
chromosome as it showed a very close relationship to the haplotype
carried by Chinese Meishan pigs. This explains the large genetic
distance observed between Q- and q-haplotypes (FIG. 4). However, it
is an open question as to whether the Q mutation occurred before or
after the Asian chromosome was introduced into European pigs.
[0081] This study provides new insights in IGF2 biology. The role
of IGF2 on prenatal development is well documented (18, 19). It has
been observed that the Q mutation does not upregulate IGF2
expression in fetal tissue but after birth, demonstrates that IGF2
has an important role for regulating postnatal myogenesis. The
finding that the sequence around the mutation does not match any
known DNA-binding site suggests that this sequence may bind an
unknown nuclear factor (14). These results also mean that
pharmacological intervention of the interaction between this DNA
segment and the corresponding nuclear factor opens up new
strategies for promoting muscle growth in human patients with
muscle deficiencies or for stimulating muscle development at the
cost of adipose tissue in obese patients.
Materials and Methods
DNA Sequencing
[0082] Animals that were homozygous for 13 of the haplotypes of
interest were identified using flanking microsatellite markers and
pedigree information. A 28.6 kb chromosome segment containing the
last exon of TH, INS, and IGF2 was amplified from genomic DNA in
seven long-range PCR products using the Expand Long Template PCR
system (Roche Diagnostics GmbH). The same procedure was used to
amplify the remaining M220 and LW197 haplotypes from two BAC clones
isolated from a genomic library that was made from a Meishan/Large
White F.sub.1 individual (20). The long template PCR products were
subsequently purified using Geneclean (Polylab) and sequenced using
the Big Dye Terminator Sequencing or dGTP Big Dye Terminator kits
(Perkin Elmer). The primers used for PCR amplification and
sequencing are available as supplementary information. The sequence
traces were assembled and analyzed for DNA sequence polymorphism
using the Polyphred/Phrap/Consed suite of programs (21).
SNP Analysis of IGF2-intron3-nt3072
[0083] The genotype was determined by pyrosequencing with a Luc 96
instrument (Pyrosequencing AB). A 231 bp DNA fragment was PCR
amplified using Hot Star Taq DNA polymerase and Q-Solution (QIAGEN)
with the primers pyrol8274F (5'-Biotine-GGGCCGCGGCTTCGCCTAG-3')
(SEQ ID NO: 2) and pyro18274R (5'-CGCACGCTTCTCCTGCCACTG-3' (SEQ ID
NO:3)). The sequencing primer (pyro18274seq:
5'-CCCCACGCGCTCCCGCGCT-3' SEQ ID NO:4)) was designed on the reverse
strand because of a palindrome located 5' to the QTN.
Electrophoretic Mobility Shift Analyses (EMSA)
[0084] DNA-binding proteins were extracted from C2C12, HEK293, and
HepG2 cells as described (22). Gel shift assays were performed with
40 fmole .sup.32P-labeled ds-oligonucleotide, 10 .mu.g nuclear
extract, and 2 .mu.g poly dI-dC in binding buffer (15 mM Hepes pH
7.65, 30.1 mM KCl, 2 mM MgCl.sub.2, 2 mM spermidine, 0.1 mM EDTA,
0.63 mM DTT, 0.06% NP-40, 7.5% glycerol). For competition assays, a
10-fold, 20-fold, 50-fold, and 100-fold molar excess of cold
ds-oligonucleotide were added. Reactions were incubated for 20
minutes on ice before .sup.32P-labeled ds-oligonucleotide was
added. Binding was then allowed to proceed for 30 minutes at room
temperature. DNA-protein complexes were resolved on a 5% native
polyacrylamide gel run in TBE 0.5.times. at room temperature for
two hours at 150 V and visualized by autoradiography. The following
two oligonucleotides were used: Q/q:
5'-GATCCTTCGCCTAGGCTC(A/G)CAGCGCGGGAGCGA-3' (SEQ ID NO:6 and SEQ ID
NO:5).
Northern Blot Analysis and Real-Time RT-PCR
[0085] Total RNA was prepared from porcine muscle (gluteus) and
liver tissues using Trizol (Invitrogen) and treated with DNase I
(Ambion). The products from the first-strand cDNA synthesis
(Amersham Biosciences) were column purified with QIAquick columns
(Qiagen). Poly (A).sup.+ RNA was purified from total RNA using the
Oligotex mRNA kit (Qiagen). Approximately 75 ng poly(A).sup.+ mRNA
from each sample was separated by electrophoresis in a
MOPS/formaldehyde agarose gel and transferred o/n to a
Hybond-N+nylon membrane (Amersham Biosciences). The membrane was
hybridized with pig-specific IGF2 and GAPDH cDNA probes using
ExpressHyb hybridization solution (Clontech). The quantification of
the transcripts was performed with a Phosphor Imager 425 (Molecular
Dynamics). Real-time PCR were performed with an ABI PRISM 7700
Sequence Detection System (Applied Biosystems). TaqMan probes and
primers were designed with the Primer Express software (version
1.5); primer and probe sequences are available as supplementary
material. PCR reactions were performed in triplicate using the
Universal PCR Master Mix (Applied Biosystems). The mRNA was
quantified as copy number using a standard curve. For each
amplicon, a ten-point calibration curve was established by a
dilution series of the cloned PCR product.
Bisulphite-Based Methylation Analysis.
[0086] Bisulphite sequencing was performed according to Engemann et
al. (23). Briefly, high molecular weight genomic DNA was isolated
from tissue samples using standard procedures based on proteinase K
digestion, phenol-chloroform extraction, and ethanol precipitation.
The DNA was digested with EcoRI, denatured, and embedded in low
melting point agarose beads. Non-methylated cytosine residues were
converted to uracil using a standard bisulphite reaction.
[0087] The region of interest was amplified using a two-step PCR
reaction with primers complementary to the bisulphite-converted DNA
sequence (PCR1-UP: 5'-TTGAGTGGGGATTGTTGAAGTTTT-3' (SEQ ID NO:7),
PCR1-DN: 5'-ACCCACTTATAATCTAAAAAAATAATAAATATATCTAA-3' (SEQ ID
NO:8), PCR2-UP: 5'-GGGGATTGTTGAAGTTTT-3' (SEQ ID NO:9), PCR2-DN:
5'-CTTCTCCTACCACTAAAAA-3' (SEQ ID NO:10)). The amplified strand was
chosen in order to be able to differentiate the Q and q alleles.
The resulting PCR products were cloned in the pCR2.1 vector
(Invitrogen). Plasmid DNA was purified using the modified Plasmid
Mini Kit (QIAGEN) and sequenced using the Big Dye Terminator Kit
(Perkin Elmer) and an ABI3100 sequence analyzer.
Transient transfection assay
[0088] C2C12 myoblast cells were plated in six-well plates and
grown to .about.80% confluence. Cells were transiently
co-transfected with a Firefly luciferase reporter construct (4
.mu.g) and a Renilla luciferase control vector (phRG-TK, Promega;
80 ng) using 10 .mu.g Lipofectamine 2000 (Invitrogen). The cells
were incubated for 24 hours before lysis in 100 .mu.l Triton Lysis
Solution. Luciferase activities were measured with a Mediators PhL
luminometer (Diagnostic Systems) using the Dual-Luciferase reporter
Assay System (Promega).
Analysis of the IGF2 Imprinting Status
[0089] RT-PCR analysis of the highly polymorphic SWC9
microsatellite (located in IGF2 3'UTR) was used to determine the
IGF2 imprinting status. The analysis involved progeny groups from
heterozygous sires. Total RNA was extracted from the gluteus muscle
using Trizol Reagent (Life Technology) and treated with RNase-free
DNase I (Roche Diagnostics GmbH). cDNA was synthesized using the
1.sup.st Strand cDNA Synthesis Kit (Roche Diagnostics GmbH). The
SWC9 marker was amplified using the primers UP
(5'-AAGCACCTGTACCCACACG-3' (SEQ ID NO:11)) and DN
(5'-GGCTCAGGGATCCCACAG-3' (SEQ ID NO:12)). The .sup.32P-labeled
RT-PCR products were separated by denaturing PAGE and revealed by
autoradiography.
EXAMPLE 3
The Mutation has an Effect on Teat Number
[0090] Sires of two commercial lines were genotyped for the
mutation. Shortly after birth, the number of teats was counted in
all piglets. Piglet counts ranged from 12 to 18 teats and included
4477 individuals from 22 sires. A statistical analysis of teat
number in piglets was performed by accounting for the following
effects: 1) genetic line (lines A and B), 2) genotype of the sire
for the mutation (QQ, Qq or qq) and 3) sex of the piglet
(male/female). Analysis of variance was performed using Proc Mixed
(SAS), assuming normality of dependent variable teat number.
Estimates of some contrasts are given in Table 4.
[0091] The effect of genotype on teat number in piglets is -0.28
teats. This effect is opposite to the one described by Hirooka et
al. 2001. An effect of genetic line could not be demonstrated. The
sex of the piglet had a significant effect on teat number with
female pigs having an average of 0.05 teat more than males. Mean
values per genotype and per line are given in Table 5.
TABLE-US-00001 TABLE 4 Analysis of variance of teat number counted
in piglets of two commercial lines (n = 4477). Effect P-value
Contrast Estimate(s.e) Genotype of sire <0.001 QQ-qq -0.28
(0.05) Qq-qq -0.22 (0.03) Genetic line 0.081 Sex 0.043 M-F -0.05
(0.03)
[0092] TABLE-US-00002 TABLE 5 Descriptive statistics of teat number
counted in piglets of two commercial lines (n = 4477) descending
from sires of three different genotypes with respect to the
mutation. Average N N descending teat Genotype sires piglets number
Stdev A QQ 2 144 14.51 0.76 Qq 5 1720 14.53 0.82 qq 3 735 14.74
0.86 B QQ 2 277 14.41 1.00 Qq 7 1054 14.48 0.81 qq 3 547 14.73
0.93
[0093] The statistical analysis confirms that the mutation
influences teat number. The Q allele that is favorable with respect
to muscle mass and reduced back fat is the unfavorable allele for
teat number. This strengthens the possibility of using the paternal
imprinting character of this QTL in breeding programs. Selecting
maternal lines for the q allele will enhance teat number, a
characteristic that is favorable for the maternal side. On the
other hand, paternal lines can be selected for the Q allele that
will increase muscle mass and reduce back fat, characteristics that
are of more importance in the paternal lines. Terminal sires that
are homozygous QQ will pass the full effect of increased muscle
mass and reduced back fat to the slaughter pigs, while selection of
parental sows that express the q allele will have more teats
without affecting slaughter quality.
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BOVENHUIS, 2001. A whole-genome scan for quantitative trait loci
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[0128] 9. QTL genotyping of the Pietrain/Large White, Wild
Boar/Large White, and Hampshire/Landrace crosses by marker-assisted
segregation analysis was performed as described (5). Briefly, the
likelihood of the pedigree data was computed under two hypothesis:
H0, postulating that the corresponding boar was homozygous at the
QTL (Q/Q or q/q), and H1, postulating that the boar was
heterozygous at the QTL (Q/q). Likelihoods were computed using "%
lean meat" as phenotype (as the effect of the QTL was shown to be
most pronounced on this trait in previous analyses), and assuming a
Q to q allele substitution effect of 3.0% (1). If the odds in favor
of one of the hypotheses were superior or equal to 100:1, the most
likely hypothesis was considered to be true. For the
Hampshire/Landrace cross, 75 offspring from four boars with
identical H254/H205 genotype were merged in a single analysis. The
odds in favor of the H0 hypothesis was 103.6:1, indicating that
these boars were either Q/Q or q/q. [0129] 10. T. F. C. Mackay,
Nature Rev. Genet. 2, 11-21 (2001). [0130] 11. J. M. Greally, M. E.
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(2000). [0132] 13. S. Eden et al., EMBO J. 20, 3518-3525 (2001).
[0133] 14. The nucleotide sequence of the conserved footprint
surrounding the QTN was analyzed in silico for potential binding
sites using the following transcription factor binding site
databases (TFSEARCH, http://www.cbrcjp/research/db/TFSEARCH.html;
Tess, http://www.cbil.upenn.edu/tess/; Signal Scan,
http://bimas.dcrt.nih.gov/molbio/signal (24); and alibaba2,
http://www.gene-regulation.de/). The sequence immediately flanking
the QTN did not show any convincing match with known binding sites.
However, the entire 94 bp fragment is highly GC-rich and
consequently, several potential binding sites for the Sp-(eight
GC-boxes), ZF5 (one consensus binding site), EGR/WT1 (three
GSG-elements), and AP2 (three AP-2-boxes) families of transcription
factors were identified in sites flanking the QTN. Both activators
and repressors are known to competitively or cooperatively interact
with such GC-rich motifs. Thus, the high density of potential
regulatory elements identified in this fragment is consistent with
the obtained EMSA and transfection results. [0134] 15. Relative
expression of IGF2/HPRT in skeletal muscle from three-week-old pigs
was as follows: Q:260.2.+-.70.8 and q:59.6.+-.12.1 (P<0.05,
Kruskal-Wallis rank sum test, two sided). [0135] 16. G. A. Walling
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Nature 389, 809-815 (1997). [0139] 20. S. I. Anderson, N. L.
Lopez-Corrales, B. Gorick, and A. L. Archibald, Mammalian Genome
11, 811-814 (2000). [0140] 21. D. Nickerson, V. O. Tobe, and S. L.
Taylor, Nucleic Acids Res. 25, 2745-2751 (1997). [0141] 22. N. C.
Andrews and D. V. Faller, Nucleic Acids Res. 19, 2499 (1991).
[0142] 23. S. Engemann, O. El-Maarri, P. Hajkova, J. Oswald, and J.
Walter, in Methods in Molecular Biology, vol. 181: Genomic
imprinting: Methods and Protocols A. Ward, Ed. (Humana Press Inc.,
Totowa, N.J., 2002). [0143] 24. D. S. Prestridge, Comput. Appl.
Biosci. 7, 203-206 (1991). [0144] 25. C. Kashuk, S. Sengupta, E.
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[0145] 26. S. Kumar, K. Tamura, I. B. Jakobsen, and M. Nei,
Bioinformatics 17, 1244-1245 (2001). TABLE-US-00003 TABLE 1
Utilized sequence tagged sites (STS) and corresponding DNA sequence
polymorphisms (DSP). STS Source UP-primer (5'-3') DN-primer (5'-3')
DSP.sup.2 DSP (5'-3') TSSC5 B1183986 TCATCCAGGGCCTGGTCAT (SEQ ID
NO:13) TGTCTGAGGCCGACACGGCC (SEQ ID NO:14) T1 CCCCCTCCC(C/T)GGCCCCC
(SEQ ID NO:15) (I1).sup.1 CG T2 ACCCAGGGC(C/T)CCTTGAG (SEQ ID
NO:16) SWR2516 gi7643973 GTGCATTATCGGGAGGTATG (SEQ ID NO:17)
ACCCTGTATGATACTGTAACTCT (SEQ ID NO:18) SSR ATAGGGTTA(GT)nAGATCAGTC
(SEQ ID NO:19) GG KVLQT1 BAC956B11 CTTTGAGGTCCATCATGTTC (SEQ ID
NO:20) GGACGTACATCCCATCGATGA (SEQ ID NO:21) SSR (SSR) CA KVLQT1
BF198846 ATGGTTGTCCTCTGCGTGG (SEQ ID NO:22) TGGCGGTCGACGTGCAGCATC
(SEQ ID NO:23) T1 TGGGTGGGGG(C/T)GCAGCCCC (SEQ ID NO:24) (I12) GC
TGGG T2 GCTGGGA(C/T)CAGACC(G/A)TC (SEQ ID NO:25) T3
GCTGGGA(C/T)CAGACC(G/A) (SEQ ID NO:25) TCTGGG T4
CTGTCTGCTCAT(C/T)CGGGGGCTG (SEQ ID NO:26) T5
GGCTGCGGGAGC(C/T)TGGGGCCAC (SEQ ID NO:27) T6
GCCACCCCCGCC(C/T)TGACCCTGA (SEQ ID NO:28) KVLQT1 BF198846
ATCCGCTTCCTCCAGATCC (SEQ ID NO:29) GCCGATGTACAGCGTGGTGA (SEQ ID
NO:30) V1 TCTGGGCCGG(G/T)GTCCCCG (SEQ ID NO:31) (I11) TG T1
AAAAGGGTCC(A/G)GGAAGCT (SEQ ID NO:32) T2 TTGCAAACAGC(C/T)CCCAGAAGG
(SEQ ID NO:33) T3 AGAAGGCGCAG(C/T)CTGCAGGGG (SEQ ID NO:34) T4
AGGGCGCTGG(C/T)TGCAGGGGTG (SEQ ID NO:35) T5
TTTATGAGTC(A/G)CAAAAACGAG (SEQ ID NO:36) T6
TGATGTCCGCC(C/T)(G/T)GGCA (SEQ ID NO:37) GACT V2
TGATGTCCGCC(C/T)(G/T)GGCA (SEQ ID NO:37) GACT KVLQT1 BF198846
GCCCCAAGCCCAAGAAGTC (SEQ ID NO:38) CCAGAATTGTCACAGCCATCC (SEQ ID
NO:39) T TCCGGGGCAT(A/G)TAGGACTGG (SEQ ID NO:40) (I7) TG 389B2T7
BAC389B2 GGAGTACCTGCTGTGGCTTA (SEQ ID NO:41)
CGTCCTATATCCATCAGGAATAT (SEQ ID NO:42) T1 AGTAGTAT(C/T)CATGAGCAC
(SEQ ID NO:43) GTG TG T2 CCCAGGCCTC(G/A)ATCAGGTGGT (SEQ ID NO:44)
TG V TATATGCCA(C/A)ACATGTGGCCCT (SEQ ID NO:45) CD81(I3) F23061
GGGGCCATCCAGGAGTCAC (SEQ ID NO:46) CAAAGAGGATCACGAGGCAGG (SEQ ID
NO:47) AG INRA370SP6 BACINRA370 TGCGTAGCCATGGCGATGG (SEQ ID NO:48)
AGTGTGGAACCCTGGGGGGGGAA (SEQ ID NO:49) GG GG 370C17T7 BAC370C17
AGAGGGTACAGAAGCCCTG (SEQ ID NO:50) TTTGGTGTGGTGTCTGCTGACCC (SEQ ID
NO:51) PULGE3 BACINRA370 AGGCTTTCTATCTGCAGGA (SEQ ID NO:52)
ACCGTGTGGCCATCTGGGTG (SEQ ID NO:53) SSR TCTCTGTAT(CA)n CGCACGCAC
(SEQ ID NO:54) GG PULGE1 BACINRA370 GCGTTGCAGTGGCTCTGGCG (SEQ ID
NO:55) GACACGGCCGCATGAATGTGC (SEQ ID NO:56) SSR ACCCCAACA(TA)n
ATTATGGTA (SEQ ID NO:57) TH(I13A) AY044828 GCCCGTCTACTTCGTGTCTG
(SEQ ID NO:58) ATCTCTGCCTTCATCGCACCCCC (SEQ ID NO:59) V
AGGATCCAGCC(A/T) GCAGCCCCG (SEQ ID NO:60) AG ID TCACAACCCCC(C)
TCCCACAGC (SEQ ID NO:61 and SEQ ID NO:62) T
CTGCGGAGGGG(A/G)GACCTGCAG (SEQ ID NO:63) TH(I13B) AY044828
GCTGCGGACCCCACCGTCAC (SEQ ID NO:64) AGACTTCACCCCTAAAAGCCTGG (SEQ ID
NO:65) ID GCCAGGT(CAAGGCCAGGT)CGAGG (SEQ ID NO:66 CC and SEQ ID
NO:67) INS(5') AY044828 AGCAGGCTGCTGTGCTGGG (SEQ ID NO:68)
AGCCCAGACCCAGCTGACGG (SEQ ID NO:69) T1 GGCGCTTATGG(G/A)GCCGGGAGC
(SEQ ID NO:70) V CAAGCCCGG(G/T)CGGTTTGGCCT (SEQ ID NO:71) T2
CTAATGACCTC(A/G)AGGCCCCCA (SEQ ID NO:72) INS(I1, AY044828
TGATGACCCACGGAGATGAT (SEQ ID NO:73) GCAGTAGTTCTCCAGCTGGTAGA (SEQ ID
NO:74) T1 GGGACCAGCTG(C/T)GTTCCCAGG (SEQ ID NO:75) E2,I2) CC GGGAA
V GCCCTGCTGGC(C/G)CTCTGGGCG (SEQ ID NO:76) T2
CTCCCACGCCC(C/T)GGTCCCGCT (SEQ ID NO:77) INS(3') AY044828
GCTCTCGCCACATCGGCTGC (SEQ ID NO:78) GGCGCCCAGCTCTAGGCCCGGC (SEQ ID
NO:79) T GGGCTGGCTGC(G/A)GTCTGGGAG (SEQ ID NO:80) IGF2(E3) AY044828
CCCCTGAACTTGAGGACGAG (SEQ ID NO:81) CGCTGTGGGCTGGGTGGGCTGCC (SEQ ID
NO:82) T GCTGCCCCCCA(A/G)CCTGAGCTG (SEQ ID NO:83) CAGCC IGF2(E5)
AY044828 CTTGCCTCCAACTCCCTCCC (SEQ ID NO:84) AGTGAACGTGAAACGGGGGG
(SEQ ID NO:85) SSR CTCTC GCT GTC (CT)n CGCCCT (SEQ ID NO:86) CTT
IGF2(I8) AY044828 TGCGCCACCCCCGCCAAGT (SEQ ID NO:87)
GCTTCCAGGTGTCATAGCGGAAG (SEQ ID NO:88) V AGCCGGCTCCT(G/C)GGCTTCAAG
(SEQ ID NO:89) CC T AGAGGTTGTTG(C/T)TCTGGGACA (SEQ ID NO:90) SWC9
AY044828 AAGCACCTGTACCCACACG (SEQ ID NO:11) GGCTCAGGGATCCCACAG (SEQ
ID NO:12) SSR (CA)n IG(IGF2- AY044828 CAAGCCAGGTCCTGTCGAGG (SEQ ID
NO:91) GGACCCTGGGGGCTGTGG (SEQ ID NO:92) T
CGGCCTGTGGC(A/G)GGGAAGCTG (SEQ ID NO:93) H19) HI9(??) AY044828
ACGGTCCCGGGTCAGCAGG (SEQ ID NO:94) CAGAGCAAGTGGGCACCCAG (SEQ ID
NO:95) T1 CGCGGGTTTGG(C/T)CAGCGGCAG (SEQ ID NO:96) T2
CACAGAGGACA(C/T)GGCCGCTTC (SEQ ID NO:97) T3
TCCTGGGGGCC(C/T)GCGGCTCGT (SEQ ID NO:98) IG(H19- AY044828
GAGCACAGCCAAAGAACGGC (SEQ ID NO:99) CTTCACCCACGGACATGGCCGC (SEQ ID
NO:100) T CACCCAGGCTG(C/T)GCCCTGCGT (SEQ ID NO:101) RL23 CG mrp)A
IG(H19- AY044828 CGGGGGCACTGGGGGTCC (SEQ ID NO:102)
CCGAGACCCTCCTCAAGTCC (SEQ ID NO:103) T GTTCGCCCTCC(A/G)CTCTCAGCA
(SEQ ID NO:104) RL23 mrp)B IG(H19- AY044828 TGAGCTGCTGAGCCCACAGG
(SEQ ID NO:105) CAAGGGAAAGGTGTGCCGACC (SEQ ID NO:106) T
GGCCGGGCGCT(C/T)CGCCTTCCC (SEQ ID NO:107) RL23 mrp)C IG(H19-
AY044828 AGGCAGAGGGCAGAGAGGGG (SEQ ID NO:108) CTCCAGCCCCACACTCTGC
(SEQ ID NO:109) T GCGTCCAGCGC(C/T)GAATCAGGC (SEQ ID NO:110) RL23
mrp)D .sup.1I = intron; E = exon. .sup.2DSP: type of DNA sequence
polymorphism: T = transition, V = transversion, ID = insertion /
deletion, SSR = simple sequence repeat.
[0146] TABLE-US-00004 TABLE 2 Definition of the multisite
haplotypes corresponding to the different markers shown in FIGS. 2
and 3. STS MH1 MH2 MH3 MH4 MH5 TSSC5 T-T C-C (I1) KVLQT1
C-C-C-G-C-C C-T-T-A-T-C T-C-C-A-T-T (I12) KVLQT1 T-C-G-C-T-T-G-T
G-T-A-T-C-C-A-T G-C-G-C-C-C-G-G (I11) 389B2T7 C-G-C T-A-A TH1 +
T-C-G- (SEQ ID NO: A-(-)-A-(-) A-C-G- (SEQ ID NO: TH2 (CAAGGCCAGGT)
111) (CAAGGCCAGGT) 112) INS(5') + G-G-A-C-C-C-G A-T-G-T-G-T-A
G-G-G-T-G-C-G INS (I1,E2,I2) + INS(3') IGF2(E3) + G-2-G-T A-2-C-C
A-1-G-T A-2-G-T G-2-G-T IGF2(E5) + IGF2(I8) H19 C-C-T C-C-C C-T-C
T-T-C IG C-A-T-T T-G-C-C (H19- RL23MRP) A,B,C,D
[0147] TABLE-US-00005 TABLE 3 Distribution of genotypes at the
Quantitative Trait Nucleotide IGF2-intron3-nt3072G.fwdarw.A among
pig populations strongly selected (+) or not strongly selected (-)
for lean growth. Genotype Breed Lean G/G G/A A/A Total European
Wild Boar - 5 0 0 5 European Wild Boar - Uppsala.sup.a - 2 0 0 2
Japanese Wild Boar - 5 0 0 5 Meishan - Roslin.sup.b - 11 0 0 11
Large White - Uppsala.sup.a + 0 1 7 8 Large White - Roslin.sup.b +
6 1 0 7 Large White - Liege.sup.c + 7 0 0 7 Swedish Large
White.sup.d + 0 0 5 5 Swedish Hampshire.sup.d + 0 0 6 6 Swedish
Landrace.sup.d + 0 0 5 5 Pietrain - Liege.sup.c + 0 1 6 7 Duroc + 0
0 1 1 Total 45 3 30 78 .sup.aFounder animals in a Wild Boar .times.
Large White intercross (2). .sup.bFounder animals in a Large White
.times. Meishan intercross (16). .sup.cFounder animals in a
Pietrain .times. Large White intercross (1). .sup.dBreeding boars
that have been tested for QTL segregation in a previous study (8).
The lack of evidence for QTL segregation shows that they can all be
considered homozygous at the IGF2 locus.
[0148]
Sequence CWU 1
1
116 1 33 DNA Artificial Sequence Stretch of nucleotides which in
the wild type pig, mouse or human IGF2 gene is part of an
evolutionary conserved CpG island 1 gatccttcgc ctaggctcnc
agcgcgggag cga 33 2 19 DNA Artificial Sequence Primer pyro18274F 2
gggccgcggc ttcgcctag 19 3 21 DNA Artificial Sequence Primer
pyro18274R 3 cgcacgcttc tcctgccact g 21 4 19 DNA Artificial
Sequence Primer pyro18274seq 4 ccccacgcgc tcccgcgct 19 5 33 DNA
Artificial Sequence Oligonucleotide Q 5 gatccttcgc ctaggctcac
agcgcgggag cga 33 6 33 DNA Artificial Sequence Oligonucleotide q 6
gatccttcgc ctaggctcgc agcgcgggag cga 33 7 24 DNA Artificial
Sequence Primer PCR1-UP 7 ttgagtgggg attgttgaag tttt 24 8 38 DNA
Artificial Sequence Primer PCR1-DN 8 acccacttat aatctaaaaa
aataataaat atatctaa 38 9 18 DNA Artificial Sequence Primer PCR2-UP
9 ggggattgtt gaagtttt 18 10 19 DNA Artificial Sequence Primer
PCR2-DN 10 cttctcctac cactaaaaa 19 11 19 DNA Artificial Sequence
Primer UP 11 aagcacctgt acccacacg 19 12 18 DNA Artificial Sequence
Primer DN 12 ggctcaggga tcccacag 18 13 21 DNA Artificial Sequence
Primer UP 13 tcatccaggg cctggtcatc g 21 14 20 DNA Artificial
Sequence Primer DN 14 tgtctgaggc cgacacggcc 20 15 17 DNA Sus scrofa
misc_feature (10)..(10) "n" stands for C/T 15 ccccctcccn ggccccc 17
16 17 DNA Sus scrofa misc_feature (10)..(10) "n" stands for C/T 16
acccagggcn ccttgag 17 17 20 DNA Artificial Sequence Primer UP 17
gtgcattatc gggaggtatg 20 18 25 DNA Artificial Sequence Primer DN 18
accctgtatg atactgtaac tctgg 25 19 20 DNA Sus scrofa repeat_region
(10)..(11) The gt at positions 10 and 11 may be repeated any number
of times 19 atagggttag tagatcagtc 20 20 22 DNA Artificial Sequence
Primer UP 20 ctttgaggtc catcatgttc ca 22 21 21 DNA Artificial
Sequence Primer DN 21 ggacgtacat cccatcgatg a 21 22 21 DNA
Artificial Sequence Primer UP 22 atggttgtcc tctgcgtggg c 21 23 21
DNA Artificial Sequence Primer DN 23 tggcggtcga cgtgcagcat c 21 24
19 DNA Sus scrofa misc_feature (11)..(11) "n" stands for C/T 24
tgggtggggg ngcagcccc 19 25 21 DNA Sus scrofa misc_feature (8)..(15)
"n" on pos. 8 stands for C/T; "n" on pos. 15 stands for G/A 25
gctggganca gaccntctgg g 21 26 22 DNA Sus scrofa misc_feature
(13)..(13) "n" stands for C/T 26 ctgtctgctc atncgggggc tg 22 27 22
DNA Sus scrofa misc_feature (13)..(13) "n" stands for C/T 27
ggctgcggga gcntggggcc ac 22 28 22 DNA Sus scrofa misc_feature
(13)..(13) "n" stands for C/T 28 gccacccccg ccntgaccct ga 22 29 21
DNA Artificial Sequence Primer UP 29 atccgcttcc tccagatcct g 21 30
20 DNA Artificial Sequence Primer DN 30 gccgatgtac agcgtggtga 20 31
18 DNA Sus scrofa misc_feature (11)..(11) "n" stands for G/T 31
tctgggccgg ngtccccg 18 32 18 DNA Sus scrofa misc_feature (11)..(11)
"n" stands for A/G 32 aaaagggtcc nggaagct 18 33 21 DNA Sus scrofa
misc_feature (12)..(12) "n" stands for C/T 33 ttgcaaacag cncccagaag
g 21 34 21 DNA Sus scrofa misc_feature (12)..(12) "n" stands for
C/T 34 agaaggcgca gnctccacgg g 21 35 21 DNA Sus scrofa misc_feature
(11)..(11) "n" stands for C/T 35 agggcgctgg ntgcaggggt g 21 36 21
DNA Sus scrofa misc_feature (11)..(11) "n" stands for A/G 36
tttatgagtc ncaaaaacga g 21 37 21 DNA Sus scrofa misc_feature
(12)..(13) "n" on pos. 12 stands for C/T; "n" on pos. 13 stands for
G/T 37 tgatgtccgc cnnggcagac t 21 38 21 DNA Artificial Sequence
Primer UP 38 gccccaagcc caagaagtct g 21 39 21 DNA Artificial
Sequence Primer DN 39 ccagaattgt cacagccatc c 21 40 20 DNA Sus
scrofa misc_feature (11)..(11) "n" stands for A/G 40 tccggggcat
ntaggactgg 20 41 23 DNA Artificial Sequence Primer UP 41 ggagtacctg
ctgtggctta gtg 23 42 25 DNA Artificial Sequence Primer DN 42
cgtcctatat ccatcaggaa tattg 25 43 18 DNA Sus scrofa misc_feature
(9)..(9) "n" stands for C/T 43 agtagtatnc atgagcac 18 44 23 DNA Sus
scrofa misc_feature (11)..(11) "n" stands for G/A 44 cccaggcctc
natcagctgg ttg 23 45 22 DNA Sus scrofa misc_feature (10)..(10) "n"
stands for C/A 45 tatatgccan acatgtggcc ct 22 46 21 DNA Artificial
Sequence Primer UP 46 ggggccatcc aggagtcaca g 21 47 21 DNA
Artificial Sequence Primer DN 47 caaagaggat cacgaggcag g 21 48 21
DNA Artificial Sequence Primer UP 48 tgcgtagcca tggcgatggg g 21 49
25 DNA Artificial Sequence Primer DN 49 agtgtggaac cctggggggg gaagg
25 50 19 DNA Artificial Sequence Primer UP 50 agagggtaca gaagccctg
19 51 23 DNA Artificial Sequence Primer DN 51 tttggtgtgg tgtctgctga
ccc 23 52 21 DNA Artificial Sequence Primer UP 52 aggctttcta
tctgcaggag g 21 53 20 DNA Artificial Sequence Primer DN 53
accgtgtggc catctgggtg 20 54 20 DNA Sus scrofa repeat_region
(10)..(11) The ca at positions 10 and 11 may be repeated any number
of times 54 tctctgtatc acgcacgcac 20 55 20 DNA Artificial Sequence
Primer UP 55 gcgttgcagc ggctctggcg 20 56 21 DNA Artificial Sequence
Primer DN 56 gacacggccg catgaatgtg c 21 57 20 DNA Sus scrofa
repeat_region (10)..(11) The ta at positions 10 and 11 may be
repeated any number of times 57 accccaacat aattatggta 20 58 22 DNA
Artificial Sequence Primer UP 58 gcccgtctac ttcgtgtctg ag 22 59 23
DNA Artificial Sequence Primer DN 59 atctctgcct tcatcgcacc ccc 23
60 21 DNA Sus scrofa misc_feature (12)..(12) "n" stands for A/T 60
aggatccagc cngcagcccc g 21 61 21 DNA Sus scrofa 61 tcacaacccc
cctcccacag c 21 62 20 DNA Sus scrofa 62 tcacaacccc ctcccacagc 20 63
21 DNA Sus scrofa misc_feature (12)..(12) "n" stands for A/G 63
ctgcggaggg gngacctgca g 21 64 20 DNA Artificial Sequence Primer UP
64 gctgcggacc ccaccgtcac 20 65 23 DNA Artificial Sequence Primer DN
65 agacttcacc cctaaaagcc tgg 23 66 25 DNA Sus scrofa 66 gccaggtcaa
ggccaggtcg aggcc 25 67 14 DNA Sus scrofa 67 gccaggtcga ggcc 14 68
19 DNA Artificial Sequence Primer UP 68 agcaggctgc tgtgctggg 19 69
20 DNA Artificial Sequence Primer DN 69 agcccagacc cagctgacgg 20 70
21 DNA Sus scrofa misc_feature (12)..(12) "n" stands for G/A 70
ggcgcttatg gngccgggag c 21 71 21 DNA Sus scrofa misc_feature
(10)..(10) "n" stands for G/T 71 caagcccggn cggtttggcc t 21 72 21
DNA Sus scrofa misc_feature (12)..(12) "n" stands for A/G 72
ctaatgacct cnaggccccc a 21 73 22 DNA Artificial Sequence Primer UP
73 tgatgaccca cggagatgat cc 22 74 28 DNA Artificial Sequence Primer
DN 74 gcagtagttc tccagctggt agagggaa 28 75 21 DNA Sus scrofa
misc_feature (12)..(12) "n" stands for C/T 75 gggaccagct gngttcccag
g 21 76 21 DNA Sus scrofa misc_feature (12)..(12) "n" stands for
C/G 76 gccctgctgg cnctctgggc g 21 77 21 DNA Sus scrofa misc_feature
(12)..(12) "n" stands for C/T 77 ctcccacgcc cnggtcccgc t 21 78 20
DNA Artificial Sequence Primer UP 78 gctctcgcca catcggctgc 20 79 22
DNA Artificial Sequence Primer DN 79 ggcgcccagc tctaggcccg gc 22 80
21 DNA Sus scrofa misc_feature (12)..(12) "n" stands for G/A 80
gggctggctg cngtctggga g 21 81 25 DNA Artificial Sequence Primer UP
81 cccctgaact tgaggacgag cagcc 25 82 23 DNA Artificial Sequence
Primer DN 82 cgctgtgggc tgggtgggct gcc 23 83 21 DNA Sus scrofa
misc_feature (12)..(12) "n" stands for A/G 83 gctgcccccc ancctgagct
g 21 84 20 DNA Artificial Sequence Primer UP 84 cttgcctcca
actccctccc 20 85 20 DNA Artificial Sequence Primer DN 85 agtgaacgtg
aaacgggggg 20 86 24 DNA Sus scrofa repeat_region (12)..(13) The ct
at positions 12 and 13 may be repeated any number of times 86
ctctcgctgt cctcgccctc tctt 24 87 21 DNA Artificial Sequence Primer
UP 87 tgcgccaccc ccgccaagtc c 21 88 23 DNA Artificial Sequence
Primer DN 88 gcttccaggt gtcatagcgg aag 23 89 21 DNA Sus scrofa
misc_feature (12)..(12) "n" stands for G/C 89 agccggctcc tnggcttcaa
g 21 90 21 DNA Sus scrofa misc_feature (12)..(12) "n" stands for
C/T 90 agaggttgtt gntctgggac a 21 91 20 DNA Artificial Sequence
Primer UP 91 caagccaggt cctgtcgagg 20 92 18 DNA Artificial Sequence
Primer DN 92 ggaccctggg ggctgtgg 18 93 21 DNA Sus scrofa
misc_feature (12)..(12) "n" stands for A/G 93 cggcctgtgg cngggaagct
g 21 94 19 DNA Artificial Sequence Primer UP 94 acggtcccgg
gtcagcagg 19 95 20 DNA Artificial Sequence Primer DN 95 cagagcaagt
gggcacccag 20 96 21 DNA Sus scrofa misc_feature (12)..(12) "n"
stands for C/T 96 cgcgggtttg gncagcggca g 21 97 21 DNA Sus scrofa
misc_feature (12)..(12) "n" stands for C/T 97 cacagaggac anggccgctt
c 21 98 21 DNA Sus scrofa misc_feature (12)..(12) "n" stands for
C/T 98 tcctgggggc cngcggctcg t 21 99 22 DNA Artificial Sequence
Primer UP 99 gagcacagcc aaagaacggc cg 22 100 22 DNA Artificial
Sequence Primer DN 100 cttcacccac ggacatggcc gc 22 101 21 DNA Sus
scrofa misc_feature (12)..(12) "n" stands for C/T 101 cacccaggct
gngccctgcg t 21 102 18 DNA Artificial Sequence Primer UP 102
cgggggcact gggggtcc 18 103 20 DNA Artificial Sequence Primer DN 103
ccgagaccct cctcaagtcc 20 104 21 DNA Sus scrofa misc_feature
(12)..(12) "n" stands for A/G 104 gttcgccctc cnctctcagc a 21 105 20
DNA Artificial Sequence Primer UP 105 tgagctgctg agcccacagg 20 106
21 DNA Artificial Sequence Primer DN 106 caagggaaag gtgtgccgac c 21
107 21 DNA Sus scrofa misc_feature (12)..(12) "n" stands for C/T
107 ggccgggcgc tncgccttcc c 21 108 20 DNA Artificial Sequence
Primer UP 108 aggcagaggg cagagagggg 20 109 19 DNA Artificial
Sequence Primer DN 109 ctccagcccc acactctgc 19 110 21 DNA Sus
scrofa misc_feature (12)..(12) "n" stands for C/T 110 gcgtccagcg
cngaatcagg c 21 111 14 DNA Artificial Sequence MH1 111 tcgcaaggcc
aggt 14 112 14 DNA Artificial Sequence MH3 112 acgcaaggcc aggt 14
113 94 DNA Sus scrofa 113 agccagggac gagcctgccc gcggcggcag
ccgggccgcg gcttcgccta ggctcgcagc 60 gcgggagcgc gtggggcgcg
gcggcggcgg ggag 94 114 94 DNA Sus scrofa 114 agccagggac gagcctgccc
gcggcggcag ccgggccgcg gcttcgccta ggctcacagc 60 gcgggagcgc
gtggggcgcg gcggcggcgg ggag 94 115 94 DNA Homo sapiens 115
agccggggac tagcctgctc ccggtggcgg ctcggccgcg gcttcgccta ggctcgcagc
60 gcggaggcga gtggggcgca gtggcgaggg ggag 94 116 94 DNA Mus sp. 116
agctagggac gagtctgccc ccggcggctg cctggccccg acttcgccta ggctcgcggc
60 gtctgagcgc gtggggcgca ggggcggcgg ggag 94
* * * * *
References