U.S. patent application number 10/339279 was filed with the patent office on 2004-03-11 for novel calpastatin (cast) alleles.
This patent application is currently assigned to Iowa State University Foundation, Inc.. Invention is credited to Ciobanu, Daniel C., Rothschild, Max F..
Application Number | 20040048267 10/339279 |
Document ID | / |
Family ID | 23362763 |
Filed Date | 2004-03-11 |
United States Patent
Application |
20040048267 |
Kind Code |
A1 |
Rothschild, Max F. ; et
al. |
March 11, 2004 |
Novel calpastatin (CAST) alleles
Abstract
Disclosed herein are novel alleles characterized by
polymorphisms in the CAST gene. The alleles may be used to
genetically type animals. In a preferred embodiment, the alleles
may be used as markers for animal meat quality and/or growth.
Methods for identifying such markers, and methods of screening
animals to determine those more likely to produce desired meat
quality and/or growth and preferably selecting those animals for
future breeding purposes are also disclosed.
Inventors: |
Rothschild, Max F.; (Ames,
IA) ; Ciobanu, Daniel C.; (Ames, IA) |
Correspondence
Address: |
MCKEE, VOORHEES & SEASE, P.L.C.
801 GRAND AVENUE
SUITE 3200
DES MOINES
IA
50309-2721
US
|
Assignee: |
Iowa State University Foundation,
Inc.
Ames
IA
|
Family ID: |
23362763 |
Appl. No.: |
10/339279 |
Filed: |
January 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60347209 |
Jan 9, 2002 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/6.1; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/8107 20130101;
C12Q 1/6876 20130101; C07K 14/8139 20130101; C12Q 2600/156
20130101 |
Class at
Publication: |
435/006 ;
530/350; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/47 |
Goverment Interests
[0002] This invention was supported at least in part by USDA/CREES
contract number 2001-31200-06019 (IAHAEES project number IOW03600).
The United States government may have certain rights in this
invention.
Claims
What is claimed is:
1. A nucleotide sequence which encodes a CAST skeletal muscle
protein, having an asparagine at amino acid 66 of SEQ ID NO: 2 or
its equivalent as determined by BLAST, said nucleotide sequence
comprising one or more of the following: (a) SEQ ID NO: 3, (b) a
sequence which will hybridize under conditions of high stringency
to SEQ ID NO: 3, or (c) a sequence with at least about 80% homology
to SEQ ID NO: 3.
2. A nucleotide sequence which encodes a CAST skeletal muscle
protein, having an lysine at amino acid 249 of SEQ ID NO: 2 or its
equivalent as determined by BLAST said nucleotide sequence
comprising one of the following: (a) SEQ ID NO: 5, (b) a sequence
which will hybridize under conditions of high stringency to SEQ ID
NO: 5, or (c) a sequence with at least about 80% homology to SEQ ID
NO: 5.
3. A nucleotide sequence which encodes a CAST skeletal muscle
protein, having an threonine at amino acid 504 of SEQ ID NO: 2 or
its equivalent as determined by BLAST said nucleotide sequence
comprising one of the following: (a) SEQ ID NO: 7, (b) a sequence
which will hybridize under conditions of high stringency to SEQ ID
NO: 7, or (c) a sequence with at least about 80% homology to SEQ ID
NO: 7.
4. A nucleotide sequence which encodes a CAST skeletal muscle
protein, having a serine at amino acid 638 of SEQ ID NO: 2 or its
equivalent as determined by BLAST said nucleotide sequence
comprising one of the following: (a) SEQ ID NO: 9, (b) a sequence
which will hybridize under conditions of high stringency to SEQ ID
NO: 9, or (c) a sequence with at least about 80% homology to SEQ ID
NO: 9.
5. A nucleotide sequence which encodes upon expression an CAST
protein, said CAST protein characterized by one of more of the
following: a lysine at amino acid position number 249, a threonine
at amino acid position number 504, a serine at amino acid position
638, an asparagine at amino acid position number 66 or their
equivalents as determine by a BLAST comparison of SEQ ID NO: 4, 6,
8, or 10.
6. A CAST protein according to claim 5.
7. A CAST skeletal muscle protein, said protein comprising an amino
acid sequence comprising one of the following: (a) SEQ ID NO: 4,
(b) conservatively modified variant of SEQ ID NO: 4, or (c) a
sequence with at least about 80% homology to SEQ ID NO: 4.
8. A CAST skeletal protein, said protein comprising an amino acid
sequence comprising one of the following: (a) SEQ ID NO: 6, (b)
conservatively modified variant of SEQ ID NO: 6, or (c) a sequence
with at least about 80% homology to SEQ ID NO: 6.
9. A CAST skeletal protein, said protein comprising an amino acid
sequence comprising one of the following: (a) SEQ ID NO: 8, (b)
conservatively modified variant of SEQ ID NO: 8, or (c) a sequence
with at least about 80% homology to SEQ ID NO: 8.
10. A CAST skeletal protein, said protein comprising an amino acid
sequence comprising one of the following: (a) SEQ ID NO: 10, (b)
conservatively modified variant of SEQ ID NO: 10, or (c) a sequence
with at least about 80% homology to SEQ ID NO: 10.
11. A method of genetically typing animals comprising: obtaining a
sample of genetic material from said animal; assaying for the
presence of an allele characterized by a polymorphism in a CAST
gene present in said sample, and correlating said allele with said
animal.
12. The method of claim 11 wherein said polymorphism results in an
amino acid change from arginine to lysine at amino acid number 249
of the CAST gene product or its equivalent as determined by a BLAST
comparison of SEQ ID NO: 2.
13. The method of claim 11 wherein said polymorphism is a
transition of a adenine to a guanine at nucleotide position 812 or
its equivalent.
14. The method of claim 11 wherein said genotype is a Hpy188I
polymorphism.
15. The method of claim 11 wherein said polymorphism results in an
amino acid change from alanine to threonine at amino acid number
504 of the CAST gene product or its equivalent as determined by a
BLAST comparison of SEQ ID NO: 2.
16. The method of claim 11 wherein said polymorphism is a
transition of a adenine to a cytosine at nucleotide position 1980
or its equivalent.
17. The method of claim 11 wherein said genotype is a PvuII
polymorphism.
18. The method of claim 11 wherein said polymorphism results in an
amino acid change from arginine to serine at amino acid number 638
of the CAST gene or its equivalent as determined by a BLAST
comparison of SEQ ID NO: 2.
19. The method of claim 11 wherein said polymorphism is a
transition of a adenine to a guanine at nucleotide position 1576 or
its equivalent.
20. The method of claim 11 wherein said genotype is a AciI
polymorphism.
21. The method of claim 11 wherein said polymorphism results in an
amino acid change from serine to asparagine at amino acid number 66
of the CAST gene or its equivalent as determined by a BLAST
comparison of SEQ ID NO: 2.
22. The method of claim 11 wherein said polymorphism is a
transition of a adenine to a guanine at nucleotide position 263 or
its equivalent.
23. The method of claim 11 wherein said genotype is a ApaLI
polymorphism.
24. The method of claim 11 wherein said step of assaying is
selected from the group consisting of: restriction fragment length
polymorphism (RFLP) analysis, minisequencing, MALD-TOF, SINE,
heteroduplex analysis, one base extension methods, single strand
conformational polymorphism (SSCP), denaturing gradient gel
electrophoresis (DGGE) and temperature gradient gel electrophoresis
(TGGE).
25. The method of claim 11 wherein said animal is a pig.
26. The method of claim 11 wherein said animal is a cow.
27. The method of claim 11 further comprising the step of
amplifying the amount of CAST gene or a portion thereof which
contains said polymorphism.
28. The method of claim 27 wherein said amplification includes the
steps of: selecting a forward and a reverse sequence primer capable
of amplifying a region of the CAST gene which contains a
polymorphic Hpy188I site.
29. The method of claim 28 wherein said forward and reverse primers
are selected from and based upon primer SEQ ID NO: 11 and primer
SEQ ID NO: 12.
30. The method of claim 27 wherein said amplification includes the
steps of: selecting a forward and a reverse sequence primer capable
of amplifying a region of the CAST gene which contains a
polymorphic PvuII site.
31. The method of claim 30 wherein said forward and reverse primers
are selected from and based upon primer SEQ ID NO: 13 and primer
SEQ ID NO:14.
32. The method of claim 27 wherein said amplification includes the
steps of: selecting a forward and a reverse sequence primer capable
of amplifying a region of the CAST gene which contains a
polymorphic AciI site.
33. The method of claim 30 wherein said forward and reverse primers
are selected from and based upon primer SEQ ID NO: 15 and primer
SEQ ID NO: 16.
34. The method of claim 32 wherein said amplification includes the
steps of: selecting a forward and a reverse sequence primer capable
of amplifying a region of the CAST gene which contains a
polymorphic ApaLI site.
35. The method of claim 34 wherein said forward and reverse primers
are selected from and based upon primer SEQ ID NO: 17 and primer
SEQ ID NO: 18.
36. A method of screening animals to determine those more likely to
exhibit improved meat quality and/or growth traits comprising:
obtaining a biological sample of material from said animal; and
assaying for the presence of a genotype in said animal which is
associated with improved meat quality and/or growth traits said
genotype characterized by the following: a) a polymorphism in the
CAST gene, said polymorphism resulting in and characterized by an
amino acid at a position selected from the group consisting of
lysine at position 249, threonine at position 504, serine at
position 638, or asparagine at position 66 or their equivalents as
determined by a BLAST comparison of SEQ ID NO: 2.
37. The method of claim 36 wherein said polymorphism results in and
is characterized by a guanine at position 812, a cytosine at
position 1980, a guanine at position 1576, a guanine at position
263 or their equivalents as determined by a BLAST comparison of SEQ
ID NO: 1.
38. A method for screening animals to determine those with a
favorable combination of traits for meat quality and/or growth,
which method comprises of the steps: determining the alleles of
CAST present in a animal said alleles comprising those which
include one or more of the following a polymorphic Hpy188I, PvuII,
AciI, or ApaLI site in the CAST gene; determining the alleles of
other markers for genes known to affect meat quality and/or growth;
and selecting for animals with favorable combinations of alleles
and against those carrying unfavorable combinations.
39. The method of claim 36 wherein the determination of CAST
alleles comprises determining the presence of at least one allele
associated with at least one DNA marker linked either directly or
indirectly to CAST.
40. The method as claimed in claim 38 wherein the DNA marker is a
microsatellite.
41. A method of screening animals to determine those more likely to
produce desired meat quality and/or growth comprising: obtaining a
sample of genetic material from said animal; and assaying for the
presence of a genotype in said animal which is associated with
improved meat quality and/or growth, said genotype characterized by
the following: a) a polymorphism in the CAST gene, said
polymorphism located in one of the following areas: exon 13 domain
1, exon 27 domain 4, exon 22, domain 3, or exon 6 domain L.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. .sctn.
119(e) of provisional application 60/347,209 filed Jan. 9,
2002.
FIELD OF THE INVENTION
[0003] This invention relates generally to the detection of genetic
differences among animals. More particularly, the invention relates
to genetic markers that are indicative of heritable phenotypes
associated with improved growth, meat quality and other such
economic traits in animals. Methods and compositions for use of
these markers in genotyping of animals and selection are also
disclosed as well as novel sequences.
BACKGROUND OF THE INVENTION
[0004] Genetic differences exist among individual animals as well
as among breeds which can be exploited by breeding techniques to
achieve animals with desirable characteristics. For example,
Chinese breeds are known for reaching puberty at an early age and
for their large litter size, while American breeds are known for
their greater growth rates and leanness. However, heritability for
desired traits is often low, and standard breeding methods which
select individuals based upon phenotypic variations do not take
fully into account genetic variability or complex gene interactions
which exist.
[0005] Restriction fragment length polymorphism (RFLP) analysis has
been used by several groups to study pig DNA. Jung et al., Theor.
Appl. Genet., 77:271-274 (1989), incorporated herein by reference,
discloses the use of RFLP techniques to show genetic variability
between two pig breeds. Polymorphism was demonstrated for swine
leukocyte antigen (SLA) Class I genes in these breeds. Hoganson et
al., Abstract for Annual Meeting of Midwestern Section of the
American Society of Animal Science, Mar. 26-28, 1990, incorporated
herein by reference, reports on the polymorphism of swine major
histocompatibility complex (MHC) genes for Chinese pigs, also
demonstrated by RFLP analysis. Jung et al., Theor. Appl. Genet.,
77:271-274 (1989), incorporated herein by reference, reports on
RFLP analysis of SLA Class I genes in certain boars. The authors
state that the results suggest that there may be an association
between swine SLA/MHC Class I genes and production and performance
traits. They further state that the use of SLA Class I restriction
fragments, as genetic markers, may have potential in the future for
improving pig growth performance.
[0006] The ability to follow a specific favorable genetic allele
involves a novel and lengthy process of the identification of a DNA
molecular marker for a major effect gene. The marker may be linked
to a single gene with a major effect or linked to a number of genes
with additive effects. DNA markers have several advantages;
segregation is easy to measure and is unambiguous, and DNA markers
are co-dominant, i.e., heterozygous and homozygous animals can be
distinctively identified. Once a marker system is established
selection decisions could be made very easily, since DNA markers
can be assayed any time after a tissue or blood sample can be
collected from the individual infant animal, or even an embryo.
[0007] The use of genetic differences in receptor genes has become
a valuable marker system for selection. For example, U.S. Pat. Nos.
5,550,024 and 5,374,526 issued to Rothschild et al. disclose a
polymorphism in the pig estrogen receptor gene which is associated
with larger litter size, the disclosure of which is incorporated
herein by reference. U.S. Pat. No. 5,935,784 discloses polymorphic
markers in the pig prolactin receptor gene which are associated
with larger litter size and overall reproductive efficiency.
[0008] The quality of raw pig meat is influenced by a large number
of genetic and non-genetic factors. The latter include farm,
transport, slaughter and processing conditions. Meat scientists
have performed a substantial amount of research on these factors,
which has led to considerable quality improvement. Part of the
research has also been dedicated to the genetic background of the
animals, and several studies have revealed the importance of
genetic factors. This has made the industry aware that selective
breeding of animals and the use of gene technology can play an
important role in enhancing pork quality.
[0009] Information at DNA level can help to fix a specific major
gene, but it can also assist the selection of a quantitative trait
for which we already select. Molecular information in addition to
phenotypic data can increase the accuracy of selection and
therefore the selection response. The size of the extra response in
such a Marker Assisted Selection (MAS) program has been considered
by many workers from a theoretical point of view. In general terms,
MAS is more beneficial for traits with a low heritability and which
are expensive to measure phenotypically. Although traits such as
meat quality and/or growth are not typically considered in this way
there are still significant advantages for the use of markers for
these traits. For example, Meuwissen and Goddard considered the
impact of MAS for different types of traits. The biggest impacts
were for traits such as meat quality, where the trait is measured
after slaughter and an additional response of up to 64% could be
achieved with the incorporation of marker information. This figure
was relatively small, 8%, for growth traits, that can be measured
on the live animal. However, once the association has been
demonstrated this marker information can be used before the animals
are tested or selected phenotypically (see below) and in this
situation a response of up to 38% was predicted.
[0010] Indeed, the best approach to genetically improve economic
traits is to find relevant DNA-markers directly in the population
under selection. Phenotypic measurements can be performed
continuously on some animals from the, nucleus populations of
breeding organizations. Since a full assessment of most of these
traits can only be done after slaughter, the data must be collected
on culled animals and cannot be obtained on potential breeding
animals.
[0011] This phenotypic data is collected in order to enable the
detection of relevant DNA markers, and to validate markers
identified using experimental populations or to test candidate
genes. Significant markers or genes can then be included directly
in the selection process. An advantage of the molecular information
is that we can obtain it already at very young age of the breeding
animal, which means that animals can be preselected based on DNA
markers before the growing performance test is completed. This is a
great advantage for the overall testing and selection system.
[0012] It can be seen from the foregoing that a need exists for
identification of markers which may be used to improve economically
beneficial characteristics in animals by identifying and selecting
animals with the improved characteristics at the genetic level.
[0013] An object of the present invention is to provide genetic
markers based on or within the calpastatin (CAST) gene which are
indicative of favorable economic characteristics such as meat
quality and/or growth.
[0014] Another object of the invention is to provide an assay for
determining the presence of these genetic markers.
[0015] A further object of the invention is to provide a method of
evaluating animals that increases accuracy of selection and
breeding methods for the desired traits.
[0016] Yet another object of the invention is to provide a PCR
amplification test which will greatly expedite the determination of
presence of the markers.
[0017] Additional objects and advantages of the invention will be
set forth in part in the description that follows, and in part will
be obvious from the description, or may be learned by the practice
of the invention. The objects and advantages of the invention will
be attained by means of the instrumentality's and combinations
particularly pointed out in the appended claims.
SUMMARY OF THE INVENTION
[0018] This invention relates to the discovery of alternate forms
of the calpastatin or CAST gene which are useful to genetically
type animals. The may be used for following lineages in breeding,
or in a preferred embodiment the novel gene forms may be used as
genetic markers associated with phenotypic differences which may be
selected for or against. In an even more preferred embodiement the
phenotypic differences are meat quality and growth traits. To the
extent that this gene is conserved among species and animals, and
it is expected that the different alleles disclosed herein will
also correlate with variability in this gene in other economic or
meat-producing animals such as bovine, sheep, chicken, etc.
[0019] To achieve the objects and in accordance with the purpose of
the invention, as embodied and broadly described herein, the
present invention provides the discovery of alternate genotypes
which provide a method for genetically typing animals, preferrably
for screening animals to determine those more likely to possess
favorable meat quality and/or growth traits or to select against
pigs which have alleles indicating less favorable growth and/or
meat quality traits. As used herein "favorable growth or meat
quality trait" means a significant improvement (increase or
decrease) in one of many measurable meat quality or growth traits
above the mean of a given population, so that this information can
be used in breeding to achieve a uniform population which is
optimized for meat quality and/or growth, this may include an
increase in some traits or a decrease in others depending on the
desired characteristics. These factors for meat quality which may
be considered include but are not limited to the following:
[0020] Loin Minolta Lightness (L*): The range of 43-47 units (from
darker to lighter color) is acceptable, but L* of 43 is better;
i.e., has higher economic value, in general in this range (this may
be dependent upon market, for example in Japan darker pork is
preferred).
[0021] Loin Japanese Color Score (JCS): The range of 2.5-5.0 units
(from lighter to darker color) is acceptable, but JCS of 3-4 is
better.
[0022] Loin Marbling (level of intramuscular fat): Generally,
higher marbling is better as it is associated with improved meat
eating quality characteristics.
[0023] Loin pH: (ultimate meat acidity measured 24 hours
post-mortem; this attribute is the single most important trait of
pork quality); --The range of 5.50-5-80 is desirable, but 5.80 is
better as it positively influences the color and (low) purge of the
meat.
[0024] Ham Minolta lightness (L*) The range of 43-52 units is
acceptable, but lower (43) is better.
[0025] Ham pHu: higher; i.e., 5.80, is better.
[0026] Drip loss or purge: the range of 1%-3% is acceptable, but
lower is better.
[0027] These measures of meat quality are examples of those
generally accepted by those of skill in the art. For a review of
meat quality traits the following may be consulted: Sosnicki, A.
A., E. R. Wilson, E. B. Sheiss, A. deVries, 1998 "Is there a cost
effective way to produce high quality pork?", Reciprocal Meat
Conference Proceedings, Vol. 51.
[0028] Growth can be measured by any of a number of standard means
such as average daily gain, weight at slaughter, etc.
[0029] Thus, the present invention provides a method for screening
pigs to identify those more likely to produce favorable meat
quality and/or growth, and/or those less likely to produce
favorable meat quality and/or growth to optimize breeding and
selection techniques for the best meat quality and/or growth.
[0030] Methods for assaying for these traits generally comprises
the steps 1) obtaining a biological sample from a pig; and 2)
analyzing the genomic DNA or protein obtained in 1) to determine
which CAST allele(s) is/are present. Also included herein are
haplotype data which allows for a series of polymorphisms in the
CAST gene to be combined in a selection or identification protocol
to maximize the benefits of each of these markers.
[0031] Since several of the polymorphisms involve changes in amino
acid composition of the CAST protein, assay methods may even
involve ascertaining the amino acid composition of the CAST
protein. Methods for this type or purification and analysis
typically involve isolation of the protein through means including
fluorescence tagging with antibodies, separation and purification
of the protein (i.e. through reverse phase HPLC system), and use of
an automated protein sequencer to identify the amino acid sequence
present. Protocols for this assay are standard and known in the art
and are disclosed in Ausubel et. al.(eds.), Short Protocols in
Molecular Biology Fourth ed. John Wiley and Sons 1999.
[0032] In a preferred embodiment a genetic sample is analyzed.
Briefly, a sample of genetic material is obtained from an animal,
and the sample is analyzed to determine the presence or absence of
a polymorphism in the CAST gene that is correlated with improved
meat quality and/or growth or both traits depending on the gene
form.
[0033] As is well known to those of skill in the art, a variety of
techniques may be utilized when comparing nucleic acid molecules
for sequence differences. These include by way of example,
restriction fragment length polymorphism analysis, heteroduplex
analysis, single strand conformation polymorphism analysis,
denaturing gradient electrophoresis and temperature gradient
electrophoresis.
[0034] In a preferred embodiment the polymorphism is a restriction
fragment length polymorphism and the assay comprises identifying
the CAST gene from isolated genetic material; exposing the gene to
a restriction enzyme that yields restriction fragments of the gene
of varying length; separating the restriction fragments to form a
restriction pattern, such as by electrophoresis or HPLC separation;
and comparing the resulting restriction fragment pattern from a
CAST gene that is either known to have or not to have the desired
marker. If an animal tests positive for the markers, such animal
can be considered for inclusion in the breeding program. If the
animal does not test positive for the marker genotype the animal
can be culled from the group and otherwise used. Use of haplotype
data can also be incorporated with the screening for multiple
alleles for different aspects of meat quality and/or growth.
[0035] In a most preferred embodiment the gene is isolated by the
use of primers and DNA polymerase to amplify a specific region of
the gene which contains the polymorphism. Next the amplified region
is digested with a restriction enzyme and fragments are again
separated. Visualization of the RFLP pattern is by simple staining
of the fragments, or by labeling the primers or the nucleoside
triphosphates used in amplification.
[0036] In another embodiment, the invention comprises a method for
identifying a genetic marker for meat quality and/or growth in a
particular population. Male and female animals of the same breed or
breed cross or similar genetic lineage is bred, and meat quality
and/or growth produced by each pig is determined. A polymorphism in
the CAST gene of each pig is identified and associated with the
meat quality and/or growth. Preferably, RFLP analysis is used to
determine the polymorphism.
[0037] In another embodiment, the invention comprises a method for
identifying a genetic marker for meat quality and/or growth in any
particular economic animal other than a pig. Based upon the highly
conserved nature of this gene among different animals and the
location of the polymorphisms within these highly conserved
regions, is it expected that with no more than routine testing as
described herein this marker can be applied to different animal
species to select for meat quality and/or growth based on the
teachings herein. Male and female animals of the same breed or
breed cross or similar genetic lineage are bred, and the meat
quality and/or growth produced by each animal is determined and
correlated. For other animals in which sequences are available a
BLAST comparison of sequences may be used to ascertain whether the
particular allele is analogous to the one disclosed herein. The
analogous polymorphism will be present in other animals and in
other closely related genes. The term "analogous polymorphism"
shall be a polymorphism which is the same as any of those disclosed
herein as determined by BLAST comparisons.
[0038] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence", (b) "comparison window", (c) "sequence
identity", (d) "percentage of sequence identity", and (e)
"substantial identity".
[0039] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. In this case the
Reference CAST sequence. A reference sequence may be a subset or
the entirety of a specified sequence; for example, as a segment of
a full-length cDNA or gene sequence, or the complete cDNA or gene
sequence.
[0040] (b) As used herein, "comparison window" includes reference
to a contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence may be compared to a reference
sequence and wherein the portion of the polynucleotide sequence in
the comparison window may comprise additions or deletions (i.e.,
gaps) compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Generally, the comparison window is at least 20 contiguous
nucleotides in length, and optionally can be 30, 40, 50, 100, or
longer. Those of skill in the art understand that to avoid a high
similarity to a reference sequence due to inclusion of gaps in the
polynucleotide sequence, a gap penalty is typically introduced and
is subtracted from the number of matches.
[0041] I Methods of alignment of sequences for comparison are
well-known in the art. Optimal alignment of sequences for
comparison may be conducted by the local homology algorithm of
Smith and Waterman, Adv. Appl. Math. 2:482 (1981); by the homology
alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443
(1970); by the search for similarity method of Pearson and Lipman,
Proc. Natl. Acad. Sci. 85:2444 (1988); by computerized
implementations of these algorithms, including, but not limited to:
CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View,
Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group (GCG), 575
Science Dr., Madison, Wis., USA; the CLUSTAL program is well
described by Higgins and Sharp, Gene 73:237-244 (1988); Higgins and
Sharp, CABIOS 5:151-153 (1989); Corpet, et al., Nucleic Acids
Research 16:10881-90 (1988); Huang, et al., Computer Applications
in the Biosciences 8:155-65 (1992), and Pearson, et al., Methods in
Molecular Biology 24:307-331 (1994). The BLAST family of programs
which can be used for database similarity searches includes: BLASTN
for nucleotide query sequences against nucleotide database
sequences; BLASTX for nucleotide query sequences against protein
database sequences; BLASTP for protein query sequences against
protein database sequences; TBLASTN for protein query sequences
against nucleotide database sequences; and TBLASTX for nucleotide
query sequences against nucleotide database sequences. See, Current
Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds.,
Greene Publishing and Wiley-Interscience, New York (1995).
[0042] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using the BLAST 2.0
suite of programs using default parameters. Altschul et a., Nucleic
Acids Res. 25:3389-3402 (1997). Software for performing BLAST
analyses is publicly available, e.g., through the National Center
for Biotechnology-Informatio- n (http://www.hcbi.nlm.nih.gov/.
[0043] This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the
query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both
strands. For amino acid sequences, the BLASTP program uses as
defaults a wordlength (W) of 3, an expectation (E) of 10, and the
BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc.
Natl. Acad. Sci. USA 89:10915).
[0044] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul,
Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance.
[0045] BLAST searches assume that proteins can be modeled as random
sequences. However, many real proteins comprise regions of
nonrandom sequences which may be homopolymeric tracts, short-period
repeats, or regions enriched in one or more amino acids. Such
low-complexity regions may be aligned between unrelated proteins
even though other regions of the protein are entirely dissimilar. A
number of low-complexity filter programs can be employed to reduce
such low-complexity alignments. For example, the SEG (Wooten and
Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie and
States, Comput. Chem., 17:191-201 (1993)) low-complexity filters
can be employed alone or in combination.
[0046] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences includes
reference to the residues in the two sequences which are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g. charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. Where sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences which differ by such conservative substitutions are said
to have "sequence similarity" or "similarity". Means for making
this adjustment are well-known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., according to the algorithm of
Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988)
e.g., as implemented in the program PC/GENE (Intelligenetics,
Mountain View, Calif., USA).
[0047] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0048] (e)(I) The term "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70% sequence identity, preferably at least 80%, more
preferably at least 90% and most preferably at least 95%, compared
to a reference sequence using one of the alignment programs
described using standard parameters. One of skill will recognize
that these values can be appropriately adjusted to determine
corresponding identity of proteins encoded by two nucleotide
sequences by taking into account codon degeneracy, amino acid
similarity, reading frame positioning and the like. Substantial
identity of amino acid sequences for these purposes normally means
sequence identity of at least 60%, or preferably at least 70%, 80%,
90%, and most preferably at least 95%.
[0049] These programs and algorithms can ascertain the analogy of a
particular polymorphism in a target gene to those disclosed herein.
It is expected that this polymorphism will exist in other animals
and use of the same in other animals than disclosed herein involved
no more than routine optimization of parameters using the teachings
herein.
[0050] It is also possible to establish linkage between specific
alleles of alternative DNA markers and alleles of DNA markers known
to be associated with a particular gene (e.g. the CAST gene
discussed herein), which have previously been shown to be
associated with a particular trait. Thus, in the present situation,
taking the CAST gene, it would be possible, at least in the short
term, to select for animals likely to produce desired meat quality
and/or growth, or alternatively against pigs likely to produce less
desirable meat quality and/or growth, indirectly, by selecting for
certain alleles of a CAST associated marker through the selection
of specific alleles of alternative chromosome markers. As used
herein the term "genetic marker" shall include not only the
polymorphism disclosed by any means of assaying for the protein
changes associated with the polymorphism, be they linked markers,
use of microsatellites, or even other means of assaying for the
causative protein changes indicated by the marker and the use of
the same to influence the meat quality and/or growth of an
animal.
[0051] As used herein, often the designation of a particular
polymorphism is made by the name of a particular restriction
enzyme. This is not intended to imply that the only way that the
site can be identified is by the use of that restriction enzyme.
There are numerous databases and resources available to those of
skill in the art to identify other restriction enzymes which can be
used to identify a particular polymorphism, for example
http://darwin.bio.geneseo.edu which can give restriction enzymes
upon analysis of a sequence and the polymorphism to be identified.
In fact as disclosed in the teachings herein there are numerous
ways of identifying a particular polymorphism or allele with
alternate methods which may not even include a restriction enzyme,
but which assay for the same genetic or proteomic alternative
form.
[0052] In yet another embodiment of this invention novel porcine
nucleotide sequences have been identified and are disclosed which
encode porcine CAST. The cDNA of the porcine CAST gene as well as
some intronic DNA sequences are disclosed. These sequences may be
used for the design of primers to assay for the SNP's of the
invention or for production of recombinant CAST. The invention is
intended to include these sequences as well as all conservatively
modified variants thereof as well as those sequences which will
hybridize under conditions of high stringency to the sequences
disclosed. The term CAST as used herein shall be interpreted to
include these conservatively modified variants as well as those
hybridized sequences.
[0053] The term "conservatively modified variants" applies to both
amino acid and nucleic acid sequences. With respect to particular
nucleic acid sequences, conservatively modified variants refers to
those nucleic acids which encode identical or conservatively
modified variants of the amino acid sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations" and represent one species
of conservatively modified variation. Every nucleic acid sequence
herein that encodes a polypeptide also, by reference to the genetic
code, describes every possible silent variation of the nucleic
acid. One of ordinary skill will recognize that each codon in a
nucleic acid (except AUG, which is ordinarily the only codon for
methionine; and UGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical
molecule. Accordingly, each silent variation of a nucleic acid
which encodes a polypeptide of the present invention is implicit in
each described polypeptide sequence and is within the scope of the
present invention.
[0054] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Thus, any number of amino acid
residues selected from the group of integers consisting of from 1
to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7, or 10
alterations can be made. Conservatively modified variants typically
provide similar biological activity as the unmodified polypeptide
sequence from which they are derived. For example, substrate
specificity, enzyme activity, or ligand/receptor binding is
generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the
native protein for its native substrate. Conservative substitution
tables providing functionally similar amino acids are well known in
the art.
[0055] The following six groups each contain amino acids that are
conservative substitutions for one another:
[0056] 1) Alanine (A), Serine (S), Threonine (T);
[0057] 2) Aspartic acid (D), Glutamic acid (E);
[0058] 3) Asparagine (N), Glutamine (Q);
[0059] 4) Arginine (R), Lysine (K);
[0060] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0061] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0062] See also, Creighton (1984) Proteins W. H. Freeman and
Company.
[0063] By "encoding" or "encoded", with respect to a specified
nucleic acid, is meant comprising the information for translation
into the specified protein. A nucleic acid encoding a protein may
comprise non-translated sequences (e.g., introns) within translated
regions of the nucleic acid, or may lack such intervening
non-translated sequences (e.g., as in cDNA). The information by
which a protein is encoded is specified by the use of codons.
Typically, the amino acid sequence is encoded by the nucleic acid
using the "universal" genetic code. However, variants of the
universal code, such as are present in some plant, animal, and
fungal mitochondria, the bacterium Mycoplasma capricolum, or the
ciliate Macronucleus, may be used when the nucleic acid is
expressed therein.
[0064] The term "stringent conditions" or "stringent hybridization
conditions" includes reference to conditions under which a probe
will hybridize to its target sequence, to a detectably greater
degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and be
different in different circumstances. By controlling the stringency
of the hybridization and/or washing conditions, target sequences
can be identified which are 100% complementary to the probe
(homologous probing). Alternatively, stringency conditions can be
adjusted to allow some mismatching in sequences so that lower
degrees of similarity are detected (heterologous probing).
Generally, a probe is less than about 1000 nucleotides in length,
optionally less than 500 nucleotides in length.
[0065] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree.
C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M
NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary
moderate stringency conditions include hybridization in 40 to 45%
formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.5.times. to 1.times.SSC at 55 to 50.degree. C. Exemplary high
stringency conditions include hybridization in 50% formnamide, 1 M
NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to
65.degree. C.
[0066] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the
T.sub.m can be approximated from the equation of Meinkoth and Wahl,
Anal. Biochem., 138:267-284 (1984): T.sub.m=81.5.degree. C. +16.6
(log M)+0.41 (% GC)-0.61 (1% form)-500/L; where M is the molarity
of monovalent cations, % GC is the percentage of guanosine and
cytosine nucleotides in the DNA, % form is the percentage of
formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the complementary target
sequence hybridizes to a perfectly matched probe. T.sub.m is
reduced by about 1.degree. C. for each 1% of mismatching; thus,
T.sub.m, hybridization and/or wash conditions can be adjusted to
hybridize to sequences of the desired identity. For example, if
sequences with .gtoreq.90% identity are sought, the T.sub.m can be
decreased 10.degree. C. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence and its complement at a
defined ionic strength and pH. However, severely stringent
conditions can utilize a hybridization and/or wash at 1, 2, 3, or
4.degree. C. lower than the thermal melting point (T.sub.m);
moderately stringent conditions can utilize a hybridization and/or
wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting
point (T.sub.m); low stringency conditions can utilize a
hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C.
lower than the thermal melting point (T.sub.m). Using the equation,
hybridization and wash compositions, and desired T.sub.m, those of
ordinary skill will understand that variations in the stringency of
hybridization and/or wash solutions are inherently described. If
the desired degree of mismatching results in a T.sub.m of less than
45.degree. C. (aqueous solution) or 32.degree. C. (formamide
solution) it is preferred to increase the SSC concentration so that
a higher temperature can be used. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, Laboratory
Techniques in Biochemistry and Molecular Biology-Hybridization with
Nucleic Acids Probes, Part I, Chapter 2, Ausubel, et al., Eds.,
Greene Publishing and Wiley-Interscience, New York (1995).
[0067] The accompanying figures, which are incorporated herein and
which constitute a part of this specification, illustrates one
embodiment of the invention and, together with the description,
serve to explain the principles of the invention.
DESCRIPTION OF THE FIGURES
[0068] FIG. 1a is the porcine skeletal calpastatin cDNA sequence
including the polymorphisms of the invention. FIG. 1b is the amino
acid sequence.
[0069] FIG. 2a is a sketch of the expected banding pattern of the
different genotypes for the Hpy188I polymorphism. FIG. 2b is the
sequence around the Hpy188I polymorphism (SEQ ID NO: 21).
[0070] FIG. 3a is a sketch of the expected banding pattern of the
different genotypes for the PvuII polymorphism. FIG. 3b is the
sequence around the PvuII polymorphism (SEQ ID NO: 22).
[0071] FIG. 4a is a sketch of the expected banding pattern of the
different genotypes for the AciI polymorphism. FIG. 4b is the
sequence around the AciI polymorphism (SEQ ID NO: 23).
[0072] FIG. 5a is a sketch of the expected banding pattern of the
different genotypes for the ApaLI polymorphism. FIG. 5b is the
sequence around the ApaLI polymorphism (SEQ ID NO: 24 and 25).
[0073] FIG. 6 is a graph showing calpastin activity at 24hr and
cast Hpy188I genotypes 11 and 12.
[0074] FIG. 7 is an alignment between the Bos taurus (SEQ ID NO:
19) and Sus scrofa (SEQ ID NO: 20) sequences for the exon 6
polymorphism using the Clustal L program. The polymorphism is in
the same codon for each. The Bos taurus nucloetide change is in the
second nucleotide triplet codon, while in the Sus scrofa the change
is in the last nucleotide triplet codon. Both changes result in a
synonomous amino acid substitution.
DETAILED DESCRIPTION OF THE INVENTION
[0075] Reference will now be made in detail to the presently
referred embodiments of the invention, which together with the
following examples, serve to explain the principles of the
invention.
[0076] In mammalian skeletal and cardiac muscle, calcium activated
proteinases have been implicated in processes that regulate limited
proteolysis of myofibrillar and cytoskeletal proteins and maintain
the intracellular architecture of the muscle fiber. Many muscle
wasting diseases such as muscular dystrophy are accompanied by
changes in calpain (calcium-dependent cysteine protease) activity.
Calpastatin (CAST) is a calpain specific endogenous protein
inhibitor that is coexpressed with calpain. CAST has been
hypothesized to be involved in muscle protein degradation in living
tissue, and has also been shown to play a key role in post mortem
tenderization of meat. Calpastatins isolated from different tissues
are heterogenous in size due to alternative splicing of gene
transcripts as well as posttranslational processing. The
physiological significance of calpastatin diversity is not
understood but is postulated to be related to intracellular
compartmentalization or differential inhibitor specificity against
calpain isoforms. There are four domains in the CAST protein, each
one having inhibitory function.
[0077] According to the invention, applicants have identified
several different alleles of the CAST gene which are correlated
with improved growth and meat quality in animals. Applicants have
also identified novel porcine skeletal cDNA sequences.
[0078] FIG. 1a depicts the Calpastatin cDNA sequence SEQ ID NO: 1
and depicts the alternate polymorphisms of the invention, (SEQ ID
NO: 3, 5, 7, and 9),. FIG. 1b depicts the amino acid sequence of
porcine skeletal CAST SEQ ID NO: 2 showing the alternate forms (SEQ
ID NOS 4, 6, 8, and 10). These new markers have been shown to
correlate with meat firmness, meat juiciness, meat tenderness,
average Instron force, average drip loss, weight before slaughter,
loin weight, loin pH, and ham pH, hpromeat (Henessey probe loin
depth). According to the invention, the association of these
polymorphisms with theses trait(s) enables genetic markers to be
identified for specific breeds or genetic lines or animals, with
favorable meat quality and or growth early in the animals life.
[0079] One of the single nucleotide polymorphisms identified
according to the invention represents a shift from an arginine
codon (AAA, Allele 2) to lysine (AGA, Allele 1) in exon 13 domain 1
of the CAST gene (SEQ ID NO: 5 and 6)(position 812 of the cDNA
sequence, FIG. 1). This polymorphism was shown to have effects on
subjective juciness, firmness, instron force, drip loss, cooking
loss, crumbliness, fibrosity, guminess, hardness, acceptance,
loinminl, loinpH, days on test, hamminl, boneless weight of the
loin, LDG, and TDG. There was also several assocaitions with growth
traits including live daily gain, daily gain on test, and weight at
the end of test. According to one embodiment of the invention, a
PCR-RFLP test has been developed to identify the presence of either
of these particular alleles in a genetic sample using the
restriction enzyme Hpy188I.
[0080] Yet another single nucleotide polymorphism identified
according to the invention represents a change from an arginine
codon (AGA) to a serine codon (AGC Allele 1) in exon 28 (domain 4)
of the CAST gene (SEQ ID NO: 9 and 10)(position 1980 of the cDNA
sequence, FIG. 1). Variation at this position was correlated with
subjective juciness, firmness, instron force, drip loss,
cumbliness, fibrosity, guminess, hardness, acceptance, loinminl,
loin pH, hpromeat, aloca backfat, days on test, drip percentage,
US_MD, and bone in weight of the ham. There was also an asociation
with growth traits such as live daily gain, daily gain on test,
Henessey probe loin depth, and weight at the end of test. According
to one embodiment of the invention, a PCR-RFLP test was developed
to identify the presence of one of these particular alleles in a
genetic sample using the restriction enzyme PvuII.
[0081] Another single nucleotide polymorphism identified according
to the invention represents a change from a threonine codon (ACT,
Allele 1) to an alanine codon (GCT) in exon 22 (domain 3) of the
CAST gene (SEQ ID NO: 7 and 8)(position 1576 of the cDNA sequence,
FIG. 1). According to yet anther embodiment of the invention, a
PCR-RFLP test was developed to identify the presence of one of
these particular alleles in a genetic sample using the restriction
enzyme AciI.
[0082] Yet another single nucleotide polymorphism identified
according to the invention results in a change from a asparagine
codon (AAT, Allele 1) to a serine codon (AGT) in exon 6 (domain L)
of the CAST gene (SEQ ID NO: 3 and 4)(position 263 of the cDNA
sequence, FIG. 1). A test for this polymorphism was developed using
the restriction enzyme ApaLI. This polymorphism was found to be in
complete linkage disequilibrium with the Hpy188I marker. Also
according to the invention, a polymorphism in the same codon was
found to be present in cattle. FIG. 6 depicts the Clustal L
alignment of the Bos taurus (cow) and Sus scrofa (pig) exon 6
sequences. Due to the highly conserved nature of this gene it is
expected that the polymorphisms disclosed herein, particularly in
that they all exist in the coding regions of the gene will be
present in other animals, breeds, lines, or populations.
[0083] Further, haplotype analysis was conducted to identify
favorable combinations of the markers identified in CAST gene and
sufficiently informative to be able to detect the poissible effect
of them.
[0084] The invention thus relates to genetic markers for
economically valuable traits in animals. The markers represent
alleles that are associated significantly with a meat quality
and/or growth trait and thus provides a method of screening animals
to determine those more likely to produce desired meat quality
and/or growth (levels of one or all of these) when bred by
identifying the presence or absence of a polymorphism in the CAST
gene that is so correlated.
[0085] Thus, the invention relates to genetic markers and methods
of identifying those markers in an animal of a particular animal,
breed, strain, population, or group, whereby the animal is more
likely to yield meat of desired meat quality and/or growth.
[0086] Any method of identifying the presence or absence of these
markers may be used, including for example single-strand
conformation polymorphism (SSCP) analysis, base excision sequence
scanning (BESS), RFLP analysis, heteroduplex analysis, denaturing
gradient gel electrophoresis, and temperature gradient
electrophoresis, allelic PCR, ligase chain reaction direct
sequencing, mini sequencing, nucleic acid hybridization,
micro-array-type detection of the CAST gene, or other linked
sequences of the CAST gene. Also within the scope of the invention
includes assaying for protein conformational or sequences changes
which occur in the presence of this polymorphism. The polymorphism
may or may not be the causative mutation but will be indicative of
the presence of this change and one may assay for the genetic or
protein bases for the phenotypic difference.
[0087] The following is a general overview of techniques which can
be used to assay for the polymorphisms of the invention.
[0088] In the present invention, a sample of genetic material is
obtained from an animal. Samples can be obtained from blood,
tissue, semen, etc. Generally, peripheral blood cells are used as
the source, and the genetic material is DNA. A sufficient amount of
cells are obtained to provide a sufficient amount of DNA for
analysis. This amount will be known or readily determinable by
those skilled in the art. The DNA is isolated from the blood cells
by techniques known to those skilled in the art.
[0089] Isolation and Amplification of Nucleic Acid
[0090] Samples of genomic DNA are isolated from any convenient
source including saliva, buccal cells, hair roots, blood, cord
blood, amniotic fluid, interstitial fluid, peritoneal fluid,
chorionic villus, and any other suitable cell or tissue sample with
intact interphase nuclei or metaphase cells. The cells can be
obtained from solid tissue as from a fresh or preserved organ or
from a tissue sample or biopsy. The sample can contain compounds
which are not naturally intermixed with the biological material
such as preservatives, anticoagulants, buffers, fixatives,
nutrients, antibiotics, or the like.
[0091] Methods for isolation of genomic DNA from these various
sources are described in, for example, Kirby, DNA Fingerprinting,
An Introduction, W. H. Freeman & Co. New York (1992). Genomic
DNA can also be isolated from cultured primary or secondary cell
cultures or from transformed cell lines derived from any of the
aforementioned tissue samples.
[0092] Samples of animal RNA can also be used. RNA can be isolated
from tissues expressing the CAST gene as described in Sambrook et
al., supra. RNA can be total cellular RNA, mRNA, poly A+ RNA, or
any combination thereof. For best results, the RNA is purified, but
can also be unpurified cytoplasmic RNA. RNA can be reverse
transcribed to form DNA which is then used as the amplification
template, such that the PCR indirectly amplifies a specific
population of RNA transcripts. See, e.g., Sambrook, supra, Kawasaki
et al., Chapter 8 in PCR Technology, (1992) supra, and Berg et al.,
Hum. Genet. 85:655-658 (1990).
[0093] PCR Amplification
[0094] The most common means for amplification is polymerase chain
reaction (PCR), as described in U.S. Pat. Nos. 4,683,195,
4,683,202, 4,965,188 each of which is hereby incorporated by
reference. If PCR is used to amplify the target regions in blood
cells, heparinized whole blood should be drawn in a sealed vacuum
tube kept separated from other samples and handled with clean
gloves. For best results, blood should be processed immediately
after collection; if this is impossible, it should be kept in a
sealed container at 4.degree. C. until use. Cells in other
physiological fluids may also be assayed. When using any of these
fluids, the cells in the fluid should be separated from the fluid
component by centrifugation.
[0095] Tissues should be roughly minced using a sterile, disposable
scalpel and a sterile needle (or two scalpels) in a 5 mm Petri
dish. Procedures for removing paraffin from tissue sections are
described in a variety of specialized handbooks well known to those
skilled in the art.
[0096] To amplify a target nucleic acid sequence in a sample by
PCR, the sequence must be accessible to the components of the
amplification system. One method of isolating target DNA is crude
extraction which is useful for relatively large samples. Briefly,
mononuclear cells from samples of blood, amniocytes from amniotic
fluid, cultured chorionic villus cells, or the like are isolated by
layering on sterile Ficoll-Hypaque gradient by standard procedures.
Interphase cells are collected and washed three times in sterile
phosphate buffered saline before DNA extraction. If testing DNA
from peripheral blood lymphocytes, an osmotic shock (treatment of
the pellet for 10 sec with distilled water) is suggested, followed
by two additional washings if residual red blood cells are visible
following the initial washes. This will prevent the inhibitory
effect of the heme group carried by hemoglobin on the PCR reaction.
If PCR testing is not performed immediately after sample
collection, aliquots of 10.sup.6 cells can be pelleted in sterile
Eppendorf tubes and the dry pellet frozen at -20.degree. C. until
use.
[0097] The cells are resuspended (10.sup.6 nucleated cells per 100
.mu.l) in a buffer of 50 mM Tris-HCl (pH 8.3), 50 mM KCl 1.5 mM
MgCl.sub.2, 0.5% Tween 20, 0.5% NP40 supplemented with 100 .mu.g/ml
of proteinase K. After incubating at 56.degree. C. for 2 hr. the
cells are heated to 95.degree. C. for 10 min to inactivate the
proteinase K and immediately moved to wet ice (snap-cool). If gross
aggregates are present, another cycle of digestion in the same
buffer should be undertaken. Ten .mu.l of this extract is used for
amplification.
[0098] When extracting DNA from tissues, e.g., chorionic villus
cells or confluent cultured cells, the amount of the above
mentioned buffer with proteinase K may vary according to the size
of the tissue sample. The extract is incubated for 4-10 hrs at
50.degree.-60.degree. C. and then at 95.degree. C. for 10 minutes
to inactivate the proteinase. During longer incubations, fresh
proteinase K should be added after about 4 hr at the original
concentration.
[0099] When the sample contains a small number of cells, extraction
may be accomplished by methods as described in Higuchi, "Simple and
Rapid Preparation of Samples for PCR", in PCR Technology, Ehrlich,
H. A. (ed.), Stockton Press, New York, which is incorporated herein
by reference. PCR can be employed to amplify target regions in very
small numbers of cells (1000-5000) derived from individual colonies
from bone marrow and peripheral blood cultures. The cells in the
sample are suspended in 20 .mu.l of PCR lysis buffer (10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM Mg Cl.sub.2, 0.1 mg/ml
gelatin, 0.45% NP40, 0.45% Tween 20) and frozen until use. When PCR
is to be performed, 0.6 .mu.l of proteinase K (2 mg/ml) is added to
the cells in the PCR lysis buffer. The sample is then heated to
about 60.degree. C. and incubated for 1 hr. Digestion is stopped
through inactivation of the proteinase K by heating the samples to
95.degree. C. for 10 min and then cooling on ice.
[0100] A relatively easy procedure for extracting DNA for PCR is a
salting out procedure adapted from the method described by Miller
et al., Nucleic Acids Res. 16:1215 (1988), which is incorporated
herein by reference. Mononuclear cells are separated on a
Ficoll-Hypaque gradient. The cells are resuspended in 3 ml of lysis
buffer (10 mM Tris-HCl, 400 mM NaCl, 2 mM Na.sub.2 EDTA, pH 8.2).
Fifty .mu.l of a 20 mg/ml solution of proteinase K and 150 .mu.l of
a 20% SDS solution are added to the cells and then incubated at
37.degree. C. overnight. Rocking the tubes during incubation will
improve the digestion of the sample. If the proteinase K digestion
is incomplete after overnight incubation (fragments are still
visible), an additional 50 .mu.l of the 20 mg/ml proteinase K
solution is mixed in the solution and incubated for another night
at 37.degree. C. on a gently rocking or rotating platform.
Following adequate digestion, one ml of a 6M NaCl solution is added
to the sample and vigorously mixed. The resulting solution is
centrifuged for 15 minutes at 3000 rpm. The pellet contains the
precipitated cellular proteins, while the supernatant contains the
DNA. The supernatant is removed to a 15 ml tube that contains 4 ml
of isopropanol. The contents of the tube are mixed gently until the
water and the alcohol phases have mixed and a white DNA precipitate
has formed. The DNA precipitate is removed and dipped in a solution
of 70% ethanol and gently mixed. The DNA precipitate is removed
from the ethanol and air-dried. The precipitate is placed in
distilled water and dissolved.
[0101] Kits for the extraction of high-molecular weight DNA for PCR
include a Genomic Isolation Kit A.S.A.P. (Boehringer Mannheim,
Indianapolis, Ind.), Genomic DNA Isolation System (GIBCO BRL,
Gaithersburg, Md.), Elu-Quik DNA Purification Kit (Schleicher &
Schuell, Keene, N.H.), DNA Extraction Kit (Stratagene, LaJolla,
Calif.), TurboGen Isolation Kit (Invitrogen, San Diego, Calif.),
and the like. Use of these kits according to the manufacturer's
instructions is generally acceptable for purification of DNA prior
to practicing the methods of the present invention.
[0102] The concentration and purity of the extracted DNA can be
determined by spectrophotometric analysis of the absorbance of a
diluted aliquot at 260 nm and 280 nm. After extraction of the DNA,
PCR amplification may proceed. The first step of each cycle of the
PCR involves the separation of the nucleic acid duplex formed by
the primer extension. Once the strands are separated, the next step
in PCR involves hybridizing the separated strands with primers that
flank the target sequence. The primers are then extended to form
complementary copies of the target strands. For successful PCR
amplification, the primers are designed so that the position at
which each primer hybridizes along a duplex sequence is such that
an extension product synthesized from one primer, when separated
from the template (complement), serves as a template for the
extension of the other primer. The cycle of denaturation,
hybridization, and extension is repeated as many times as necessary
to obtain the desired amount of amplified nucleic acid.
[0103] In a particularly useful embodiment of PCR amplification,
strand separation is achieved by heating the reaction to a
sufficiently high temperature for a sufficient time to cause the
denaturation of the duplex but not to cause an irreversible
denaturation of the polymerase (see U.S. Pat. No. 4,965,188,
incorporated herein by reference). Typical heat denaturation
involves temperatures ranging from about 80.degree. C. to
105.degree. C. for times ranging from seconds to minutes. Strand
separation, however, can be accomplished by any suitable denaturing
method including physical, chemical, or enzymatic means. Strand
separation may be induced by a helicase, for example, or an enzyme
capable of exhibiting helicase activity. For example, the enzyme
RecA has helicase activity in the presence of ATP. The reaction
conditions suitable for strand separation by helicases are known in
the art (see Kuhn Hoffman-Berling, 1978, CSH-Quantitative Biology,
43:63-67; and Radding, 1982, Ann. Rev. Genetics 16:405-436, each of
which is incorporated herein by reference).
[0104] Template-dependent extension of primers in PCR is catalyzed
by a polymerizing agent in the presence of adequate amounts of four
deoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP, and
dTTP) in a reaction medium comprised of the appropriate salts,
metal cations, and pH buffering systems. Suitable polymerizing
agents are enzymes known to catalyze template-dependent DNA
synthesis. In some cases, the target regions may encode at least a
portion of a protein expressed by the cell. In this instance, mRNA
may be used for amplification of the target region. Alternatively,
PCR can be used to generate a cDNA library from RNA for further
amplification, the initial template for primer extension is RNA.
Polymerizing agents suitable for synthesizing a complementary,
copy-DNA (cDNA) sequence from the RNA template are reverse
transcriptase (RT), such as avian myeloblastosis virus RT, Moloney
murine leukemia virus RT, or Thermus thermophilus (Tth) DNA
polymerase, a thermostable DNA polymerase with reverse
transcriptase activity marketed by Perkin Elmer Cetus, Inc.
Typically, the genomic RNA template is heat degraded during the
first denaturation step after the initial reverse transcription
step leaving only DNA template. Suitable polymerases for use with a
DNA template include, for example, E. coli DNA polymerase I or its
Klenow fragment, T4 DNA polymerase, Tth polymerase, and Taq
polymerase, a heat-stable DNA polymerase isolated from Thermus
aquaticus and commercially available from Perkin Elmer Cetus, Inc.
The latter enzyme is widely used in the amplification and
sequencing of nucleic acids. The reaction conditions for using Taq
polymerase are known in the art and are described in Gelfand, 1989,
PCR Technology, supra.
[0105] Allele Specific PCR
[0106] Allele-specific PCR differentiates between target regions
differing in the presence of absence of a variation or
polymorphism. PCR amplification primers are chosen which bind only
to certain alleles of the target sequence. This method is described
by Gibbs, Nucleic Acid Res. 17:12427-2448 (1989).
[0107] Allele Specific Oligonucleotide Screening Methods
[0108] Further diagnostic screening methods employ the
allele-specific oligonucleotide (ASO) screening methods, as
described by Saiki et al., Nature 324:163-166 (1986).
Oligonucleotides with one or more base pair mismatches are
generated for any particular allele. ASO screening methods detect
mismatches between variant target genomic or PCR amplified DNA and
non-mutant oligonucleotides, showing decreased binding of the
oligonucleotide relative to a mutant oligonucleotide.
Oligonucleotide probes can be designed that under low stringency
will bind to both polymorphic forms of the allele, but which at
high stringency, bind to the allele to which they correspond.
Alternatively, stringency conditions can be devised in which an
essentially binary response is obtained, i.e., an ASO corresponding
to a variant form of the target gene will hybridize to that allele,
and not to the wildtype allele.
[0109] Ligase Mediated Allele Detection Method
[0110] Target regions of a test subject's DNA can be compared with
target regions in unaffected and affected family members by
ligase-mediated allele detection. See Landegren et al., Science
241:107-1080 (1988). Ligase may also be used to detect point
mutations in the ligation amplification reaction described in Wu et
al., Genomics 4:560-569 (1989). The ligation amplification reaction
(LAR) utilizes amplification of specific DNA sequence using
sequential rounds of template dependent ligation as described in
Wu, supra, and Barany, Proc. Nat. Acad. Sci. 88:189-193 (1990).
[0111] Denaturing Gradient Gel Electrophoresis
[0112] Amplification products generated using the polymerase chain
reaction can be analyzed by the use of denaturing gradient gel
electrophoresis. Different alleles can be identified based on the
different sequence-dependent melting properties and electrophoretic
migration,of DNA in solution. DNA molecules melt in segments,
termed melting domains, under conditions of increased temperature
or denaturation. Each melting domain melts cooperatively at a
distinct, base-specific melting temperature (TM). Melting domains
are at least 20 base pairs in length, and may be up to several
hundred base pairs in length.
[0113] Differentiation between alleles based on sequence specific
melting domain differences can be assessed using polyacrylamide gel
electrophoresis, as described in Chapter 7 of Erlich, ed., PCR
Technology, Principles and Applications for DNA Amplification, W.
H. Freeman and Co., New York (1992), the contents of which are
hereby incorporated by reference.
[0114] Generally, a target region to be analyzed by denaturing
gradient gel electrophoresis is amplified using PCR primers
flanking the target region. The amplified PCR product is applied to
a polyacrylamide gel with a linear denaturing gradient as described
in Myers et al., Meth. Enzymol. 155:501-527 (1986), and Myers et
al., in Genomic Analysis, A Practical Approach, K. Davies Ed. IRL
Press Limited, Oxford, pp. 95-139 (1988), the contents of which are
hereby incorporated by reference. The electrophoresis system is
maintained at a temperature slightly below the Tm of the melting
domains of the target sequences.
[0115] In an alternative method of denaturing gradient gel
electrophoresis, the target sequences may be initially attached to
a stretch of GC nucleotides, termed a GC clamp, as described in
Chapter 7 of Erlich, supra. Preferably, at least 80% of the
nucleotides in the GC clamp are either guanine or cytosine.
Preferably, the GC clamp is at least 30 bases long. This method is
particularly suited to target sequences with high Tm's.
[0116] Generally, the target region is amplified by the polymerase
chain reaction as described above. One of the, oligonucleotide PCR
primers carries at its 5' end, the GC clamp region, at least 30
bases of the GC rich sequence, which is incorporated into the 5'
end of the target region during amplification. The resulting
amplified target region is run on an electrophoresis gel under
denaturing gradient conditions as described above. DNA fragments
differing by a single base change will migrate through the gel to
different positions, which may be visualized by ethidium bromide
staining.
[0117] Temperature Gradient Gel Electrophoresis
[0118] Temperature gradient gel electrophoresis (TGGE) is based on
the same underlying principles as denaturing gradient gel
electrophoresis, except the denaturing gradient is produced by
differences in temperature instead of differences in the
concentration of a chemical denaturant. Standard TGGE utilizes an
electrophoresis apparatus with a temperature gradient running along
the electrophoresis path. As samples migrate through a gel with a
uniform concentration of a chemical denaturant, they encounter
increasing temperatures. An alternative method of TGGE, temporal
temperature gradient gel electrophoresis (TTGE or tTGGE) uses a
steadily increasing temperature of the entire electrophoresis gel
to achieve the same result. As the samples migrate through the gel
the temperature of the entire gel increases, leading the samples to
encounter increasing temperature as they migrate through the gel.
Preparation of samples, including PCR amplification with
incorporation of a GC clamp, and visualization of products are the
same as for denaturing gradient gel electrophoresis.
[0119] Single-Strand Conformation Polymorphism Analysis
[0120] Target sequences or alleles at the CAST locus can be
differentiated using single-strand conformation polymorphism
analysis, which identifies base differences by alteration in
electrophoretic migration of single stranded PCR products, as
described in Orita et al., Proc. Nat. Acad. Sci. 85:2766-2770
(1989). Amplified PCR products can be generated as described above,
and heated or otherwise denatured, to form single stranded
amplification products. Single-stranded nucleic acids may refold or
form secondary structures which are partially dependent on the base
sequence. Thus, electrophoretic mobility of single-stranded
amplification products can detect base-sequence difference between
alleles or target sequences.
[0121] Chemical or Enzymatic Cleavage of Mismatches
[0122] Differences between target sequences can also be detected by
differential chemical cleavage of mismatched base pairs, as
described in Grompe et al., Am. J. Hum. Genet. 48:212-222 (1991).
In another method, differences between target sequences can be
detected by enzymatic cleavage of mismatched base pairs, as
described in Nelson et al., Nature Genetics 4:11-18 (1993).
Briefly, genetic material from an animal and a phenotypicallt
different family member may be used to generate mismatch free
heterohybrid DNA duplexes. As used herein, "heterohybrid" means a
DNA duplex strand comprising one strand of DNA from one animal, and
a second DNA strand from another animal, usually an animal
differing in the phenotype for the trait of interest. Positive
selection for heterohybrids free of mismatches allows determination
of small insertions, deletions or other polymorphisms that may be
associated with CAST polymorphisms.
[0123] Non-gel Systems
[0124] Other possible techniques include non-gel systems such as
TaqMan.TM. (Perkin Elmer). In this system oligonucleotide PCR
primers are designed that flank the mutation in question and allow
PCR amplification of the region. A third oligonucleotide probe is
then designed to hybridize to the region containing the base
subject to change between different alleles of the gene. This probe
is labeled with fluorescent dyes at both the 5' and 3' ends. These
dyes are chosen such that while in this proximity to each other the
fluorescence of one of them is quenched by the other and cannot be
detected. Extension by Taq DNA polymerase from the PCR primer
positioned 5' on the template relative to the probe leads to the
cleavage of the dye attached to the 5' end of the annealed probe
through the 5' nuclease activity of the Taq DNA polymerase. This
removes the quenching effect allowing detection of the fluorescence
from the dye at the 3' end of the probe. The discrimination between
different DNA sequences arises through the fact that if the
hybridization of the probe to the template molecule is not
complete, i.e. there is a mismatch of some form, the cleavage of
the dye does not take place. Thus only if the nucleotide sequence
of the oligonucleotide probe is completely complimentary to the
template molecule to which it is bound will quenching be removed. A
reaction mix can contain two different probe sequences each
designed against different alleles that might be present thus
allowing the detection of both alleles in one reaction.
[0125] Yet another technique includes an Invader Assay which
includes isothermic amplification that relies on a catalytic
release of fluorescence. See Third Wave Technology at
www.twt.com.
[0126] Non-PCR Based DNA Diagnostics
[0127] The identification of a DNA sequence linked to CAST can be
made without an amplification step, based on polymorphisms
including restriction fragment length polymorphisms in an animal
and a family member. Hybridization probes are generally
oligonucleotides which bind through complementary base pairing to
all or part of a target nucleic acid. Probes typically bind target
sequences lacking complete complementarity with the probe sequence
depending on the stringency of the hybridization conditions. The
probes are preferably labeled directly or indirectly, such that by
assaying for the presence or absence of the probe, one can detect
the presence or absence of the target sequence. Direct labeling
methods include radioisotope labeling, such as with 32P or 35S.
Indirect labeling methods include fluorescent tags, biotin
complexes which may be bound to avidin or streptavidin, or peptide
or protein tags. Visual detection methods include
photoluminescents, Texas red, rhodamine and its derivatives, red
leuco dye and 3,3',5,5'-tetramethylbenzidine (TMB), fluorescein,
and its derivatives, dansyl, umbelliferone and the like or with
horse radish peroxidase, alkaline phosphatase and the like.
[0128] Hybridization probes include any nucleotide sequence capable
of hybridizing to the porcine chromosome where CAST resides, and
thus defining a genetic marker linked to CAST, including a
restriction fragment length polymorphism, a hypervariable region,
repetitive element, or a variable number tandem repeat.
Hybridization probes can be any gene or a suitable analog. Further
suitable hybridization probes include exon fragments or portions of
cDNAs or genes known to map to the relevant region of the
chromosome.
[0129] Preferred tandem repeat hybridization probes for use
according to the present invention are those that recognize a small
number of fragments at a specific locus at high stringency
hybridization conditions, or that recognize a larger number of
fragments at that locus when the stringency conditions are
lowered.
[0130] One or more additional restriction enzymes and/or probes
and/or primers can be used. Additional enzymes, constructed probes,
and primers can be determined by routine experimentation by those
of ordinary skill in the art and are intended to be within the
scope of the invention.
[0131] Although the methods described herein may be in terms of the
use of a single restriction enzyme and a single set of primers, the
methods are not so limited. One or more additional restriction
enzymes and/or probes and/or primers can be used, if desired.
Indeed in some situations it may be preferable to use combinations
of markers giving specific haplotypes. Additional enzymes,
constructed probes and primers can be determined through routine
experimentation, combined with the teachings provided and
incorporated herein.
[0132] According to the invention, polymorphisms in the CAST gene
have been identified which have an association with meat quality
and/or growth. The presence or absence of the markers, in one
embodiment may be assayed by PCR RFLP analysis using the
restriction endonucleases and amplification primers may be designed
using analogous human, pig or other CAST sequences due to the high
homology in the region surrounding the polymorphisms, or may be
designed using known CAST gene sequence data as exemplified in
GenBank or even designed from sequences obtained from linkage data
from closely surrounding genes based upon the teachings and
references herein. The sequences surrounding the polymorphism will
facilitate the development of alternate PCR tests in which a primer
of about 4-30 contiguous bases taken from the sequence immediately
adjacent to the polymorphism is used in connection with a
polymerase chain reaction to greatly amplify the region before
treatment with the desired restriction enzyme. The primers need not
be the exact complement; substantially equivalent sequences are
acceptable. The design of primers for amplification by PCR is known
to those of skill in the art and is discussed in detail in Ausubel
(ed.), "Short Protocols in Molecular Biology, Fourth Edition" John
Wiley and Sons 1999. The following is a brief description of primer
design.
[0133] Primer Design Strategy
[0134] Increased use of polymerase chain reaction (PCR) methods has
stimulated the development of many programs to aid in the design or
selection of oligonucleotides used as primers for PCR. Four
examples of such programs that are freely available via the
Internet are: PRIMER by Mark Daly and Steve Lincoln of the
Whitehead Institute (UNIX, VMS, DOS, and Macintosh),
Oligonucleotide Selection Program (OSP) by Phil Green and LaDeana
Hiller of Washington University in St. Louis (UNIX, VMS, DOS, and
Macintosh), PGEN by Yoshi (DOS only), and Amplify by Bill Engels of
the University of Wisconsin (Macintosh only). Generally these
programs help in the design of PCR primers by searching for bits of
known repeated-sequence elements and then optimizing the T.sub.m by
analyzing the length and GC content of a putative primer.
Commercial software is also available and primer selection
procedures are rapidly being included in most general sequence
analysis packages.
[0135] Sequencing and PCR Primers
[0136] Designing oligonucleotides for use as either sequencing or
PCR primers requires selection of an appropriate sequence that
specifically recognizes the target, and then testing the sequence
to eliminate the possibility that the oligonucleotide will have a
stable secondary structure. Inverted repeats in the sequence can be
identified using a repeat-identification or RNA-folding program
such as those described above (see prediction of Nucleic Acid
Structure). If a possible stem structure is observed, the sequence
of the primer can be shifted a few nucleotides in either direction
to minimize the predicted secondary structure. The sequence of the
oligonucleotide should also be compared with the sequences of both
strands of the appropriate vector and insert DNA. Obviously, a
sequencing primer should only have a single match to the target
DNA. It is also advisable to exclude primers that have only a
single mismatch with an undesired target DNA sequence. For PCR
primers used to amplify genomic DNA, the primer sequence should be
compared to the sequences in the GenBank database to determine if
any significant matches occur. If the oligonucleotide sequence is
present in any known DNA sequence or, more importantly, in any
known repetitive elements, the primer sequence should be
changed.
[0137] The methods and materials of the invention may also be used
more generally to evaluate animal DNA, genetically type individual
animals, and detect genetic differences in animals. In particular,
a sample of genomic DNA may be evaluated by reference to one or
more controls to determine if a polymorphism in the CAST gene is
present. Preferably, RFLP analysis is performed with respect to the
CAST gene, and the results are compared with a control. The control
is the result of a RFLP analysis of the CAST gene of a different
animal where the polymorphism of the animal's CAST gene is known.
Similarly, the CAST genotype of an animal may be determined by
obtaining a sample of its genomic DNA, conducting RFLP analysis of
the CAST gene in the DNA, and comparing the results with a control.
Again, the control is the result of RFLP analysis of the CAST gene
of a different animal. The results genetically type the animal by
specifying the polymorphism(s) in its CAST genes. Finally, genetic
differences among animals can be detected by obtaining samples of
the genomic DNA from at least two animals, identifying the presence
or absence of a polymorphism in the CAST gene, and comparing the
results to trace lineage, to track animals and the like.
[0138] These assays are useful for identifying the genetic markers
relating to meat quality and/or growth, as discussed above, for
identifying other polymorphisms in the CAST gene that may be
correlated with other characteristics, such as litter size and for
the general scientific analysis of pig genotypes and
phenotypes.
[0139] The examples and methods herein disclose a certain gene
which has been identified to have a polymorphism which is
associated either positively or negatively with a beneficial trait
that will have an effect on meat quality and/or growth for animals
carrying this polymorphism. The identification of the existence of
a polymorphism within a gene is often made by a single base
alternative that results in a restriction site in certain allelic
forms. A certain allele, however, as demonstrated and discussed
herein, may have a number of base changes associated with it that
could be assayed for which are indicative of the same polymorphism
(allele). Further, other genetic markers or genes may be linked to
the polymorphisms disclosed herein so that assays may involve
identification of other genes or gene fragments, but which
ultimately rely upon genetic characterization of animals for the
same polymorphism. Any assay which sorts and identifies animals
based upon the allelic differences disclosed herein are intended to
be included within the scope of this invention.
[0140] One of skill in the art, once a polymorphism has been
identified and a correlation to a particular trait established,
will understand that there are many ways to genotype animals for
this polymorphism. The design of such alternative tests merely
represent optimization of parameters known to those of skill in the
art and are intended to be within the scope of this invention as
fully described herein.
EXAMPLE 1
[0141] CAST Hpy188I PCR-RFLP Test
[0142] Hpy188I polymorphism
[0143] Exon: 13 (domain 1)
[0144] Non-synonymous change: Arg-Lys
[0145] Allele 1-Lys (K)-AAA
[0146] Allele 2-Arg (R)-AGA
1 Primers C14F2: 5' AAA TCT ACT GGA GAG GTT TTG AA 3' SEQ ID NO:11
C14R2: 5' GAC TTC TCC CGA ATC AGT TCC 3' SEQ ID NO:12
[0147] PCR Conditions
[0148] Mix 1
2 10x PCR buffer 1.0 .mu.l MgCl.sub.2 (15 mM) 1.0 .mu.l dNTPs (2
mM) 1.0 .mu.l CI4F2 primer (10 pm/.mu.l) 0.25 .mu.l CI4R2 primer
(10 pM/.mu.l) 0.25 .mu.l Taq polymerase (5 U/.mu.l) 0.07 .mu.l
ddH.sub.20 5.43 .mu.l genomic DNA 1 .mu.l
[0149] Combine the Mix 1 and DNA in a reaction tube. Overlay with
mineral oil. Run the following PCR program: 94.degree. C. for 4
min.; 35 cycles of 94.degree. C. for 45 sec., 54.degree. C. for 45
sec and 72.degree. C. for 30 sec; followed by a final extension at
72.degree. C. for 12 min.
[0150] Check 3 .mu.l of the PCR on a 2% agarose gel to confirm
amplification. Product size is 182 bp.
[0151] Hpy188I digestion
3 1X Volume (.mu.l) NEB Buffer 4 10X* 1.0 Hpy188I (10 units/l) 0.4
ddWater 5.6 Mix Final Volume 7.0 *NEB
[0152] Aliquot 7 .mu.l Hpy188I mix and add 3 .mu.l PCR product.
[0153] Incubate at 37.degree. C.
[0154] Gel Electrophoresis
[0155] Add 2 .mu.l orange G loading buffer and load on a 4.0%
Nusieve/Me (3:1) agarose.
[0156] Run at 150V. Products should be resolved in about 30
minutes.
[0157] The expected band pattern and sequence around the
polymorphism are shown in FIG. 2.
[0158] CAST PvuII PCR-RFLP Test
[0159] PvuII polymorphism
[0160] Exon: 27 (domain 4)
[0161] Non-synonymous change: Arg-Ser
[0162] Allele 1-Arg (R)-AGA
[0163] Allele 2-Ser (S)-AGC
4 Primers CS26F: 5' AGG GCA AAT CPA CGA AGC CAC 3' SEQ ID NO:13
C27R2: 5' CCT TTG TTG TGT TCT CTG AGG 3' SEQ ID NO:14
[0164] PCR Conditions
[0165] Mix 1
5 10x PCR buffer 1.0 .mu.l MgCl.sub.2 (15 mM) 1.0 .mu.l dNTPs (2
mM) 1.0 .mu.l CS26F primer (10 pm/.mu.l) 0.25 .mu.l C27R2 primer
(10 pM/.mu.l) 0.25 .mu.l Taq polymerase (5 U/.mu.l) 0.07 .mu.l
ddH.sub.20 5.43 .mu.l genomic DNA 1 .mu.l
[0166] Combine the Mix 1 and DNA in a reaction tube. Overlay with
mineral oil. Run the following PCR program: 94.degree. C. for 4
min.; 35 cycles of 94.degree. C. for 45 sec., 54.degree. C. for 45
sec and 72.degree. C. for 30 sec; followed by a final extension at
72.degree. C. for 12 min.
[0167] Check 3 .mu.l of the PCR on a 2% agarose gel to confirm
amplification. Product size is 539 bp.
[0168] PvuII digestion
6 1X Volume (.mu.l) NEB Buffer 2 10X* 1.0 PvuII (10 units/l) 0.4
ddWater 5.6 Mix Final Volume 7.0 *NEB
[0169] Aliquot 7 .mu.l PvuII mix and add 3 .mu.l PCR product.
[0170] Incubate at 37.degree. C.
[0171] Gel Electrophoresis
[0172] Add 2 .mu.l orange G loading buffer and load on a 4.0%
Nusieve/Me (3:1) agarose.
[0173] Run at 150V. Products should be resolved in about 30
minutes.
[0174] FIG. 3 shows the expected banding pattern and sequence
around the PvuII polymorphism.
[0175] CAST AciI PCR-RFLP Test
[0176] AciI polymorphism
[0177] Exon: 22 (domain 3)
[0178] Non-synonymous change: Thr-Ala.
[0179] Allele 1-Thr (T)-ACT
[0180] Allele 2-Ala (A)-GCT
7 Primers CS22F: 5' AGA CTT CGT CCT TGA TGC TTT G 3' SEQ ID NO:15
CS22R: 5' TAA TGG CTA TGA TGG GTT GAG G 3' SEQ ID NO:16
[0181] PCR Conditions
[0182] Mix 1
8 10x PCR buffer 1.0 .mu.l MgCl.sub.2 (15 mM) 1.0 .mu.l dNTPs (2
mM) 1.0 .mu.l CS22F primer (10 pm/.mu.l) 0.25 .mu.l CS22R primer
(10 pM/.mu.l) 0.25 .mu.l Taq polymerase (5 U/.mu.l) 0.07 .mu.l
ddH.sub.20 5.43 .mu.l genomic DNA 1 .mu.l
[0183] Combine the Mix 1 and DNA in a reaction tube. Overlay with
mineral oil. Run the following PCR program: 94.degree. C. for 4
min.; 35 cycles of 94.degree. C. for 45 sec., 54.degree. C. for 35
sec and 72.degree. C. for 30 sec; followed by a final extension at
72.degree. C. for 12 min.
[0184] Check 3 .mu.l of the PCR on a 2% agarose gel to confirm
amplification. Product size is 196 bp.
[0185] AciI digestion
9 1X Volume (.mu.l) NEB Buffer 3 10X* 1.0 AciI (10 units/l) 0.4
ddWater 5.6 Mix Final Volume 7.0 *NEB
[0186] Aliquot 7 .mu.l AciI mix and add 3 .mu.l PCR product.
[0187] Incubate at 37.degree. C.
[0188] Gel Electrophoresis
[0189] Add 2 .mu.l orange G loading buffer and load on a 4.0%
Nusieve/Me (3:1) agarose.
[0190] Run at 150V. Products should be resolved in about 30
minutes.
[0191] FIG. 4 shows the expected banding pattern and the sequence
around the CAST-AciI polymorphism.
[0192] CAST ApaLl PCR-RFLP Test
[0193] ApaLI polymorphism
[0194] Exon: 6 (domain L)
[0195] Non-synonymous change: Ser-Asn
[0196] Allele 1-Asn (N)-AAT
[0197] Allele 2-Ser (S)-AGT
10 Primers C282F: 5' GTA AAG CCA AAG GAA CAC CCA G 3' (SEQ ID
NO:17) C28MR: 5' TTT TTA TTT CTC TGA TGT TGG CTG TGC A 3' (SEQ ID
NO:18)
[0198] PCR Conditions
[0199] Mix 1
11 10x PCR buffer 1.0 .mu.l MgCl.sub.2 (15 mM) 1.0 .mu.l dNTPs (2
mM) 1.0 .mu.l C282F primer (10 pm/.mu.l) 0.25 .mu.l C28MR primer
(10 pM/.mu.l) 0.25 .mu.l Taq polymerase (5 U/.mu.l) 0.07 .mu.l
ddH.sub.2O 5.43 .mu.l genomic DNA 1 .mu.l
[0200] Combine the Mix 1 and DNA in a reaction tube. Overlay with
mineral oil. Run the following PCR program: 94.degree. C. for 4
min.; 35 cycles of 94.degree. C. for 45 sec., 54.degree. C. for 60
sec and 72.degree. C. for 50 sec; followed by a final extension at
72.degree. C. for 12 min.
[0201] Check 2 .mu.l of the PCR on a 2% agarose gel to confirm
amplification. Product size is 535 bp.
[0202] ApaLI digestion.
[0203] The reverse primer (C28MR) was modified (engineered)
comparing with the original cDNA sequence and a ApaLI restriction
site was added in order to be able to differentiate the
alleles.
12 1X Volume (.mu.l) NEB Buffer 4 10X 1.0 ApaLI (10 units/.mu.l)
0.4 BSA 100X 0.1 ddWater 5.5 Mix Final Volume 7.0
[0204] Aliquot 7 .mu.l ApaLI mix and add 3 .mu.l PCR product.
[0205] Incubate at 37.degree. C.
[0206] Gel Electrophoresis
[0207] Add 2 .mu.l orange G loading buffer and load on a 4.0%
Nusieve/Me (3: 1) agarose.
[0208] Run at 150V. Products should be resolved in about 30
minutes.
[0209] FIG. 5 shows the expected band pattern and sequence around
the CAST ApaLI polymorphism.
EXAMPLE 2
[0210] A. CAST Linkage Mapping
[0211] For linkage mapping, the B.times.Y resource family (Malek et
al. 2001) was genotyped using the CAST-MspI marker previously
reported by Ernst et al. (1998) and two-point and multipoint
linkage analysis was performed using the CRI-MAP program (Green et
al. 1990).
[0212] The results of the linkage analysis showed that CAST gene
was significantly linked to five markers on SSC2 (the two point
recombination frequencies and LOD scores are given in parentheses):
SW766 (0.02, 112.92), SW1408 (0.12, 67.34), SW2157 (0.15, 25.99)
SW1844 (0.27, 13.16) and SW2445 (0.31, 11.36). These results and
multipoint linkage analysis show that the CAST gene is most likely
located between SW766 and SW1408, at approximately 73.1 Kosambi
cM.
[0213] B. Polymorphism Discovery
[0214] We sequenced the entire CAST gene using RT-PCR and analyzing
samples from the B.times.Y F.sub.3 generation family but also
samples from Duroc and Meishan pig breeds, in order to find
causative polymorphisms responsible for the phenotypic variation in
Plant/abbatoir (24 hr) loin Minolta, water holding capacity and
firmness in pigs (Malek et al., 2001). The primers used for
sequencing of the entire CAST coding region were designed based on
the published cDNA sequence of the CAST heart isoform (GenBank
Accession no. M20160). We found four unsynonymous substitutions
(see FIG. 1).
[0215] Unsynonymous Polymorphisms:
[0216] 1. CAST-Hpy188I:
[0217] AGA-AAA: Arg-Lys; position, exon 13 (domain 1);
[0218] Pig: Arg/Lys; rabbit, sheep, bovine, human and mouse,
cercopithecus: Arg.
[0219] 2. CAST-AciI
[0220] ACT-GCT: Thr-Ala; position, exon 22 (domain 3);
[0221] Pig: Ala/Thr; rabbit, human: Ala; sheep and bovine: Thr;
mouse: Ile.
[0222] 3. CAST-PvuII
[0223] AGA-AGC: Arg-Ser; position, exon 27 (domain 4);
[0224] Pig: Arg/Ser; rabbit, bovine, human, mouse: Ser.
[0225] 4. CAST-ApaLI
[0226] AGT-AAT: Ser-Asn; position, exon 6 (domain L)
[0227] The engineered CAST-ApaLI SNP is important because is in
complete linkage disequilibrium with CAST-Hpy188I even though there
are about 11 kb distance between them based on human genomic
sequence. This fact was revealed by genotyping over 200 animals
from different commercial lines. CAST-ApaLI SNP could substitute
CAST-Hpy188I which has some effects on meat tenderness.
[0228] The repetitive multidomain structure of human Calpastatin is
disclosed in Takano & Maki, 1999, the disclosure of which is
hereby incorporated herein by reference.
[0229] The CAST PvuII and AciI are in a very conserved region of
the subdomains. CAST Hpy188I is in exon 13 (TVRSAAP) in the second
part of subdomain C, domain 1. All four domains have inhibitory
functions independently (Maki et al., 1987; Emori et al., 1988).
Subdomain B is the inhibitory center (Ma et al., 1993). Subdomains
A and C are important for the potential of the inhibition activity
of CAST (Maki et al., 1988); Kawasaki et al., 1989; Uemori et al.,
1990). Ma et al. (1994) reported that single mutations in the
inhibitory center (subdomain B) or even in either subdomains A or C
(involved in the potentiation of the inhibition activity) affects
CAST activity.
EXAMPLE 3
[0230] Association Analysis Between the CAST Unsynonymous
Polymorphisms and Meat Quality and Growth Traits in Pigs.
[0231] We designed a PCR-RFLP test for each of the unsynonymous
polymorphism we discovered and we genotyped samples from the
following resources:
[0232] a) The F.sub.2 generation of the B.times.Y family.
[0233] b) 14 Duroc DNA samples with Calpastatin activity data from
Longisimus dorsi muscle measured at 0, 6 and 24 hr after
slaughter.
[0234] c) 64 samples of a F.sub.1 generation Duroc.times.Yorkshire
cross with meat quality data.
[0235] d) Three PIC commercial populations as follows: Large White
and Duroc synthetic based lines and also a Composite line.
[0236] a) The F.sub.2 generation of the B.times.Y family was used
for an association study between the CAST-Hpy188I and PvuII
substitutions with the traits with QTL in the area where CAST was
mapped (Table 2 and 15). CAST-AciI was not polymorphic in this
population. This polymorphism we found to be highly informative in
Meishan breed and the results are shown in Table 2. The table shows
that for CAST Hpy188I, the 11 (KK) genotype is associated with a
meat less firm, more juicy, tender and easier to chew and a lower
average Instron force. Similar results were obtained for CAST PvuII
association study(Table 15).
[0237] A haplotype analysis was run in order to be able to dissect
which polymorphism has real effects on the traits measured. There
are 3 haplotypes present: haplotype 1: Hpy188I-1 and PvuII-1;
haplotype 2: Hpy188I -2 and PvuII-1; haplotype 3: Hpy188I-2 and
PvuII-2 (Table 16). There are significant differences between the
effects of haplotype 1 and 3 for juiceness, instron force and chew
score. Haplotype 3 is associated with higher average instron force,
the meat is less tender and has a higher chew score. For firmness
there are significant differences between the effects of haplotype
1 and 2 and between 2 and 3, both sites being involved in the
phenotypic variation of this trait.
[0238] Using NetPhos2 software a prediction analysis was performed
of possible calpastatin phosphorylation substrates recognized by
PKA (cAMP-dependent Protein Kinase). PKA phosphorylates calpastatin
and agregates it near the nucleus (Alverna et al., 2001). Same
authors consider that this intracellular reversible mechanism
regulates the level of cytosolic CAST. Based on this, probably
during earlier steps of its inactivation calpains can escape
calpastatin inhibition. The prediction analysis revealed that PvuII
and eApaLI affect two phosphorylation consensus sequences and
finally possible change in calpastatin localization and ability to
inhibit calpains.
[0239] CAST ApaLI is in linkage disequilibrium with Hpy188I even
they are at about 11 kb. distance apart based on human CAST genomic
DNA sequence. A test for one of these SNPs can be used to genotype
actually both.
13TABLE 2 Association results between the genotypes of CAST Hpy1881
and some meat quality traits in F.sub.2 Berkshire .times. Yorkshire
family Least Square Means* Traits 11(KK) 12(KR) 22(RR) P Firmness
3.21.sup.g,e 3.44.sup.h 3.43.sup.f 0.0012 136 233 134 Juiceness
6.23.sup.c 6.05.sup.a 5.76.sup.d,b 0.0449 136 228 129 Tenderness
8.01.sup.c,a 7.74.sup.d 7.75.sup.b 0.1060 136 228 129 Chew score
2.32.sup.a 2.51.sup.b 2.54.sup.b 0.1084 136 228 129 Ave Instron
Force 4.39.sup.c 4.45.sup.c 4.63.sup.d 0.0457 127 213 128
*Significant differences: a - b p < .1; c - d p < .05; e - f
p < .005, g - h p < .0005
[0240] b) A total number of 14 Duroc DNA samples with CAST activity
data at 0, 6 and 24 hours were genotyped for the CAST-Hpy188I and
PvuII. In the case of Hpy188I where we have a similar number of
animals in the two genotype classes we identified a small
difference between the means in the favor of 11 genotype at 24 hr
(Table 3, FIG. 6). For the same polymorphism there were no
differences between the 11 and 12 genotypes regarding CAST activity
at 0 and 6 hr. The CAST-AciI polymorphism was not informative in
this set of samples. See FIG 6.
14TABLE 3 The mean values of the calpastatin activity in two of the
CAST Hpy188I and PvuII genotypes. Calpastatin activity Calpastatin
activity Calpastatin activity SNP/ 0 hr units/g 6 hr Units/g 24 hr
units/g Genotypes 11 12 11 12 11 12 Hpy188I 2.17 2.16 1.69 1.64
1.04 0.96 n 8 6 8 5 8 6 PvuII 2.20 1.99 1.67 1.72 0.97 1.27 n 12 2
12 1 12 2
[0241] c) A total number of 64 samples from a F.sub.1 generation
Duroc.times.Yorkshire cross with meat quality data, were genotyped
for the CAST-Hpy188I and PvuII polymorphisms. The CAST-AciI
polymorphism was not informative in this set of samples.
[0242] Associations were detected between CAST-Hpy188I polymorphism
with firmness (P=0.0936), average drip loss at day 1 (P=0.0701) and
WBS force at day 3 (P=0.0315) and 5 (P=0.0045) (Table 4).
Significant associations were detected between the genotypes of
this polymorphism and the traits mentioned before. The highest
association was for WBS force at day 5 (P=0.0045). Except the day 7
the highest variability of this trait is at day 5 and there is
possible that CAST-Hpy188I to be associated with CAST activity with
final effect on meat tenderness.
15TABLE 4 Association analysis of CAST Hpy188I marker with meat
quality, body composition and growth traits in a F.sub.1 Duroc
.times. Yorkshire cross No. Mean animals LSmeans (s.e.) Geno Trait
(s.e.) .sigma..sub.p 11 12 22 11 12 22 P Live 250.09 9.72 27 31 6
253.32 (3.78) 253.04 (3.46) 246.85 (10.16) 0.8722 Hot 193.49 7.67
27 31 6 194.70 (2.88) 195.18 (2.64) 194.48 (7.74) 0.9844
Dressing(%) 0.774 0.015 27 31 6 0.769 (0.0053) a 0.771 (0.0049)
0.787 (0.0143) b 0.5305 Loin Temp 45 min 35.831 1.38 27 31 6 35.27
(0.40) a 35.78 (0.37) b 36.79 (1.08) 0.2797 Loin pH 45 min 6.244
0.22 27 31 6 6.26 (0.09) 6.33 (0.08) 6.16 (0.23) 0.6611 Ham pH 45
min 6.26 0.31 27 31 6 6.32 (0.11) 6.39 (0.10) 6.07 (0.30) 0.6156
Loin pH 2 hr 5.88 0.32 27 31 6 5.86 (0.12) a 6.00 (0.11) b 5.71
(0.32) 0.4172 Ham pH 2 hr 5.87 0.31 27 31 6 5.89 (0.12) 6.00 (0.11)
5.77 (0.32) 0.5909 Loin Temp 4 hr 15.400 2.11 27 31 6 14.75 (0.59)
15.06 (0.54) 16.45 (1.58) 0.6308 Loin ph 4 hr 5.76 0.27 27 31 6
5.79 (0.11) 5.88 (0.10) 5.53 (0.28) 0.4686 Ham temp 4 hr 21.52 2.12
26 25 6 21.86 (0.50) c 21.66 (0.43) a 19.17 (1.27) d, b 0.2406 Ham
pH 4 hr 5.65 0.21 27 31 6 5.70 (0.07) 5.70 (0.06) 5.50 (0.18)
0.6587 Loin Temp 6 hr 10.682 2.70 25 22 5 10.24 (0.51) a 10.42
(0.47) 12.13 (1.37) b 0.5245 Loin pH 6 hr 5.69 0.20 27 31 6 5.67
(0.08) 5.75 (0.07) 5.58 (0.21) 0.5121 Ham Temp 6 hr 17.02 2.71 27
31 6 17.75 (0.42) 17.31 (0.36) 17.50 (1.27) 0.6618 Ham pH 6 hr 5.56
0.13 27 31 6 5.54 (0.04) 5.55 (0.04) 5.55 (0.11) 0.9874 Loin pH 24
hr 5.56 0.08 27 31 6 5.60 (0.03) a 5.56 (0.03) b 5.56 (0.08) 0.3814
Loin Temp 24 hr 2.095 0.52 27 31 6 2.04 (0.11) 2.15 (0.10) a 1.77
(0.30) b 0.4081 Temp BF 24 hr 2.948 0.49 27 31 6 3.00 (0.13) 3.09
(0.12) a 2.64 (0.35) b 0.4691 pH BF 24 hr 5.62 0.13 27 31 6 5.63
(0.04) 5.60 (0.04) 5.67 (0.12) 0.5856 Temp SM 24 hr 2.811 0.53 27
31 6 2.84 (0.14) 2.93 (0.13) 2.47 (0.38) 0.5354 pH SM 24 hr 5.60
0.13 27 31 6 5.60 (0.05) 5.58 (0.04) 5.59 (0.12) 0.8663 Last rib
fat 0.914 0.15 27 31 6 0.937 (0.041) 0.964 (0.038) a 0.819 (0.111)
b 0.4933 NPPC Color 2.55 0.60 27 31 6 2.42 (0.21) a 2.67 (0.20) b
2.74 (0.58) 0.4841 Marbling 1.633 0.58 27 31 6 1.65 (0.17) a 1.83
(0.16) b 1.39 (0.46) 0.4549 JCS 2.383 0.58 27 31 6 2.199 (0.20)
2.390 (0.18) 2.453 (0.53) 0.5960 LD L 49.69 3.31 27 31 6 50.49
(1.22) 49.70 (1.12) 47.87 (3.28) 0.6906 Ld a 4.12 1.14 27 31 6 4.64
(0.37) a 4.21 (0.34) b 3.80 (1.00) 0.4363 LD b 10.62 1.08 27 31 6
11.13 (0.35) a 10.84 (0.32) a 9.38 (0.94) b 0.2694 LD L* 56.59 3.19
27 31 6 57.36 (1.18) 56.59 (1.08) 54.87 (3.16) 0.6860 Ld a* 3.17
1.23 27 31 6 3.69 (0.39) a 3.27 (0.36) b 2.93 (1.06) 0.5109 SM L
44.92 2.64 27 31 6 44.45 (0.94) 45.13 (0.86) 44.93 (2.53) 0.7626 SM
a 6.73 1.27 27 31 6 7.28 (0.39) a 6.92 (0.36) a 5.16 (1.06) b
0.2132 SM b 10.44 0.99 27 31 6 10.60 (0.33) a 10.65 (0.30) a 9.37
(0.87) b 0.4897 SM L* 51.83 2.67 27 31 6 51.34 (0.95) 52.03 (0.87)
51.88 (2.56) 0.7595 SM a* 6.46 1.47 27 31 6 7.11 (0.45) a 6.64
(0.41) b, a 4.73 (1.22) b 0.2104 SM b* 15.90 1.43 27 31 6 16.23
(0.43) a 16.21 (0.40) a 14.13 (1.17) b 0.3352 BF L 45.87 3.70 27 31
6 45.98 (1.36) a 44.39 (1.25) b 45.64 (3.66) 0.4920 BF a 7.83 1.29
27 31 6 8.47 (0.39) 8.12 (0.35) 7.36 (1.04) 0.5067 BF b 10.93 1.24
27 31 6 11.13 (0.43) 10.71 (0.39) 10.18 (1.15) 0.5258 BF L* 52.79
3.69 27 31 6 52.85 (1.40) a 51.27 (1.23) b 52.57 (3.62) 0.4842 BF
a* 7.61 1.46 27 31 6 8.35 (0.42) 8.06 (0.38) 7.17 (1.13) 0.5956 BF
b* 16.57 1.65 27 31 6 16.88 (0.54) 16.42 (0.49) 15.29 (1.44) 0.5092
Drip Loss Day 1-5 0.0194 0.0067 27 31 6 0.020 (0.0015)a 0.019
(0.0013)b 0.016 (0.0039) 0.4072 Sirloin % Purge 0.0337 0.0148 27 31
6 0.031 (0.0043) 0.029 (0.0039) a 0.046 (0.0116)b 0.5071 BF % Purge
0.0313 0.0155 26 30 6 0.031 (0.0047) a 0.026 (0.0044) b 0.035
(0.0128) 0.4910 Day 1 WBS 3.143 27 31 6 2.940 (0.22) a 3.172 (0.20)
b 3.29 (0.59) 0.5329 Day 3 WBS 3.585 27 31 6 3.079 (0.22) a, e
3.426 (0.20) b, c 4.847 (0.59) f, d 0.0315 Day 7 WBS 3.586 27 31 6
3.301 (0.22) a 3.407 (0.20) a 4.279 (0.60) b 0.3953 LSmeans
significance levels: a-b p < .3 c-d p < .1 e-f p < .05 g-h
p < .01 i-j p < .005 k-l p < .001 m-n p < .0005 o-p p
< .0001
[0243] Associations were detected between CAST-PvuII polymorphism
and sirloin % purge (P=0.0121) and WBS force at day 5 (P=0.025)
(Table 5). Because there were only 2 animals with the 22 genotype,
we concentrated on the differences between 11 and 12 genotypes. For
WBS force at day 5 (overall P value=0.0045) we detected a
significant difference between 11 and 12 genotypes (p<0.01).
16TABLE 5 Association analysis of CAST PvuII marker with meat
quality, body composition and growth traits in a Duroc .times.
Yorkshire cross No. Mean animals LSmeans (s.e.) Geno Trait (s.e.)
.sigma..sub.p 11 12 22 11 12 22 P Live 250.09 9.72 40 22 2 254.46
(3.78) a 253.96 (3.36) a 233.68 (14.68) b 0.4522 Hot 193.49 7.67 40
22 2 197.11 (2.83) a 197.06 (2.52) c 176.93 (10.99) b, d 0.2515
Dressing(%) 0.774 0.015 40 22 2 0.7748 (0.0054) 0.7758 (0.0048)
0.7588 (0.0211) 0.7445 Loin Temp 45 min 35.831 1.38 40 22 2 35.484
(0.416) a 36.021 (0.370) b 36.286 (1.615) 0.5729 Loin pH 45 min
6.244 0.22 40 22 2 6.246 (0.088) 6.291 (0.078) 6.330 (0.342) 0.9139
Ham Temp 45 min 36.29 1.34 40 22 2 35.78 (0.41) a 36.57 (0.37) b
36.52 (1.61) 0.2990 Ham pH 45 min 6.26 0.31 40 22 2 6.31 (0.12)
6.29 (0.10) 6.35 (0.45) 0.9864 Loin temp 2 hr 26.669 1.85 40 22 2
25.847 (0.648) c 27.176 (0.577) d 25.960 (2.517) 0.2169 Loin pH 2
hr 5.88 0.32 40 22 2 5.86 (0.12) 5.91 (0.11) 6.02 (0.47) 0.9352 Ham
Temp 2 hr 28.08 3.03 40 22 2 28.75 (0.93) 27.77 (0.83) 27.86 (3.60)
0.6886 Ham pH 2 hr 5.87 0.31 40 22 2 5.92 (0.12) 5.85 (0.11) 6.18
(0.47) 0.7090 Loin Temp 4 hr 15.400 2.11 40 22 2 15.537 (0.599)
15.299 (0.533) 13.518 (2.328) 0.7535 Loin ph 4 hr 5.76 0.27 40 22 2
5.77 (0.11) 5.77 (0.097) 5.93 (0.425) 0.9375 Ham temp 4 hr 21.52
2.12 37 18 2 21.63 (0.52) 21.16 (0.46) 20.04 (1.92) 0.7013 Ham pH 4
hr 5.65 0.21 40 22 2 5.67 (0.07) 5.60 (0.06) 5.88 (0.27) 0.3920
Loin Temp 6 hr 10.682 2.70 40 22 2 10.977 (0.525) 10.559 (0.467)
9.880 (2.039) 0.7830 Loin pH 6 hr 5.69 0.20 40 22 2 567 (0.08) 5.70
(0.07) 5.74 (0.31) 0.9453 Ham pH 6 hr 5.56 0.13 40 22 2 5.55 (0.04)
5.53 (0.04) 5.57 (0.16) 0.8614 Loin pH 24 hr 5.56 0.08 40 22 2 5.61
(0.03) a 5.57 (0.03) b, a 5.42 (0.11) b 0.2774 Loin Temp 24 hr
2.095 0.52 40 22 2 1.944 (0.113) 2.061 (0.100) 2.387 (0.438) 0.5798
Temp BF 24 hr 2.948 0.49 40 22 2 2.90 (0.13) 3.05 (0.12) 3.05
(0.51) 0.6404 pH BF 24 hr 5.62 0.13 40 22 2 5.66 (0.04) a 5.62
(0.04) a 5.40 (0.17) b 0.4029 Temp SM 24 hr 2.811 0.53 40 22 2 2.64
(0.14) a 2.88 (0.13) b 3.26 (0.55) 0.3434 pH SM 24 hr 5.60 0.13 40
22 2 5.62 (0.05) 5.58 (0.04) 5.48 (0.18) 0.6812 Last rib fat 0.914
0.15 40 22 2 0.904 (0.042) 0.937 (0.038) 0.995 (0.165) 0.7831 NPPC
Color 2.55 0.60 40 22 2 2.55 (0.22) 2.69 (0.20) 2.48 (0.86) 0.8471
Firmness 1.875 0.42 40 22 2 1.873 (0.146) 1.938 (0.130) 1.857
(0.567) 0.9240 Wetness 1.945 0.50 40 22 2 1.833 (0.182) 2.022
(0.162) 2.325 (0.708) 0.6643 Marbling 1.633 0.58 40 22 2 1.56
(0.18) 1.72 (0.16) 2.11 (0.69) 0.6936 JCS 2.383 0.58 40 22 2 2.337
(0.200) 2.489 (0.178) 1.753 (0.777) 0.5415 LD L 49.69 3.31 40 22 2
49.65 (1.24) 48.83 (1.10) 52.61 (4.81) 0.6459 Ld a 4.12 1.14 40 22
2 4.33 (0.38) 4.20 (0.34) 4.35 (1.49) 0.9526 LD b 10.62 1.08 40 22
2 10.65 (0.36) 10.38 (0.32) 11.88 (1.40) 0.4931 LD L* 56.59 3.19 40
22 2 56.53 (1.19) 55.75 (1.06) 59.55 (4.63) 0.6336 Ld a* 3.17 1.23
40 22 2 3.41 (0.41) 3.29 (0.36) 3.25 (1.57) 0.9720 LD b* 15.53 1.37
40 22 2 15.56 (0.42) 15.26 (0.37) a 17.22 (1.62) b 0.4343 SM L
44.92 2.64 40 22 2 44.19 (0.95) 44.90 (0.85) 47.63 (3.70) 0.6821 SM
a 6.73 1.27 40 22 2 6.57 (0.41) 6.81 (0.37) 6.95 (1.61) 0.8942 SM b
10.44 0.99 40 22 2 10.10 (0.33) 10.40 (0.29) 11.64 (1.29) 0.5488 SM
L* 51.83 2.67 40 22 2 51.07 (0.96) 51.80 (0.86) 54.68 (3.74) 0.6642
SM a* 6.46 1.47 40 22 2 6.36 (0.48) 6.55 (0.42) 6.36 (1.85) 0.9396
SM b* 15.90 1.43 40 22 2 15.42 (0.45) a 15.82 (0.40) 17.63 (1.73) b
0.5111 BF L 45.87 3.70 40 22 2 44.56 (1.39) 44.38 (1.24) a 50.60
(5.40) b 0.5625 BF a 7.83 1.29 40 22 2 8.19 (0.40) 8.18 (0.36) 7.35
(1.55) 0.8861 BF b 10.93 1.24 40 22 2 10.65 (0.44) 10.62 (0.39)
11.66 (1.71) 0.8516 BF L* 52.79 3.69 40 22 2 51.45 (1.37) 51.26
(1.22) a 57.50 (5.34) b 0.5536 BF a* 7.61 1.46 40 22 2 8.16 (0.43)
8.13 (0.38) 6.73 (1.68) 0.7469 BF b* 16.57 1.65 40 22 2 16.27
(0.55) 16.24 (0.49) 17.22 (2.14) 0.9111 Ave Drip Loss 0.0212 0.0114
40 22 2 0.0219 (0.0045) 0.0217 (0.0040) 0.0197 (0.0174) 0.9939 Day
1 Drip Loss Day 1-5 0.0194 0.0067 40 22 2 0.0191 (0.0015) 0.0189
(0.0014) 0.0173 (0.0059) 0.9665 Ave Drip Loss 0.0402 0.0149 40 22 2
0.0405 (0.0052) 0.0402 (0.0046) 0.0365 (0.0201) 0.9849 SM % Purge
0.0344 0.0126 40 22 2 0.0340 (0.0041) 0.0328 (0.0037) 0.0490
(0.0160) 0.6198 BF % Purge 0.0313 0.0155 38 22 2 0.0327 (0.0049)
0.0311 (0.0043) 0.0105 (0.0189) 0.5984 Ave % Purge 0.0331 0.0104 40
22 2 0.0304 (0.0030) a 0.0309 (0.0027) a 0.0478 (0.0117) b 0.4199
Day 3 WBS 3.585 40 22 2 3.465 (0.236) a 3.860 (0.210) b 2.917
(0.917) 0.2339 Day 5 WBS 3.524 40 22 2 3.083 (0.208) g 3.802
(0.185) h 3.883 (0.807) 0.0250 Day 7 WBS 3.586 40 22 2 3.711
(0.227) a 3.385 (0.202) b 3.375 (0.881) 0.5032 LSmeans significance
levels: a-b p < .3 c-d p < .1 e-f p < .05 g-h p < .01
i-j p < .005 k-l p < .001 m-n p < .0005 o-p p <
.0001
[0244] d) We used several PIC commercial populations for an
association study in order to verify some of the significant
results we obtained using the F.sub.2 generation of the B.times.Y
family. The samples we used have data for meat quality, body
composition and growth traits. For these samples we did not have
Instron force data, but we had firmness (a subjective score) and
also drip percentage. We again used only CAST-Hpy199I and PvuII
markers. CAST AciI was not sufficiently polymorphic for an
association study (see Table 6).
17TABLE 6 Genotype and allelic frequency for the porcine
CAST-Hpy188I, PvuII and AciI polymorphisms in several PIC
commercial lines. Large Duroc Landrace White Berkshire Duroc
Synthetic Hampshire Pietrain Composite CastHpy188I 11 5 4 21 12 6
11 16 6 12 14 9 2 12 16 8 8 15 22 5 10 2 5 3 N 24 23 23 24 24 24 24
24 P 0.5 0.37 0.95 0.75 0.58 0.63 0.83 0.56 Cast-PvuII 11 21 7 19
21 9 11 24 19 12 10 2 3 14 7 5 22 5 1 5 N 21 22 21 24 24 23 24 24 P
1 0.55 0.95 0.94 0.67 0.63 1 0.9 Cast-AciI 11 21 24 22 22 24 22 22
24 12 3 1 1 2 22 N 24 24 23 22 24 23 24 24 P 0.94 1 0.98 1 1 0.98
0.96 1 p - frequency of allele 1
[0245] In the Large White based line we saw some significant
associations (Table 7 and 8 ). For both markers, firmness has the
same trend as in the B.times.Y population. The 11 (KK) CAST Hpy188I
genotype is associated with higher drip percentage. In the same
direction are also the effects of CAST-PvuII genotypes. Regarding
the other traits we can see that 22 genotype (for both of the
markers) is associated with slower growth rate and is leaner than
11. There are significant differences between the genotype on test
daily gain (TDG) and US-muscle depth (US_MD) for both of the
markers.
18TABLE 7 Association results between the genotypes of CAST Hpy188I
and some meat quality and growth traits in PIC Large White based
population. Least Square Means* Traits 11(KK) 12(KR) 22(RR) P
Firmness 2.74 (29).sup.a 2.88 (60) 2.98 (26).sup.b 0.36 29 60 26
Hprofat 14.28 (81).sup.a 13.89 (176).sup.b 13.87 (82) 0.48 81 176
82 LDG, g/d 648.7 (90).sup.a 649.5 (193).sup.a 643.0 (101).sup.b
0.32 90 193 101 TDG, g/d 846.1 (86).sup.c 846.7 (189).sup.e 830.0
(101).sup.d f 0.09 86 189 101 US_MD 57.90 (85).sup.e 58.50
(185).sup.e 60.33 (98).sup.f 0.03 85 185 98 *Significant
differences: a - b p < .3; c - d p < .1; e - f p < .05
[0246]
19TABLE 8 Association results between the genotypes of CAST PvuII
and some meat quality and growth traits in PIC Large White based
population Least Square Means* Traits 11 (RR) 12 (RS) 22 (SS) P
Firmness 2.80 (44) 2.87 (53) 2.97 (10) 0.69 44 53 10 Hprofat 13.96
(111) 14.06 (164) 13.38 (41) 0.28 111 164 41 LDG, g/d 649.2
(123).sup.a 648.5 (185).sup.c 637.9 (50).sup.d 0.13 123 185 50 TDG,
g/d 846.1 (118).sup.e 846.7 (182).sup.c 830.0 (50).sup.f,d 0.06 118
182 50 US_MD 57.69 (116).sup.e,a 59.56 (176).sup.f 59.44 (50).sup.b
0.05 116 176 50 *Significant differences: .sup.a-bp < .3;
.sup.c-dp < .1; .sup.e-fp < .05
[0247] In a joint analysis including both markers with and without
interaction between them, some associations are suggested. There
are interactions detected only for boneless weight of the loin
(P=0.03), loinpH (0.04) and hampH (0.02) but the number of
individuals in four of the classes is very low making it difficult
to draw any conclusions. Including both genotypes in the model, the
overall probability was <0.10 for US_MD for Hpy188I, and for
loinminb, LMprct, US_MD for PvuII respectively.
[0248] In the Duroc Synthetic line we saw some significant
associations (Tables 9 and 10). For both markers, the most
important association was for drip percentage: P=0.004 for PvuII
and P=0.03 for Hpy188I. The difference between LS means of
homozygous classes was 1.56% for PvuII and 0.74% for Hpy188I.
[0249] In the Composite line for the CAST-Hpy188I an important
association was detected for firmness (Table 11). The 11 genotype
is associated with lower firmness, as for Large White population
(both markers), and for B.times.Y (CAST-Hpy188I) and the F1
generation Duroc.times.Yorkshire for CAST-Hpy188I.
[0250] For the CAST-PvuII an interesting association was found for
h_binwt (bone in weight of the ham), the 11 genotype being
associated with the higher value (Table 12). Also an association
was detected for hpromeat (Henessey probe loin depth), with the 11
genotype having the highest value. This association was also
confirmation by haplotype analysis in same line (contrast P is 0.06
between haplotype 2 and 3, difference between them is due to PvuII
site).
[0251] Across the lines we detected for CAST Hpy188I some
associations for firmness (0.07) (11 genotype-lower firmness like
in B.times.YF2 population), hamminl (P=0.05), hprorib (P=0.14),
endwt (P=0.02), LDG (P=0.06), TDG (0.04) and for US_MD
(P=0.05).
[0252] For the last four traits the same associations were observed
and they were in the same direction as was obtained for Large White
population (Table 13).
[0253] For the CAST-PvuII some associations were observed for
loinminb (0.001), for hammina (P=0.01), endwt (P=0.03), LDG
(P=0.13) and for TDG (0.12). For the last three traits the same
associations were observed and were in the same direction as was
obtained for Large White population and also across lines analysis
for CAST Hpy188I (Table 14)
20TABLE 9 Analysis of meat quality and production traits with CAST
Hpy-188I in PIC Duroc Synthetic based line. No. animals Lsmeans
(s.e.) geno .alpha. .delta. Trait Mean (s.e.) .sigma..sub.p 11 12
22 11 12 22 p trait (s.e.) p trait (s.e.) p Marbling 2.50 (0.05)
0.71 75 104 23 2.47 (0.09) a 2.62 (0.07)b e 2.26 (0.14)b f 0.04
-0.10 (0.08) a 0.17 (0.07) c LoinpH 5.73 (0.01) 0.14 106 176 55
5.74 (0.01) e 5.73 (0.01) c 5.69 (0.02)f d 0.11 -0.02 (0.01) c 0.01
(0.01) Hamminl 47.98 (0.30) 4.34 75 105 25 48.60 (0.59) a c 47.62
(0.53) b 46.84 (0.89) d 0.15 -0.88 (0.50) b -0.07 (0.39)
[0254]
21TABLE 10 Analysis of meat quality and production traits with CAST
PvuII in PIC Duroc Synthetic based line. No. animals Lsmeans (s.e.)
geno .alpha. .delta. Trait Mean (s.e.) .sigma..sub.p 11 12 22 11 12
22 p trait (s.e.) p trait (s.e.) p L_binwt 20.64 (0.15) 2.12 113 64
8 20.80 (0.14) e 20.20 (0.18)f a 21.08 (0.51) b 0.02 0.14 (0.27)
-0.49 (0.21) c Dripprct 2.25 (0.10) 1.37 103 51 7 2.10 (0.12)a i
2.44 (0.17)b e 3.66 (0.45)j f 0.004 0.78 (0.24) e -0.29 (0.19)
a
[0255]
22TABLE 11 Analysis of meat quality and production traits with CAST
Hpy-188I in PIC Composite line. No. animals Lsmeans (s.e.) geno
.alpha. .delta. Trait Mean (s.e.) .sigma..sub.p 11 12 22 11 12 22 p
trait (s.e.) p trait (s.e.) p Firmness 2.71 (0.11) 0.94 26 28 14
2.57 (0.13) c i 2.87 (0.11) d 3.29 (0.17) j c 0.005 0.36 (0.11) e
-0.04 (0.11) Endwt 113.3 (0.47) 7.88 82 145 40 111.8 (1.02) e 111.6
(0.80) e 108.6 (1.38) f 0.10 -1.63 (0.80) c 0.89 (0.69) a
[0256]
23TABLE 12 Analysis of meat quality and production traits with CAST
PvuII in PIC Composite line. No. animals Lsmeans (s.e.) geno
.alpha. .delta. Trait Mean (s.e.) .sigma..sub.p 11 12 22 11 12 22 p
trait (s.e.) p trait (s.e.) p h_binwt 25.43 (0.18) 1.98 76 23 1
25.56 (0.22)e a 24.80 (0.34) f 23.61 (1.39) b 0.05 -0.97 (0.69) a
0.15 (0.49) Hpromeat 63.19 (0.64) 9.74 149 49 4 60.97 (0.89) e
58.16 (1.35) f 58.89 (4.10) 0.13 -1.04 (2.05) -1.18 (1.54)
[0257]
24TABLE 13 Analysis of meat quality and production traits with CAST
Hpy-188I Across All lines No. animals LSmeans (s.e.) Trait Mean
(s.e.) .sigma..sub.p 11 12 22 11 12 22 Marbling 2.18 (0.03) 0.78
179 310 104 2.23 (0.05) 2.28 (0.04) a 2.20 (0.07) b Firmness 2.86
(0.06) 1.06 118 163 58 2.95 (0.06)c e 3.10 (0.05) d 3.17 (0.09) f
Hamminl 46.89 (0.20) 4.52 156 253 86 47.62 (0.39) e 47.24 (0.33) e
46.19 (0.49) f Hprorib 14.59 (0.21) 4.07 130 182 61 15.22 (0.42)c a
14.30 (0.37) d 14.30 (0.56) b Endwt 111.6 (0.24) 7.62 285 518 201
111.4 (0.46) g 110.9 (0.37) e 109.6 (0.54)h f LDG, g/d 664.5 (1.55)
49.7 285 518 201 665.6 (2.91) e 663.3 (2.26) c 656.8 (3.16)f d TDG,
g/d 862.1 (2.53) 75.4 225 450 185 870.7 (5.11) e 867.8 (3.94) e
854.3 (5.56) f US_MD 61.52 (0.27) 8.09 226 449 183 59.48 (0.56) e
59.71 (0.46) e 60.95 (0.60) f .alpha. .delta. geno Line * geno p p
Trait p p trait (s.e.) 1 2 trait (s.e.) 1 2 Marbling 0.40 0.009
-0.02 (0.04) c 0.05 (0.03) a c Firmness 0.07 0.02 0.11 (0.05) c e
0.03 (0.05) Hamminl 0.05 0.69 -0.71 (0.29) c 0.22 (0.24) Hprorib
0.14 0.80 -0.46 (0.33) a -0.31 (0.29) a Endwt 0.02 0.38 -0.91
(0.33) d 0.30 (0.29) a LDG, g/d 0.06 0.89 -4.37 (1.93) c 1.42
(1.72) TDG, g/d 0.04 0.75 -8.17 (3.44) c 3.54 (3.07) a US_MD 0.05
0.39 0.74 (0.33) c a -0.33 (0.28) a
[0258]
25TABLE 14 Analysis of meat quality and production traits with CAST
PvuII Across All lines No. animals LSmeans (s.e.) Trait Mean (s.e.)
.sigma..sub.p 11 12 22 11 12 22 Loinminb 3.17 (0.04) 1.23 480 337
71 3.35 (0.05)e a 3.52 (0.06)f i 3.16 (0.11)b j Hamminl 46.89
(0.20) 4.52 254 167 31 47.25 (0.33) a 47.23 (0.39) a 46.05 (0.80) b
Hammina 8.59 (0.08) 1.8 254 166 31 8.60 (0.12)e a 8.97 (0.14) f
8.27 (0.29)b e Endwt 111.6 (0.24) 7.62 506 351 77 111.2 (0.39)c e
110.3 (0.46)da 109.0 (0.86)f b LDG, g/d 664.5 (1.55) 49.7 506 351
77 663.1 (2.43) c 662.9 (2.70) c 653.4 (4.81) d TDG, g/d 862.1
(2.53) 75.4 422 313 73 869.2 (4.20) e 865.5 (4.70) c 850.8 (8.50) f
d .alpha. .delta. geno Line * geno p p Trait p p trait (s.e.) 1 2
trait (s.e.) 1 2 Loinminb 0.001 0.38 -0.09 (0.06) a a 0.18 (0.05) g
Hamminl 0.32 0.76 -0.60 (0.41) a 0.39 (0.34) a Hammina 0.01 0.40
-0.16 (0.15) a 0.36 (0.12) e Endwt 0.03 0.65 -1.08 (0.45) c 0.11
(0.38) LDG, g/d 0.13 0.47 -4.82 (2.53) b 3.08 (2.13) a TDG, g/d
0.12 0.77 -9.18 (4.49) c 3.66 (3.81)
[0259] Haplotype analysis for each population revealed several
interesting associations. This analysis allows us to estimate the
impact of the different polymorphisms. In order to do this we are
interested in the differences between effects of haplotypes that
are different at only one polymorphic site so as to detect each
site effect. For example in the Duroc Synthetic and Composite Line
where we have three haplotypes the unique difference between
haplotype 2 (2-1) and 3 (2-2) is at the PvuII site. In this way it
is possible to estimate the potential effect of the PvuII
polymorphism. In summary based on haplotype analysis we revealed
the following effects (taking in account contrast P values lower
than 0.10):
[0260] CAST Hpy188I has effects (or is in linkage disequilibrium
with QTL) on: loinminl (Large White), loinpH (Large White), days on
test (Large White), hamminl (Large White, all lines), boneless
weight of the loin (Duroc), firmness (Composite), LDG (Composite,
all lines), TDG (all lines).
[0261] CAST-PvuII has effects (or is in linkage disequilibrium with
QTL) on: loinminl (Large White, Duroc and all lines), loin pH
(Large White), hpromeat (Large White, all lines), aloca backfat
(Large White), days on test (Large White), drip percentage (Duroc),
US_MD (Composite) and bone in weight of the ham (Composite).
[0262] Both CAST Hpy188I and PvuII have an effect (or are in
linkage disequilibrium with a QTL) on % drip loss. We also ran an
analysis including both the Duroc and Composite line because there
are just 3 haplotypes present in both lines in order to attempt a
better estimate of the effects. We obtained a highly significant
difference for % drip loss (larger as in Duroc) (Table 17) between
the effects of haplotype 1 and 3. Haplotype 3 is associated with
higher drip loss as expected based on the other analyses. Also
significant differences between haplotype 2 and 3 were revealed,
again similar to the Duroc population, suggesting an effect of
PvuII alone. When allele 2 is present for both sites (haplotype 3)
the change in % drip loss is significant. When we compared the
haplotype results with single association study we obtained the
same direction in the phenotype variation. For example in the case
of firmness, allele CAST-Hpy188I-1 is associated with lower
firmness compared with the 2 allele. The same result was revealed
by haplotype analysis. The same situation applies for another
trait--drip percentage with a strong association that was revealed
in Duroc population for CAST-PvuII polymorphism. (as indicated we
only consider here the effects based on the differences between the
haplotypes and only the differences where P<0.10 taken in
account).
26TABLE 15 Association results between the genotypes of CAST PvuII
and some meat quality traits in F.sub.2 Berkshire .times. Yorkshire
family TRAITS 11(RR) 12(RS) 22(SS) P Leanness 35.37.sup.a
36.10.sup.b 35.92 0.232 Firmness 3.33 3.42 3.41 0.301 24 h loin pH
5.73.sup.a,c 5.76.sup.b 5.78.sup.d 0.087 Juiceness 6.19.sup.c 6.03
5.76.sup.d 0.093 Tenderness 7.98.sup.a 7.76.sup.b 7.76 0.151 Chew
score 2.37 2.51 2.53 0.238 InstronForce 4.39.sup.a 4.49 4.62.sup.b
0.105 WHC 0.203 0.199 0.178 0.277 Significant differences:
.sup.a-bp < .1; .sup.c-dp < .05; .sup.e-fp < .005,
.sup.g-hp < .0005. n = 168 (11), 209-216 (12) and 98-104
(22).
[0263]
27TABLE 16 CAST haplotype substitution effects for some meat
quality traits in B .times. Y Haplotype* effect Contrast p value
Trait 1 2 3 1 vs 2 1 vs 3 2 vs 3 Juiciness 0.22 0.06 0 0.43 0.01
0.75 Tenderness 0.14 0.10 0 0.82 0.07 0.55 Chew score -0.12 -0.02 0
0.43 0.03 0.87 InstronForce (kg) -0.14 -0.21 0 0.54 0.008 0.07
Firmness -0.06 0.18 0 0.01 0.10 0.04 frequency *haplotype 1:
Hpy188I - 1 and PvuII - 1 0.50 haplotype 2: Hpy188I - 2 and PvuII -
1 0.07 haplotype 3: Hpy188I - 2 and PvuII - 2 0.43 n = 448-482
[0264]
28TABLE 17 CAST haplotype substitution effects for % drip loss in
two PIC populations Haplotype* freq. Haplotype effect Contrast p
value Line n 1 2 3 1 2 3 1 vs 2 1 vs 3 2 vs 3 DS 154 0.61 0.19 0.20
-0.55 -0.46 0 0.66 0.004 0.04 C 93 0.62 0.28 0.10 -0.47 -0.24 0
0.28 0.17 0.51 DS + C 297 0.61 0.22 0.17 -0.58 -0.40 0 0.23 0.0004
0.03 *haplotype 1: Hpy188I - 1 and PvuII - 1. haplotype 2: Hpy188I
- 2 and PvuII - 1. haplotype 3: Hpy188I - 2 and PvuII - 2.
EXAMPLE 4
[0265] In order to demonstrate further the effect of the markers
discovered in the Calpastatin gene on meat quality and growth
traits, we tested their effects in additional populations of
pigs:
[0266] 1. Meat Quality Data Set A.
[0267] Phenotypic data (meat quality, body composition and growth
traits) were collected on three commercial populations or lines.
Statistical analysis was undertaken to determine associations
between CAST genotype and variation in the phenotypic traits.
[0268] The associations between the CAST polymorphisms and the
traits considered were tested using mixed model procedures
(SAS.RTM. procedure MIXED, SAS Institute Inc., Cary, N.C.) with a
model which always included sire as a random effect and slaughter
date and marker genotype(s) as fixed effects. Line was added as a
fixed effect for across line analyses. Sex and farm were not
included because all traits were measured on females only and no
more than one farm was represented on each slaughter date. While
males were not used in this portion of the analysis our results in
the B.times.Y suggest no sex by genotype effect (see example
3).
[0269] The number of animals used in association analyses varied
based on the trait measured, and are listed in the tables.
[0270] Results for relevant meat quality and growth traits are
shown in Tables 1 and 2 for a Large White based line for two of the
markers (CAST Hpy188I and CAST PvuII).
[0271] These samples did not have instron force data, but firmness
(a subjective score) and drip percentage were recorded. For both
markers, firmness shows the same trend as was observed for the
B.times.Y population (Example 3)--genotype 11 being associated with
lower firmness (a lower value is considered preferable). This
difference did not reach statistical significance (p=0.36 for CAST
Hpy188I) however this is a subjective score which is a less
powerful measure. However, the trend is in the expected direction
and it is expected to be a significant difference when larger
numbers are recorded. The 22 genotype (for both of the markers) is
associated with slower growth rate than the 11 and 12 genotypes and
there is a trend for animals of this genotype to be leaner.
[0272] Results with a Duroc based population are shown in Tables 3
and 4. Significant associations were found for both markers for
Henessey probe loin depth (genotype 11 associated with higher least
square (LS) mean value); CAST-PvuII and marbling (P=0.010) where
genotype 11 has a lower value.
[0273] The third population was a synthetic or composite population
originally made by crossing several different breeds. Results are
shown in Tables 5 and 6. For the CAST-Hpy188I, a highly significant
association was detected for firmness (P=0.005 1; Table 5). The 11
genotype is associated with lower firmness, exactly as in the
B.times.Y F.sub.2 experiment (see Example 3) or in the other
populations in this example. For CAST-PvuII a highly significant
association was discovered again for Henessey probe loin depth
(genotype 11 has a higher LS value; P=0.006) (Table 6). Drip loss
also tends to be lower with genotype 11 for both of the markers.
The difference is statistically significant for CAST Hpy188I
(P=0.03).
[0274] In order to improve the estimate of the marker effects, the
data from the all populations were combined and analysed using the
same model (this was deemed to be appropriate as there was no
evidence of a significant interaction of genotype by population)
(Table 7 and 8).
[0275] The most significant association for both of the markers was
(as expected from the individual results) with firmness. There are
also significant differences between the LS means of the genotypes
(p<0.05). There is a 0.26 unit difference between the homozygous
genotypes for CAST PvuII and 0.1 3 units for CAST Hpy188I--and
again the 11 genotype was associated with lower firmness for both
markers (Table 7 and 8).
29TABLE 1 Association results between CAST Hpy188I genotype and
some meat quality and growth traits in a Large White based
population. Least Square Traits 11 (KK) 12 (KR) 22 (RR) P Means*
Firmness 2.74 (.14).sup.a 2.88 (.11) 2.98 (.15).sup.b 0.36 29 60 26
Hprofat 14.28 (.35).sup.a 13.89 (.29).sup.b 13.87 (.37) 0.48 81 176
82 LDG, g/d 648.7 (4.42).sup.a 649.5 (3.36).sup.a 643.0
(4.29).sup.b 0.32 90 193 101 TDG, g/d 846.1 (7.84).sup.c 846.7
(5.94).sup.e 830.0 (7.60).sup.d f 0.09 86 189 101 US_MD 57.90
(.77).sup.e 58.50 (.57).sup.e 60.33 (.74).sup.f 0.03 85 185 98
*Significant differences: .sup.a-bp < .3; .sup.c-dp < .1;
.sup.e-fp < .05
[0276]
30TABLE 2 Association results between CAST PvuII genotype and some
meat quality and growth traits in a Large White based population
Least Square Means* Traits 11 (RR) 12 (RS) 22 (SS) P Firmness 2.80
(.12) 2.87 (.12) 2.97 (.21) 0.69 44 53 10 Hprofat 13.96 (.31).sup.a
14.06 (.28).sup.a 13.38 (.45).sup.b 0.28 111 164 41 LDG, g/d 649.2
(3.93).sup.c 648.5 (3.42).sup.c 637.9 (5.53).sup.d 0.13 123 185 50
TDG, g/d 849.1 (6.94).sup.e 842.6 (6.03).sup.c 823.2 (9.82).sup.f,d
0.06 118 182 50 US_MD 57.69 (.69).sup.e,a 59.56 (.58).sup.f 59.44
(.98).sup.b 0.05 116 176 50 *Significant differences: .sup.a-bp
< .3; .sup.c-dp < .1; .sup.e-fp < .05
[0277]
31TABLE 3 Association results between CAST Hpy188I genotype and
some meat quality and growth traits in a Duroc based population.
Least Square Traits 11 (KK) 12 (KR) 22 (RR) P Means* Dripprct 1.81
(.13).sup.a 2.01 (.15).sup.b 1.87 (.30).sup.b 0.43 137 100 17
Hpromeat 53.15 (.82).sup.c,k 51.22 (.92).sup.d,e 46.38
(1.92).sup.l,f 0.002 129 95 17 aloca backfat 12.47 (.28).sup.e
12.77 (.31).sup.c 13.92 (.64).sup.f,d 0.09 136 99 17 *Significant
differences: .sup.a-bp < .3; .sup.c-dp < .1; .sup.e-fp <
.05; .sup.k-1p < .001
[0278]
32TABLE 4 Association results between CAST PvuII genotype and some
meat quality and growth traits in a Duroc based population Least
Square Means* Least Square Traits 11 (RR) 12 (RS) 22 (SS) P Means*
Firmness 3.21 (.05).sup.a 3.27 (.09) 3.60 (.33).sup.b 0.45 152 54 3
Dripprct 1.84 (.12).sup.a 2.03 (.17).sup.b 1.83 (.48) 0.50 175 76 6
Hpromeat 52.94 (.75).sup.i,a 49.43 (1.10).sup.j 48.04 (3.08).sup.b
0.0045 166 72 6 Marbling 2.76 (.08).sup.e,c 2.56 (0.11).sup.f 3.29
(0.28).sup.d,e 0.01 175 76 6 *Significant differences: .sup.a-bp
< .3; .sup.c-dp < .1; .sup.e-fp < .05; .sup.i-jp <
.005
[0279]
33TABLE 5 Association results between CAST Hpy188I genotype and
some meat quality and growth traits in a Composite population.
Least Square Traits 11 (KK) 12 (KR) 22 (RR) P Means * Firmness 2.89
3.19 (.10).sup.f,c 3.50 (.14).sup.j,d 0.0051 33 44 21 (.13).sup.e,i
Dripprct 2.06 2.41 (.22).sup.b 2.46 (.28).sup.b 0.33 47 64 27
(.26).sup.a *Significant differences: .sup.a-bp < .3; .sup.c-dp
< .1; .sup.e-fp < .05; .sup.i-j p < .005
[0280]
34TABLE 6 Association results between CAST PvuII genotype and some
meat quality and growth traits in a Composite population Least
Square Means* Least Square Traits 11 (RR) 12 (RS) 22 (SS) P Means*
Firmness 3.17 (.11) 3.21 (.13) 3.40 (.34) 0.81 67 28 3 Dripprct
2.11 (.20).sup.g 2.84 (.26).sup.h 2.41 (.72) 0.03 103 34 3 Hpromeat
62.57 (.87).sup.e,g 59.80 (1.19).sup.f,c 53.65 (3.35).sup.h,d 0.006
175 64 6 LMprct 47.26 (.23).sup.a,i 46.81 (.29).sup.b,e 45.05
(.74).sup.j,f 0.014 62 27 3 Marbling 2.14 (.08).sup.g 2.25
(.11).sup.e 3.21 (.39).sup.h,f 0.02 125 46 3 US_MD 63.21
(.7).sup.c,a 61.63 (.91).sup.d 59.78 (2.52).sup.b 0.13 215 72 6
*Significant differences: .sup.a-bp < .3; .sup.c-dp < .1;
.sup.e-fp < .05; .sup.g-hp < .01; .sup.i-jp < .005.
[0281]
35TABLE 7 Association results between CAST Hpy188I genotype and
some meat quality and growth traits across all lines/populations.
Least Square Traits 11 (KK) 12 (KR) 22 (RR) P Means* Firmness 2.96
3.06 (.05).sup.f 3.09 (.07) 0.06 319 359 102 (.06).sup.e,c Dripprct
2.06 2.11 (.10) 2.14 (.14) 0.82 367 421 123 (.11) *Significant
differences: .sup.a-bp < .3; .sup.c-dp < .1; .sup.e-fp <
.05
[0282]
36TABLE 8 Association results between CAST PvuII genotype and some
meat quality and growth traits across all lines/populations. Least
Square Means* Least Traits 11 (RR) 12 (RS) 22 (SS) P Square Means*
Firmness 3.03 (.05).sup.e 3.04 (.05).sup.e 3.29 (.11).sup.f 0.06
495 249 30 Dripprct 2.07 (.1).sup.a 2.21 (.11).sup.b 2.04 (.22)
0.32 575 295 37 *Significant differences: .sup.a-bp < .3;
.sup.c-dp < .1; .sup.e-fp < .05
[0283] Haplotype Analysis
[0284] In order to estimate the effect of both markers, we
constructed haplotypes and repeated the analysis. Three common
haplotypes were identified: 1 (1.sub.--1), 2 (2.sub.--1) and 3
(2.sub.--2). The combined effects of the three substitutions were
estimated as haplotype substitution effects. Contrasts between
haplotypes were estimated from a model including sire (random),
slaughter date and one variable for each haplotype with values -1,
0 and 1 corresponding to the animal having 0, 1 or 2 copies of the
haplotype in question. The haplotype substitution effects were
presented as deviations from the effect of haplotype 3 which was
set arbitrary to 0.
[0285] Haplotype analysis on each population and across the lines
revealed several interesting associations (Table 9). The difference
between haplotype 1 and 2 reflects the effect of the Hpy188I site
and the differences between haplotype 2 and 3 are due to the PvuII
site. In summary, based on haplotype analysis we revealed the
following effects (taking in account contrast P values lower than
0.10):
[0286] CAST Hpy188I has effects (or is in linkage disequilibrium
with QTL) on: days on test (Large White), Henessey probe backfat
thickness, aloca backfat (Duroc), firmness (Composite--P=0.0008),
life time daily gain (LDG) (Duroc, all lines), Henessey probe rib
thickness (Composite, all lines), daily gain while on test (TDG)
(Duroc, all lines) and the weight of the end of the test (Duroc,
all lines).
[0287] CAST-PvuII has effects (or is in linkage disequilibrium with
QTL) on: Henessey probe loin depth (Large White, Composite), aloca
backfat (Large White), days on test (Large White), life time daily
gain (Duroc), daily gain while on test (Duroc) and the weight of
the end of the test (Duroc), Henessey probe backfat thickness
(Duroc), Henessey probe rib thickness, muscle depth at the end of
the test and lean meat % of the carcass (Composite).
[0288] Effects of both markers on: marbling (Composite), percentage
drip loss (Composite; P=0.04; haplotype 1 is associated with a
lower substitution effect), Henessey probe loin depth (Composite,
P=0.008; haplotype 1 is associated with a high substitution effect;
Duroc, haplotype 1 is associated with a higher substitution
effect), firmness (all; P=0.06; haplotype 1 is associated with a
lower substitution effect), life time daily gain (Large White),
daily gain while on test (Large White) and the weight of the end of
the test (Large White--P=0.009--haplotype 1 is associated with a
higher substitution effect; all lines--P=0.07--haplotype 1 is
associated with a higher substitution effect), lean meat % of the
carcass (Composite) and muscle depth at the end of the test (Large
White).
37TABLE 9 Haplotype analysis. Meat quality data set A. estimate
contrast p values line trait mean (s.e.) s.d. hap1 hap2 Hap3 hap1
vs 2 hap1 vs 3 Hap2 vs 3 Large hpromeat 50.30 (0.70) 12.3 -0.39
-1.65 0 0.13 0.52 0.05 White aloc_f 13.33 (0.17) 3.14 0.31 0.69 0
0.32 0.28 0.09 endwt 109.2 (0.36) 6.53 1.52 0.81 0 0.37 0.009 0.31
days 171.0 (0.79) 10.2 -0.34 -2.86 0 0.10 0.78 0.08 ldg 644.7
(2.24) 41.2 6.89 5.25 0 0.73 0.05 0.28 tdg 844.1 (3.83) 69.6 13.35
7.03 0 0.42 0.02 0.38 us_md 59.02 (0.39) 7.04 -1.42 -0.92 0 0.54
0.02 0.27 Duroc hprofat 14.03 (0.21) 3.3 0.22 1.20 0 0.09 0.60 0.09
hpromeat 51.19 (0.46) 7.2 3.30 1.77 0 0.25 0.0008 0.27 aloc_f 12.78
(0.18) 2.92 -0.24 0.80 0 0.02 0.45 0.12 endwt 106.0 (0.67) 10.6
-0.63 -4.58 0 0.011 0.58 0.014 ldg 646.4 (3.78) 60 -4.75 -28.08 0
0.008 0.46 0.008 tdg 823.6 (6.80) 102 -9.00 -33.10 0 0.08 0.39 0.05
Composite firmness 2.92 (0.10) 1.01 -0.20 0.19 0 0.0008 0.11 0.18
dripprct 2.14 (0.11) 1.32 -0.52 -0.39 0 0.50 0.04 0.15 hpromeat
63.33 (0.64) 9.91 3.10 3.92 0 0.39 0.008 0.002 marbling 2.21 (0.07)
0.87 -0.25 -0.12 0 0.19 0.04 0.34 hprorib 14.30 (0.44) 4.25 -0.93
-2.52 0 0.08 0.34 0.03 LMprct 47.24 (0.14) 1.33 0.64 0.93 0 0.27
0.03 0.006 us_md 65.96(0.50) 8.41 1.14 1.90 0 0.26 0.19 0.04 All
lines firmness 3.05 (0.04) 1.03 -0.07 -0.01 0 0.13 0.06 0.92
hprorib 14.90 (0.14) 4.04 0.24 -0.29 0 0.09 0.38 0.42 endwt 110.2
(0.23) 8.89 0.67 -0.21 0 0.04 0.07 0.66 ldg 662.7 (1.46) 55.4 2.84
-3.11 0 0.02 0.21 0.30 tdg 852.3 (2.44) 83.1 4.20 -6.03 0 0.02 0.27
0.23
[0289] Comparing the haplotype results with the single marker
association results we can see as expected for the significant
traits the same direction in the phenotype variation. For example,
in the case of firmness, allele CAST-Hpy188I-1 is associated with
lower firmness compared to the 2 allele. The same result was
revealed by haplotype analysis. Haplotype 1 was also found to be
the preferred haplotype in the B.times.Y population (see Example
3).
[0290] Trait Description--Data Set A
[0291] Firmness--subjective score of loin firmness (1 to 3)--lower
is better.
[0292] Percentage drip loss (Drpprct)--amount of moisture lost from
the longissimus muscle during 48 h.--lower is better.
[0293] Henessey probe loin depth (hpromeat)--higher is better.
[0294] Muscle depth at the end of the test (us_md)--higher is
better
[0295] Daily gain while on test (TDG)--g/day--higher is better
[0296] Life time daily gain (LDG)--g/day--higher is better
[0297] Weight of the end of the test (endwt)
[0298] Lean meat % of the carcass (LMprct)
[0299] Days on test (days)
[0300] Henessey probe backfat thickness (hprofat)
[0301] Aloca backfat (aloc_f)--backfat thickness p2 position.
[0302] Henessey probe rib thickness (hprorib)
[0303] 2. Meat Quality Data Set B.
[0304] The individuals sampled for this study represent common
commercial (slaughter pig) pigs resulting from crosses involving
three or more pure lines. Animals were harvested in a commercial
abattoir. Phenotypic data was collected for several subjective and
non-subjective meat quality traits. Loins from each individual were
aged for exactly 14 days from the date when the carcasses were cut
into primals. After 14 days of aging, purge loss, cooking loss,
drip loss, moisture %, intramuscular fat % (IMF) and shear force
were measured.
[0305] For the individual marker analysis a linear model was used
with product (combination of sireline and damline) as fixed effect.
The genotype was entered as fixed effect to estimate Least Squares
Means. Single marker association analysis revealed significant
association for tenderness and tenderness related traits:
[0306] CAST Hpy188I has effects on: cooking loss_% (P=0.0004;
genotype 11 has a lower least square mean value; there is a 4.26%
difference between the homozygotes LS means); moisture % (P=0.08;
11 has a lower LS mean value); subjective juiciness and tenderness
score (P=0.06-0.07; genotype 11 has a higher value). There is also
evidence for an effect on shear force (P=0.16 ) difference between
the homozygotes=0.23 (Table 10).
[0307] CAST-PvuII has effects on: loin pH (P=0.06) and juiciness
score (P=0.04; genotype 11 has a higher LS mean value) (Table
11).
38TABLE 10 Analysis of meat quality and production traits with CAST
Hpy 188I - Meat quality data set B. No. animals LSmeans (s.e.)
Trait Mean (s.e.) .sigma..sub.p 11 12 22 11 12 22 geno p drip_%
1.72 (0.04) 0.6 39 105 48 1.62 (0.11) 1.66 (0.07) 1.72 (0.10) 0.73
Shear Force 1.91 (0.04) 0.54 39 106 47 1.73 (0.10) a c 1.90 (0.06)
b 1.96 (0.09) d 0.16 Cooking 24.09 (0.35) 4.96 39 106 48 22.29
23.83 (0.57) b i 26.55 (0.82) n j 0.0004 loss_% (0.90) am
Moisture_% 74.87 (0.05) 0.74 39 106 48 74.57 (0.13) ec 74.88 (0.08)
f 74.88 (0.12) d 0.08 Subjective 7.39 (0.09) 1.34 39 106 46 7.68
(0.24) e a 7.09 (0.15) f 7.21 (0.22) b 0.07 Tenderness score
Subjective 8.09 (0.08) 1.17 39 106 46 8.32 (0.21) c e 7.91 (0.13) d
7.72 (0.19) f 0.06 Juiciness score *Significant differences: a-b p
< .3; c-d p < .1; e-f p < .05; g-h p < .01; i-j p <
.005; m-n p < .0005
[0308]
39TABLE 11 Analysis of meat quality and production traits with CAST
PvuII - Meat quality data set B. No. animals LSmeans (s.e.) Trait
Mean (s.e.) .sigma..sub.p 11 12 22 11 12 22 geno p LoinpH 5.73
(0.01) 0.18 78 64 16 5.72 (0.03) e 5.70 (0.03) e 5.60 (0.05) f 0.06
Drip_% 1.72 (0.04) 0.6 95 86 23 1.64 (0.08) 1.68 (0.08) 1.77 (0.14)
0.66 Shear Force 1.91 (0.04) 0.54 96 86 22 1.87 (0.07) 1.90 (0.07)
1.89 (0.12) 0.93 Cooking 24.09 (0.35) 4.96 96 86 23 23.67 (0.63) e
24.04 (0.63) c 26.00 (1.11) f d 0.14 loss_% Fat_% 2.00 (0.05) 0.68
78 71 18 1.87 (0.10) 1.95 (0.09) 1.91 (0.17) 0.77 Subjective 7.39
(0.09) 1.34 96 85 22 7.44 (0.17) c 7.08 (0.17) d 7.15 (0.30) 0.18
Tenderness score Subjective 8.09 (0.08) 1.17 96 85 22 8.22 (0.15) e
7.84 (0.15) f 7.66 (0.27) f 0.04 Juiciness score *Significant
differences: a-b p < .3; c-d p < .1; e-f p < .05; g-h p
< .01; i-j p < .005.
[0309] Haplotype Analysis
[0310] Three common haplotypes were identified: 1, 2 and 3. The
combined effects of the three substitutions were estimated as
haplotype substitution effects. A linear model was used with
product (combination of sireline and damline) as fixed effect.
Contrasts between haplotypes were estimated from a model in which
we used one variable for each haplotype with values -1, 0 and 1
corresponding to the animal having 0, 1 or 2 copies of the
haplotype in question. The haplotype substitution effects were
presented as deviations from the effect of haplotype 3 which was
set arbitrary to 0 (Table 12).
[0311] CAST Hpy188I has effects on: shear force (P=0.04; haplotype
1 has lower substitution effect), cooking loss (P=0.0004; haplotype
1 has a lower substitution effect).
[0312] effects of both markers on: cooking loss (P=0.002; haplotype
1 has a lower substitution effect), subjective tenderness (P=0.09;
haplotype 1 is associated with a higher substitution effect) and
juiciness score (P=0.008; haplotype 1 has a higher substitution
effect).
40TABLE 12 Haplotype analysis - Meat quality data set B. estimate
contrast p values Trait mean (s.e.) s.d. hap1 hap2 hap3 hap1 vs 2
hap1 vs 3 Hap2 vs 3 Loin pH 5.73 (0.02) 0.18 0.055 0.026 0 0.34
0.03 0.37 Shear Force 1.92 (0.04) 0.55 -0.074 0.097 0 0.04 0.28
0.22 Cooking loss_% 24.23 (0.36) 5.01 -1.916 0.727 0 0.0004 0.002
0.30 Moisture_% 74.87 (0.05) 0.76 -0.151 -0.001 0 0.18 0.10 0.99
Subjective Tenderness score 7.33 (0.10) 1.32 0.276 0.199 0 0.69
0.09 0.29 Subjective Juiciness score 8.02 (0.08) 1.14 0.381 0.253 0
0.45 0.008 0.12
[0313] Trait Description--Data Set B
[0314] Cooking loss %--measured in the 14 days aged longissimus
muscle at 80.degree. C.
[0315] Juiciness--moisture feeling inside the mouth as a result of
the chewing (subjective).
[0316] Tenderness--force required to bite through the loin sample
(subjective).
[0317] Shear Force--measurement of the tenderness of the broiled
chops-lower is better.
[0318] 3. Meat Quality Data Set C.
[0319] This set consists in gilts from 10 different commercial
lines. A large number of meat quality traits were measured
including a large set of sensory and texture traits, starting with
juiciness, fibrosity, etc., and ending up with acceptance.
[0320] For the individual marker association analysis a mixed model
was used with line and slaughterdate as fixed effects and sire as
random effect. The genotype was entered as fixed effect to estimate
Least Squares Means. (Table 13 and 14). Single marker association
analysis revealed the following significant associations:
[0321] CAST Hpy188I has effects on the sensory and texture traits
like: crumbliness (P=0.05; genotype 11 is associated with a higher
LS mean value) and fibrosity (P=0.03; genotype 11 has a lower
value); cooking loss was not significant (P=0.18) but genotype 11
has a lower value and the difference between homozygotes is close
to significance (P<0.10).
[0322] CAST-PvuII has effects on: intramuscular fat (Gluteus
medius) (P=0.04; genotype 11 is significantly leaner) and firmness
(P=0.05).
41TABLE 13 Analysis of meat quality and production traits with CAST
Hpy 188I - Meat quality data set C. No. animals LSmeans (s.e.)
Trait Mean (s.e.) .sigma..sub.p 11 12 22 11 12 22 geno p s_crumbli
4.38 (0.03) 0.8 162 235 183 4.54 4.40 4.31 (0.06) f b 0.05 (0.07) c
e (0.05) d a cooking loss 33.70 (0.12) 2.29 96 136 103 33.37 33.71
(0.16) b 33.94 (0.20) d 0.18 (0.21) a c s_fibrosity 3.35 (0.03)
0.81 162 235 183 3.24 (0.06) e 3.43 (0.05) f 3.42 (0.06) f 0.03
t_gumines 6.25 (0.07) 1.17 70 108 83 6.01 (0.15) e 6.45 (0.12) f a
6.23 (0.15) b 0.05 *Significant differences: a-b p < .3; c-d p
< .1; e-f p < .05; g-h p < .01; i-j p < .005.
[0323]
42TABLE 14 Analysis of meat quality and production traits with CAST
PvuII - Meat quality data set C. No. animals LSmeans (s.e.) Trait
Mean (s.e.) .sigma..sub.p 11 12 22 (s.e.) geno p IMFGm 1.29 (0.03)
0.48 151 110 58 1.24 (0.04) e a 1.39 (0.04) f 1.33 (0.06) b 0.04
Firmness 2.93 (0.05) 0.83 111 118 46 2.91 (0.08) c 2.87 (0.07) e
3.17 (0.11) d f 0.05 *Significant differences: a-b p < .3; c-d p
< .1; e-f p < .05; g-h p < .01; i-j p < .005.
[0324] Haplotype Analysis
[0325] Three common haplotypes were identified: 1, 2 and 3. The
combined effects of the three substitutions were estimated as
haplotype substitution effects. A mixed model was used with line
and slaughterdate as fixed effects and sire as random effect.
Contrasts between haplotypes were estimated from a model in which
we used one variable for each haplotype with values -1, 0 and 1
corresponding to the animal having 0, 1 or 2 copies of the
haplotype in question. The haplotype substitution effects were
presented as deviations from the effect of haplotype 3 which was
set arbitrary to 0 (Table 15). Based on the contrast between the
haplotype effects we were able to reveal the effect of each marker
on several meat quality traits, and it is attributed separately,
based on the contrast between the haplotype's effects:
[0326] CAST Hpy188I has effects on several sensory and texture
traits: hardness (P=0.004); crumbliness (P<0.0001, haplotype 1
is associated with a higher substitution effect); juiciness
(P=0.07, haplotype 1 has a higher substitution effect); fibrosity
(P=0.003); acceptance (P=0.005); guminess (P=0.02); cooking loss
(P=0.l 1; haplotype 1 has a lower substitution effect).
[0327] CAST-PvuII has effects on: hardness (P=0.0005); crumbliness
(P=0.0005); fibrosity (P=0.02); acceptance (P=0.003) and guminess
(P=0.02).
43TABLE 15 Haplotype analysis - Meat quality data set C. estimate
contrast p values Trait mean (s.e.) s.d. hap1 hap3 hap4 hap1 vs 2
hap1 vs 3 Hap2 vs 3 S_hardness 4.18 (0.04) 0.88 0.075 0.284 0 0.004
0.23 0.0005 S_crumbliness 4.39 (0.03) 0.8 0.007 -0.256 0 <.0001
0.91 0.0005 cooking loss 33.73 (0.12) 2.23 -0.200 0.140 0 0.11 0.28
0.57 S_juiciness 3.11 (0.03) 0.74 0.016 -0.095 0 0.07 0.75 0.16
S_fibrosity 3.35 (0.03) 0.8 -0.019 0.164 0 0.003 0.72 0.02
S_acceptance 4.39 (0.04) 0.85 -0.037 -0.239 0 0.005 0.54 0.003
t_guminess 6.22 (0.07) 1.17 0.021 0.401 0 0.02 0.87 0.02
[0328] Trait Description--Data Set C
[0329] Cooking loss--mesured in the longissimus muscle at
80.degree. C.--lower is better.
[0330] Intramuscular fat (IMFGm)--measured by NIT Gluteus Medius
muscle.
[0331] Hardness--force required to bite through the loin
sample--lower is better.
[0332] Fibrosity--textural property measured by ease with which a
substance can be separated--lower is better.
[0333] Juiciness--moisture feeling inside the mouth as a result of
the chewing--higher is better.
[0334] Acceptability--or acceptance: an experience characterized by
a positive attitude--higher is better.
[0335] Crumbliness--textural property characterized by ease with
which a substance can be separated into smaller particles during
the chewing--higher is better.
[0336] Guminess--was defined as the product of
hardness.times.cohesiviness- --lower is better.
[0337] These results further support the findings (Example 3)
indicating that the CAST Hpy188I and PvuII polymorphisms are useful
as markers in selection programs for tenderness and/or related meat
quality traits. Haplotype I is the preferred haplotype for
juiciness, tenderness and firmness; CAST Hpy188I seems to have
slightly larger effects than PvuII.
[0338] In addition, there are effects on growth/loin depth in some
populations and in the across lines analysis (dataset A). Haplotype
1 is associated with faster growth. These effects could be related
to the effect of Calpastatin/calpain system on protein turnover or
reflect linkage disequilibrium with another locus directly
impacting these traits. It is possible to utilize the CAST markers
(through linkage disequilibrium) to select for these traits.
[0339] In the Composite line and the across lines analysis (data
set A), a significant effect was found for firmness. Haplotype 1
was associated with lower substitution effect for firmness, the
same haplotype was also found to be the preferred haplotype in the
B.times.Y F.sub.2 resource population (Example 3).
[0340] In the Composite line (data set A) a significant effect was
found for drip loss; signs of associations for this particular
trait were also found in other lines/data sets.
[0341] Meat quality data set B revealed very significant effects on
cooking loss, significant differences in instron force and
subjective tenderness measures. Using haplotype analysis we were
able to detect a highly significant 5.29% difference in cooking
loss between the homozygote classes.
[0342] Both markers have significant effects on several tenderness
and tenderness related measures on meat quality in set C. For one
of these traits, for example,--acceptability--a highly significant
difference was revealed between the substitution effects of the
worst and the best haplotype.
[0343] In general, haplotype 1 is associated with a more tender,
juicy meat; less cooking loss and firmness and more acceptable pork
and can therefore be used to select for improved meat quality.
[0344] References
[0345] All references cited herein are hereby incorporated in their
entirety by reference. This includes but is not limited to:
[0346] Alverna, M., De Tulio, R., Passalacqua, M., Salamino, F.,
Pontremoli, S., Melloni, E., 2001, Changes in intracellular
calpastatin localization are mediated by reversible
phosphorylation, Biochem. J. (2001) 354, 25-30.
[0347] Ernst C. W., Robic A., Yerle M., Wang L., Rothschild M. F.,
1998 Mapping of calpastatin and three microsatellites to porcine
chromosome 2q2.1-q2.4., Anim. Genet. 29 212-215.
[0348] Ma, H., Yang, H. Q, Takano, E., Hatanaka, M., Maki, M.,
1994, Amino-terminal conserved region in proteinase inhibitor
domain of calpastatin potentiates its calpain inhibitory activity
by interacting with calmodulin-like domain of proteinase. J. Biol.
Chem. 268:24430-24436.
[0349] Malek M., J. C. M. Dekkers, H. K. Lee, T. J. Baas, K. Prusa,
E. Huff-Lonergan, M. F. Rothschild (2001). A molecular genome scan
analysis to identify chromosomal region influencing economic traits
in the pig. II. Meat and muscle composition. Mammalian Genome 12,
637-645.
Sequence CWU 1
1
25 1 2159 DNA Sus scrofa CDS (67)..(2139) 1 tctctcggcc gggaagccag
aagcagagta tcgccttcct ctgcttcaac gagcaagtct 60 tccagt atg aat ccc
aca gaa acc aag gct gta aaa aca gaa cct gaa 108 Met Asn Pro Thr Glu
Thr Lys Ala Val Lys Thr Glu Pro Glu 1 5 10 aag aag tca caa tca act
aag cca tct gtg gtt cat gag aaa aaa acc 156 Lys Lys Ser Gln Ser Thr
Lys Pro Ser Val Val His Glu Lys Lys Thr 15 20 25 30 caa gaa gta aag
cca aag gaa cac cca gag cca aaa agc cta ccc acg 204 Gln Glu Val Lys
Pro Lys Glu His Pro Glu Pro Lys Ser Leu Pro Thr 35 40 45 cac tca
gca gat gca ggg agc aag cgt gct cat aaa gaa aaa gca gtt 252 His Ser
Ala Asp Ala Gly Ser Lys Arg Ala His Lys Glu Lys Ala Val 50 55 60
tcc aga tct aat gag cag cca aca tca gag aaa tca aca aaa cca aag 300
Ser Arg Ser Asn Glu Gln Pro Thr Ser Glu Lys Ser Thr Lys Pro Lys 65
70 75 gct aaa cca cag gac ccg acc ccc agt gat gga aag ctt tct gtt
act 348 Ala Lys Pro Gln Asp Pro Thr Pro Ser Asp Gly Lys Leu Ser Val
Thr 80 85 90 ggt gta tct gca gca tct ggc aaa cca gct gag acg aaa
aaa gat gat 396 Gly Val Ser Ala Ala Ser Gly Lys Pro Ala Glu Thr Lys
Lys Asp Asp 95 100 105 110 aaa tca tta aca tcg tct gta cca gct gaa
tcc aaa tca agt aaa cca 444 Lys Ser Leu Thr Ser Ser Val Pro Ala Glu
Ser Lys Ser Ser Lys Pro 115 120 125 tca gga aag tca gat atg gat gct
gct ttg gat gac tta ata gac act 492 Ser Gly Lys Ser Asp Met Asp Ala
Ala Leu Asp Asp Leu Ile Asp Thr 130 135 140 tta gga gga cct gaa gaa
act gag gaa gat aat aca aca tat act gga 540 Leu Gly Gly Pro Glu Glu
Thr Glu Glu Asp Asn Thr Thr Tyr Thr Gly 145 150 155 cct gaa gtt ttg
gat cca atg agt tct acc tat ata gag gaa ttg ggt 588 Pro Glu Val Leu
Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly 160 165 170 aaa aga
gaa gtc aca ctt cct cca aaa tat agg gaa ttg ttg gat aaa 636 Lys Arg
Glu Val Thr Leu Pro Pro Lys Tyr Arg Glu Leu Leu Asp Lys 175 180 185
190 aaa gaa ggg att cca gtg cct cct cca gac act tcg aaa ccc ctg ggg
684 Lys Glu Gly Ile Pro Val Pro Pro Pro Asp Thr Ser Lys Pro Leu Gly
195 200 205 ccc gat gat gcc atc gat gcc ttg tca tta gac ttg acc tgc
agt tct 732 Pro Asp Asp Ala Ile Asp Ala Leu Ser Leu Asp Leu Thr Cys
Ser Ser 210 215 220 cct aca gct gat ggg aag aaa acc gag aaa gag aaa
tct act ggg gag 780 Pro Thr Ala Asp Gly Lys Lys Thr Glu Lys Glu Lys
Ser Thr Gly Glu 225 230 235 gtt ttg aaa gct cag tct gtt ggg gta atc
aga agc gct gct gct cca 828 Val Leu Lys Ala Gln Ser Val Gly Val Ile
Arg Ser Ala Ala Ala Pro 240 245 250 ccc cac gag aaa aaa aga agg gtg
gaa gag gac acg atg agt gat caa 876 Pro His Glu Lys Lys Arg Arg Val
Glu Glu Asp Thr Met Ser Asp Gln 255 260 265 270 gca ctg gag gct ttg
tca gct tcc ctg ggc agc cgg aag tca gaa ccc 924 Ala Leu Glu Ala Leu
Ser Ala Ser Leu Gly Ser Arg Lys Ser Glu Pro 275 280 285 gag ctt gac
ctc agc tcc att aag gaa att gat gag gca aaa gcc aaa 972 Glu Leu Asp
Leu Ser Ser Ile Lys Glu Ile Asp Glu Ala Lys Ala Lys 290 295 300 gaa
gag aaa cta aag aag tgt ggt gaa gat gac gaa acg gtc ccg cca 1020
Glu Glu Lys Leu Lys Lys Cys Gly Glu Asp Asp Glu Thr Val Pro Pro 305
310 315 gag tat aga ttg aaa cca gcc atg gat aaa gat gga aaa cca ctc
ttg 1068 Glu Tyr Arg Leu Lys Pro Ala Met Asp Lys Asp Gly Lys Pro
Leu Leu 320 325 330 cca gag gct gaa gaa aaa ccc aag ccc ctg agt gaa
tca gaa ctc att 1116 Pro Glu Ala Glu Glu Lys Pro Lys Pro Leu Ser
Glu Ser Glu Leu Ile 335 340 345 350 gac gaa ctt tcg gaa gat ttt gac
cag tct aag cgt aaa gaa aaa caa 1164 Asp Glu Leu Ser Glu Asp Phe
Asp Gln Ser Lys Arg Lys Glu Lys Gln 355 360 365 tct aag cca act gaa
aaa aca aaa gag tct cag gcc act gcc cct act 1212 Ser Lys Pro Thr
Glu Lys Thr Lys Glu Ser Gln Ala Thr Ala Pro Thr 370 375 380 cct gtg
gga gag gcc gtg tct cgg acc tcc ttg tgc tgt gtg cag tcg 1260 Pro
Val Gly Glu Ala Val Ser Arg Thr Ser Leu Cys Cys Val Gln Ser 385 390
395 gca ccc cca aag cca gct acg ggc atg gtg cca gat gat gct gta gaa
1308 Ala Pro Pro Lys Pro Ala Thr Gly Met Val Pro Asp Asp Ala Val
Glu 400 405 410 gcc ttg gct gga agc ctg ggg aaa aag gaa gca gat cca
gaa gat gga 1356 Ala Leu Ala Gly Ser Leu Gly Lys Lys Glu Ala Asp
Pro Glu Asp Gly 415 420 425 430 aag cct gtg gag gat aaa gtc aag gag
aaa gcc aaa gaa gag gat cgt 1404 Lys Pro Val Glu Asp Lys Val Lys
Glu Lys Ala Lys Glu Glu Asp Arg 435 440 445 gaa aaa ctt ggt gaa aag
gaa gaa acg att cct cct gat tat aga tta 1452 Glu Lys Leu Gly Glu
Lys Glu Glu Thr Ile Pro Pro Asp Tyr Arg Leu 450 455 460 gaa gag gtc
aag gac aaa gat gga aaa act ctc ccg cac aaa gac ccc 1500 Glu Glu
Val Lys Asp Lys Asp Gly Lys Thr Leu Pro His Lys Asp Pro 465 470 475
aag gaa cca gtc ctg ccc ttg agt gaa gac ttc gtc ctt gat gct ttg
1548 Lys Glu Pro Val Leu Pro Leu Ser Glu Asp Phe Val Leu Asp Ala
Leu 480 485 490 tcc cag gac ttt gcc ggt ccc cca gcc gct tca tct ctt
ttt gaa gat 1596 Ser Gln Asp Phe Ala Gly Pro Pro Ala Ala Ser Ser
Leu Phe Glu Asp 495 500 505 510 gct aaa ctt tca gct gcc gtc tct gaa
gtg gtt tcc caa acc tca gct 1644 Ala Lys Leu Ser Ala Ala Val Ser
Glu Val Val Ser Gln Thr Ser Ala 515 520 525 cca acc acc cac tct gca
ggt cca ccc cct gac act gtg agt gat gac 1692 Pro Thr Thr His Ser
Ala Gly Pro Pro Pro Asp Thr Val Ser Asp Asp 530 535 540 aaa aaa ctt
gac gat gcc ctg gat cag ctt tct gac agt ctg ggg caa 1740 Lys Lys
Leu Asp Asp Ala Leu Asp Gln Leu Ser Asp Ser Leu Gly Gln 545 550 555
aga cag cct gac cca gat gag aac aag ccc ata gag gat aaa gtc aag
1788 Arg Gln Pro Asp Pro Asp Glu Asn Lys Pro Ile Glu Asp Lys Val
Lys 560 565 570 gaa aaa gct gaa gct gaa cat aga gac aag ctg gga gaa
aga gat gac 1836 Glu Lys Ala Glu Ala Glu His Arg Asp Lys Leu Gly
Glu Arg Asp Asp 575 580 585 590 act atc ccg cct gaa tat aga cat ctc
ttg gat aag gat gag gag ggc 1884 Thr Ile Pro Pro Glu Tyr Arg His
Leu Leu Asp Lys Asp Glu Glu Gly 595 600 605 aaa tca acg aag cca ccc
aca aag aaa cct gag gca cca aag aaa cct 1932 Lys Ser Thr Lys Pro
Pro Thr Lys Lys Pro Glu Ala Pro Lys Lys Pro 610 615 620 gaa gct gcc
caa gat ccc att gat gcc ctc tca ggg gat ttt gac aga 1980 Glu Ala
Ala Gln Asp Pro Ile Asp Ala Leu Ser Gly Asp Phe Asp Arg 625 630 635
tgt cca tca act aca gaa acc tca gag aac aca aca aag gac aaa gac
2028 Cys Pro Ser Thr Thr Glu Thr Ser Glu Asn Thr Thr Lys Asp Lys
Asp 640 645 650 aag aag acg gct tcc aag tcc aaa gca ccc aag aat ggg
ggt aaa gca 2076 Lys Lys Thr Ala Ser Lys Ser Lys Ala Pro Lys Asn
Gly Gly Lys Ala 655 660 665 670 aag gat tcc aca aag gca aag gag gaa
act tcc aaa caa aaa tct gat 2124 Lys Asp Ser Thr Lys Ala Lys Glu
Glu Thr Ser Lys Gln Lys Ser Asp 675 680 685 gga aag agt aca agt
taaaagttca cactattttc 2159 Gly Lys Ser Thr Ser 690 2 691 PRT Sus
scrofa 2 Met Asn Pro Thr Glu Thr Lys Ala Val Lys Thr Glu Pro Glu
Lys Lys 1 5 10 15 Ser Gln Ser Thr Lys Pro Ser Val Val His Glu Lys
Lys Thr Gln Glu 20 25 30 Val Lys Pro Lys Glu His Pro Glu Pro Lys
Ser Leu Pro Thr His Ser 35 40 45 Ala Asp Ala Gly Ser Lys Arg Ala
His Lys Glu Lys Ala Val Ser Arg 50 55 60 Ser Asn Glu Gln Pro Thr
Ser Glu Lys Ser Thr Lys Pro Lys Ala Lys 65 70 75 80 Pro Gln Asp Pro
Thr Pro Ser Asp Gly Lys Leu Ser Val Thr Gly Val 85 90 95 Ser Ala
Ala Ser Gly Lys Pro Ala Glu Thr Lys Lys Asp Asp Lys Ser 100 105 110
Leu Thr Ser Ser Val Pro Ala Glu Ser Lys Ser Ser Lys Pro Ser Gly 115
120 125 Lys Ser Asp Met Asp Ala Ala Leu Asp Asp Leu Ile Asp Thr Leu
Gly 130 135 140 Gly Pro Glu Glu Thr Glu Glu Asp Asn Thr Thr Tyr Thr
Gly Pro Glu 145 150 155 160 Val Leu Asp Pro Met Ser Ser Thr Tyr Ile
Glu Glu Leu Gly Lys Arg 165 170 175 Glu Val Thr Leu Pro Pro Lys Tyr
Arg Glu Leu Leu Asp Lys Lys Glu 180 185 190 Gly Ile Pro Val Pro Pro
Pro Asp Thr Ser Lys Pro Leu Gly Pro Asp 195 200 205 Asp Ala Ile Asp
Ala Leu Ser Leu Asp Leu Thr Cys Ser Ser Pro Thr 210 215 220 Ala Asp
Gly Lys Lys Thr Glu Lys Glu Lys Ser Thr Gly Glu Val Leu 225 230 235
240 Lys Ala Gln Ser Val Gly Val Ile Arg Ser Ala Ala Ala Pro Pro His
245 250 255 Glu Lys Lys Arg Arg Val Glu Glu Asp Thr Met Ser Asp Gln
Ala Leu 260 265 270 Glu Ala Leu Ser Ala Ser Leu Gly Ser Arg Lys Ser
Glu Pro Glu Leu 275 280 285 Asp Leu Ser Ser Ile Lys Glu Ile Asp Glu
Ala Lys Ala Lys Glu Glu 290 295 300 Lys Leu Lys Lys Cys Gly Glu Asp
Asp Glu Thr Val Pro Pro Glu Tyr 305 310 315 320 Arg Leu Lys Pro Ala
Met Asp Lys Asp Gly Lys Pro Leu Leu Pro Glu 325 330 335 Ala Glu Glu
Lys Pro Lys Pro Leu Ser Glu Ser Glu Leu Ile Asp Glu 340 345 350 Leu
Ser Glu Asp Phe Asp Gln Ser Lys Arg Lys Glu Lys Gln Ser Lys 355 360
365 Pro Thr Glu Lys Thr Lys Glu Ser Gln Ala Thr Ala Pro Thr Pro Val
370 375 380 Gly Glu Ala Val Ser Arg Thr Ser Leu Cys Cys Val Gln Ser
Ala Pro 385 390 395 400 Pro Lys Pro Ala Thr Gly Met Val Pro Asp Asp
Ala Val Glu Ala Leu 405 410 415 Ala Gly Ser Leu Gly Lys Lys Glu Ala
Asp Pro Glu Asp Gly Lys Pro 420 425 430 Val Glu Asp Lys Val Lys Glu
Lys Ala Lys Glu Glu Asp Arg Glu Lys 435 440 445 Leu Gly Glu Lys Glu
Glu Thr Ile Pro Pro Asp Tyr Arg Leu Glu Glu 450 455 460 Val Lys Asp
Lys Asp Gly Lys Thr Leu Pro His Lys Asp Pro Lys Glu 465 470 475 480
Pro Val Leu Pro Leu Ser Glu Asp Phe Val Leu Asp Ala Leu Ser Gln 485
490 495 Asp Phe Ala Gly Pro Pro Ala Ala Ser Ser Leu Phe Glu Asp Ala
Lys 500 505 510 Leu Ser Ala Ala Val Ser Glu Val Val Ser Gln Thr Ser
Ala Pro Thr 515 520 525 Thr His Ser Ala Gly Pro Pro Pro Asp Thr Val
Ser Asp Asp Lys Lys 530 535 540 Leu Asp Asp Ala Leu Asp Gln Leu Ser
Asp Ser Leu Gly Gln Arg Gln 545 550 555 560 Pro Asp Pro Asp Glu Asn
Lys Pro Ile Glu Asp Lys Val Lys Glu Lys 565 570 575 Ala Glu Ala Glu
His Arg Asp Lys Leu Gly Glu Arg Asp Asp Thr Ile 580 585 590 Pro Pro
Glu Tyr Arg His Leu Leu Asp Lys Asp Glu Glu Gly Lys Ser 595 600 605
Thr Lys Pro Pro Thr Lys Lys Pro Glu Ala Pro Lys Lys Pro Glu Ala 610
615 620 Ala Gln Asp Pro Ile Asp Ala Leu Ser Gly Asp Phe Asp Arg Cys
Pro 625 630 635 640 Ser Thr Thr Glu Thr Ser Glu Asn Thr Thr Lys Asp
Lys Asp Lys Lys 645 650 655 Thr Ala Ser Lys Ser Lys Ala Pro Lys Asn
Gly Gly Lys Ala Lys Asp 660 665 670 Ser Thr Lys Ala Lys Glu Glu Thr
Ser Lys Gln Lys Ser Asp Gly Lys 675 680 685 Ser Thr Ser 690 3 2159
DNA Sus scrofa CDS (67)..(2139) 3 tctctcggcc gggaagccag aagcagagta
tcgccttcct ctgcttcaac gagcaagtct 60 tccagt atg aat ccc aca gaa acc
aag gct gta aaa aca gaa cct gaa 108 Met Asn Pro Thr Glu Thr Lys Ala
Val Lys Thr Glu Pro Glu 1 5 10 aag aag tca caa tca act aag cca tct
gtg gtt cat gag aaa aaa acc 156 Lys Lys Ser Gln Ser Thr Lys Pro Ser
Val Val His Glu Lys Lys Thr 15 20 25 30 caa gaa gta aag cca aag gaa
cac cca gag cca aaa agc cta ccc acg 204 Gln Glu Val Lys Pro Lys Glu
His Pro Glu Pro Lys Ser Leu Pro Thr 35 40 45 cac tca gca gat gca
ggg agc aag cgt gct cat aaa gaa aaa gca gtt 252 His Ser Ala Asp Ala
Gly Ser Lys Arg Ala His Lys Glu Lys Ala Val 50 55 60 tcc aga tct
aat gag cag cca aca tca gag aaa tca aca aaa cca aag 300 Ser Arg Ser
Asn Glu Gln Pro Thr Ser Glu Lys Ser Thr Lys Pro Lys 65 70 75 gct
aaa cca cag gac ccg acc ccc agt gat gga aag ctt tct gtt act 348 Ala
Lys Pro Gln Asp Pro Thr Pro Ser Asp Gly Lys Leu Ser Val Thr 80 85
90 ggt gta tct gca gca tct ggc aaa cca gct gag acg aaa aaa gat gat
396 Gly Val Ser Ala Ala Ser Gly Lys Pro Ala Glu Thr Lys Lys Asp Asp
95 100 105 110 aaa tca tta aca tcg tct gta cca gct gaa tcc aaa tca
agt aaa cca 444 Lys Ser Leu Thr Ser Ser Val Pro Ala Glu Ser Lys Ser
Ser Lys Pro 115 120 125 tca gga aag tca gat atg gat gct gct ttg gat
gac tta ata gac act 492 Ser Gly Lys Ser Asp Met Asp Ala Ala Leu Asp
Asp Leu Ile Asp Thr 130 135 140 tta gga gga cct gaa gaa act gag gaa
gat aat aca aca tat act gga 540 Leu Gly Gly Pro Glu Glu Thr Glu Glu
Asp Asn Thr Thr Tyr Thr Gly 145 150 155 cct gaa gtt ttg gat cca atg
agt tct acc tat ata gag gaa ttg ggt 588 Pro Glu Val Leu Asp Pro Met
Ser Ser Thr Tyr Ile Glu Glu Leu Gly 160 165 170 aaa aga gaa gtc aca
ctt cct cca aaa tat agg gaa ttg ttg gat aaa 636 Lys Arg Glu Val Thr
Leu Pro Pro Lys Tyr Arg Glu Leu Leu Asp Lys 175 180 185 190 aaa gaa
ggg att cca gtg cct cct cca gac act tcg aaa ccc ctg ggg 684 Lys Glu
Gly Ile Pro Val Pro Pro Pro Asp Thr Ser Lys Pro Leu Gly 195 200 205
ccc gat gat gcc atc gat gcc ttg tca tta gac ttg acc tgc agt tct 732
Pro Asp Asp Ala Ile Asp Ala Leu Ser Leu Asp Leu Thr Cys Ser Ser 210
215 220 cct aca gct gat ggg aag aaa acc gag aaa gag aaa tct act ggg
gag 780 Pro Thr Ala Asp Gly Lys Lys Thr Glu Lys Glu Lys Ser Thr Gly
Glu 225 230 235 gtt ttg aaa gct cag tct gtt ggg gta atc aga agc gct
gct gct cca 828 Val Leu Lys Ala Gln Ser Val Gly Val Ile Arg Ser Ala
Ala Ala Pro 240 245 250 ccc cac gag aaa aaa aga agg gtg gaa gag gac
acg atg agt gat caa 876 Pro His Glu Lys Lys Arg Arg Val Glu Glu Asp
Thr Met Ser Asp Gln 255 260 265 270 gca ctg gag gct ttg tca gct tcc
ctg ggc agc cgg aag tca gaa ccc 924 Ala Leu Glu Ala Leu Ser Ala Ser
Leu Gly Ser Arg Lys Ser Glu Pro 275 280 285 gag ctt gac ctc agc tcc
att aag gaa att gat gag gca aaa gcc aaa 972 Glu Leu Asp Leu Ser Ser
Ile Lys Glu Ile Asp Glu Ala Lys Ala Lys 290 295 300 gaa gag aaa cta
aag aag tgt ggt gaa gat gac gaa acg gtc ccg cca 1020 Glu Glu Lys
Leu Lys Lys Cys Gly Glu Asp Asp Glu Thr Val Pro Pro 305 310 315 gag
tat aga ttg aaa cca gcc atg gat aaa gat gga aaa cca ctc ttg 1068
Glu Tyr Arg Leu Lys Pro Ala Met Asp Lys Asp Gly Lys Pro Leu Leu 320
325 330 cca gag gct gaa gaa aaa ccc aag ccc ctg agt gaa tca gaa ctc
att 1116 Pro Glu Ala Glu Glu Lys Pro Lys Pro Leu Ser Glu Ser Glu
Leu Ile 335 340 345 350 gac gaa ctt tcg gaa gat ttt gac cag tct aag
cgt aaa gaa aaa caa 1164 Asp Glu Leu Ser Glu Asp Phe Asp Gln Ser
Lys Arg Lys Glu Lys Gln 355 360 365 tct aag cca act gaa aaa aca aaa
gag tct cag gcc act gcc cct act 1212 Ser Lys Pro Thr
Glu Lys Thr Lys Glu Ser Gln Ala Thr Ala Pro Thr 370 375 380 cct gtg
gga gag gcc gtg tct cgg acc tcc ttg tgc tgt gtg cag tcg 1260 Pro
Val Gly Glu Ala Val Ser Arg Thr Ser Leu Cys Cys Val Gln Ser 385 390
395 gca ccc cca aag cca gct acg ggc atg gtg cca gat gat gct gta gaa
1308 Ala Pro Pro Lys Pro Ala Thr Gly Met Val Pro Asp Asp Ala Val
Glu 400 405 410 gcc ttg gct gga agc ctg ggg aaa aag gaa gca gat cca
gaa gat gga 1356 Ala Leu Ala Gly Ser Leu Gly Lys Lys Glu Ala Asp
Pro Glu Asp Gly 415 420 425 430 aag cct gtg gag gat aaa gtc aag gag
aaa gcc aaa gaa gag gat cgt 1404 Lys Pro Val Glu Asp Lys Val Lys
Glu Lys Ala Lys Glu Glu Asp Arg 435 440 445 gaa aaa ctt ggt gaa aag
gaa gaa acg att cct cct gat tat aga tta 1452 Glu Lys Leu Gly Glu
Lys Glu Glu Thr Ile Pro Pro Asp Tyr Arg Leu 450 455 460 gaa gag gtc
aag gac aaa gat gga aaa act ctc ccg cac aaa gac ccc 1500 Glu Glu
Val Lys Asp Lys Asp Gly Lys Thr Leu Pro His Lys Asp Pro 465 470 475
aag gaa cca gtc ctg ccc ttg agt gaa gac ttc gtc ctt gat gct ttg
1548 Lys Glu Pro Val Leu Pro Leu Ser Glu Asp Phe Val Leu Asp Ala
Leu 480 485 490 tcc cag gac ttt gcc ggt ccc cca gcc gct tca tct ctt
ttt gaa gat 1596 Ser Gln Asp Phe Ala Gly Pro Pro Ala Ala Ser Ser
Leu Phe Glu Asp 495 500 505 510 gct aaa ctt tca gct gcc gtc tct gaa
gtg gtt tcc caa acc tca gct 1644 Ala Lys Leu Ser Ala Ala Val Ser
Glu Val Val Ser Gln Thr Ser Ala 515 520 525 cca acc acc cac tct gca
ggt cca ccc cct gac act gtg agt gat gac 1692 Pro Thr Thr His Ser
Ala Gly Pro Pro Pro Asp Thr Val Ser Asp Asp 530 535 540 aaa aaa ctt
gac gat gcc ctg gat cag ctt tct gac agt ctg ggg caa 1740 Lys Lys
Leu Asp Asp Ala Leu Asp Gln Leu Ser Asp Ser Leu Gly Gln 545 550 555
aga cag cct gac cca gat gag aac aag ccc ata gag gat aaa gtc aag
1788 Arg Gln Pro Asp Pro Asp Glu Asn Lys Pro Ile Glu Asp Lys Val
Lys 560 565 570 gaa aaa gct gaa gct gaa cat aga gac aag ctg gga gaa
aga gat gac 1836 Glu Lys Ala Glu Ala Glu His Arg Asp Lys Leu Gly
Glu Arg Asp Asp 575 580 585 590 act atc ccg cct gaa tat aga cat ctc
ttg gat aag gat gag gag ggc 1884 Thr Ile Pro Pro Glu Tyr Arg His
Leu Leu Asp Lys Asp Glu Glu Gly 595 600 605 aaa tca acg aag cca ccc
aca aag aaa cct gag gca cca aag aaa cct 1932 Lys Ser Thr Lys Pro
Pro Thr Lys Lys Pro Glu Ala Pro Lys Lys Pro 610 615 620 gaa gct gcc
caa gat ccc att gat gcc ctc tca ggg gat ttt gac aga 1980 Glu Ala
Ala Gln Asp Pro Ile Asp Ala Leu Ser Gly Asp Phe Asp Arg 625 630 635
tgt cca tca act aca gaa acc tca gag aac aca aca aag gac aaa gac
2028 Cys Pro Ser Thr Thr Glu Thr Ser Glu Asn Thr Thr Lys Asp Lys
Asp 640 645 650 aag aag acg gct tcc aag tcc aaa gca ccc aag aat ggg
ggt aaa gca 2076 Lys Lys Thr Ala Ser Lys Ser Lys Ala Pro Lys Asn
Gly Gly Lys Ala 655 660 665 670 aag gat tcc aca aag gca aag gag gaa
act tcc aaa caa aaa tct gat 2124 Lys Asp Ser Thr Lys Ala Lys Glu
Glu Thr Ser Lys Gln Lys Ser Asp 675 680 685 gga aag agt aca agt
taaaagttca cactattttc 2159 Gly Lys Ser Thr Ser 690 4 691 PRT Sus
scrofa 4 Met Asn Pro Thr Glu Thr Lys Ala Val Lys Thr Glu Pro Glu
Lys Lys 1 5 10 15 Ser Gln Ser Thr Lys Pro Ser Val Val His Glu Lys
Lys Thr Gln Glu 20 25 30 Val Lys Pro Lys Glu His Pro Glu Pro Lys
Ser Leu Pro Thr His Ser 35 40 45 Ala Asp Ala Gly Ser Lys Arg Ala
His Lys Glu Lys Ala Val Ser Arg 50 55 60 Ser Asn Glu Gln Pro Thr
Ser Glu Lys Ser Thr Lys Pro Lys Ala Lys 65 70 75 80 Pro Gln Asp Pro
Thr Pro Ser Asp Gly Lys Leu Ser Val Thr Gly Val 85 90 95 Ser Ala
Ala Ser Gly Lys Pro Ala Glu Thr Lys Lys Asp Asp Lys Ser 100 105 110
Leu Thr Ser Ser Val Pro Ala Glu Ser Lys Ser Ser Lys Pro Ser Gly 115
120 125 Lys Ser Asp Met Asp Ala Ala Leu Asp Asp Leu Ile Asp Thr Leu
Gly 130 135 140 Gly Pro Glu Glu Thr Glu Glu Asp Asn Thr Thr Tyr Thr
Gly Pro Glu 145 150 155 160 Val Leu Asp Pro Met Ser Ser Thr Tyr Ile
Glu Glu Leu Gly Lys Arg 165 170 175 Glu Val Thr Leu Pro Pro Lys Tyr
Arg Glu Leu Leu Asp Lys Lys Glu 180 185 190 Gly Ile Pro Val Pro Pro
Pro Asp Thr Ser Lys Pro Leu Gly Pro Asp 195 200 205 Asp Ala Ile Asp
Ala Leu Ser Leu Asp Leu Thr Cys Ser Ser Pro Thr 210 215 220 Ala Asp
Gly Lys Lys Thr Glu Lys Glu Lys Ser Thr Gly Glu Val Leu 225 230 235
240 Lys Ala Gln Ser Val Gly Val Ile Arg Ser Ala Ala Ala Pro Pro His
245 250 255 Glu Lys Lys Arg Arg Val Glu Glu Asp Thr Met Ser Asp Gln
Ala Leu 260 265 270 Glu Ala Leu Ser Ala Ser Leu Gly Ser Arg Lys Ser
Glu Pro Glu Leu 275 280 285 Asp Leu Ser Ser Ile Lys Glu Ile Asp Glu
Ala Lys Ala Lys Glu Glu 290 295 300 Lys Leu Lys Lys Cys Gly Glu Asp
Asp Glu Thr Val Pro Pro Glu Tyr 305 310 315 320 Arg Leu Lys Pro Ala
Met Asp Lys Asp Gly Lys Pro Leu Leu Pro Glu 325 330 335 Ala Glu Glu
Lys Pro Lys Pro Leu Ser Glu Ser Glu Leu Ile Asp Glu 340 345 350 Leu
Ser Glu Asp Phe Asp Gln Ser Lys Arg Lys Glu Lys Gln Ser Lys 355 360
365 Pro Thr Glu Lys Thr Lys Glu Ser Gln Ala Thr Ala Pro Thr Pro Val
370 375 380 Gly Glu Ala Val Ser Arg Thr Ser Leu Cys Cys Val Gln Ser
Ala Pro 385 390 395 400 Pro Lys Pro Ala Thr Gly Met Val Pro Asp Asp
Ala Val Glu Ala Leu 405 410 415 Ala Gly Ser Leu Gly Lys Lys Glu Ala
Asp Pro Glu Asp Gly Lys Pro 420 425 430 Val Glu Asp Lys Val Lys Glu
Lys Ala Lys Glu Glu Asp Arg Glu Lys 435 440 445 Leu Gly Glu Lys Glu
Glu Thr Ile Pro Pro Asp Tyr Arg Leu Glu Glu 450 455 460 Val Lys Asp
Lys Asp Gly Lys Thr Leu Pro His Lys Asp Pro Lys Glu 465 470 475 480
Pro Val Leu Pro Leu Ser Glu Asp Phe Val Leu Asp Ala Leu Ser Gln 485
490 495 Asp Phe Ala Gly Pro Pro Ala Ala Ser Ser Leu Phe Glu Asp Ala
Lys 500 505 510 Leu Ser Ala Ala Val Ser Glu Val Val Ser Gln Thr Ser
Ala Pro Thr 515 520 525 Thr His Ser Ala Gly Pro Pro Pro Asp Thr Val
Ser Asp Asp Lys Lys 530 535 540 Leu Asp Asp Ala Leu Asp Gln Leu Ser
Asp Ser Leu Gly Gln Arg Gln 545 550 555 560 Pro Asp Pro Asp Glu Asn
Lys Pro Ile Glu Asp Lys Val Lys Glu Lys 565 570 575 Ala Glu Ala Glu
His Arg Asp Lys Leu Gly Glu Arg Asp Asp Thr Ile 580 585 590 Pro Pro
Glu Tyr Arg His Leu Leu Asp Lys Asp Glu Glu Gly Lys Ser 595 600 605
Thr Lys Pro Pro Thr Lys Lys Pro Glu Ala Pro Lys Lys Pro Glu Ala 610
615 620 Ala Gln Asp Pro Ile Asp Ala Leu Ser Gly Asp Phe Asp Arg Cys
Pro 625 630 635 640 Ser Thr Thr Glu Thr Ser Glu Asn Thr Thr Lys Asp
Lys Asp Lys Lys 645 650 655 Thr Ala Ser Lys Ser Lys Ala Pro Lys Asn
Gly Gly Lys Ala Lys Asp 660 665 670 Ser Thr Lys Ala Lys Glu Glu Thr
Ser Lys Gln Lys Ser Asp Gly Lys 675 680 685 Ser Thr Ser 690 5 2159
DNA Sus scrofa CDS (67)..(2139) 5 tctctcggcc gggaagccag aagcagagta
tcgccttcct ctgcttcaac gagcaagtct 60 tccagt atg aat ccc aca gaa acc
aag gct gta aaa aca gaa cct gaa 108 Met Asn Pro Thr Glu Thr Lys Ala
Val Lys Thr Glu Pro Glu 1 5 10 aag aag tca caa tca act aag cca tct
gtg gtt cat gag aaa aaa acc 156 Lys Lys Ser Gln Ser Thr Lys Pro Ser
Val Val His Glu Lys Lys Thr 15 20 25 30 caa gaa gta aag cca aag gaa
cac cca gag cca aaa agc cta ccc acg 204 Gln Glu Val Lys Pro Lys Glu
His Pro Glu Pro Lys Ser Leu Pro Thr 35 40 45 cac tca gca gat gca
ggg agc aag cgt gct cat aaa gaa aaa gca gtt 252 His Ser Ala Asp Ala
Gly Ser Lys Arg Ala His Lys Glu Lys Ala Val 50 55 60 tcc aga tct
aat gag cag cca aca tca gag aaa tca aca aaa cca aag 300 Ser Arg Ser
Asn Glu Gln Pro Thr Ser Glu Lys Ser Thr Lys Pro Lys 65 70 75 gct
aaa cca cag gac ccg acc ccc agt gat gga aag ctt tct gtt act 348 Ala
Lys Pro Gln Asp Pro Thr Pro Ser Asp Gly Lys Leu Ser Val Thr 80 85
90 ggt gta tct gca gca tct ggc aaa cca gct gag acg aaa aaa gat gat
396 Gly Val Ser Ala Ala Ser Gly Lys Pro Ala Glu Thr Lys Lys Asp Asp
95 100 105 110 aaa tca tta aca tcg tct gta cca gct gaa tcc aaa tca
agt aaa cca 444 Lys Ser Leu Thr Ser Ser Val Pro Ala Glu Ser Lys Ser
Ser Lys Pro 115 120 125 tca gga aag tca gat atg gat gct gct ttg gat
gac tta ata gac act 492 Ser Gly Lys Ser Asp Met Asp Ala Ala Leu Asp
Asp Leu Ile Asp Thr 130 135 140 tta gga gga cct gaa gaa act gag gaa
gat aat aca aca tat act gga 540 Leu Gly Gly Pro Glu Glu Thr Glu Glu
Asp Asn Thr Thr Tyr Thr Gly 145 150 155 cct gaa gtt ttg gat cca atg
agt tct acc tat ata gag gaa ttg ggt 588 Pro Glu Val Leu Asp Pro Met
Ser Ser Thr Tyr Ile Glu Glu Leu Gly 160 165 170 aaa aga gaa gtc aca
ctt cct cca aaa tat agg gaa ttg ttg gat aaa 636 Lys Arg Glu Val Thr
Leu Pro Pro Lys Tyr Arg Glu Leu Leu Asp Lys 175 180 185 190 aaa gaa
ggg att cca gtg cct cct cca gac act tcg aaa ccc ctg ggg 684 Lys Glu
Gly Ile Pro Val Pro Pro Pro Asp Thr Ser Lys Pro Leu Gly 195 200 205
ccc gat gat gcc atc gat gcc ttg tca tta gac ttg acc tgc agt tct 732
Pro Asp Asp Ala Ile Asp Ala Leu Ser Leu Asp Leu Thr Cys Ser Ser 210
215 220 cct aca gct gat ggg aag aaa acc gag aaa gag aaa tct act ggg
gag 780 Pro Thr Ala Asp Gly Lys Lys Thr Glu Lys Glu Lys Ser Thr Gly
Glu 225 230 235 gtt ttg aaa gct cag tct gtt ggg gta atc aaa agc gct
gct gct cca 828 Val Leu Lys Ala Gln Ser Val Gly Val Ile Lys Ser Ala
Ala Ala Pro 240 245 250 ccc cac gag aaa aaa aga agg gtg gaa gag gac
acg atg agt gat caa 876 Pro His Glu Lys Lys Arg Arg Val Glu Glu Asp
Thr Met Ser Asp Gln 255 260 265 270 gca ctg gag gct ttg tca gct tcc
ctg ggc agc cgg aag tca gaa ccc 924 Ala Leu Glu Ala Leu Ser Ala Ser
Leu Gly Ser Arg Lys Ser Glu Pro 275 280 285 gag ctt gac ctc agc tcc
att aag gaa att gat gag gca aaa gcc aaa 972 Glu Leu Asp Leu Ser Ser
Ile Lys Glu Ile Asp Glu Ala Lys Ala Lys 290 295 300 gaa gag aaa cta
aag aag tgt ggt gaa gat gac gaa acg gtc ccg cca 1020 Glu Glu Lys
Leu Lys Lys Cys Gly Glu Asp Asp Glu Thr Val Pro Pro 305 310 315 gag
tat aga ttg aaa cca gcc atg gat aaa gat gga aaa cca ctc ttg 1068
Glu Tyr Arg Leu Lys Pro Ala Met Asp Lys Asp Gly Lys Pro Leu Leu 320
325 330 cca gag gct gaa gaa aaa ccc aag ccc ctg agt gaa tca gaa ctc
att 1116 Pro Glu Ala Glu Glu Lys Pro Lys Pro Leu Ser Glu Ser Glu
Leu Ile 335 340 345 350 gac gaa ctt tcg gaa gat ttt gac cag tct aag
cgt aaa gaa aaa caa 1164 Asp Glu Leu Ser Glu Asp Phe Asp Gln Ser
Lys Arg Lys Glu Lys Gln 355 360 365 tct aag cca act gaa aaa aca aaa
gag tct cag gcc act gcc cct act 1212 Ser Lys Pro Thr Glu Lys Thr
Lys Glu Ser Gln Ala Thr Ala Pro Thr 370 375 380 cct gtg gga gag gcc
gtg tct cgg acc tcc ttg tgc tgt gtg cag tcg 1260 Pro Val Gly Glu
Ala Val Ser Arg Thr Ser Leu Cys Cys Val Gln Ser 385 390 395 gca ccc
cca aag cca gct acg ggc atg gtg cca gat gat gct gta gaa 1308 Ala
Pro Pro Lys Pro Ala Thr Gly Met Val Pro Asp Asp Ala Val Glu 400 405
410 gcc ttg gct gga agc ctg ggg aaa aag gaa gca gat cca gaa gat gga
1356 Ala Leu Ala Gly Ser Leu Gly Lys Lys Glu Ala Asp Pro Glu Asp
Gly 415 420 425 430 aag cct gtg gag gat aaa gtc aag gag aaa gcc aaa
gaa gag gat cgt 1404 Lys Pro Val Glu Asp Lys Val Lys Glu Lys Ala
Lys Glu Glu Asp Arg 435 440 445 gaa aaa ctt ggt gaa aag gaa gaa acg
att cct cct gat tat aga tta 1452 Glu Lys Leu Gly Glu Lys Glu Glu
Thr Ile Pro Pro Asp Tyr Arg Leu 450 455 460 gaa gag gtc aag gac aaa
gat gga aaa act ctc ccg cac aaa gac ccc 1500 Glu Glu Val Lys Asp
Lys Asp Gly Lys Thr Leu Pro His Lys Asp Pro 465 470 475 aag gaa cca
gtc ctg ccc ttg agt gaa gac ttc gtc ctt gat gct ttg 1548 Lys Glu
Pro Val Leu Pro Leu Ser Glu Asp Phe Val Leu Asp Ala Leu 480 485 490
tcc cag gac ttt gcc ggt ccc cca gcc gct tca tct ctt ttt gaa gat
1596 Ser Gln Asp Phe Ala Gly Pro Pro Ala Ala Ser Ser Leu Phe Glu
Asp 495 500 505 510 gct aaa ctt tca gct gcc gtc tct gaa gtg gtt tcc
caa acc tca gct 1644 Ala Lys Leu Ser Ala Ala Val Ser Glu Val Val
Ser Gln Thr Ser Ala 515 520 525 cca acc acc cac tct gca ggt cca ccc
cct gac act gtg agt gat gac 1692 Pro Thr Thr His Ser Ala Gly Pro
Pro Pro Asp Thr Val Ser Asp Asp 530 535 540 aaa aaa ctt gac gat gcc
ctg gat cag ctt tct gac agt ctg ggg caa 1740 Lys Lys Leu Asp Asp
Ala Leu Asp Gln Leu Ser Asp Ser Leu Gly Gln 545 550 555 aga cag cct
gac cca gat gag aac aag ccc ata gag gat aaa gtc aag 1788 Arg Gln
Pro Asp Pro Asp Glu Asn Lys Pro Ile Glu Asp Lys Val Lys 560 565 570
gaa aaa gct gaa gct gaa cat aga gac aag ctg gga gaa aga gat gac
1836 Glu Lys Ala Glu Ala Glu His Arg Asp Lys Leu Gly Glu Arg Asp
Asp 575 580 585 590 act atc ccg cct gaa tat aga cat ctc ttg gat aag
gat gag gag ggc 1884 Thr Ile Pro Pro Glu Tyr Arg His Leu Leu Asp
Lys Asp Glu Glu Gly 595 600 605 aaa tca acg aag cca ccc aca aag aaa
cct gag gca cca aag aaa cct 1932 Lys Ser Thr Lys Pro Pro Thr Lys
Lys Pro Glu Ala Pro Lys Lys Pro 610 615 620 gaa gct gcc caa gat ccc
att gat gcc ctc tca ggg gat ttt gac aga 1980 Glu Ala Ala Gln Asp
Pro Ile Asp Ala Leu Ser Gly Asp Phe Asp Arg 625 630 635 tgt cca tca
act aca gaa acc tca gag aac aca aca aag gac aaa gac 2028 Cys Pro
Ser Thr Thr Glu Thr Ser Glu Asn Thr Thr Lys Asp Lys Asp 640 645 650
aag aag acg gct tcc aag tcc aaa gca ccc aag aat ggg ggt aaa gca
2076 Lys Lys Thr Ala Ser Lys Ser Lys Ala Pro Lys Asn Gly Gly Lys
Ala 655 660 665 670 aag gat tcc aca aag gca aag gag gaa act tcc aaa
caa aaa tct gat 2124 Lys Asp Ser Thr Lys Ala Lys Glu Glu Thr Ser
Lys Gln Lys Ser Asp 675 680 685 gga aag agt aca agt taaaagttca
cactattttc 2159 Gly Lys Ser Thr Ser 690 6 691 PRT Sus scrofa 6 Met
Asn Pro Thr Glu Thr Lys Ala Val Lys Thr Glu Pro Glu Lys Lys 1 5 10
15 Ser Gln Ser Thr Lys Pro Ser Val Val His Glu Lys Lys Thr Gln Glu
20 25 30 Val Lys Pro Lys Glu His Pro Glu Pro Lys Ser Leu Pro Thr
His Ser 35 40 45 Ala Asp Ala Gly Ser Lys Arg Ala His Lys Glu Lys
Ala Val Ser Arg 50 55 60 Ser Asn Glu Gln Pro Thr Ser Glu Lys Ser
Thr Lys Pro Lys Ala Lys 65 70 75 80 Pro Gln Asp Pro Thr Pro Ser Asp
Gly Lys Leu Ser Val Thr Gly Val 85 90 95 Ser Ala Ala Ser Gly
Lys Pro Ala Glu Thr Lys Lys Asp Asp Lys Ser 100 105 110 Leu Thr Ser
Ser Val Pro Ala Glu Ser Lys Ser Ser Lys Pro Ser Gly 115 120 125 Lys
Ser Asp Met Asp Ala Ala Leu Asp Asp Leu Ile Asp Thr Leu Gly 130 135
140 Gly Pro Glu Glu Thr Glu Glu Asp Asn Thr Thr Tyr Thr Gly Pro Glu
145 150 155 160 Val Leu Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu
Gly Lys Arg 165 170 175 Glu Val Thr Leu Pro Pro Lys Tyr Arg Glu Leu
Leu Asp Lys Lys Glu 180 185 190 Gly Ile Pro Val Pro Pro Pro Asp Thr
Ser Lys Pro Leu Gly Pro Asp 195 200 205 Asp Ala Ile Asp Ala Leu Ser
Leu Asp Leu Thr Cys Ser Ser Pro Thr 210 215 220 Ala Asp Gly Lys Lys
Thr Glu Lys Glu Lys Ser Thr Gly Glu Val Leu 225 230 235 240 Lys Ala
Gln Ser Val Gly Val Ile Lys Ser Ala Ala Ala Pro Pro His 245 250 255
Glu Lys Lys Arg Arg Val Glu Glu Asp Thr Met Ser Asp Gln Ala Leu 260
265 270 Glu Ala Leu Ser Ala Ser Leu Gly Ser Arg Lys Ser Glu Pro Glu
Leu 275 280 285 Asp Leu Ser Ser Ile Lys Glu Ile Asp Glu Ala Lys Ala
Lys Glu Glu 290 295 300 Lys Leu Lys Lys Cys Gly Glu Asp Asp Glu Thr
Val Pro Pro Glu Tyr 305 310 315 320 Arg Leu Lys Pro Ala Met Asp Lys
Asp Gly Lys Pro Leu Leu Pro Glu 325 330 335 Ala Glu Glu Lys Pro Lys
Pro Leu Ser Glu Ser Glu Leu Ile Asp Glu 340 345 350 Leu Ser Glu Asp
Phe Asp Gln Ser Lys Arg Lys Glu Lys Gln Ser Lys 355 360 365 Pro Thr
Glu Lys Thr Lys Glu Ser Gln Ala Thr Ala Pro Thr Pro Val 370 375 380
Gly Glu Ala Val Ser Arg Thr Ser Leu Cys Cys Val Gln Ser Ala Pro 385
390 395 400 Pro Lys Pro Ala Thr Gly Met Val Pro Asp Asp Ala Val Glu
Ala Leu 405 410 415 Ala Gly Ser Leu Gly Lys Lys Glu Ala Asp Pro Glu
Asp Gly Lys Pro 420 425 430 Val Glu Asp Lys Val Lys Glu Lys Ala Lys
Glu Glu Asp Arg Glu Lys 435 440 445 Leu Gly Glu Lys Glu Glu Thr Ile
Pro Pro Asp Tyr Arg Leu Glu Glu 450 455 460 Val Lys Asp Lys Asp Gly
Lys Thr Leu Pro His Lys Asp Pro Lys Glu 465 470 475 480 Pro Val Leu
Pro Leu Ser Glu Asp Phe Val Leu Asp Ala Leu Ser Gln 485 490 495 Asp
Phe Ala Gly Pro Pro Ala Ala Ser Ser Leu Phe Glu Asp Ala Lys 500 505
510 Leu Ser Ala Ala Val Ser Glu Val Val Ser Gln Thr Ser Ala Pro Thr
515 520 525 Thr His Ser Ala Gly Pro Pro Pro Asp Thr Val Ser Asp Asp
Lys Lys 530 535 540 Leu Asp Asp Ala Leu Asp Gln Leu Ser Asp Ser Leu
Gly Gln Arg Gln 545 550 555 560 Pro Asp Pro Asp Glu Asn Lys Pro Ile
Glu Asp Lys Val Lys Glu Lys 565 570 575 Ala Glu Ala Glu His Arg Asp
Lys Leu Gly Glu Arg Asp Asp Thr Ile 580 585 590 Pro Pro Glu Tyr Arg
His Leu Leu Asp Lys Asp Glu Glu Gly Lys Ser 595 600 605 Thr Lys Pro
Pro Thr Lys Lys Pro Glu Ala Pro Lys Lys Pro Glu Ala 610 615 620 Ala
Gln Asp Pro Ile Asp Ala Leu Ser Gly Asp Phe Asp Arg Cys Pro 625 630
635 640 Ser Thr Thr Glu Thr Ser Glu Asn Thr Thr Lys Asp Lys Asp Lys
Lys 645 650 655 Thr Ala Ser Lys Ser Lys Ala Pro Lys Asn Gly Gly Lys
Ala Lys Asp 660 665 670 Ser Thr Lys Ala Lys Glu Glu Thr Ser Lys Gln
Lys Ser Asp Gly Lys 675 680 685 Ser Thr Ser 690 7 2159 DNA Sus
scrofa CDS (67)..(2139) 7 tctctcggcc gggaagccag aagcagagta
tcgccttcct ctgcttcaac gagcaagtct 60 tccagt atg aat ccc aca gaa acc
aag gct gta aaa aca gaa cct gaa 108 Met Asn Pro Thr Glu Thr Lys Ala
Val Lys Thr Glu Pro Glu 1 5 10 aag aag tca caa tca act aag cca tct
gtg gtt cat gag aaa aaa acc 156 Lys Lys Ser Gln Ser Thr Lys Pro Ser
Val Val His Glu Lys Lys Thr 15 20 25 30 caa gaa gta aag cca aag gaa
cac cca gag cca aaa agc cta ccc acg 204 Gln Glu Val Lys Pro Lys Glu
His Pro Glu Pro Lys Ser Leu Pro Thr 35 40 45 cac tca gca gat gca
ggg agc aag cgt gct cat aaa gaa aaa gca gtt 252 His Ser Ala Asp Ala
Gly Ser Lys Arg Ala His Lys Glu Lys Ala Val 50 55 60 tcc aga tct
aat gag cag cca aca tca gag aaa tca aca aaa cca aag 300 Ser Arg Ser
Asn Glu Gln Pro Thr Ser Glu Lys Ser Thr Lys Pro Lys 65 70 75 gct
aaa cca cag gac ccg acc ccc agt gat gga aag ctt tct gtt act 348 Ala
Lys Pro Gln Asp Pro Thr Pro Ser Asp Gly Lys Leu Ser Val Thr 80 85
90 ggt gta tct gca gca tct ggc aaa cca gct gag acg aaa aaa gat gat
396 Gly Val Ser Ala Ala Ser Gly Lys Pro Ala Glu Thr Lys Lys Asp Asp
95 100 105 110 aaa tca tta aca tcg tct gta cca gct gaa tcc aaa tca
agt aaa cca 444 Lys Ser Leu Thr Ser Ser Val Pro Ala Glu Ser Lys Ser
Ser Lys Pro 115 120 125 tca gga aag tca gat atg gat gct gct ttg gat
gac tta ata gac act 492 Ser Gly Lys Ser Asp Met Asp Ala Ala Leu Asp
Asp Leu Ile Asp Thr 130 135 140 tta gga gga cct gaa gaa act gag gaa
gat aat aca aca tat act gga 540 Leu Gly Gly Pro Glu Glu Thr Glu Glu
Asp Asn Thr Thr Tyr Thr Gly 145 150 155 cct gaa gtt ttg gat cca atg
agt tct acc tat ata gag gaa ttg ggt 588 Pro Glu Val Leu Asp Pro Met
Ser Ser Thr Tyr Ile Glu Glu Leu Gly 160 165 170 aaa aga gaa gtc aca
ctt cct cca aaa tat agg gaa ttg ttg gat aaa 636 Lys Arg Glu Val Thr
Leu Pro Pro Lys Tyr Arg Glu Leu Leu Asp Lys 175 180 185 190 aaa gaa
ggg att cca gtg cct cct cca gac act tcg aaa ccc ctg ggg 684 Lys Glu
Gly Ile Pro Val Pro Pro Pro Asp Thr Ser Lys Pro Leu Gly 195 200 205
ccc gat gat gcc atc gat gcc ttg tca tta gac ttg acc tgc agt tct 732
Pro Asp Asp Ala Ile Asp Ala Leu Ser Leu Asp Leu Thr Cys Ser Ser 210
215 220 cct aca gct gat ggg aag aaa acc gag aaa gag aaa tct act ggg
gag 780 Pro Thr Ala Asp Gly Lys Lys Thr Glu Lys Glu Lys Ser Thr Gly
Glu 225 230 235 gtt ttg aaa gct cag tct gtt ggg gta atc aga agc gct
gct gct cca 828 Val Leu Lys Ala Gln Ser Val Gly Val Ile Arg Ser Ala
Ala Ala Pro 240 245 250 ccc cac gag aaa aaa aga agg gtg gaa gag gac
acg atg agt gat caa 876 Pro His Glu Lys Lys Arg Arg Val Glu Glu Asp
Thr Met Ser Asp Gln 255 260 265 270 gca ctg gag gct ttg tca gct tcc
ctg ggc agc cgg aag tca gaa ccc 924 Ala Leu Glu Ala Leu Ser Ala Ser
Leu Gly Ser Arg Lys Ser Glu Pro 275 280 285 gag ctt gac ctc agc tcc
att aag gaa att gat gag gca aaa gcc aaa 972 Glu Leu Asp Leu Ser Ser
Ile Lys Glu Ile Asp Glu Ala Lys Ala Lys 290 295 300 gaa gag aaa cta
aag aag tgt ggt gaa gat gac gaa acg gtc ccg cca 1020 Glu Glu Lys
Leu Lys Lys Cys Gly Glu Asp Asp Glu Thr Val Pro Pro 305 310 315 gag
tat aga ttg aaa cca gcc atg gat aaa gat gga aaa cca ctc ttg 1068
Glu Tyr Arg Leu Lys Pro Ala Met Asp Lys Asp Gly Lys Pro Leu Leu 320
325 330 cca gag gct gaa gaa aaa ccc aag ccc ctg agt gaa tca gaa ctc
att 1116 Pro Glu Ala Glu Glu Lys Pro Lys Pro Leu Ser Glu Ser Glu
Leu Ile 335 340 345 350 gac gaa ctt tcg gaa gat ttt gac cag tct aag
cgt aaa gaa aaa caa 1164 Asp Glu Leu Ser Glu Asp Phe Asp Gln Ser
Lys Arg Lys Glu Lys Gln 355 360 365 tct aag cca act gaa aaa aca aaa
gag tct cag gcc act gcc cct act 1212 Ser Lys Pro Thr Glu Lys Thr
Lys Glu Ser Gln Ala Thr Ala Pro Thr 370 375 380 cct gtg gga gag gcc
gtg tct cgg acc tcc ttg tgc tgt gtg cag tcg 1260 Pro Val Gly Glu
Ala Val Ser Arg Thr Ser Leu Cys Cys Val Gln Ser 385 390 395 gca ccc
cca aag cca gct acg ggc atg gtg cca gat gat gct gta gaa 1308 Ala
Pro Pro Lys Pro Ala Thr Gly Met Val Pro Asp Asp Ala Val Glu 400 405
410 gcc ttg gct gga agc ctg ggg aaa aag gaa gca gat cca gaa gat gga
1356 Ala Leu Ala Gly Ser Leu Gly Lys Lys Glu Ala Asp Pro Glu Asp
Gly 415 420 425 430 aag cct gtg gag gat aaa gtc aag gag aaa gcc aaa
gaa gag gat cgt 1404 Lys Pro Val Glu Asp Lys Val Lys Glu Lys Ala
Lys Glu Glu Asp Arg 435 440 445 gaa aaa ctt ggt gaa aag gaa gaa acg
att cct cct gat tat aga tta 1452 Glu Lys Leu Gly Glu Lys Glu Glu
Thr Ile Pro Pro Asp Tyr Arg Leu 450 455 460 gaa gag gtc aag gac aaa
gat gga aaa act ctc ccg cac aaa gac ccc 1500 Glu Glu Val Lys Asp
Lys Asp Gly Lys Thr Leu Pro His Lys Asp Pro 465 470 475 aag gaa cca
gtc ctg ccc ttg agt gaa gac ttc gtc ctt gat gct ttg 1548 Lys Glu
Pro Val Leu Pro Leu Ser Glu Asp Phe Val Leu Asp Ala Leu 480 485 490
tcc cag gac ttt gcc ggt ccc cca gcc act tca tct ctt ttt gaa gat
1596 Ser Gln Asp Phe Ala Gly Pro Pro Ala Thr Ser Ser Leu Phe Glu
Asp 495 500 505 510 gct aaa ctt tca gct gcc gtc tct gaa gtg gtt tcc
caa acc tca gct 1644 Ala Lys Leu Ser Ala Ala Val Ser Glu Val Val
Ser Gln Thr Ser Ala 515 520 525 cca acc acc cac tct gca ggt cca ccc
cct gac act gtg agt gat gac 1692 Pro Thr Thr His Ser Ala Gly Pro
Pro Pro Asp Thr Val Ser Asp Asp 530 535 540 aaa aaa ctt gac gat gcc
ctg gat cag ctt tct gac agt ctg ggg caa 1740 Lys Lys Leu Asp Asp
Ala Leu Asp Gln Leu Ser Asp Ser Leu Gly Gln 545 550 555 aga cag cct
gac cca gat gag aac aag ccc ata gag gat aaa gtc aag 1788 Arg Gln
Pro Asp Pro Asp Glu Asn Lys Pro Ile Glu Asp Lys Val Lys 560 565 570
gaa aaa gct gaa gct gaa cat aga gac aag ctg gga gaa aga gat gac
1836 Glu Lys Ala Glu Ala Glu His Arg Asp Lys Leu Gly Glu Arg Asp
Asp 575 580 585 590 act atc ccg cct gaa tat aga cat ctc ttg gat aag
gat gag gag ggc 1884 Thr Ile Pro Pro Glu Tyr Arg His Leu Leu Asp
Lys Asp Glu Glu Gly 595 600 605 aaa tca acg aag cca ccc aca aag aaa
cct gag gca cca aag aaa cct 1932 Lys Ser Thr Lys Pro Pro Thr Lys
Lys Pro Glu Ala Pro Lys Lys Pro 610 615 620 gaa gct gcc caa gat ccc
att gat gcc ctc tca ggg gat ttt gac aga 1980 Glu Ala Ala Gln Asp
Pro Ile Asp Ala Leu Ser Gly Asp Phe Asp Arg 625 630 635 tgt cca tca
act aca gaa acc tca gag aac aca aca aag gac aaa gac 2028 Cys Pro
Ser Thr Thr Glu Thr Ser Glu Asn Thr Thr Lys Asp Lys Asp 640 645 650
aag aag acg gct tcc aag tcc aaa gca ccc aag aat ggg ggt aaa gca
2076 Lys Lys Thr Ala Ser Lys Ser Lys Ala Pro Lys Asn Gly Gly Lys
Ala 655 660 665 670 aag gat tcc aca aag gca aag gag gaa act tcc aaa
caa aaa tct gat 2124 Lys Asp Ser Thr Lys Ala Lys Glu Glu Thr Ser
Lys Gln Lys Ser Asp 675 680 685 gga aag agt aca agt taaaagttca
cactattttc 2159 Gly Lys Ser Thr Ser 690 8 691 PRT Sus scrofa 8 Met
Asn Pro Thr Glu Thr Lys Ala Val Lys Thr Glu Pro Glu Lys Lys 1 5 10
15 Ser Gln Ser Thr Lys Pro Ser Val Val His Glu Lys Lys Thr Gln Glu
20 25 30 Val Lys Pro Lys Glu His Pro Glu Pro Lys Ser Leu Pro Thr
His Ser 35 40 45 Ala Asp Ala Gly Ser Lys Arg Ala His Lys Glu Lys
Ala Val Ser Arg 50 55 60 Ser Asn Glu Gln Pro Thr Ser Glu Lys Ser
Thr Lys Pro Lys Ala Lys 65 70 75 80 Pro Gln Asp Pro Thr Pro Ser Asp
Gly Lys Leu Ser Val Thr Gly Val 85 90 95 Ser Ala Ala Ser Gly Lys
Pro Ala Glu Thr Lys Lys Asp Asp Lys Ser 100 105 110 Leu Thr Ser Ser
Val Pro Ala Glu Ser Lys Ser Ser Lys Pro Ser Gly 115 120 125 Lys Ser
Asp Met Asp Ala Ala Leu Asp Asp Leu Ile Asp Thr Leu Gly 130 135 140
Gly Pro Glu Glu Thr Glu Glu Asp Asn Thr Thr Tyr Thr Gly Pro Glu 145
150 155 160 Val Leu Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly
Lys Arg 165 170 175 Glu Val Thr Leu Pro Pro Lys Tyr Arg Glu Leu Leu
Asp Lys Lys Glu 180 185 190 Gly Ile Pro Val Pro Pro Pro Asp Thr Ser
Lys Pro Leu Gly Pro Asp 195 200 205 Asp Ala Ile Asp Ala Leu Ser Leu
Asp Leu Thr Cys Ser Ser Pro Thr 210 215 220 Ala Asp Gly Lys Lys Thr
Glu Lys Glu Lys Ser Thr Gly Glu Val Leu 225 230 235 240 Lys Ala Gln
Ser Val Gly Val Ile Arg Ser Ala Ala Ala Pro Pro His 245 250 255 Glu
Lys Lys Arg Arg Val Glu Glu Asp Thr Met Ser Asp Gln Ala Leu 260 265
270 Glu Ala Leu Ser Ala Ser Leu Gly Ser Arg Lys Ser Glu Pro Glu Leu
275 280 285 Asp Leu Ser Ser Ile Lys Glu Ile Asp Glu Ala Lys Ala Lys
Glu Glu 290 295 300 Lys Leu Lys Lys Cys Gly Glu Asp Asp Glu Thr Val
Pro Pro Glu Tyr 305 310 315 320 Arg Leu Lys Pro Ala Met Asp Lys Asp
Gly Lys Pro Leu Leu Pro Glu 325 330 335 Ala Glu Glu Lys Pro Lys Pro
Leu Ser Glu Ser Glu Leu Ile Asp Glu 340 345 350 Leu Ser Glu Asp Phe
Asp Gln Ser Lys Arg Lys Glu Lys Gln Ser Lys 355 360 365 Pro Thr Glu
Lys Thr Lys Glu Ser Gln Ala Thr Ala Pro Thr Pro Val 370 375 380 Gly
Glu Ala Val Ser Arg Thr Ser Leu Cys Cys Val Gln Ser Ala Pro 385 390
395 400 Pro Lys Pro Ala Thr Gly Met Val Pro Asp Asp Ala Val Glu Ala
Leu 405 410 415 Ala Gly Ser Leu Gly Lys Lys Glu Ala Asp Pro Glu Asp
Gly Lys Pro 420 425 430 Val Glu Asp Lys Val Lys Glu Lys Ala Lys Glu
Glu Asp Arg Glu Lys 435 440 445 Leu Gly Glu Lys Glu Glu Thr Ile Pro
Pro Asp Tyr Arg Leu Glu Glu 450 455 460 Val Lys Asp Lys Asp Gly Lys
Thr Leu Pro His Lys Asp Pro Lys Glu 465 470 475 480 Pro Val Leu Pro
Leu Ser Glu Asp Phe Val Leu Asp Ala Leu Ser Gln 485 490 495 Asp Phe
Ala Gly Pro Pro Ala Thr Ser Ser Leu Phe Glu Asp Ala Lys 500 505 510
Leu Ser Ala Ala Val Ser Glu Val Val Ser Gln Thr Ser Ala Pro Thr 515
520 525 Thr His Ser Ala Gly Pro Pro Pro Asp Thr Val Ser Asp Asp Lys
Lys 530 535 540 Leu Asp Asp Ala Leu Asp Gln Leu Ser Asp Ser Leu Gly
Gln Arg Gln 545 550 555 560 Pro Asp Pro Asp Glu Asn Lys Pro Ile Glu
Asp Lys Val Lys Glu Lys 565 570 575 Ala Glu Ala Glu His Arg Asp Lys
Leu Gly Glu Arg Asp Asp Thr Ile 580 585 590 Pro Pro Glu Tyr Arg His
Leu Leu Asp Lys Asp Glu Glu Gly Lys Ser 595 600 605 Thr Lys Pro Pro
Thr Lys Lys Pro Glu Ala Pro Lys Lys Pro Glu Ala 610 615 620 Ala Gln
Asp Pro Ile Asp Ala Leu Ser Gly Asp Phe Asp Arg Cys Pro 625 630 635
640 Ser Thr Thr Glu Thr Ser Glu Asn Thr Thr Lys Asp Lys Asp Lys Lys
645 650 655 Thr Ala Ser Lys Ser Lys Ala Pro Lys Asn Gly Gly Lys Ala
Lys Asp 660 665 670 Ser Thr Lys Ala Lys Glu Glu Thr Ser Lys Gln Lys
Ser Asp Gly Lys 675 680 685 Ser Thr Ser 690 9 2159 DNA Sus scrofa
CDS (67)..(2139) 9 tctctcggcc gggaagccag aagcagagta tcgccttcct
ctgcttcaac gagcaagtct 60 tccagt atg aat ccc aca gaa acc aag gct gta
aaa aca gaa cct gaa 108 Met
Asn Pro Thr Glu Thr Lys Ala Val Lys Thr Glu Pro Glu 1 5 10 aag aag
tca caa tca act aag cca tct gtg gtt cat gag aaa aaa acc 156 Lys Lys
Ser Gln Ser Thr Lys Pro Ser Val Val His Glu Lys Lys Thr 15 20 25 30
caa gaa gta aag cca aag gaa cac cca gag cca aaa agc cta ccc acg 204
Gln Glu Val Lys Pro Lys Glu His Pro Glu Pro Lys Ser Leu Pro Thr 35
40 45 cac tca gca gat gca ggg agc aag cgt gct cat aaa gaa aaa gca
gtt 252 His Ser Ala Asp Ala Gly Ser Lys Arg Ala His Lys Glu Lys Ala
Val 50 55 60 tcc aga tct aat gag cag cca aca tca gag aaa tca aca
aaa cca aag 300 Ser Arg Ser Asn Glu Gln Pro Thr Ser Glu Lys Ser Thr
Lys Pro Lys 65 70 75 gct aaa cca cag gac ccg acc ccc agt gat gga
aag ctt tct gtt act 348 Ala Lys Pro Gln Asp Pro Thr Pro Ser Asp Gly
Lys Leu Ser Val Thr 80 85 90 ggt gta tct gca gca tct ggc aaa cca
gct gag acg aaa aaa gat gat 396 Gly Val Ser Ala Ala Ser Gly Lys Pro
Ala Glu Thr Lys Lys Asp Asp 95 100 105 110 aaa tca tta aca tcg tct
gta cca gct gaa tcc aaa tca agt aaa cca 444 Lys Ser Leu Thr Ser Ser
Val Pro Ala Glu Ser Lys Ser Ser Lys Pro 115 120 125 tca gga aag tca
gat atg gat gct gct ttg gat gac tta ata gac act 492 Ser Gly Lys Ser
Asp Met Asp Ala Ala Leu Asp Asp Leu Ile Asp Thr 130 135 140 tta gga
gga cct gaa gaa act gag gaa gat aat aca aca tat act gga 540 Leu Gly
Gly Pro Glu Glu Thr Glu Glu Asp Asn Thr Thr Tyr Thr Gly 145 150 155
cct gaa gtt ttg gat cca atg agt tct acc tat ata gag gaa ttg ggt 588
Pro Glu Val Leu Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Gly 160
165 170 aaa aga gaa gtc aca ctt cct cca aaa tat agg gaa ttg ttg gat
aaa 636 Lys Arg Glu Val Thr Leu Pro Pro Lys Tyr Arg Glu Leu Leu Asp
Lys 175 180 185 190 aaa gaa ggg att cca gtg cct cct cca gac act tcg
aaa ccc ctg ggg 684 Lys Glu Gly Ile Pro Val Pro Pro Pro Asp Thr Ser
Lys Pro Leu Gly 195 200 205 ccc gat gat gcc atc gat gcc ttg tca tta
gac ttg acc tgc agt tct 732 Pro Asp Asp Ala Ile Asp Ala Leu Ser Leu
Asp Leu Thr Cys Ser Ser 210 215 220 cct aca gct gat ggg aag aaa acc
gag aaa gag aaa tct act ggg gag 780 Pro Thr Ala Asp Gly Lys Lys Thr
Glu Lys Glu Lys Ser Thr Gly Glu 225 230 235 gtt ttg aaa gct cag tct
gtt ggg gta atc aga agc gct gct gct cca 828 Val Leu Lys Ala Gln Ser
Val Gly Val Ile Arg Ser Ala Ala Ala Pro 240 245 250 ccc cac gag aaa
aaa aga agg gtg gaa gag gac acg atg agt gat caa 876 Pro His Glu Lys
Lys Arg Arg Val Glu Glu Asp Thr Met Ser Asp Gln 255 260 265 270 gca
ctg gag gct ttg tca gct tcc ctg ggc agc cgg aag tca gaa ccc 924 Ala
Leu Glu Ala Leu Ser Ala Ser Leu Gly Ser Arg Lys Ser Glu Pro 275 280
285 gag ctt gac ctc agc tcc att aag gaa att gat gag gca aaa gcc aaa
972 Glu Leu Asp Leu Ser Ser Ile Lys Glu Ile Asp Glu Ala Lys Ala Lys
290 295 300 gaa gag aaa cta aag aag tgt ggt gaa gat gac gaa acg gtc
ccg cca 1020 Glu Glu Lys Leu Lys Lys Cys Gly Glu Asp Asp Glu Thr
Val Pro Pro 305 310 315 gag tat aga ttg aaa cca gcc atg gat aaa gat
gga aaa cca ctc ttg 1068 Glu Tyr Arg Leu Lys Pro Ala Met Asp Lys
Asp Gly Lys Pro Leu Leu 320 325 330 cca gag gct gaa gaa aaa ccc aag
ccc ctg agt gaa tca gaa ctc att 1116 Pro Glu Ala Glu Glu Lys Pro
Lys Pro Leu Ser Glu Ser Glu Leu Ile 335 340 345 350 gac gaa ctt tcg
gaa gat ttt gac cag tct aag cgt aaa gaa aaa caa 1164 Asp Glu Leu
Ser Glu Asp Phe Asp Gln Ser Lys Arg Lys Glu Lys Gln 355 360 365 tct
aag cca act gaa aaa aca aaa gag tct cag gcc act gcc cct act 1212
Ser Lys Pro Thr Glu Lys Thr Lys Glu Ser Gln Ala Thr Ala Pro Thr 370
375 380 cct gtg gga gag gcc gtg tct cgg acc tcc ttg tgc tgt gtg cag
tcg 1260 Pro Val Gly Glu Ala Val Ser Arg Thr Ser Leu Cys Cys Val
Gln Ser 385 390 395 gca ccc cca aag cca gct acg ggc atg gtg cca gat
gat gct gta gaa 1308 Ala Pro Pro Lys Pro Ala Thr Gly Met Val Pro
Asp Asp Ala Val Glu 400 405 410 gcc ttg gct gga agc ctg ggg aaa aag
gaa gca gat cca gaa gat gga 1356 Ala Leu Ala Gly Ser Leu Gly Lys
Lys Glu Ala Asp Pro Glu Asp Gly 415 420 425 430 aag cct gtg gag gat
aaa gtc aag gag aaa gcc aaa gaa gag gat cgt 1404 Lys Pro Val Glu
Asp Lys Val Lys Glu Lys Ala Lys Glu Glu Asp Arg 435 440 445 gaa aaa
ctt ggt gaa aag gaa gaa acg att cct cct gat tat aga tta 1452 Glu
Lys Leu Gly Glu Lys Glu Glu Thr Ile Pro Pro Asp Tyr Arg Leu 450 455
460 gaa gag gtc aag gac aaa gat gga aaa act ctc ccg cac aaa gac ccc
1500 Glu Glu Val Lys Asp Lys Asp Gly Lys Thr Leu Pro His Lys Asp
Pro 465 470 475 aag gaa cca gtc ctg ccc ttg agt gaa gac ttc gtc ctt
gat gct ttg 1548 Lys Glu Pro Val Leu Pro Leu Ser Glu Asp Phe Val
Leu Asp Ala Leu 480 485 490 tcc cag gac ttt gcc ggt ccc cca gcc gct
tca tct ctt ttt gaa gat 1596 Ser Gln Asp Phe Ala Gly Pro Pro Ala
Ala Ser Ser Leu Phe Glu Asp 495 500 505 510 gct aaa ctt tca gct gcc
gtc tct gaa gtg gtt tcc caa acc tca gct 1644 Ala Lys Leu Ser Ala
Ala Val Ser Glu Val Val Ser Gln Thr Ser Ala 515 520 525 cca acc acc
cac tct gca ggt cca ccc cct gac act gtg agt gat gac 1692 Pro Thr
Thr His Ser Ala Gly Pro Pro Pro Asp Thr Val Ser Asp Asp 530 535 540
aaa aaa ctt gac gat gcc ctg gat cag ctt tct gac agt ctg ggg caa
1740 Lys Lys Leu Asp Asp Ala Leu Asp Gln Leu Ser Asp Ser Leu Gly
Gln 545 550 555 aga cag cct gac cca gat gag aac aag ccc ata gag gat
aaa gtc aag 1788 Arg Gln Pro Asp Pro Asp Glu Asn Lys Pro Ile Glu
Asp Lys Val Lys 560 565 570 gaa aaa gct gaa gct gaa cat aga gac aag
ctg gga gaa aga gat gac 1836 Glu Lys Ala Glu Ala Glu His Arg Asp
Lys Leu Gly Glu Arg Asp Asp 575 580 585 590 act atc ccg cct gaa tat
aga cat ctc ttg gat aag gat gag gag ggc 1884 Thr Ile Pro Pro Glu
Tyr Arg His Leu Leu Asp Lys Asp Glu Glu Gly 595 600 605 aaa tca acg
aag cca ccc aca aag aaa cct gag gca cca aag aaa cct 1932 Lys Ser
Thr Lys Pro Pro Thr Lys Lys Pro Glu Ala Pro Lys Lys Pro 610 615 620
gaa gct gcc caa gat ccc att gat gcc ctc tca ggg gat ttt gac agc
1980 Glu Ala Ala Gln Asp Pro Ile Asp Ala Leu Ser Gly Asp Phe Asp
Ser 625 630 635 tgt cca tca act aca gaa acc tca gag aac aca aca aag
gac aaa gac 2028 Cys Pro Ser Thr Thr Glu Thr Ser Glu Asn Thr Thr
Lys Asp Lys Asp 640 645 650 aag aag acg gct tcc aag tcc aaa gca ccc
aag aat ggg ggt aaa gca 2076 Lys Lys Thr Ala Ser Lys Ser Lys Ala
Pro Lys Asn Gly Gly Lys Ala 655 660 665 670 aag gat tcc aca aag gca
aag gag gaa act tcc aaa caa aaa tct gat 2124 Lys Asp Ser Thr Lys
Ala Lys Glu Glu Thr Ser Lys Gln Lys Ser Asp 675 680 685 gga aag agt
aca agt taaaagttca cactattttc 2159 Gly Lys Ser Thr Ser 690 10 691
PRT Sus scrofa 10 Met Asn Pro Thr Glu Thr Lys Ala Val Lys Thr Glu
Pro Glu Lys Lys 1 5 10 15 Ser Gln Ser Thr Lys Pro Ser Val Val His
Glu Lys Lys Thr Gln Glu 20 25 30 Val Lys Pro Lys Glu His Pro Glu
Pro Lys Ser Leu Pro Thr His Ser 35 40 45 Ala Asp Ala Gly Ser Lys
Arg Ala His Lys Glu Lys Ala Val Ser Arg 50 55 60 Ser Asn Glu Gln
Pro Thr Ser Glu Lys Ser Thr Lys Pro Lys Ala Lys 65 70 75 80 Pro Gln
Asp Pro Thr Pro Ser Asp Gly Lys Leu Ser Val Thr Gly Val 85 90 95
Ser Ala Ala Ser Gly Lys Pro Ala Glu Thr Lys Lys Asp Asp Lys Ser 100
105 110 Leu Thr Ser Ser Val Pro Ala Glu Ser Lys Ser Ser Lys Pro Ser
Gly 115 120 125 Lys Ser Asp Met Asp Ala Ala Leu Asp Asp Leu Ile Asp
Thr Leu Gly 130 135 140 Gly Pro Glu Glu Thr Glu Glu Asp Asn Thr Thr
Tyr Thr Gly Pro Glu 145 150 155 160 Val Leu Asp Pro Met Ser Ser Thr
Tyr Ile Glu Glu Leu Gly Lys Arg 165 170 175 Glu Val Thr Leu Pro Pro
Lys Tyr Arg Glu Leu Leu Asp Lys Lys Glu 180 185 190 Gly Ile Pro Val
Pro Pro Pro Asp Thr Ser Lys Pro Leu Gly Pro Asp 195 200 205 Asp Ala
Ile Asp Ala Leu Ser Leu Asp Leu Thr Cys Ser Ser Pro Thr 210 215 220
Ala Asp Gly Lys Lys Thr Glu Lys Glu Lys Ser Thr Gly Glu Val Leu 225
230 235 240 Lys Ala Gln Ser Val Gly Val Ile Arg Ser Ala Ala Ala Pro
Pro His 245 250 255 Glu Lys Lys Arg Arg Val Glu Glu Asp Thr Met Ser
Asp Gln Ala Leu 260 265 270 Glu Ala Leu Ser Ala Ser Leu Gly Ser Arg
Lys Ser Glu Pro Glu Leu 275 280 285 Asp Leu Ser Ser Ile Lys Glu Ile
Asp Glu Ala Lys Ala Lys Glu Glu 290 295 300 Lys Leu Lys Lys Cys Gly
Glu Asp Asp Glu Thr Val Pro Pro Glu Tyr 305 310 315 320 Arg Leu Lys
Pro Ala Met Asp Lys Asp Gly Lys Pro Leu Leu Pro Glu 325 330 335 Ala
Glu Glu Lys Pro Lys Pro Leu Ser Glu Ser Glu Leu Ile Asp Glu 340 345
350 Leu Ser Glu Asp Phe Asp Gln Ser Lys Arg Lys Glu Lys Gln Ser Lys
355 360 365 Pro Thr Glu Lys Thr Lys Glu Ser Gln Ala Thr Ala Pro Thr
Pro Val 370 375 380 Gly Glu Ala Val Ser Arg Thr Ser Leu Cys Cys Val
Gln Ser Ala Pro 385 390 395 400 Pro Lys Pro Ala Thr Gly Met Val Pro
Asp Asp Ala Val Glu Ala Leu 405 410 415 Ala Gly Ser Leu Gly Lys Lys
Glu Ala Asp Pro Glu Asp Gly Lys Pro 420 425 430 Val Glu Asp Lys Val
Lys Glu Lys Ala Lys Glu Glu Asp Arg Glu Lys 435 440 445 Leu Gly Glu
Lys Glu Glu Thr Ile Pro Pro Asp Tyr Arg Leu Glu Glu 450 455 460 Val
Lys Asp Lys Asp Gly Lys Thr Leu Pro His Lys Asp Pro Lys Glu 465 470
475 480 Pro Val Leu Pro Leu Ser Glu Asp Phe Val Leu Asp Ala Leu Ser
Gln 485 490 495 Asp Phe Ala Gly Pro Pro Ala Ala Ser Ser Leu Phe Glu
Asp Ala Lys 500 505 510 Leu Ser Ala Ala Val Ser Glu Val Val Ser Gln
Thr Ser Ala Pro Thr 515 520 525 Thr His Ser Ala Gly Pro Pro Pro Asp
Thr Val Ser Asp Asp Lys Lys 530 535 540 Leu Asp Asp Ala Leu Asp Gln
Leu Ser Asp Ser Leu Gly Gln Arg Gln 545 550 555 560 Pro Asp Pro Asp
Glu Asn Lys Pro Ile Glu Asp Lys Val Lys Glu Lys 565 570 575 Ala Glu
Ala Glu His Arg Asp Lys Leu Gly Glu Arg Asp Asp Thr Ile 580 585 590
Pro Pro Glu Tyr Arg His Leu Leu Asp Lys Asp Glu Glu Gly Lys Ser 595
600 605 Thr Lys Pro Pro Thr Lys Lys Pro Glu Ala Pro Lys Lys Pro Glu
Ala 610 615 620 Ala Gln Asp Pro Ile Asp Ala Leu Ser Gly Asp Phe Asp
Ser Cys Pro 625 630 635 640 Ser Thr Thr Glu Thr Ser Glu Asn Thr Thr
Lys Asp Lys Asp Lys Lys 645 650 655 Thr Ala Ser Lys Ser Lys Ala Pro
Lys Asn Gly Gly Lys Ala Lys Asp 660 665 670 Ser Thr Lys Ala Lys Glu
Glu Thr Ser Lys Gln Lys Ser Asp Gly Lys 675 680 685 Ser Thr Ser 690
11 23 DNA synthetic 11 aaatctactg gagaggtttt gaa 23 12 21 DNA
synthetic 12 gacttctccc gaatcagttc c 21 13 21 DNA synthetic 13
agggcaaatc aacgaagcca c 21 14 21 DNA synthetic 14 cctttgttgt
gttctctgag g 21 15 22 DNA synthetic 15 agacttcgtc cttgatgctt tg 22
16 22 DNA synthetic 16 taatggctat gatgggttga gg 22 17 22 DNA
synthetic 17 gtaaagccaa aggaacaccc ag 22 18 28 DNA synthetic 18
tttttatttc tctgatgttg gctgtgca 28 19 114 DNA Bos taurus 19
ccaaaaagcc tacccaagca ctcatcagat acaggaagca agcatgctcc taaggaaaaa
60 gccgtttcca aatcaagyga gcagccacca tcagagaaat caacaaaacc aaag 114
20 164 DNA Sus scrofa 20 agccatctgt ggttcatgag aaaaaaaccc
aagaagtaaa gccaaaggaa cacccagagc 60 caaaaagcct acccacgcac
tcagcagatg cagggagcaa gcgtgctcat aaagaaaaag 120 cagtttccag
atctartgag cagccaacat cagagaaatc aaca 164 21 399 DNA Sus scrofa 21
aaatctactg gagaggtttt gaaagctcag tctgttgggg taatcaaaag cgctgctgct
60 ccaccccacg agaaaaaaag aagggtggaa gaggtataaa tcattacttc
tttgcaacga 120 agcatggtcc gcctgacagc agatgctttc ctgaggctta
tggaactgat tcgggagaag 180 tccgattgtg catcacactt gatgagtgtc
tttgcgctcc tggtcctgtg tggagtagtg 240 aaaccagtca gggttcactc
ggtcatctcc aggcaggcct tctctttctg caaatgcttg 300 tgggtgattt
cagcacactt gccttgattg tggagtaaga ctatctcaag attctactgc 360
tcagaagggc aggaccccag agcagctgca ctcttgctt 399 22 539 DNA Sus
scrofa 22 agggcaaatc aacgaagcca cccacaaaga aacctgaggc accaaaggta
aatacttttt 60 ttacactctt gctgcaactc ttaaatttta gaaatagaaa
atttattgaa ttcttacctt 120 gtgctttatg atcccaaagg gtttgtataa
gaatgtatta tttctgtttt cccgagagcc 180 attcaagata ggcagttcca
ttttccagat tagaaaattg aagctgagyg aatactaagc 240 aatttgtata
aaagagtaga agaaaataga gctgtcaaga ttttcctgtt ttaatatctc 300
ttttgtaata cactactttg ttaggaaagg aatgacagca aggctttatt ttaaacaaac
360 ctattttcag ggatatggga aaatatccca cagagcattc tctcctttgc
ctcttcattg 420 atggccattt ctattaatat ctcagaaacc tgaagctgcc
caagatccca ttgatgccct 480 ctcaggggat tttgacagat gtccatcaac
tacagaaacc tcagagaaca caacaaagg 539 23 195 DNA Sus scrofa 23
agacttcgtc cttgatgctt tgtcccagga ctttgccggt cccccagccg cttcatctct
60 tgtaagtctt tggagattcc tggtttaatt tccttagttt tagagtagca
cgaaatagat 120 ggaaacttgg gacttagaat ctgatgtggg agctgaggaa
acaaaattaa tggccctcaa 180 cccatcatag ccatt 195 24 164 DNA Sus
scrofa 24 agccatctgt ggttcatgag aaaaaaaccc aagaagtaaa gccaaaggaa
cacccagagc 60 caaaaagcct acccacgcac tcagcagatg cagggagcaa
gcgtgctcat aaagaaaaag 120 cagtttccag atctartgag cagccaacat
cagagaaatc aaca 164 25 164 DNA Sus scrofa 25 agccatctgt ggttcatgag
aaaaaaaccc aagaagtaaa gccaaaggaa cacccagagc 60 caaaaagcct
acccacgcac tcagcagatg cagggagcaa gcgtgctcat aaagaaaaag 120
cagtttccag atctartgca cagccaacat cagagaaata aaaa 164
* * * * *
References