U.S. patent application number 14/935546 was filed with the patent office on 2016-09-29 for methods of genotyping livestock.
This patent application is currently assigned to CY O'CONNOR ERADE VILLAGE FOUNDATION. The applicant listed for this patent is CY O'Connor ERADE Village Foundation. Invention is credited to Patrick Robert Carnegie, Roger Letts Dawkins, John Anthony Millman, Joseph Frederick Williamson.
Application Number | 20160281162 14/935546 |
Document ID | / |
Family ID | 43757950 |
Filed Date | 2016-09-29 |
United States Patent
Application |
20160281162 |
Kind Code |
A1 |
Dawkins; Roger Letts ; et
al. |
September 29, 2016 |
Methods of Genotyping Livestock
Abstract
The present invention relates to methods of genotyping for
selecting an animal with a desired trait such as the level
monounsaturated fats in muscle tissue, the types and/or ratio of
different monounsaturated fats in muscle tissue, marbling, carcass
weight, meat quality, speed of finishing, feedlot efficiency and/or
consumer preference. In particular the invention relates to methods
of selecting an animal with a desired trait by analysing the M-RIP,
NT5M and/or TCAP genes for one or more polymorphisms.
Inventors: |
Dawkins; Roger Letts;
(Forrestdale, AU) ; Millman; John Anthony;
(Shoalwater Bay, AU) ; Williamson; Joseph Frederick;
(Victoria Park, AU) ; Carnegie; Patrick Robert;
(Salter Point, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CY O'Connor ERADE Village Foundation |
Canning Vale South |
|
AU |
|
|
Assignee: |
CY O'CONNOR ERADE VILLAGE
FOUNDATION
Canning Vale South
AU
|
Family ID: |
43757950 |
Appl. No.: |
14/935546 |
Filed: |
November 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13496607 |
Aug 20, 2012 |
9181585 |
|
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PCT/AU2010/001214 |
Sep 17, 2010 |
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14935546 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/124 20130101;
C12Q 2600/156 20130101; C12Q 1/6883 20130101; C12Q 1/6876 20130101;
C12Q 2600/172 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 17, 2009 |
AU |
2009904511 |
Claims
1. An oligonucleotide primer for use in amplifying a polymorphic
region of the M-RIP, NT5M and/or TCAP genes of an animal.
2. The oligonucleotide primer of claim 1, wherein the primer is
selected from: a) an oligonucleotide comprising a sequence selected
from: TABLE-US-00008 5'-AGG GGT GCT GAG TCT ACA GG-3', (SEQ ID NO:
1) 5'-CTC CAG GAG GCA GGA GAA G-3', (SEQ ID NO: 2) 5'-GGA AGG CCA
GTT ACA TGG CA-3', (SEQ ID NO: 3) 5'-CAC AAC CAA GGC CAA AAT CGC
A-3', (SEQ ID NO: 4) 5'-AGT ACC AGC TGC CCT ACC A-3', (SEQ ID NO:
5) and 5'-CTG AGA CAT GGA GCG AGC CA-3', (SEQ ID NO: 6)
b) an oligonucleotide comprising a sequence which is the reverse
complement of any oligonucleotide provided in a), and c) a variant
of a) or b) which can be used to amplify the same region of the
animal genome as any one of the oligonucleotides of a) or b).
3. A composition comprising an oligonucleotide of claim 1 and an
acceptable carrier.
4. A kit comprising an oligonucleotide of claim 1.
5. A method of detecting the haplotype of a Wagyu cow, comprising:
a. obtaining a sample containing DNA from a Wagyu cow; and b.
detecting whether a NT5M-MRIP-TCAP-GH haplotype selected from the
group consisting of 22-40-20 B/C, 10-30-20 A and 10-40-20 B/C is
present in the DNA by genotyping the DNA.
6. The method of claim 5, wherein the haplotype is 22-40-20
B/C.
7. The method of claim 5, wherein the haplotype is 10-30-20 A.
8. The method of claim 5, wherein the haplotype is 10-40-20
B/C.
9. The method of claim 6, wherein a genotyping fragment for
genotyping the DNA is between 428 and 432 basepairs long.
10. The method of claim 7, wherein a genotyping fragment for
genotyping the DNA is between 428 and 432 basepairs long or between
343 and 346 basepairs long.
11. The method of claim 8, wherein a genotyping fragment for
genotyping the DNA is between 428 and 432 basepairs long or between
343 and 346 basepairs long.
12. A method of detecting a haplotype of a Wagyu cow, comprising:
a. obtaining a sample containing DNA from a Wagyu cow; and b.
detecting whether a TCAP-GH selected from the group consisting of
20-C, 10-C and 10-B is present in the DNA by genotyping the
DNA.
13. The method of claim 9, wherein the haplotype is 20-C.
14. The method of claim 9, wherein the haplotype is 10-C.
15. The method of claim 9, wherein the haplotype is 10-B.
16. A method of detecting a haplotype of a Wagyu cow, comprising:
a. obtaining a sample containing DNA from a Wagyu cow; and b.
detecting whether a MRIP-TCAP-GH haplotype selected from the group
consisting of 30-10-B and 40-20-A is present in the DNA by
genotyping the DNA.
17. The method of claim 13, wherein the haplotype is 30-10-B.
18. The method of claim 13, wherein the haplotype is 40-20-A.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of U.S. patent
application Ser. No. 13/496,607, filed on Aug. 20, 2012, which is a
U.S. national phase patent application under 35 U.S.C. 371 of
International Patent Application No. PCT/AU2010/001214 entitled
"Methods of Genotyping Livestock" filed Sep. 17, 2010, which claims
benefit of priority of Australian Patent Application No. 2009904511
filed on Sep. 17, 2009. Each of these applications are hereby
incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to methods of genotyping for
selecting an animal with a desired trait such as the level of
monounsaturated fats in muscle tissue, the types and/or ratio of
different monounsaturated fats in muscle tissue, marbling, carcass
weight, meat quality, speed of finishing, feedlot efficiency and/or
consumer preference.
BACKGROUND OF THE INVENTION
[0003] It has been claimed that DNA testing can provide an
indication of alleles associated with thyroglobulin gene
polymorphism. However, Rincker et al. (2006) found the GeneSTAR
marbling marker was not associated with intramuscular fat
deposition in early weaned steers fed a high-concentrate diet and
the thyroglobulin gene was not associated with other carcass,
performance parameters, or marbling expected progeny difference.
Similarly, Van Eenennaam et al. (2007) could not validate the
utility of the markers.
[0004] By contrast, US 2008/0010696 shows that SCD SREB has
relevance in marbling in Japanese cattle but not others. SREBP is a
transcription factor which regulates gene expression levels of
stearoyl-CoA desaturase (SCDl) and other genes relevant to lipid
and fatty acid metabolism in tissue (Hoashi et al., 2007). The
content of unsaturated fatty acid in beef is an important factor in
taste and texture (Hoashi et al., 2007; EP 1845159) and the degree
of marbling. A recent study revealed that the high SCD activities
were positively correlated with beef marbling score, amount of
monounsaturated fatty acid and conjugated linoleic acid, but
negatively correlated with the amount of saturated fatty acid
(Jiang et al., 2008).
[0005] U.S. Pat. No. 7,157,231 shows that bovine growth hormone (GH
Exon 5) has an important role with the B & C alleles
contributing to marbling, again this effect is confined to Japanese
cattle. U.S. Pat. No. 7,238,479 shows that genetic testing for
alleles of the gene encoding calcium activated neutral protease
effecting meat tenderness in bovines is useful in selective
breeding. US 2004/0261138 shows that genetic markers in the porcine
melanocortin-4 receptor (MC4R) gene are associated with meat
quality.
[0006] However, there is a need for further methods of genetic
analysis for identifying livestock with desirable traits.
SUMMARY OF THE INVENTION
[0007] The present inventors have identified new polymorphic
regions linked to desired traits.
[0008] In one aspect, the present invention provides a method of
selecting an animal with a desired trait, the method comprising
[0009] i) analysing the M-RIP, NTSM and/or TCAP genes of the animal
for one or more polymorphisms associated with the desired trait,
and [0010] ii) selecting an animal with one or more polymorphisms
associated with the desired trait.
[0011] In a preferred embodiment, the one or more polymorphisms,
more preferably a combination of polymorphisms, are characteristic
of a haplotype associated with the desired trait.
[0012] In a further embodiment, step i) further comprises analysing
the SREBP1 and/or GH genes, preferably at least the SREBP1 gene, of
the animal for one or more polymorphisms associated with the
desired trait.
[0013] In a preferred embodiment, the M-RIP gene is analysed by
[0014] a) amplifying a region of the gene using an oligonucleotide
primer comprising the sequence 5'-AGG GGT GCT GAG TCT ACA GG-3'
(SEQ ID NO:1) and an oligonucleotide comprising the sequence 5'-CTC
CAG GAG GCA GGA GAA G-3' (SEQ ID NO:2), or a variant of one or both
primers which can be used to amplify the same region of the genome,
and
[0015] b) analysing the amplification products for the one or more
polymorphisms. In an embodiment, the M-RIP gene is analysed for an
allele designated 10, 30, 40 or 60 as defined herein.
[0016] In another preferred embodiment, the NT5M gene is analysed
by
[0017] a) amplifying a region of the gene using an oligonucleotide
primer comprising the sequence 5'-GGA AGG CCA GTT ACA TGG CA-3'
(SEQ ID NO:3) and an oligonucleotide primer comprising the sequence
5'-CAC AAC CAA GGC CAA AAT CGC A-3' (SEQ ID NO:4), or a variant of
one or both primers which can be used to amplify the same region of
the genome, and
[0018] b) analysing the amplification products for the one or more
polymorphisms.
[0019] In an embodiment, the NT5M gene is analysed for an allele
designated 10, 20 or 22 as defined herein.
[0020] In a further preferred embodiment, the TCAP gene is analysed
by
[0021] a) amplifying a region of the gene using an oligonucleotide
primer comprising the sequence 5'-AGT ACC AGC TGC CCT ACC A-3' (SEQ
ID NO:5) and an oligonucleotide primer comprising the sequence
5'-CTG AGA CAT GGA GCG AGC CA-3' (SEQ ID NO:6), or a variant of one
or both primers which can be used to amplify the same region of the
genome, and
[0022] b) analysing the amplification products for the one or more
polymorphisms. In an embodiment, the TCAP gene is analysed for an
allele designated 10 or 20 as defined herein.
[0023] In another preferred embodiment, the SREBP1 gene is analysed
by
[0024] a) amplifying a region of the gene using an oligonucleotide
primer comprising the sequence 5'-CCA CAA CGC CAT CGA GAA ACG CTA
C-3' (SEQ ID NO:7) and an oligonucleotide primer comprising the
sequence 5'-GGC CTT CCC TGA CCA CCC AAC TTA G-3' (SEQ ID NO:8), or
a variant of one or both primers which can be used to amplify the
same region of the genome, and
[0025] b) analysing the amplification products for the one or more
polymorphisms.
[0026] In an embodiment, the SREBP1 gene is analysed for an allele
designated S or L as defined herein and US 2008/0010696.
[0027] In an embodiment, the SREBP1 gene is analysed for an allele
designated S or L as defined herein and US 2008/0010696.
[0028] In yet a further preferred embodiment, the GH gene is
analysed by
[0029] a) amplifying a region of the gene using [0030] 1) an
oligonucleotide primer comprising the sequence 5'-TCT ATG AGA AGC
TGA AGG ACC TGG AGG AA-3' (SEQ ID NO:9) or a variant thereof which
20 can be used to amplify the same region of the genome, and [0031]
2) an oligonucleotide primer comprising the sequence 5'-CGG GGG GTG
CCA TCT TCC AG-3' (SEQ ID NO:10) or an oligonucleotide primer
comprising the sequence 5'-CGG GGG GTG CCA TCT TCC AC-3' (SEQ ID
NO:11) or a variant of one or both primers which can be used to
amplify the same region of the genome, and [0032] 3) an
oligonucleotide primer comprising the sequence 5'-ATG ACC CTC TGG
TAC GTC TCC G-3' (SEQ ID NO:12) or an oligonucleotide primer
comprising the sequence 5'-CAT GAC CCT CTG GTA CGT CTC CA-3' (SEQ
ID NO:13) or a variant of one or both primers which can be used to
amplify the same region of the genome, and
[0033] b) analysing the amplification products for the one or more
polymorphisms.
[0034] In an embodiment, the GH gene is analysed for an allele
designated A, B, C or D as defined herein and U.S. Pat. No.
7,157,231.
[0035] In an embodiment, at least the M-RIP gene is analysed, more
preferably at least the M-RIP and NT5M genes, at least the M-RIP
and TCAP genes, at least the M-RIP, NT5M and TCAP genes, at least
the SREBP1, M-RIP, NT5M and TCAP genes, at least the M-RIP,
NT5M,
[0036] TCAP and GH genes, or at least the SREBP1, M-RIP, NT5M, TCAP
and GH genes. In alternate embodiments, at least the TCAP and GH
genes, or at least the SREBP1 and NT5M genes, are analysed.
[0037] In an embodiment, the trait is the level of monounsaturated
fats in muscle tissue, the types and/or ratio of different
monounsaturated fats in muscle tissue, marbling, carcass weight,
meat quality, speed of finishing, feedlot efficiency and/or
consumer preference. In an embodiment, the meat quality trait is
eye muscle area and/or tenderness.
[0038] Preferably, the animal is a livestock animal. More
preferably, the livestock animal is a sheep, cow, pig, goat or
horse. Even more preferably, the livestock animal is a cow.
[0039] In an embodiment, the cow is a Wagyu, the meat quality trait
is eye muscle area (EMA) and the haplotypes (NTSM-MRIP-TCAP-GH)
22-40-20 B/C, 10-30-20 A, and 10-40-20-B/C, more preferably
22-40-20 B/C, are positively associated with higher EMA when
compared to other NT5M-MRIP-TCAP-GH haplotypes. Furthermore, the
haplotypes 20-30-20-A, 20-40-20-A and 22 40-10-B/C, more preferably
20-30-20-A and 20-40-20-A, are negatively associated with higher
EMA when compared to other NT5M-MRIP-TCAP-GH haplotypes.
[0040] In a further embodiment, the cow is a Wagyu, the trait is
carcass weight, and the haplotypes (TCAP-GH) 20-C, 10-C and 10-B,
more preferably 20-C, are positively associated with higher carcass
weight when compared to other TCAP-GH haplotypes.
[0041] In yet another embodiment, the cow is a Wagyu, the trait is
marbling, and the haplotypes (MRIP-TCAP-GH) 30-10-B and 40-20-A are
positively associated with higher marbling when compared to other
MRIP-TCAP-GH haplotypes. Furthermore, the haplotypes 40-10-B,
40-10-A and 30-10-C are negatively associated with higher marbling
when compared to other MRIP-TCAP-GH haplotypes.
[0042] In an embodiment, the 40.1 and 30.1 haplotypes of Table 2
are associated with the desired trait in cattle, preferably Wagyu,
Simmental and/or Angus cattle.
[0043] In an embodiment, step i) is performed on a sample which
comprises DNA that has been obtained from the animal.
[0044] In a further aspect, the present invention provides a method
of breeding an animal with a desired trait, the method comprising
selecting a first animal using a method of the invention, and
crossing the first animal with a second animal of the same species
but opposite sex.
[0045] In an embodiment, the second animal is also selected using a
method of the invention.
[0046] In an embodiment of the above aspect, the sample is an egg,
semen or an embryo. In relation to this embodiment, selection of an
embryo can be used to accelerate the expansion or rare genotypes.
Recovered embryo's can be stored, and preferred embryo's placed
into a third party recipient animal such as a cow. The embryo's can
also be split to speed up production of animals with the desired
genotype.
[0047] In a further embodiment, the method comprises selecting a
progeny of the cross for the desired trait using the method of the
invention.
[0048] In another aspect, the present invention provides a method
of genotyping an animal, the method comprising analysing the M-RIP,
NT5M and/or TCAP genes of the animal for one or more
polymorphisms.
[0049] This aspect of the invention can be used for a variety of
purposes such as paternity testing, exclusion testing and breed
composition testing.
[0050] With regard to the use of the above aspect for paternity
testing, preferably the method comprises
[0051] i) genotyping an animal by analysing the M-RIP, NT5M and/or
TCAP genes of the animal for one or more polymorphisms,
[0052] ii) determining the genotype of the candidate father and/or
mother of the animal by analysing the M-RIP, NT5M and/or TCAP genes
for the one or more polymorphisms, and
[0053] iii) comparing the genotypes from steps i) and ii) to
determine the probability that the candidate father and/or mother
is the parent of the animal.
[0054] As the skilled person would appreciate, steps i) and ii) can
be performed in any order.
[0055] In an embodiment, the method further comprises analysing the
SREBP1 and/or GH genes of the animal for one or more
polymorphisms.
[0056] Preferably, the one or more polymorphisms, more preferably a
combination of polymorphisms, are characteristic of a
haplotype.
[0057] In yet a further aspect, the present invention provides an
oligonucleotide primer for use in amplifying a polymorphic region
of the M-RIP, NT5M and/or TCAP genes of an animal.
[0058] In an embodiment, the primer is selected from:
[0059] a) an oligonucleotide comprising a sequence selected
from:
TABLE-US-00001 5'-AGG GGT GCT GAG TCT ACA GG-3', (SEQ ID NO: 1)
5'-CTC CAG GAG GCA GGA GAA G-3', (SEQ ID NO: 2) 5'-GGA AGG CCA GTT
ACA TGG CA-3', (SEQ ID NO: 3) 5'-CAC AAC CAA GGC CAA AAT CGC A-3',
(SEQ ID NO: 4) 5'-AGT ACC AGC TGC CCT ACC A-3', (SEQ ID NO: 5) and
5'-CTG AGA CAT GGA GCG AGC CA-3', (SEQ ID NO: 6)
[0060] b) an oligonucleotide comprising a sequence which is the
reverse complement of any oligonucleotide provided in a), and
[0061] c) a variant of a) or b) which can be used to amplify the
same region of the animal genome as any one of the oligonucleotides
of a) or b).
[0062] Also provided is a composition comprising an oligonucleotide
of the invention and an acceptable carrier.
[0063] In a further aspect, the present invention provides a kit
comprising an oligonucleotide of the invention.
[0064] As will be apparent, preferred features and characteristics
of one aspect of the invention are applicable to many other aspects
of the invention.
[0065] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0066] The invention is hereinafter described by way of the
following non-limiting Examples and with reference to the
accompanying figures.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0067] FIGS. 1 and 2 provide schematic representations of
polymorphic markers on chromosome 19 used to define the 14 Mb
haplotypes in cattle, with FIG. 1 providing a summary of the
alleles analysed.
[0068] FIG. 3 shows the stable inheritance of the haplotypes of the
invention in three different families of cattle.
[0069] FIG. 4 shows the amplification products of the M-RIP gene
using the primers provided in Table 1.
[0070] FIG. 5 shows SREBP1 to GH haplotype frequencies in different
breeds (Simm=Simmental, AA=Angus).
[0071] FIG. 6 shows the correlation between TCAP-GH haplotypes and
hot dead carcass weight.
[0072] FIG. 7 shows the correlation between SREBP1 to GH haplotype
frequencies and eye muscle area.
[0073] FIG. 8 shows the correlation between MRIP to GH haplotypes
and marbling.
DETAILED DESCRIPTION OF THE INVENTION
General Techniques and Definitions
[0074] Unless specifically defined otherwise, all technical and
scientific terms used herein shall be taken to have the same
meaning as commonly understood by one of ordinary skill in the art
(e.g., in cell culture, molecular genetics, animal breeding,
protein chemistry, and biochemistry).
[0075] Unless otherwise indicated, the recombinant protein, cell
culture, and immunological techniques utilized in the present
invention are standard procedures, well known to those skilled in
the art. Such techniques are described and explained throughout the
literature in sources such as, J. Perbal, A Practical Guide to
Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbour
Laboratory Press (1989), T. A. Brown (editor), Essential Molecular
Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991),
D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical
Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel
et al. (editors), Current Protocols in Molecular Biology, Greene
Pub. Associates and Wiley-Interscience (1988, including all updates
until present), Ed Harlow and David Lane (editors) Antibodies: A
Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.
E. Coligan et al. (editors) Current Protocols in Immunology, John
Wiley & Sons (including all updates until present).
[0076] A "haplotype" is the particular combination of alleles
(sometimes identified by single nucleotide polymorphisms (SNPs)) on
one chromosome or a part of a chromosome. Haplotypes can be
exploited for the fine mapping of traits.
[0077] Some polymorphisms may be in linkage disequilibrium and are
inherited in blocks. A "haplotype block" (also known in the art as
a "frozen block") is thus a discrete chromosome region of high
linkage disequilibrium (LD) and low haplotype diversity. It is
expected that all pairs of polymorphisms within a block will be in
strong linkage disequilibrium, whereas other pairs will show much
weaker association. Blocks are hypothesized to be regions of low
recombination flanked by recombination hotspots. Blocks may contain
a large number of polymorphisms, but a few polymorphisms are enough
to uniquely identify the haplotypes in a block.
[0078] An "ancestral haplotype" is passed from generation to
generation just like familial haplotype blocks but is found at
higher than expected frequencies in the population at large between
animals not closely related, namely all arising from some distant
ancestor. Generally, the phrases "haplotype" and "ancestral
haplotype" are used herein interchangeably.
[0079] As used herein, the term "the one or more polymorphisms are
characteristic of a haplotype" means that presence of the
polymorphism(s), typically numerous polymorphisms, is a marker of a
specific haplotype.
[0080] The term "polymorphism" refers to the coexistence of more
than one form of a locus of interest. A region of the genome of
which there are at least two different forms, i.e., two different
nucleotide sequences, is referred to as a "polymorphic region" or
"polymorphic locus". A polymorphic locus can be a single
nucleotide, the identity of which differs in the other alleles. A
polymorphic locus can also be more than one nucleotide long and/or
can be a varying length due to insertions or deletions (such as of
the number of repeats) at a given locus. The allelic form occurring
most frequently in a selected population is often referred to as
the reference and/or wild-type form. Other allelic forms are
typically designated or alternative or variant alleles.
[0081] Diploid organisms may be homozygous or heterozygous for
allelic forms. A diallelic or biallelic polymorphism has two forms.
A trialleleic polymorphism has three forms.
[0082] The term "single nucleotide polymorphism" (SNP) refers to a
polymorphic site occupied by a single nucleotide, which is the site
of variation between allelic sequences. The site is usually
preceded by and followed by highly conserved sequences of the
allele (e.g., sequences that vary in less than 1/100 or 1/1000
members of a population). SNP usually arises due to substitution of
one nucleotide for another at the polymorphic site. Typically the
polymorphic site is occupied by a base other than the reference
base. For example, where the reference allele contains the base "T"
(thymidine) at the polymorphic site, the altered allele can contain
a "C" (cytidine), "G" (guanine), or "A" (adenine) at the
polymorphic site.
[0083] The terms "linkage", "linked" or variations thereof describe
the tendency of genes, alleles, loci or genetic markers to be
inherited together as a result of their location on the same
chromosome. It can be measured by percent recombination between the
two genes, alleles, loci, or genetic markers. The term "linkage
disequilibrium" refers to a greater than random association between
specific alleles at two marker loci within a particular population.
In general, linkage disequilibrium decreases with an increase in
physical distance. If linkage disequilibrium exists between two
markers within one gene or block, then the genotypic information at
one marker can be used to make probabilistic predictions about the
genotype of the second marker.
[0084] As used herein, the term "analyse" or variations thereof
refers to determining the sequence, either directly (for example by
actual sequencing) or indirectly (for example by the analysis of
different fragment lengths following amplification and/or
restriction enzyme cleavage), of polymorphic loci of the genes
defined herein, particularly the specific alleles which have been
disclosed (see, for example, FIGS. 1 and 2). Typically, a method of
the invention will be conducted by analysing a sample obtained from
the animal.
[0085] The "sample" refers to a material which comprises the
animals genomic DNA, or RNA encoding a gene of interest. The sample
can be used as obtained directly from the source or following at
least one step to at least partially purify DNA or RNA from the
sample obtained directly from the source. Preferably, the sample
comprises genomic DNA. The sample can be prepared in any convenient
medium which does not interfere with the methods of the invention.
Typically, the sample is an aqueous solution or biological fluid as
described in more detail below. The sample can be derived from any
source, such as a physiological fluid, including blood, serum,
plasma, saliva, sputum, ocular lens fluid, sweat, faeces urine,
milk, ascites fluid, mucous, synovial fluid, peritoneal fluid,
transdermal exudates, pharyngeal exudates, bronchoalveolar lavage,
tracheal aspirations, cerebrospinal fluid, semen, cervical mucus,
vaginal or urethral secretions, buccal swab, amniotic fluid, and
the like. Herein, fluid homogenates of cellular tissues such as,
for example, hair, skin and nail scrapings, meat extracts are also
considered biological fluids. An important use of the invention is
that it enables one skilled in the art to select rare alleles and
haplotypes prior to expansion. Since these may be demonstrated by
embryo biopsy it is possible to expand a very rare genotype. Thus,
in a further embodiment the sample may be a biopsy from an embryo
(preferably at the blastocyst stage), an egg or individual semen.
Pretreatment may involve preparing plasma from blood, diluting
viscous fluids, and the like. Methods of treatment can involve
filtration, distillation, separation, concentration, inactivation
of interfering components, and the addition of reagents. The
selection and pretreatment of biological samples prior to testing
is well known in the art and need not be described further.
[0086] As used herein, the term "gene" is to be taken in its
broadest context and includes the deoxyribonucleotide sequences
comprising the protein coding region of a structural gene and
including sequences located adjacent to the coding region on both
the 5' and 3' ends for a distance of about 1 kb on either end such
that the gene corresponds to the length of the full-length mRNA.
Regions further distances (than about 1 kb) from the coding region
may also comprise part of a gene if they directly influence
transcription. The sequences which are located 5' of the coding
region and which are present on the mRNA are referred to as 5'
non-translated sequences. The sequences which are located 3' or
downstream of the coding region and which are present on the mRNA
are referred to as 3' non-translated sequences. A genomic form or
clone of a gene contains the coding region which is interrupted
with non-coding sequences termed "introns" or "intervening regions"
or "intervening sequences". Introns are segments of a gene which
are transcribed into nuclear RNA (nRNA); introns may contain
regulatory elements such as enhancers. Introns are removed or
"spliced out" from the nuclear or primary transcript; introns
therefore are absent in the messenger RNA (mRNA) transcript. The
mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide.
[0087] The term "livestock animal" refers to any breed or
population of animal kept by humans for a useful, commercial
purpose. As used herein, a livestock animal can be mammal or avian.
Generally, the livestock animal is an agricultural mammal, for
example, bovine, equine, ovine, porcine. Livestock animals raised
for the production of meat find use with the present invention, for
example, beef cattle, pigs, goats, sheep, bison, chickens, turkeys,
etc. The livestock animals can be in all stages of development,
including embryonic, fetal, neonate, yearling, juvenile and adult
stages.
[0088] The term "bovine", "cow", "cattle" or variations thereof
refer to a domesticated (purebred or crossbreeds) or wild mammal
that is a Bovinae, for example, of the genera Bos (e g , cattle or
oxen) or Bison (e.g. American buffalo). Exemplary mammals of the
genus Bos include without limitation Bos taurus, Bos bovis, Bos
frontalis (gayal), Bos gaurus (gaur), Bos grunniens (domestic yak),
Bos grunniens.times.Bos taurus (dzo), Bos indicus (zebu cattle),
Bos indicus gudali (Gudali zebu), Bos indicus.times.Bos taurus
(hybrid cattle), Bos javanicus (banteng), Bos primigenius
(aurochs), and Bos sauveli (kouprey). Bos species for the
production of meat products, for example beef cattle, which are of
particular relevance to the invention include, but not necessarily
limited to, Black Angus, Red Angus, Horned Hereford, Polled
Hereford, Murray Gray, Charolais, Simmental, Limousine, Chianina,
Brahman, Santa Gertrudis, Texas Longhorn and Wagyu. Exemplary dairy
cattle breeds of Bos include without limitation Ayrshire, Brown
Swiss, Canadiennem, Dutch Belted, Guernsey, Holstein
(Holstein-Friesian), Jersey, Kerry, Milking Devon, Milking
Shorthorn and Norwegian Red.
Polymorphisms and Haplotypes
[0089] The present inventors have shown that alleles of the M-RIP,
NT5M and/or TCAP genes, particularly combinations thereof which may
also include alleles of the SREBP1 and/or GH genes, represent
ancestral haplotypes which can be used for selecting and/or
genotyping animals.
[0090] As used herein, the term "M-RIP" refers to the Myosin
Phosphatase-Rho interacting protein encoding gene, the term
"SREBP-1" refers to the Sterol Regulatory Element Binding Protein-1
encoding gene (an example of the cow SREBP-1 gene is described in
US 2008/0010696), the term "GH" refers to the Growth Hormone
encoding gene (an example of the cow GH gene is described in U.S.
Pat. No. 7,157,231), the term "NT5M" refers to the 5',
3'-Nucleotidase, Mitochondrial encoding gene, and the term "TCAP"
refers to the titin-cap (telethonin) encoding gene. The location of
these genes in the cow genome on chromosome 19 is shown in FIGS. 1
and 2.
[0091] As outlined in FIG. 1, in cattle there are at least two
informative alleles of the SREBP-1 gene (designated S and L), at
least three informative alleles of the NT5M gene (designated 10, 20
and 22), at least four informative alleles of the M-RIP gene
(designated 10, 30, 40 and 60), at least two informative alleles of
the TCAP gene (designated 10 and 20), and at least four informative
alleles of the GH gene (designated A, B, C and D). Of all possible
combinations of these alleles, 41 have been identified thus far in
cattle with varying degrees of representation between different
breeds.
[0092] When referring to the specific haplotypes, when all 5 genes
are analysed the same order is used (SREBP1-NT5M-MRIP-TCAP-GH) as
the genes exist on the cow genome. Thus, L-20-30-20-A means the
animal has the L allele of SREBP1, the 20 allele of NT5M, the 30
allele of M-RIP, the 20 allele of TCAP and the A allele of GH.
[0093] As used herein, the L and S designations for SREBP1
correspond to the same nomenclature used in US 2008/0010696. More
specifically, "L" refers to the larger (428-432 bp) fragment, and
"S" refers to the smaller (343-346 bp) fragment, following
amplification with primers comprising the sequence 5'-CCA CAA CGC
CAT CGA GAA ACG CTA C-3' (SEQ ID
[0094] NO:7) and 5'-GGC CTT CCC TGA CCA CCC AAC TTA G-3' (SEQ ID
NO:8).
[0095] As used herein, the 10, 20 and 22 designations for NT5M
correspond to the 321 bp, 331 bp and 334 bp, respectively, products
following amplification with primers comprising the sequence 5'-GGA
AGG CCA GTT ACA TGG CA-3' (SEQ ID NO:3) and 5'-CAC AAC CAA GGC CAA
AAT CGC A-3' (SEQ ID NO:4).
[0096] As used herein, the 10, 30, 40 and 60 designations for M-RIP
correspond to the 192.about.193 bp, 201.about.203 bp, 208.about.209
bp, 218.about.220 bp, respectively, products following
amplification with primers comprising the sequence 5'-AGG GGT GCT
GAG TCT ACA GG-3' (SEQ ID NO:1) and 5'-CTC CAG GAG GCA GGA GAA G-3'
(SEQ ID NO:2). As used herein, the 10 and 20 designations for TCAP
correspond to the smaller (approximately .about.230.about.235 bp)
and the larger (about 245 bp), respectively, products following
amplification with primers comprising the sequence 5'-AGT ACC AGC
TGC CCT ACC A-3' (SEQ ID NO:5) and sequence 5'-CTG AGA CAT GGA GCG
AGC CA-3' (SEQ ID NO:6).
[0097] As used herein, the A, B and C designations for GH
correspond to the same nomenclature used in U.S. Pat. No.
7,157,231. The D designation is defined as in the Examples herein.
These designation and the corresponding genotypes are summarized in
Table 4, and relate to various combinations of polymorphisms in the
codons encoding amino acids 127 and 172 of the GH protein.
Oligonucleotide Primers
[0098] As those skilled in the art would be aware, the sequence of
the oligonucleotide primers described herein can be varied to some
degree without effecting their usefulness for the methods of the
invention. A "variant" of an oligonucleotide disclosed herein (also
referred to herein as a "primer" or "probe" depending on its use)
useful for the methods of the invention includes molecules of
varying sizes of, and/or are capable of hybridising to the genome
close to that of, the specific oligonucleotide molecules defined
herein. For example, variants may comprise additional nucleotides
(such as 1, 2, 3, 4, or more), or less nucleotides as long as they
still hybridise to the target region. Furthermore, a few
nucleotides may be substituted without influencing the ability of
the oligonucleotide to hybridise the target region. In addition,
variants may readily be designed which hybridise close (for
example, but not limited to, within 50 nucleotides or within 100
nucleotides) to the region of the genome where the specific
oligonucleotides defined herein hybridise. Oligonucleotides can be
naturally occurring or synthetic, but are typically prepared by
synthetic means.
[0099] The term "primer" as used herein, is meant to encompass any
nucleic acid that is capable of priming the synthesis of a nascent
nucleic acid in a template-dependent process. Primers may be
provided in double-stranded or single-stranded form, although the
single-stranded form is preferred. Primers act as a point of
initiation of template-directed DNA synthesis under appropriate
conditions (e.g., in the presence of four different nucleoside
triphosphates and as agent for polymerization, such as DNA or RNA
polymerase or reverse transcriptase) in an appropriate buffer and
at a suitable temperature. Methods of primer design are well-known
in the art, based on the design of complementary sequences obtained
from standard Watson-Crick base-pairing (i.e., binding of adenine
to thymine or uracil and binding of guanine to cytosine). A primer
need not match the exact sequence of a template, but must be
sufficiently complementary to hybridize with the template.
Computerized programs, when provided with suitable information
regarding a target region, for selection and design of
amplification primers are available from commercial and/or public
sources well known to the skilled artisan. The length of a primer
may vary but typically ranges from 15 to 30 nucleotides.
[0100] Primers used in the methods of the invention can have one or
more modified nucleotides. Many modified nucleotides (nucleotide
analogs) are known and can be used in oligonucleotides. A
nucleotide analog is a nucleotide which contains some type of
modification to either the base, sugar, or phosphate moieties.
Modifications to the base moiety would include natural and
synthetic modifications of A, C, G, and T/U as well.
[0101] Chimeric primers can also be used. Chimeric primers are
primers having at least two types of nucleotides, such as both
deoxyribonuucleotides and ribonucleotides, ribonucleotides and
modified nucleotides, two or more types of modified nucleotides,
deoxyribonucleotides and two or more different types of modified
nucleotides, ribonucleotides and two or more different types of
modified nucleotides, or deoxyribonucleotides, ribonucleotides and
two or more different types of modified nucleotides. One form of
chimeric primer is peptide nucleic acid/nucleic acid primers. For
example, 5'-PNA-DNA-3' or 5'-PNA-RNA-3' primers may be used for
more efficient strand invasion and polymerization invasion. Other
forms of chimeric primers are, for example, 5'-(2'-O-Methyl)
RNA-RNA-3' or 5'-(2'-O-Methyl) RNA-DNA-3'.
[0102] The term "primer pair" refers to a set of primers including
an upstream primer that hybridizes with the 3' end of the
complement of the nucleic acid to be amplified and a downstream
primer that hybridizes with the 3' end of the sequence to be
amplified as different purine or pyrimidine bases. Such
modifications are well known in the art.
[0103] Primers may be chemically synthesized by methods well known
within the art. Chemical synthesis methods allow for the placement
of detectable labels such as fluorescent labels, radioactive
labels, etc. to be placed virtually anywhere within the sequence.
Solid phase methods as well as other methods of oligonucleotide or
polynucleotide synthesis known to one of ordinary skill may used
within the context of the disclosure.
Genetic Screening
[0104] Genetic assay methods useful for the invention include, but
are not limited to, sequencing of the DNA at one or more of the
relevant positions; differential hybridisation of an
oligonucleotide probe designed to hybridise at the relevant
positions of the desired sequence; denaturing gel electrophoresis
following digestion with an appropriate restriction enzyme,
preferably following amplification of the relevant DNA regions; S1
nuclease sequence analysis; non-denaturing gel electrophoresis,
preferably following amplification of the relevant DNA regions;
conventional RFLP (restriction fragment length polymorphism)
assays; selective DNA amplification using oligonucleotides which
are matched for one sequence and unmatched for a polymorphism
thereof or vice versa; or the selective introduction of a
restriction site using a PCR (or similar) primer matched for
alleles of a polymorphic locus, followed by a restriction digest.
As indicated above, the assay may be indirect, i.e. capable of
detecting a polymorphism at another position or gene which is known
to be linked to a polymorphism of the interest. The probes and
primers may be fragments of DNA isolated from nature or may be
synthetic.
[0105] Nucleic acid amplification can be performed using any
technique known in the art such as, but not limited to, polymerase
chain reaction (PCR), strand displacement amplification (SDA),
nucleic acid sequence based amplification (NASBA), rolling circle
amplification (RCA), T7 polymerase mediated amplification, T3
polymerase mediated amplification and SP6 polymerase mediated
amplification.
[0106] In one method, a primer pair are used which hybridise to one
allele but not another, for instance as described for the GH gene
in U.S. Pat. No. 7, 157,231. Whether amplified DNA is produced will
then indicate which allele is present.
[0107] Another method employs similar primer pairs but, as well as
hybridising to only one of the alleles, they introduce a
restriction site which is not otherwise there in any known
allele.
[0108] In an alternative method, following amplification the
products are sequenced. Preferably the products are sequenced
without subcloning such that if two different alleles are present
in the individual being tested their presence can easily be
identified. If the products are subcloned a suitable number of
subclones would need to be sequenced to ensure that both alleles
have been analysed.
[0109] In order to facilitate subsequent cloning of amplified
sequences, primers may have restriction enzyme sites appended to
their 5' ends. Thus, all nucleotides of the oligonucleotide primers
are derived from the gene sequence of interest or sequences
adjacent to that gene except the few nucleotides necessary to form
a restriction enzyme site. Such enzymes and sites are well known in
the art. The primers themselves can be synthesized using techniques
which are well known in the art. Generally, the primers can be made
using synthesizing machines which are commercially available.
[0110] A non-denaturing gel may be used to detect differing lengths
of fragments resulting from digestion with an appropriate
restriction enzyme. The DNA is usually amplified before digestion,
for example using the polymerase chain reaction (PCR) method and
modifications thereof.
[0111] PCR techniques that utilize fluorescent dyes may also be
used to detect the genetic locus of interest. These include, but
are not limited to, the following five techniques.
[0112] i) Fluorescent dyes can be used to detect specific PCR
amplified double stranded DNA product (e.g. ethidium bromide, or
SYBR Green I).
[0113] ii) The 5' nuclease (TaqMan) assay can be used which
utilizes a specially constructed primer whose fluorescence is
quenched until it is released by the nuclease activity of the Taq
DNA polymerase during extension of the PCR product.
[0114] iii) Assays based on Molecular Beacon technology can be used
which rely on a specially constructed oligonucleotide that when
self-hybridized quenches fluorescence (fluorescent dye and quencher
molecule are adjacent). Upon hybridization to a specific amplified
PCR product, fluorescence is increased due to separation of the
quencher from the fluorescent molecule.
[0115] iv) Assays based on Amplifluor (Intergen) technology can be
used which utilize specially prepared primers, where again
fluorescence is quenched due to self-hybridization. In this case,
fluorescence is released during PCR amplification by extension
through the primer sequence, which results in the separation of
fluorescent and quencher molecules.
[0116] v) Assays that rely on an increase in fluorescence resonance
energy transfer can be used which utilize two specially designed
adjacent primers, which have different fluorochromes on their ends.
When these primers anneal to a specific PCR amplified product, the
two fluorochromes are brought together. The excitation of one
fluorochrome results in an increase in fluorescence of the other
fluorochrome. Such assays may also use a ligase so that the two
annealed primers joined together.
[0117] In a particularly useful embodiment, two or more
amplification reactions are performed in a single vessel, known in
the art as multiplexing. For instance, alleles described herein in
the SREBP1, NTSM, M-RIP, TCAP and GH genes can be analysed in a
single reaction.
[0118] The one or more polymorphisms of the SREBP1 gene can be
analysed as described in US 2008/0010696. Furthermore, the one or
more polymorphisms of the GH gene can be analysed as described in
U.S. Pat. No. 7,157,231, however, alternatives such as sequence
specific priming (SSP) can be used.
EXAMPLES
Example 1
Identification and Analysis of Haplotypes
[0119] The cattle genomic region shown in FIGS. 1 and 2 was
interrogated by an entwinement algorithm which revealed a highly
polymorphic locus designated M-RIP. When combined with adjacent
polymorphisms, SREBP1, NT5M, T-CAP and/or GH ancestral haplotypes
were identified.
[0120] Primers useful in the methods of the invention for
amplifying polymorphic regions of the M-RIP, NT5M and TCAP genes,
as well as the SREBP1 and GH genes, are listed in Table 1.
Depending on the individual, amplification products for the NT5M
gene are between about 320 and about 340 bp, for the M-RIP gene
between about 190 and about 250 bp, and for the TCAP gene between
about 230 and about 250 bp.
TABLE-US-00002 TABLE 1 Primers useful for the invention. Gene
Primers SREBP1 CCA CAA CGC CAT CGA GAA ACG CTA C (SEQ ID NO: 7) GGC
CTT CCC TGA CCA CCC AAC TTA G (SEQ ID NO: 8) NT5M GGA AGG CCA GTT
ACA TGG CA (SEQ ID NO: 3) CAC AAC CAA GGC CAA AAT CGC A (SEQ ID NO:
4) M-RIP AGG GGT GCT GAG TCT ACA GG (SEQ ID NO: 1) CTC CAG GAG GCA
GGA GAA G (SEQ ID NO: 2) TCAP AGT ACC AGC TGC CCT ACC A (SEQ ID NO:
5) CTG AGA CAT GGA GCG AGC CA (SEQ ID NO: 6) GH TCT ATG AGA AGC TGA
AGG ACC TGG AGG AA (SEQ ID NO: 9) CGG GGG GTG CCA TCT TCC AG (SEQ
ID NO: 10) CGG GGG GTG CCA TCT TCC AC (SEQ ID NO: 11) ATG ACC CTC
TGG TAC GTC TCC G (SEQ ID NO: 12) ATG ACC CTC TGG TAC GTC TCC A
(SEQ ID NO: 13)
[0121] The specific ancestral haplotypes which were identified are
listed in Tables 2 and 3. These haplotypes have been confirmed by
family analysis as shown in FIG. 3.
TABLE-US-00003 TABLE 2 Ancestral haplotypes - SREBP1 to TCAP 10.1
L-20-10-10 60.1 S-10-60-10 10.2 L-20-10-20 60.2 L-20-60-20 10.3
L-10-10-20 60.3 S-10-60-20 10.4 L-10-10-10 60.4 S-20-60-10 30.1
L-22-30-20 40.1 L-22-40-20 30.2 L-20-30-20 40.2 L-20-40-20 30.3
L-20-30-10 40.3 L-20-40-10 30.1 S-20-30-20 40.4 L-20-40-20 30.5
L-10-30-10 40.5 L-10-40-10 30.6 L-10-30-20 40.6 L-10-40-20 30.7
S-20-30-10 40.7 S-20-40-10 30.8 L-22-30-10 40.8 L-22-40-10 30.9
S-10-30-20 40.9 S-10-40-20
TABLE-US-00004 TABLE 3 allele frequirencies (%) of ancestrial
haplotypes - SREBP1 to TCAP SREB NT5M M-RIP TCAP GH S L 10 20 22 10
30 40 60 10 20 A B C n Jersey 5 96 23 64 14 36 23 23 18 0 100 60*
40* 0* 11 6* Simm 7 93 11 88 2 0 43 50 7 22 78 77 23 0 274 Angus 1
99 13 71 16 0 51 42 7 28 72 87 14 0 52 Wagyu 26 74 20 69 11 13 44
17 26 59 41 67 20 13 27
[0122] With regard to Table 2, the numbers 10.1, 60.1 etc
correspond to specific haplotypes. The L and S designations
correspond to the same nomenclature used in US 2008/0010696. The
numbers next to L or S correspond to specific amplification
products from the NT5M gene. The next numbers correspond to
specific amplification products from the M-RIP gene. The next
numbers in the series (for example L-20-10-10) correspond to
specific amplification products from the TCAP gene. Finally, when
present the next character in the series (for example, A, B, C or
D) corresponds to specific amplification products from the GH
gene.
[0123] SREBP1 marker, bands at 343-346 by and 428-432 by represent
short (S) and long (L), respectively. NT5M marker, bands at 321 bp,
331 by and 334 by are designated 10, 20 and 22 respectively. M-RIP,
bands at 192-193 bp, 201-203 bp, 208-209 bp, 210-212 bp, 218-220
bp, 226-227 by and 231 are designated 10, 30, 40, 50, 60, 80 and 90
respectively. When analysing genotypes at the GH loci a fourth
haplotype was identified which is not described in U.S. Pat. No.
7,157,231. This haplotype was designated D (Table 4). Guidance as
to the relative size of each amplification product for the M-RIP
gene is provided in FIG. 4. Generally, the lower the number the
smaller the amplification product.
TABLE-US-00005 TABLE 4 Genotypes of GHex5. Genotype 127 172 A CTG
ACG B GTG ACG C GTG ATG D CTG ATG
[0124] A total of 41 different haplotypes were observed for the 5
genes analysed (Table 5 and FIG. 5). Table 5 does not include the 6
haplotypes that occurred only once in the 298 cattle (152
[0125] Simmental, 31 Wagyu and 16 Angus) analysed. Two haplotypes
are common in around 20% of the cattle tested. 35 of the haplotypes
show different frequencies in different breeds. Analysis of SREBP1,
NT5M, M-RIP, T-CAP and GH was found to be more informative than
SREBP1 and GH alone. The frequency of different haplotypes in
Simmental cattle is summarized in Table 6.
TABLE-US-00006 TABLE 5 Combined frequirencies of observed
haplotypes. SREB NT5M M-RIP TCAP GH Freq. % L 20 30 20 A 18.6* L 20
40 20 A 14.7* L 20 30 10 A 9.7 L 22 40 20 A 7.4 S 10 60 10 B 7.1 L
20 30 20 B 5.0 L 20 40 10 A 4.1 L 22 40 20 C 3.5 L 20 40 20 B 3.3 L
20 10 10 A 2.9 S 10 60 10 A 2.9 L 22 40 20 B 2.6 S 20 30 20 A 2.1 L
20 60 20 A 1.6 L 22 40 10 A 1.5 L 20 40 10 A 1.5 L 20 40 20 C 1.5 L
22 40 10 B 1.4 L 20 30 10 B 1.2 L 20 10 10 C 1.0 L 20 30 10 C 1.0 L
10 30 10 A 0.9 L 20 30 20 C 0.7 L 22 30 20 C 0.7 L 10 40 20 B 0.7 S
20 40 10 B 0.7 S 10 60 20 A 0.5 S 10 60 20 B 0.4 S 10 30 20 A 0.3 S
20 40 20 B 0.3 L 10 40 10 A 0.3 L 10 40 10 B 0.2 S 20 40 20 A 0.2 S
20 30 20 B 0.1 S 22 60 20 A 0.1
TABLE-US-00007 TABLE 6 Simmental haplotype frequencies. Haplotype
ID SREBP NT5M M-RIP T-CAP GH Frequency 30.2.3 L 20 30 20 A 22.7.8%
30.2.2 L 20 30 20 B 7.8% 30.3.3 L 20 30 10 A 6.8% 30.3.2 L 20 30 10
B 1.7% 30.5.3 L 10 30 10 A 3.4% 30.6.3 L 10 30 20 A 0.4% 30.9.3 S
10 30 20 A 0.8% 30.4.2 S 20 30 20 B 0.4% 40.2.3 L 20 40 20 A 28.2%
40.2.2 L 20 40 20 B 9.1% 40.3.3 L 20 40 10 A 7.2% 40.1.3 L 22 40 20
A 0.8% 40.4.2 S 20 40 20 B 1.1% 40.5.3 L 10 40 10 A 1.1% 40.5.2 L
10 40 10 B 0.8% 60.2.3 L 20 60 20 A 2.3% 60.3.2 S 10 60 20 B 1.3%
60.3.3 S 10 60 20 A 1.9% 60.7.3 S 22 60 20 A 0.4%
Example 2
Haplotypes and Production Traits in Wagyu
[0126] The Japanese Black cattle breed "Wagyu" is the industry
bench mark for high marbling (intramuscular fat) and thus taste.
Within Wagyu phenotypic traits were assessed on carcasses from the
Australian Cattle Company (AACo). Statistical analysis of data
obtained from approximately 67 Wagyu showed significant, or near
significant, correlation signals with combinations of NT5M to GH
alleles in the SREBP-GH region on bovine Chromosome 19.
[0127] The SREB locus alleles (S/L) were found not to be relevant
in this context as Wagyu animals usually have the characteristic S
allele and are all high marbling.
[0128] The hot dead carcass weight (HDCW) is associated as expected
with alleles in the growth hormone locus (GH)--positive for the GH
C allele and negative for the B allele. When additionally looking
at TCAP alleles further useful information could be obtained from
the genetic analysis, with the TCAP 10 and 20 alleles segregating
with low carcass weight for the 20-B haplotype and improved carcass
weight for the 10-B fragment (FIG. 6). In contrast the 20-C
haplotype shows a 16 Kg carcass weight improvement in this analysis
over the 20-B haplotype.
[0129] Of particular interest are the significant correlation
signals for Eye Muscle Area (EMA) a key indicator of tenderness and
meat quality. This is associated with certain alleles at loci
extending from NT5M to GH (FIG. 7). In particular, a positive
association with EMA was noted with haplotype (NT5M-MRIP-TCAP-GH)
22-40-20 B/C and to a lesser extent with of 10-30-20 A,
10-40-20-B/C. Negative associations with EMA in this analysis noted
particularly with haplotypes 20-30-20-A and 20-40-20-A, and to a
lesser extend with 22-40-10-B/C.
[0130] For MRIP-TCAP-GH haplotypes and Marbling Score, there are
multiple contributions across the haplotype for
marbling--positively for 30-10-B and 40-20-A haplotypic fragments
and negatively for 40-10-B, 40-10-A and 30-10-C (FIG. 8).
[0131] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
[0132] The present application claims priority from AU 2009904511
filed 17 Sep. 2009, the entire contents of which are incorporated
herein by reference.
[0133] All publications discussed and/or referenced herein are
incorporated herein in their entirety.
[0134] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is solely for the purpose of providing a context for
the present invention. It is not to be taken as an admission that
any or all of these matters form part of the prior art base or were
common general knowledge in the field relevant to the present
invention as it existed before the priority date of each claim of
this application.
REFERENCES
[0135] Hoashi et al. (2007) Mammalian Genome 18:880-886. [0136]
Jiang et al. (2008) International Journal of Biological Sciences
4:345-351. [0137] Rincker et al. (2006) Journal of Animal Science
84:686-693. [0138] Van Eenennaam et al. (2007) Journal of Animal
Science 85:891-900.
Sequence CWU 1
1
13120DNAArtificial SequenceOligonucleotide primer 1aggggtgctg
agtctacagg 20219DNAArtificial SequenceOligonucleotide primer
2ctccaggagg caggagaag 19320DNAArtificial SequenceOligonucleotide
primer 3ggaaggccag ttacatggca 20422DNAArtificial
SequenceOligonucleotide primer 4cacaaccaag gccaaaatcg ca
22519DNAArtificial SequenceOligonucleotide primer 5agtaccagct
gccctacca 19620DNAArtificial SequenceOligonucleotide primer
6ctgagacatg gagcgagcca 20725DNAArtificial SequenceOligonucleotide
primer 7ccacaacgcc atcgagaaac gctac 25825DNAArtificial
SequenceOligonucleotide primer 8ggccttccct gaccacccaa cttag
25929DNAArtificial SequenceOligonucleotide primer 9tctatgagaa
gctgaaggac ctggaggaa 291020DNAArtificial SequenceOligonucleotide
primer 10cggggggtgc catcttccag 201120DNAArtificial
SequenceOligonucleotide primer 11cggggggtgc catcttccac
201222DNAArtificial SequenceOligonucleotide primer 12atgaccctct
ggtacgtctc cg 221323DNAArtificial SequenceOligonucleotide primer
13catgaccctc tggtacgtct cca 23
* * * * *