U.S. patent application number 11/686707 was filed with the patent office on 2007-10-04 for detection of lethality gene for improved fertility in mammals.
Invention is credited to Hasan Khatib.
Application Number | 20070234437 11/686707 |
Document ID | / |
Family ID | 38523176 |
Filed Date | 2007-10-04 |
United States Patent
Application |
20070234437 |
Kind Code |
A1 |
Khatib; Hasan |
October 4, 2007 |
Detection of Lethality Gene for Improved Fertility in Mammals
Abstract
Oligonucleic acid molecules comprising a SNP site at a position
corresponding to position 7480 of the bovine signal transducer and
activator of transcription (STAT5A) coding sequence (SEQ ID NO: 1).
Also disclosed are an array or a kit comprising the same, a method
for detecting the SNPs, a method for progeny testing of mammals, a
method for increasing human and non-human mammal pregnancy rate in
natural and artificial reproduction processes. Further provided are
cattle breeding methods for improved milk production traits.
Inventors: |
Khatib; Hasan; (Madison,
WI) |
Correspondence
Address: |
BAKER DONELSON BEARMAN CALDWELL & BERKOWITZ, PC
555 11TH STREET, NW
6TH FLOOR
WASHINGTON
DC
20004
US
|
Family ID: |
38523176 |
Appl. No.: |
11/686707 |
Filed: |
March 15, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60782539 |
Mar 16, 2006 |
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Current U.S.
Class: |
800/14 ;
435/6.11; 435/6.12; 800/15 |
Current CPC
Class: |
A61D 19/02 20130101;
C12Q 2600/156 20130101; C12Q 1/6888 20130101; C12Q 1/6883 20130101;
C12Q 2600/124 20130101; A61D 19/04 20130101; C12Q 2600/158
20130101; A01K 2227/101 20130101 |
Class at
Publication: |
800/014 ;
800/015; 435/006 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. An isolated single or double stranded oligonucleotide molecule
comprising a polymorphic site at a position corresponding to
position 7480 of exon 8 of bovine Signal Transducer and Activator
5A (STAT5A) coding sequence (SEQ ID NO: 1), wherein position 7480
is cytosine or guanine, and at least about 9 contiguous nucleotides
of SEQ ID NO: 1 adjacent to the polymorphic site.
2. The oligonucleotide molecule according to claim 1, which
comprises at least about 15 contiguous nucleotides adjacent to the
polymorphic site.
3. The oligonucleotide molecule according to claim 2, which
comprises at least about 20 contiguous nucleotides adjacent to the
polymorphic site.
4. The ioligonucleotide molecule according to claim 1, which
comprises not more than about 150 nucleotides.
5. The oligonucleotide molecule according to claim 1, which
comprises not more than about 100 nucleotides.
6. The oligonucleotide molecule according to claim 1, which
comprises not more than about 50 nucleotides.
7. The oligonucleotide molecule according to claim 1, wherein the
polymorphic site is within 4 nucleotides of the center of the
oligonucleotide molecule.
8. The oligonucleotide molecule according to claim 7, wherein the
polymorphic site is at the center of the oligonucleotide
molecule.
9. The oligonucleotide molecule according to claim 1, wherein the
polymorphic site is at the 3'-end of the oligonucleotide
molecule.
10. An array of nucleic acid molecules comprising the isolated
oligonucleotide molecules according to claim 1 supported on a
substrate.
11. The array according to claim 10, further comprising one or more
markers in linkage disequilibrium with the polymorphic site.
12. A kit comprising a nucleic acid molecule of claim 1, and a
suitable container.
13. A method for detecting single nucleotide polymorphism (SNP) on
STAT5A coding region in an animal cell, the method comprising
determining the identity of a nucleotide at a position
corresponding to position 7480 of exon 8 of bovine STAT5A coding
sequence (SEQ ID NO: 1) of the cell.
14. The method according to claim 13, wherein the animal is a
mammalian, avian or aquatic species.
15. The method according to claim 13, wherein the animal cell is an
adult cell, an embryo cell, a sperm, an egg, a fertilized egg, or a
zygote.
16. A method according to claim 14, wherein the animal is a
mammal.
17. A method according to claim 13, wherein the mammal is
bovine.
18. A method according to claim 13, wherein the identity of the
nucleotide is determined by sequencing a nucleic acid molecule
comprising a position corresponding to position 7480 of exon 8 of
bovine STAT5A coding sequence, or a relevant fragment thereof,
isolated from the cell.
19. A method according to claim 18, wherein the nucleic acid
molecule is isolated from the cell via amplification by the
polymerase chain reaction (PCR) of genomic DNA of the cell, or by
RT-PCR of the mRNA of the cell.
20. A method according to claim 13, wherein the identity of the
nucleotide is determined by hybridizing a suitable probe to a
preparation comprising the nucleic acid from the cell.
21. A method according to claim 20, wherein the probe is labeled
with a detectable label.
22. A method according to claim 13, wherein the identity of the
nucleotide is determined by an invasive signal amplification
assay.
23. A method according to claim 13, wherein the identity of the
nucleotide of both copies of the coding sequence of the cell is
determined.
24. A method according to claim 12, wherein the identity of the
nucleotide of both copies of the sequence is determined based on
genotypes of the parent of the cell, genotypes of the daughter of
the cell, or both.
25. A method for determining whether an individual animal is
suitable as a gamete donor for natural mating, artificial
insemination or in vitro fertilization, the method comprising
determining allele identify of the SNP site according to claim 13,
or of an allele in linkage disequilibrium with the SNP site, and
selecting as gamete donor an individual whose genotype is
homozygous with regard to C allele at the SNP site, or homozygous
with regard to an allele in linkage disequilibrium with the C
allele.
26. The method according to claim 25, wherein the animal is
selected from the group consisting of cattle, swine, equine, dog,
sheep and goat.
27. The method according to claim 25, wherein the animal is
human.
28. A method of selecting an embryo for planting in a uterus, the
method comprising determining identify of the nucleotide at a
position corresponding to position 7480 of bovine STAT5A (SEQ ID
NO: 1) of the embryo while preserving the viability of the embryo,
and selecting for planting only an embryo whose genotype is CC
homozygous at the position.
29. The method according to claim 28, wherein the animal is an
animal selected from the group consisting of cattle, swine, equine,
dog, sheep and goat.
30. The method according to claim 29, wherein multiple ovulation
and embryo transfer (MOET) is used to generate multiple fertilized
eggs.
31. The method according to claim 28, wherein the animal is
human.
32. A method for increasing successful pregnancy rate of a
non-human animal, comprising selecting a male or a female mammal
for breeding purposes that are CC homozygous at a position
correspond to position 7480 of exon 8 of bovine STAT5A gene (SEQ ID
NO: 1).
33. A method according to claim 32, wherein both male and female
parents are CC homozygous.
34. The method according to claim 33, wherein the female mammal is
in vitro fertilized.
35. A method for increasing pregnancy rate and reducing multiple
pregnancy rate in a human ART procedure, the method comprising
genotyping, via pre-implantation genetic diagnosis, the genotype of
embryos to be planted with regard to the nucleotide corresponding
to position 7480 of exon 8 of bovine STAT5A gene (SEQ ID NO: 1),
and planting not more than 3 embryos which are homozygous CC with
regard to the position.
36. A method for determining whether an individual dairy cattle is
suitable as a gamete donor for natural mating, artificial
insemination or in vitro fertilization, the method comprising
determining allele identify of the SNP site according to claim 13,
or of an allele in linkage disequilibrium with the SNP site, and
selecting as gamete donor an individual whose genotype is
heterozygous at the SNP site, or heterozygous with regard to a
locus in linkage disequilibrium with the SNP site.
37. The method according to claim 36, wherein an individual having
CC genotype at the SNP site is selected to mate with an individual
with a CG genotype.
38. The method according to claim 37, wherein gametes from the CG
individual and the CC individual are used in artificial
insemination, or in in vitro fertilization, or in multiple
ovulation and embryo transfer procedure.
39. A method of selecting a dairy cattle embryo for planting in a
uterus, the method comprising determining identify of the
nucleotide at a position corresponding to position 7480 of bovine
STAT5A (SEQ ID NO: 1) of the embryo while preserving the viability
of the embryo, and selecting for planting only an embryo whose
genotype is CG heterozygous at the position.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods and compositions
for detecting a lethality allele in an animal, especially a mammal,
and for improving fertility or increasing reproductive performance
of the animal. The present invention further relates to methods and
compositions for improving milk production of dairy cattle.
BACKGROUND OF THE INVENTION
[0002] It is highly desirable in many contexts that reproductive
performance in mammals be improved or enhanced. For example, in
farm animals, increased pregnancy rate and/or increased numbers of
live offspring often would increase profitability. In
meat-producing animals, increased litter size and birth or hatching
rates improve the overall efficiency and profitability of a farm
operation. Embryonic survival is directly relevant in avian species
to improved hatching rates and for aquatic species to improved
survival rate per spawn. Swine litter size would be positively
influenced by the elimination of conditions that are lethal for
developing embryos. Improved survival spreads the hatchery or
piggery cost over a larger number of offspring, for example day-old
chicks, post larvae shrimp, or piglets, and thus reduces the unit
cost of production.
[0003] In milk producing animals, aside from the inherent value of
young animals, periodic pregnancy and the resultant early lactation
period are necessary or desirable for the animal to have steady and
high milk yield. Tremendous efforts, such as systematic animal
breeding programs and artificial insemination, have been and
continue to be invested in ensuring that the animals have high and
sustained productivity, and that the milk produced is of high
quality or has desired composition.
[0004] While modern cattle breeding technologies have increased
consistency of herd quality or performance and generally achieved
increased milk yield, many studies have reported a decrease in
fertility in dairy cows. Cows with the highest milk production have
the lowest fertility performance. For example, it is well known
that infertility is the major reason for culling cows, and it is
estimated that in the UK alone, over 17,000 cows are culled every
year due to infertility or reproductive failure (Genus Breeding,
UK). Epidemiological studies suggest that, in addition to milk
production, other factors such as increasing levels of inbreeding
are probably decreasing reproductive efficiency in the dairy herd.
The first-service conception rate declined approximately from 65%
in 1951 to 40% in 1996 (Lucy, 2001, Reproductive loss in
high-producing dairy cattle: where will it end? J Dairy Sci.
84:1277-93.). A large number of "normal embryos" in dairy cattle
are found to undergo early embryonic death, but there is currently
no explanation for such early embryo death. Reducing embryonic loss
and achieving high rates of conception in dairy cattle would change
the way we manage the lactation cycle (Lucy, 2001, supra).
[0005] The conception rate for cattle in the U.S. at first
artificial insemination (AI) has also been decreasing for many
years, and according to one report it decreased by 0.45% per year
over a 20-year period (Butler and Smith, 1989, J. Dairy Sci.
72:767-83.). There was an increase in the number of AIs required
for conception from 1.75 to more than 3 over a period of 20 years
(Lucy, 2001, supra). Conception rates in large commercial herds
stand at only 35-40% for mature cows. A similar need for improved
reproductive performance exists with regard to many other farm
animals such as swine, equine, sheep and goat.
[0006] In humans, infertility or low fertility plagues a
significant portion of the population. It has been reported that in
Western countries, about 10-15% of couples experience some
difficulty with fertility (Evers, 2002, Female sub-fertility.
Lancet 2:151-159.) Many couples suffering from impaired
reproductive ability go to great effort and expense to successfully
give birth to a child. The economic and emotional costs of
embryonic mortality are significant, and a better understanding of
its causes and improved methods for managing it are needed.
[0007] Infertility or low reproductive performance in animals,
however, is presently poorly understood although it is known that
there are many contributing factors, both genetic and
environmental. It is nevertheless readily recognized that a genetic
factor that causes the death of the embryo will be a major
factor.
[0008] Lethal genes have been suggested as a cause of embryonic
death and, if present, could cause failures in recurrent
inseminations. However, lethal genetic factors or lethality genes
if dominant, cannot survive in a population. Consequently, little
is known about these lethal genetic factors. Identification and
characterization of lethality genes would allow animal breeders,
farmers and doctors to better understand low fertility, selectively
improve the chances of success in animal breeding, develop
strategic plans for improved fertility based on the genetics of
parents and help eliminate these lethal factors from the population
and improve overall reproductive performance in mammals.
[0009] As natural selection favors survival and reproduction of the
more advantageous variants and elimination of the less advantageous
variants, and an allele that confers lethality, even though
recessive, generally decreases reproductive fitness of the
individual carrying the lethality alleles. These recessive lethal
alleles will eventually disappear from the population, unless it is
otherwise selected for. Unnatural prevalence of a lethal allele,
that is, at a frequency higher than predicted, indicates that it is
favored by the condition under which the population is selected or
propagated. Thus, if an allele is recessively lethal
(reproductively disadvantaged), yet confers certain desirable
production traits (for example, in the case of dairy cattle, milk
yield or milk quality), this allele may be favored by breeding
programs and persist in incidences higher than expected under
natural selection conditions, even though its identity or
phenotypic characteristics are not known. Insight about the exact
nature of the phenotypic characteristics of recessive lethal
alleles will be invaluable in assisting animal breeders in
balancing reproductive performance with the animal's productive
traits, and in achieving optimal economic outcome.
SUMMARY OF THE INVENTION
[0010] It has now been surprisingly discovered that a single
nucleotide polymorphism (SNP) in the STAT5 gene is responsible for
early embryo death in many animals including mammals, especially
dairy cattle. Embryos homozygous with regard to a form of this SNP
die at very early stages. This is believed to be the first reported
gene associated with lethality at an early developmental stage in
mammals. It is further discovered that in dairy cattle, an
individual heterozygous with regard to this allele produces higher
milk yield, as well as higher milk fat content and protein content
when compared to individuals homozygous of the non-lethal
allele.
[0011] In one embodiment, the present invention provides an
isolated single or double stranded nucleic acid molecule comprising
a polymorphic site at a position corresponding to position 7480 of
exon 8 of the bovine Signal Transducer and Activator 5A (STAT5A)
coding sequence (SEQ ID NO: 1), wherein position 7480 is either
cytosine (the C allele) or guanine (the G allele), and at least
about 9 contiguous nucleotides of SEQ ID NO: 1 adjacent to the
polymorphic site. Preferably, the nucleic acid molecule comprises
at least about 10, or at least about 15, or at least about 20
contiguous nucleotides adjacent to the polymorphic site. The
isolated nucleic acid molecule of the present invention in certain
circumstances preferably comprises not more than about 150
nucleotides, or not more than about 100 nucleotides, or not more
than about 50 nucleotides.
[0012] In a preferred embodiment, the polymorphic site is within 4
nucleotides of the center of the nucleic acid molecule according to
the present invention, or at the center of the nucleic acid
molecule, or is at the 3'-end of the nucleic acid molecule.
[0013] In another embodiment, the present invention provides an
array of nucleic acid molecules comprising the above isolated
nucleic acid molecule supported on a substrate. The array may
further comprise one or more markers in linkage disequilibrium with
the polymorphic site. In another embodiment, the present invention
provides a kit comprising a nucleic acid molecule described above,
and a suitable container.
[0014] In another embodiment, the present invention provides a
method for detecting single nucleotide polymorphism (SNP) on STAT5A
coding region in an animal cell, the method comprising determining
the identity of a nucleotide at a position corresponding to
position 7480 of exon 8 of bovine STAT5A coding sequence (SEQ ID
NO: 1) of the cell. The animal may be a mammalian, avian or aquatic
species, and the animal cell may be an adult cell, an embryo cell,
a sperm, an egg, a fertilized egg, or a zygote. Preferably, the
mammal is bovine.
[0015] In a preferred embodiment, the identity of the nucleotide is
determined by sequencing nucleic acid molecule, or a relevant
fragment thereof, isolated from the cell. The nucleic acid molecule
may be isolated from the cell via amplification by the polymerase
chain reaction (PCR) of genomic DNA of the cell, or by RT-PCR of
the mRNA of the cell.
[0016] In another preferred embodiment, the identity of the
nucleotide is determined by hybridizing a suitable probe to a
nucleic acid preparation from the cell, wherein the probe is
preferably labeled with a detectable label.
[0017] In a further embodiment, the identity of the nucleotide is
determined by an invasive signal amplification assay.
[0018] Preferably, the sequence of both copies of the polymorphic
genetic locus in the cell is determined.
[0019] In another embodiment, the present invention provides a
method wherein the identity of the SNP site in the cell is
determined based on the genotypes of the parents, the genotypes of
a daughter, or both.
[0020] In another embodiment, the present invention provides a
method for determining whether an individual animal is suitable as
a gamete donor for natural mating, artificial insemination or in
vitro fertilization procedure, the method comprising determining
the allele identity of the SNP site according to the present
invention, or of an allele in linkage disequilibrium with the SNP
site, and selecting as a gamete donor only an individual whose
genotype is homozygous with regard to the C allele at the SNP site,
or homozygous with regard to an allele in linkage disequilibrium
with the C allele. Preferably, the animal is selected from the
group consisting of cattle, swine, equine, dog, sheep and goat.
[0021] In another embodiment, the present invention provides a
method of selecting an embryo for planting in a uterus, the method
comprising determining identify of the nucleotide at a position
corresponding to position 7480 of STAT5A (SEQ ID NO: 1) of the
embryo while preserving the viability of the embryo, and selecting
for planting only an embryo whose genotype is CC homozygous at the
position. Preferably, multiple ovulation and embryo transfer (MOET)
is used to generate multiple fertilized eggs.
[0022] In another embodiment, the present invention provides a
method for increasing successful pregnancy rate of a non-human
animal, comprising selecting a male or a female mammal for breeding
purposes that are CC homozygous at a position correspond to
position 7480 of exon 8 of bovine STAT5A gene (SEQ ID NO: 1).
Preferably, both the male and female parents are selected to be CC
homozygous. The female mammal may be in vitro fertilized.
[0023] In another embodiment, the present invention provides a
method for increasing pregnancy rate and reducing multiple
pregnancy rate in a human assisted reproductive technologies (ART)
procedure, the method comprising genotyping, via pre-implantation
genetic diagnosis, the genotype of embryos to be planted with
regard to the nucleotide corresponding to position 7480 of exon 8
of bovine STAT5A gene (SEQ ID NO: 1), and planting not more than 3
embryos which are homozygous CC with regard to the position.
[0024] In yet another embodiment, the present invention provides a
method for determining whether an individual dairy cattle is
suitable as a gamete donor for a natural mating, artificial
insemination or in vitro fertilization procedure, the method
comprising determining allele identify of the SNP site according to
the present invention, or of an allele in linkage disequilibrium
with the SNP site, and selecting as gamete donor an individual
whose genotype is heterozygous at the SNP site, or heterozygous
with regard to a locus in linkage disequilibrium with the C allele.
Preferably, an individual having CC genotype at the SNP site is
selected to mate with an individual with a CG genotype. Still more
preferably, gametes from the CG individual and the CC individual
are used in artificial insemination, or in in vitro fertilization,
or in multiple ovulation and embryo transfer procedure.
[0025] In another embodiment, the present invention provides a
method of selecting a dairy cattle embryo for planting in a uterus,
the method comprising determining the identity of the nucleotide at
a position corresponding to position 7480 of bovine STAT5A (SEQ ID
NO: 1) of the embryo while preserving the viability of the embryo,
and selecting for planting only an embryo whose genotype is CG
heterozygous at the position.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows the partial sequence of the coding region for
exons 5-19 of STAT5A (accession no. AJ237937) (SEQ ID NO: 3). The
two PCR primers used to amplify the fragment (bold) for initial SNP
identification are underlined, and the SNP position of 7480 is
shaded. Positions are as originally labeled in the GenBank.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The Signal Transducer and Activator (STAT) proteins are
known to play an important role in cytokine signaling pathways. The
proteins are transcription factors that are specifically activated
to regulate gene transcription when cells encounter cytokines and
growth factors, hence they act as signal transducers in the
cytoplasm and transcription activators in the nucleus (Kisseleva et
al., Signaling through the JAK/STAT pathway, recent advances and
future challenges. Gene 285: 1-24 (2002)). Binding of factors such
as cytokines and growth factors to cell-surface receptors leads to
receptor autophosphorylation at a tyrosine, the phosphotyrosine
being recognized by the STAT SH2 domain, which mediates the
recruitment of STAT proteins from the cytosol and their association
with the activated receptor. The STAT proteins are then activated
by phosphorylation via members of the JAK family of protein
kinases, causing them to dimerize and translocate to the nucleus,
where they bind to specific promoter sequences in target genes. In
mammals, STATs comprise a family of seven structurally and
functionally related proteins: Stat1, Stat2, Stat3, Stat4, Stat5a,
Stat5b, and Stat6.
[0028] Signaling through the JAK/STAT pathway is initiated when a
cytokine binds to its corresponding receptor. This leads to
conformational changes in the cytoplasmic portion of the receptor,
initiating activation of receptor associated members of the JAK
family of kinases. The JAKs, in turn, mediate phosphorylation at
the specific receptor tyrosine residues, which then serve as
docking sites for STATs and other signaling molecules. Once
recruited to the receptor, STATs also become phosphorylated by
JAKs, on a single tyrosine residue. Activated STATs dissociate from
the receptor, dimerize, translocate to the nucleus and bind to
members of the GAS (gamma activated site) family of enhancers.
[0029] The seven STAT proteins identified in mammals range in size
from 750 and 850 amino acids. The chromosomal distribution of these
STATs, as well as the identification of STATs in more primitive
eukaryotes, suggest that this family arose from a single primordial
gene. STATs share structurally and functionally conserved domains
(see e.g. Chen et al., Crystal structure of a tyrosine
phosphorylated STAT-1 dimer bound to DNA. Cell 93: 827-839
(1998)).
[0030] The STAT5A protein is also known as the mammary gland factor
(MGF). MGF knockout female mice failed to lactate. The encoding
genomic region is about 19,517 bp long, and has 19 exons. The
bovine sequence is known and is publicly available in the GenBank
(accession number AJ242522 for exons 1.about.4 and AJ237937 for
exons 5.about.19. The protein was initially identified in the
mammary gland as a prolactin-induced transcription factor. STAT5A
is a member of the IFN-tau and placental lactogen (PL) signaling
pathway. The uterus is exposed to IFN-tau, PL, as well as others
hormones including estrogen, progesterone, and placental growth
hormone. Mediated by prolactin receptor (PRLR) homodimers, and
perhaps by PRLR and growth hormone receptor (GHR) heterodimers, PL
stimulates the formation of STATS homodimers, which in turn induce
the transcription of bovine uterine milk protein (UTMP) and
osteopontin (OPN) genes (see e.g. Spencer T. E. and Bazer F. W.
2002. Biology of progesterone action during pregnancy recognition
and maintenance of pregnancy. Front. Biosci. 1, d1879-98; Stewart
M. D., Choi Y., Johnson G. A., Yu-Lee L.Y. et al. 2002. Roles of
Stat1, Stat2, and interferon regulatory factor-9 (IRF-9) in
interferon tau regulation of IRF-1. Biol Reprod. 66, 393-400;
Spencer T. E. and Bazer F. W. 2004. Conceptus signals for
establishment and maintenance of pregnancy. Reprod Biol Endocrinol.
2, 49). The UTMP gene is known to affect milk production traits in
cattle. The OPN protein was first described in 1979 as a protein
associated with malignant transformation, and has been intensively
studied in human, mouse, and sheep. It has been suggested that
human OPN has various roles in cell adhesion, chemotaxis, cell
survival, tissue remodeling, regulation of inflammation, fetal
growth and development, and in initiating and maintaining pregnancy
(Denhardt et al. 2001, Osteopontin as a means to cope with
environmental insults: regulation of inflammation, tissue
remodeling, and cell survival. J Clin Invest. 107:1055-1061;
Johnson et al., 2003. Osteopontin: roles in implantation and
placentation. Biol Reprod. 69:1458-1471).
[0031] STAT5A and STAT5B from the same species share about 96%
sequence similarity at the amino acid level. The sequence homology
among STAT5 proteins from different animal species is high, as
shown in Table 1, which summarizes the sequence similarity of STATS
proteins among various species when compared to the bovine
sequence. Table 1 also makes clear that the sequence homology among
mammalian species is especially high. TABLE-US-00001 TABLE 1
Sequence similarity among STAT5A proteins Sequence similarity to
Bovine Sequence Species Accession No. CAB52173 (%) Canine 97 Sus
Scrofa 96 Human 96 Mouse 96 Rattus 95 Ovies aries 94 Gallus gallus
90 Danio rerio 79 Takifugo rubripes 79 Xenopus laevis 87
[0032] The present inventor has identified a single nucleotide
polymorphism (SNP) in STAT5A that is associated with early embryo
death in animals. The term "polymorphism" as used herein refers to
the occurrence of two or more alternative genomic sequences or
alleles between or among different genomes or individuals.
"Polymorphic" refers to the condition in which two or more variants
of a specific genomic sequence can be found in a population. A
"polymorphic site" is the locus at which the variation occurs. A
polymorphic site generally has at least two alleles, each occurring
at a significant frequency in a selected population. A polymorphic
locus may be as small as one base pair, in which case it is
referred to as single nucleotide polymorphism (SNP). The first
identified allelic form is arbitrarily designated as the reference
form, and other allelic forms are designated as alternative or
variant alleles. The allelic form occurring most frequently in a
selected population is sometimes referred to as the wild type form.
Diploid organisms may be homozygous or heterozygous for an allelic
form. A biallelic polymorphism has two forms, and a triallelic
polymorphism has three forms, and so on.
[0033] Polymorphisms may provide functional differences in the
genetic sequence, through changes in the encoded polypeptide,
changes in mRNA stability, binding of transcriptional and
translation factors to the DNA or RNA, and the like. Polymorphisms
are also used to detect genetic linkage to phenotypic
variation.
[0034] SNPs have gained wide use for the detection of genetic
linkage recently. SNPs are generally biallelic systems, that is,
there are two alleles that an individual may have for any
particular SNP marker.
[0035] The SNP associated with early embryo death according to the
present invention is located on STAT5A, at a position corresponding
to position 7480 on exon 8 of the bovine sequence (GenBank
accession number AJ237937) (see FIG. 1). It has been discovered
that at this position, the predominant allele is cytosine (C), and
the other allele is guanine (G), and that homozygote GG genotype is
lethal and does not exist in the population.
[0036] It is well-known to those ordinarily skilled in the art that
the STAT5A genes in all animal species are derived from a common
ancestor, which is reflected in the high DNA sequence similarity of
the gene among these species. The nucleotide sequences of different
animal species can be easily aligned, using widely available
sequence comparison/alignment tools (e.g. Altschul, et al., 1990,
"Basic local alignment search tool." J. Mol. Biol. 215:403-410),
allowing maximum number of identical nucleotides on sequences from
different animals to be positioned and correspond to each other.
Similar alignment can be done based on the amino acid sequences
encoded by the nucleic acid sequence. In many instances,
appropriate gaps and insertions, determined by widely accepted
computer algorithm, are introduced to allow for genetic deletions
or insertions that are believed to have occurred during the
evolutionary history of the genes. Accordingly, by a
"corresponding" nucleotide position, as used in the present
invention, is meant a nucleotide position that is identified using
the nucleotide sequence alignment methodologies well-known in the
art, based on a reference sequence, e.g. the bovine STAT5A
sequence.
[0037] Using two PCR primers, STAT7 and STAT8, the inventor
amplified and sequenced a 712-bp fragment from more than 2100
bovine samples from different cattle breeds (Holstein, Jersey,
Brown Swiss, Bison bison, and Bos indictis). STAT7 is located on
exon 8, and STAT8 is located on exon 9. The sequences of the two
primers are: STAT7: 5'-GAGAAGTTGGCGGAGATTATC-3' (SEQ ID NO:1) and
STAT8: 5'-GTGTTCTCGTTCTTGAGCAG-3' (SEQ ID NO: 2). It was found that
at the SNP position (7480), the predominant allele was cytosine
(C), and the other allele was guanine (G), with about 70% CC
homozygote, and about 30% CG heterozygote. No cattle GG homozygote
was found. This lead to the conclusion that the GG homozygote is
lethal and does not exist in the population.
[0038] The samples include semen samples from more than 1200 bulls
and 1100 blood samples from Holstein cows. The 1100 blood samples
were obtained from a University of Wisconsin (UW) daughter design
resource population, consisting of 12 sire families. The sires used
to create this population were chosen from a large number of
candidate bulls with large numbers of daughters in production in
the year 2000. Criteria for the final selection of the 12 bulls
included large numbers of daughters in production, in total and
separately in lactations 1, 2 and 3, and relatively low pedigree
relationships among the chosen bulls in order to more broadly
sample the chromosomes of the U.S. Holstein population. The 1200
semen samples were obtained from 30 half-sib families with a
granddaughter design. Genomic DNA was extracted from semen samples
by standard methods using proteinase K and phenol/chloroform
extraction, and from blood samples using GFX Genomic Blood DNA
Purification Kit (Amersham Biosciences, Piscataway, N.J.). The DNA
concentration was measured using a spectrophotometer (Ultraspec
2100; Amersham Biosciences).
[0039] Cattle fetuses at 55-125 days were similarly genotyped and
none was found to be GG homozygous, supporting the correlation
between the GG homozygote and lethality. Fetuses were obtained from
a local slaughterhouse. DNA was extracted from fetal tissues and
genotyped with STATS gene.
[0040] Similar results were obtained from sheep samples. Two
half-sib sheep families were genotyped, where the two sires were
heterozygous. All genotyped offspring (n=60) were either GC or
CC.
[0041] Based on the results above, it was concluded that death of
GG homozygous embryo occur at the first few days after
fertilization.
[0042] The lethality of GG homozygote in cattle was further
confirmed by an experiment using IVF embryos. The inventor produced
more than 300 JFV embryos using a GC sire and GC cows, or a GC sire
and CC cows. Specifically, semen samples were obtained from seven
bulls currently used in artificial insemination (Al) in the U.S.,
and their STAT5A gene genotyped. A bull that was found to be
heterozygous (CG) was selected for the experiment. Oocytes were
aspirated from 21 heterozygous (CG) and homozygous (CC) cows and
fertilized with semen obtained from the heterozygous bull. Survival
rate of the embryos was measured at days 7-9. It was found that
embryos from the GC x CC parents had a survival rate about 19%
higher than that from the CGxCG parents. Genotyping the survived
embryos revealed that all embryos were either CC or CG. When
degenerative embryos (those that did not survive beyond day 5 or 6)
were genotyped, it was found that GG genotype was present,
indicating that the GG genotype leads to early embryo
degeneration.
[0043] The present inventors further tested the correlation between
genotypes of the lethal gene and occurrence of pregnancy of
Holstein heifers. Heifers is an ideal population to carry out this
experiment compared to lactating cows because in lactating cows,
genetic factors affecting pregnancy are believed to be diluted with
many other environmental factors which makes it hard to detect
these genetic factors. In contrast, heifers have consistently
higher pregnancy rates than lactating cows and any small increase
is of great economic importance. Records were collected of 623
inseminations between different bulls and cows. As shown in Table 2
below, the results were in agreement with previous results obtained
in the IVF, experiments. TABLE-US-00002 TABLE 2 Pregnancy Frequency
Heifers of Different Genotypes Frequency of pregnancy OPEN PREGNANT
CC genotype 0.34 0.66 GC genotype 0.45 0.55
Pregnancy rate was 66% in cows carrying genotype CC versus 55% in
cows carrying genotype GC. This the first field experiment that
confirms lab results.
[0044] The cattle and pig STAT5A proteins have 96% identity at the
amino acid level, and the identity between sheep and pig STAT5
proteins is 92%. Because of this high similarity (see Table 1,
supra), similar roles the protein plays in various organisms
(Development 130, 5257-5268 (2003)) and its evolutionary history, a
GG homozygote, or a similar SNP, in other animals, especially other
mammals also have lethal effects on the embryos.
[0045] Based on the above results, the present invention provides,
in one embodiment, a method for increasing the reproductive
performance of an mammal population, the method comprising
determining the identity of the nucleotide, on both copies of the
chromosome, at or corresponding to position 7480 of exon 8 of the
bovine STAT5A gene, and eliminating animals having a heterozygous
CG genotype as a breeding parent. As discussed above, while a GG
homozygote cannot survive in the adult animal population, a GC
heterozygote will survive and be present in the population. GC
heterozygous animals, however, are not ideal candidates as parents
in a breeding program because about 25% of progenies from such
parents will be of the GG genotype and will not survive.
[0046] Accordingly, the present invention provides a nucleic acid
based genetic marker for identifying a lethality allele. This
marker can be used for genotyping an animal and for selecting an
animal for breeding purposes.
[0047] In another embodiment, the present invention provides a
method for selecting a mammal as a parent, wherein the mammal is
genotyped as described above, and selected as a breeder only if the
animal is homozygous CC.
[0048] In another embodiment, the present invention can be used to
find markers that are in strong linkage disequilibrium with the SNP
corresponding to position 7480 of the bovine STAT5A gene. These
strongly linked markers can be used as a substitute for the
described marker. The present invention also provides, in a
preferred embodiment, polymorphisms or polymorphic sites that are
in linkage disequilibrium with the SNP corresponding to position
7480 of the bovine STAT5A gene. Linkage refers to the phenomenon
that DNA sequences closely adjacent to each other in the genome,
specifically, on a chromosome in eukaryotes, have a tendency to be
inherited together. Typically, two polymorphic sequences are
co-inherited because of the relative infrequency with which meiotic
recombination events occur within the region between the two
polymorphisms, often due to physical proximity. Two sequences may
also be linked because of some selective advantage of
co-inheritance. The co-inherited polymorphic alleles are said to be
in linkage disequilibrium with one another because, in a given
population, they tend to either both occur together or else not
occur at all in any particular member of the population. Where
multiple polymorphisms in a given chromosomal region are found to
be in linkage disequilibrium with one another, they define a
quasi-stable genetic "haplotype." In contrast, if meiotic
recombination between two polymorphisms occurs frequently enough,
the two polymorphisms will appear to segregate independently and
are said to be in linkage equilibrium.
[0049] Generally speaking, the frequency of meiotic recombination
between two markers is proportional to the physical distance
between them on the chromosome. However, there are "hot spots" and
regions of repressed chromosomal recombination that cause
discrepancies between the physical distance and the so-called
"recombinational distance." Consequently, a genetic haplotype could
cover a broad region of the chromosome, with multiple polymorphic
loci in linkage disequilibrium with one another. Where one mutation
or polymorphism is found within or in linkage with this haplotype,
another one or more polymorphic alleles of the haplotype can be
used as indicators of a phenotype known to be linked to or caused
by the mutation. Such correlation can and often are used for
prognostic or diagnostic procedures without the identification and
isolation of the actual causal genetic factor. This is significant
because the precise determination of the molecular nature involved
in the genetic cause of the phenotype of interest can be difficult
and laborious, and the availability of polymorphic markers in
linkage disequilibrium with the phenotype of interest often
facilitates in the identification of the genetic causal genetic
factor(s).
[0050] In another embodiment, the present invention can be used to
genotype relatives of the animals of interest. Gene probability
theory can then be used to predict the marker genotype of an
individual based on marker genotype information from relatives in
the population. For instance if an individual animal has CG
genotype and its female parent has CC genotype, it will be known
with 100% certainty that the male parent has CG genotype without
having genotyped that sire for the described marker.
[0051] In general, for use as markers, nucleic acid fragments,
preferably DNA fragments, will be of at least 10 to 12 nucleotides
(nt), preferably at least 15 nt, usually at least 20 nt, often at
least 50 nt. Such small DNA fragments are useful as primers for the
polymerase chain reaction (PCR), and/or probes for
hybridization-based screening.
[0052] The present invention also encompasses the complementary
sequence corresponding to the polymorphism. In order to provide an
unambiguous identification of the specific site of a polymorphism,
the numbering of the original sequences in the GenBank is shown in
FIG. 1 and is used.
[0053] The term primer refers to a single-stranded oligonucleotide
capable of acting as a point of initiation of template-directed DNA
synthesis under appropriate conditions (i.e., in the presence of
four different nucleoside triphosphates and an agent for
polymerization, such as, DNA or RNA polymerase or reverse
transcriptase) in an appropriate buffer and at a suitable
temperature. The appropriate length of a primer depends on the
intended use of the primer but typically ranges from 15 to 30
nucleotides. Short primer molecules generally require cooler
temperatures to form sufficiently stable hybrid complexes with the
template. A primer needs not reflect the exact sequence of the
template but must be sufficiently complementary to hybridize with a
template. The term primer site, or priming site, refers to the
region of the target DNA to which a primer hybridizes. The term
primer pair means a set of primers including a 5' upstream primer
that hybridizes with the 5' end of the DNA sequence to be amplified
and a 3', downstream primer that hybridizes with the complement of
the 3' end of the sequence to be amplified.
[0054] The term "probe" or "hybridization probe" denotes a defined
nucleic acid segment (or nucleotide analog segment) which can be
used to identify by hybridization a specific polynucleotide
sequence present in samples, the nucleic acid segment comprising a
nucleotide sequence complementary of the specific polynucleotide
sequence to be identified. "Probes" or "hybridization probes" are
nucleic acids capable of binding in a base-specific manner to a
complementary strand of nucleic acid.
[0055] An objective of genotyping according to the present
invention is to determine which embodiment of the polymorphism a
specific sample of DNA has. Many detection techniques are available
and well-known to those skilled in the art. For example, an
oligonucleotide probe can be used for such purpose. Preferably, the
oligonucleotide probe will have a detectable label. Experimental
conditions can be chosen such that if the sample DNA contains a C
at position 7480, then the hybridization signal can be detected
because the probe hybridizes to the corresponding complementary DNA
strand in the sample, while if the sample DNA contains an G, no
hybridization signal is detected.
[0056] Similarly, PCR primers and conditions can be devised,
whereby the oligonucleotide is used as one of the PCR primers, for
analyzing nucleic acids for the presence of a specific sequence.
These may be direct amplification of the genomic DNA, or RT-PCR
amplification of the mRNA transcript of the genes. Amplification
may be used to determine whether a polymorphism is present, by
using a primer that is specific for the polymorphism.
Alternatively, various methods are known in the art that utilize
oligonucleotide ligation as a means of detecting polymorphisms, for
examples see Riley et al (1990) Nucleic Acids Res. 18:2887-2890;
and Delahunty et al (1996) Am. J. Hum. Genet. 58:1239-1246. The
detection method may also be based on direct DNA sequencing, or
hybridization, or a combination thereof. Where large amounts of DNA
are available, genomic DNA is used directly. Alternatively, the
region of interest is cloned into a suitable vector and grown in
sufficient quantity for analysis. The nucleic acid may be amplified
by PCR, to provide sufficient amounts for analysis.
[0057] Hybridization may be performed in solution, or such
hybridization may be performed when either the oligonucleotide
probe or the target polynucleotide is covalently or noncovalently
affixed to a solid support. Attachment may be mediated, for
example, by antibody-antigen interactions, poly-L-Lys, streptavidin
or avidin-biotin, salt bridges, hydrophobic interactions, chemical
linkages, UV cross-linking baking, etc. Oligonucleotides may be
synthesized directly on the solid support or attached to the solid
support subsequent to synthesis. Solid-supports suitable for use in
detection methods of the invention include substrates made of
silicon, glass, plastic, paper and the like, which may be formed,
for example, into wells (as in 96-well plates), slides, sheets,
membranes, fibers, chips, dishes, and beads. The solid support may
be treated, coated or derivatized to facilitate the immobilization
of the allele-specific oligonucleotide or target nucleic acid. For
screening purposes, hybridization probes of the polymorphic
sequences may be used where both forms are present, either in
separate reactions, spatially separated on a solid phase matrix, or
labeled such that they can be distinguished from each other. Assays
may utilize nucleic acids that hybridize to one or more of the
described polymorphisms, and may include all or a subset of the
polymorphisms listed in Table 1.
[0058] Hybridization may also be performed with nucleic acid arrays
and subarrays such as described in WO 95/11995. The arrays would
contain a battery of allele-specific oligonucleotides representing
a plurality of the polymorphic sites. One or both polymorphic forms
may be present in the array, for example the polymorphism at
position 7480 of the STAT5A gene may be represented. Usually such
an array will include at least 2 different polymorphic sequences,
i.e. polymorphisms located at unique positions within the locus.
Arrays of interest may further comprise sequences, including
polymorphisms, of other genetic sequences, particularly other
sequences of interest. The oligonucleotide sequence on the array
will usually be at least about 12 nt in length, or may extend into
the flanking regions to generate fragments of 100 to 200 nt in
length. For examples of arrays, see Ramsay (1998) Nat. Biotech.
16:4044; Hacia et al. (1996) Nature Genetics 14:441-447; Lockhart
et al. (1996) Nature Biotechnol. 14:1675-1680; and De Risi et al.
(1996) Nature Genetics 14:457-460. As well-known to those
ordinarily skilled in the art, the presence or absence of
hybridization signals, optionally in combination of the signal
strength, will determine the presence or absence of which of the
alleles, and whether the sample is heterozygous or homozygous in
regard to the SNP.
[0059] The identity of polymorphisms may also be determined using a
mismatch detection technique, including but not limited to the
RNase protection method using riboprobes (Winter et al., Proc.
Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science
230:1242, 1985) and proteins which recognize nucleotide mismatches,
such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet.
25:229-253, 1991). Alternatively, variant alleles can be identified
by single strand conformation polymorphism (SSCP) analysis (Orita
et al., Genomics 5:874-879, 1989; Humphries et al., in Molecular
Diagnosis of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or
denaturing gradient gel electrophoresis (DGGE) (Wartell et al.,
Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al., Proc. Natl.
Acad. Sci. USA 86:232-236, 1989).
[0060] A polymerase-mediated primer extension method may also be
used to identify the polymorphism(s). Several such methods have
been described in the patent and scientific literature and include
the "Genetic Bit Analysis" method (WO 92/15712) and the
ligase/polymerase mediated genetic bit analysis (U.S. Pat. No.
5,679,524). Related methods are disclosed in WO 91/02087, WO
90/09455, WO 95/17676, U.S. Pat. Nos. 5,302,509, and 5,945,283.
Extended primers containing a polymorphism may be detected by mass
spectrometry as described in U.S. Pat. No. 5,605,798. Another
primer extension method is allele-specific PCR (Ruao et al., Nucl.
Acids Res. 17:8392, 1989; Ruao et al., Nudl. Acids Res. 19,
6877-6882, 1991; WO 93/22456; Turki et al., J. Clin. Invest.
95:1635-1641, 1995). In addition, multiple polymorphic sites may be
investigated by simultaneously amplifying multiple regions of the
nucleic acid using sets of allele-specific primers as described in
Wallace et al. (WO 89/10414).
[0061] A detectable label may be included in an amplification
reaction. Suitable labels include fluorochromes, e.g. fluorescein
isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin,
allophycocyanin, 6-carboxyfluorescein (6-FAM),
2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE),
6-carboxy-X-rhodamine (ROX),
6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX),
5-carboxyfluorescein (5-FAM) or
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive
labels, e.g. .sup.32P, .sup.35S, .sup.3H; etc. The label may be a
two stage system, where the amplified DNA is conjugated to biotin,
haptens, etc. having a high affinity binding partner, e.g. avidin,
specific antibodies, etc., where the binding partner is conjugated
to a detectable label. The label may be conjugated to one or both
of the primers. Alternatively, the pool of nucleotides used in the
amplification is labeled, so as to incorporate the label into the
amplification product.
[0062] It is readily recognized by those ordinarily skilled in the
art that in order to maximize the signal to noise ratio, in probe
hybridization detection procedure, the polymorphic site should be
at the center of the probe fragment used, whereby a mismatch has a
maximum effect on destabilizing the hybrid molecule; and in a PCR
detection procedure, the polymorphic site should be placed at the
very 3'-end of the primer, whereby a mismatch has the maximum
effect on preventing a chain elongation reaction by the DNA
polymerase. The location of nucleotides in a polynucleotide with
respect to the center of the polynucleotide is described herein in
the following manner. When a polynucleotide has an odd number of
nucleotides, the nucleotide at an equal distance from the 3' and 5'
ends of the polynucleotide is considered to be "at the center" of
the polynucleotide, and any nucleotide immediately adjacent to the
nucleotide at the center, or the nucleotide at the center itself is
considered to be "within 1 nucleotide of the center." With an odd
number of nucleotides in a polynucleotide any of the five
nucleotides positions in the middle of the polynucleotide would be
considered to be within 2 nucleotides of the center, and so on.
When a polynucleotide has an even number of nucleotides, there
would be a bond and not a nucleotide at the center of the
polynucleotide. Thus, either of the two central nucleotides would
be considered to be "within 1 nucleotide of the center" and any of
the four nucleotides in the middle of the polynucleotide would be
considered to be "within 2 nucleotides of the center," and so
on.
[0063] Alternatively, the relevant portion of the relevant genetic
locus of the sample of interest may be amplified via PCR and
directly sequenced. It is readily recognized that numerous other
primers can be devised based on the sequence of Accession No. AJ
237937 such as those shown in FIG. 1. PCR and sequencing techniques
are well known in the art and reagents and equipments are readily
available commercially. The identity of the polymorphic site in the
amplified fragment may also identified by RFLP, according to method
and techniques well-known to those skilled in the art.
[0064] Alternatively, an invasive signal amplification assay, as
described in e.g. U.S. Pat. No. 5,422,253 and Lyamichev et al.,
2000, Biochemistry 39:9523-9532, both incorporated herein by
reference in their entirety, may be used for detecting the SNP of
interest. This assay takes advantage of enzymes such as the 5'
nuclease activity of a DNA polymerase or the gene 6 product from
bacteriophage T7 in their ability to cleave polynucleotide
molecules by recognizing specific structures instead of specific
sequences. A single-stranded target molecule is annealed to a pilot
oligonucleotide such that the 5' end of the pilot forms a duplex
with the target molecule. If the 3' end of the pilot
oligonucleotide does not pair with the target, a 3' arm is formed.
When exposed to a cleavage agent such as a DNA polymerase having a
5' nuclease activity or the gene 6 product from bacteriophage T7,
the target molecule is cleaved in the 5' region, one nucleotide
into the duplex adjacent to the unpaired region of the target. If a
cut in a double-stranded molecule is required, the double-stranded
molecule is denatured. Because this unpaired 3' arm can be as short
as one nucleotide, this assay can be used for detecting a
single-nucleotide difference, e.g. in the context of SNP detection.
The pilot oligonucleotide is designed such that it pairs perfectly
with one allele, but has a 3', single nucleotide mismatch with
another allele. Cleavage only occurs if there is a mismatch between
the target molecule and the pilot. To achieve signal amplification,
the above invasive reaction is modified such that cleavage occurs
on the pilot oligonucleotide. Two oligonucleotides are annealed in
an adjacent manner to the target molecule. The resulting adjacent
duplexes overlaps by at least one nucleotide to create an efficient
substrate, called the overlapping substrate, for the 5' nucleases.
The 5' end of the downstream oligonucleotide, also called the
probe, contains an unpaired region termed the 5' arm (Lyamichev et
al., 1993, Science 260:778-783.) or flap (Harrington and Lieber,
1994, EMBO J 13: 1235-1246) that is not required for the enzyme
activity; however, very long arms can inhibit cleavage (Lyamichev
et al., 1993, Science 260:778-783). Specific cleavage of the probe,
termed invasive cleavage (Lyamichev et al., 1999, Nat. Biotechnol.
17 292-296; Kwiatkowski et al., 1999, Mol. Diagn. 4, 353-364.),
occurs at the position defined by the 3' end of the upstream
oligonucleotide, which displaces or "invades" the probe. If the
overlap between the adjacent oligonucleotides is only one
nucleotide, cleavage takes place between the first two base pairs
of the probe, thus releasing its 5' arm and one nucleotide of the
base paired region (Lyamichev et al., 1999, Proc. Natl. Acad. Sci.
USA. 96: 6143-6148, and Kaiser et al., 1999, J Biol. Chem.
274:21387-21394). If the upstream oligonucleotide and the probe are
present in large molar excess over the target nucleic acid, and
invasive cleavage is carried out near the melting temperature of
the probe, a cut probe can rapidly dissociate, and an intact probe
will anneal to the target more frequently than will a cut probe,
thus initiating a new cycle of cleavage. This allows multiple
probes to be cut for each target molecule under isothermal
conditions, resulting in linear signal amplification with respect
to target concentration and time (Lyamichev et al., 1999, Nat.
Biotechnol. 17: 292-296).
[0065] The present invention further provides a method for
genotyping the SATA5A gene of an animal, especially a mammalian, or
an individual of an avian or aquatic species, the method comprising
determining the nucleotide identity for the two copies of the
genetic locus. One embodiment of a genotyping method of the
invention involves examining both copies of the genes or coding
sequence of STAT5A, or a fragment thereof, to identify the
nucleotide pair at the polymorphic site in the two copies to assign
a genotype to the individual. In some embodiments, "examining a
gene" may include examining one or more of DNA containing the gene,
mRNA transcripts thereof, or cDNA copies thereof. As will be
readily understood by the skilled artisan, the two "copies" of a
gene, mRNA or cDNA, or fragment thereof in an individual may be the
same allele or may be different alleles.
[0066] In another embodiment, a genotyping method of the invention
comprises determining the identity of the nucleotide pair at the
polymorphic site.
[0067] The present invention further provides a kit for detecting
the SNP of the present invention or for genotyping a sample, the
kit comprising in a container a nucleic acid molecule, as described
above, designed for detecting the polymorphism, and optionally at
least another component for carrying out such detection.
Preferably, a kit comprises at least two oligonucleotides packaged
in the same or separate containers. The kit may also contain other
components such as instructional materials and reagents (e.g.
hybridization buffer where the oligonucleotides are to be used as a
probe and/or enzymes for PCR or RFLP) packaged in a separate
container. Alternatively, where the oligonucleotides are to be used
to amplify a target region, the kit may contain, preferably
packaged in separate containers, a polymerase and a reaction buffer
optimized for primer extension mediated by the polymerase, such as
PCR.
[0068] In another embodiment the present invention provides an
animal breeding method whereby genotyping as described above is
conducted on animal embryos, and based on the results, certain
embryos are either selected or removed from the breeding program.
In a preferred embodiment, where CG parent(s) are not avoidable,
the present invention provides a selective breeding method which
takes advantage of multiple ovulation and embryo transfer procedure
(MOET). The method comprises superovulating a female animal,
collecting eggs from said superovulated female, in vitro
fertilizing said eggs using semen from a suitable male animal,
implanting said fertilized eggs into suitable females allowing for
an embryo to develop, and determining the SNP of the developing
embryo as described above. For animals other than dairy cattle,
embryos other than homozygous CC are not transplanted or the
pregnancy terminated depending on the circumstances. For dairy
cattle, embryos other than CG genotype are not transplanted.
[0069] In another embodiment the present invention provides an
animal breeding method whereby genotyping as described above is
conducted on elite cows (cows to produce bulls), and based on the
results, certain elite cows are either selected or not selected to
produce the next generation of breeding bulls (young bulls). This
makes it possible to break any linkage of the non-lethal allele
with lower performance characteristics that may otherwise be
present.
[0070] In another embodiment, the present invention provides a
method for selecting a gamete donor in human assisted reproduction.
Assisted reproductive technologies (ART) include IVF (in vitro
Fertilization and embryo transfer), GIFT (gamete intrafallopian
transfer) and ZIFT (zygote intrafallopian transfer). A fertilized
egg is transferred in ZIFT at the pronuclear stage, i.e., prior to
the first cell division. The fertilized egg undergoes the first
cell division generally at about 30 hours after fertilization, and
becomes an embryo. The earliest stage embryo, generally up to four
days after fertilization, and up to the 8-cell stage, is referred
to as blastomere. From this point on, the embryo is referred to as
a morula, and is a solid mass of cells. Approximately 5-6 days
after fertilization, the embryo becomes a blastocyst which is a
hollow ball of cells, filled with fluid. Embryo hatching and
implantation follow.
[0071] When a gamete (egg or sperm) donor is needed, the candidates
are genotyped and only those who are CC homozygous with regard to
the STAT5A SNP of the present invention are selected, thereby
eliminating the generation of GG embryos that will subsequently
perish.
[0072] In the context of human ART, often one of the parents has a
CG heterozygous genotype. The present invention provides a method
and relevant compositions that can be used to screen in vitro
embryos for planting in the uterus, increasing the rate of success
while diminishing the chance of multiple pregnancy. A naturally
conceived embryo remains in the fallopian tube for 4 days before
entering the uterus and implanting on or about day 6. Ideally
embryos created through IVF should therefore be transferred on Day
5. However for the last 10 years IVF embryos transferred on Day 5,
after being cultured in conventional growth media, had lower
implantation rates than those transferred on Day 3. For this reason
the practice has been to transfer embryos into the uterus on Day 3.
Implantation rates for Day 3 embryos, however, are still only 15%
to 20%. To achieve respectable pregnancy rates 3 or more embryos
must therefore be transferred whenever possible. This unfortunately
dramatically increases the incidence of multiple pregnancies, which
is the main criticism against IVF. Currently the ability to
accurately identify day 3 embryos that would survive and implant
from those that would not is lacking.
[0073] According to another embodiment of the present invention, a
method is provided for selecting early stage embryos for transfer,
such that embryos that cannot survive, i.e. GG homozygotes, are not
selected or transferred. This will ensure a high success rate,
which would lead to avoidance of transfer of large numbers of
embryos and multiple pregnancy, which is the main criticism of
ART.
[0074] Pre-implantation genetic diagnosis (PGD) techniques are well
known to those ordinarily skilled in the art and are frequently
used to test embryos for genetic disorders before it implants in
the uterus. Typically, a single cell is removed from an 8-cell
embryo through an opening in the outer protective coat. The
procedure is carried out under the microscope without damaging the
embryo's ability to continue to develop normally (because at this
stage of development none of the embryo cells have become
specialized). The cell is then analyzed for the presence of genetic
disorders.
[0075] The present invention further discovered, surprisingly, that
dairy cattle with heterozygous CG genotype has increased milk yield
and higher milk fat and protein yield. Specifics of the experiments
that lead to this discovery are discussed below.
[0076] Populations and phenotypic data: Blood samples were obtained
from the University of Wisconsin (UW) daughter design resource
population (henceforth: UW resource population). This population
has been investigated to search for genetic markers in association
with susceptibility to mycobacterium tuberculosis. The 12 sire
families of this population were chosen from a large number of
candidate bulls with large numbers of daughters in production in
2000. Blood samples from the bulls' daughters have been collected
through cooperation with commercial dairy producers throughout the
U.S. since January 2001. Yield deviation data for the UW resource
populations for milk yield (kg), milk protein and fat yields (kg),
were obtained from the USDA Animal Improvement Programs Laboratory
(Beltsville, Md.). Summary statistics of these data for milk
production traits are given in Table 3. TABLE-US-00003 TABLE 3
Means, standard deviations (SD), minimum, and maximum yield
deviations (YD) of cows in the UW resource population for the
production traits University of Wisconsin resource population Trait
Mean (Kg) SD Min Max Milk YD 1092.6 1815.1 -5917 7344 Fat YD 29.97
68.17 -277 322 Protein YD 29.86 48.73 -159 181
[0077] Polymorphism detection and genotyping: Genomic DNA was
extracted from blood samples using GFX Genomic Blood DNA
Purification Kit (Amersham Biosciences, Piscataway, N.J.). DNA
concentration was measured using a spectrophotometer (Ultraspec
2100; Amersham Biosciences). Amplification was performed in a 25
.mu.l reaction volume, which included 50 ng genomic DNA, 50 ng each
primer, 200 .mu.M each dNTP, 2.5 .mu.l 10.times. PCR buffer
(Promega, Madison, Wis.), and 0.3 u Taq DNA polymerase (Promega).
The temperature cycles were as follows: 95.degree. C. for 5 min,
followed by 30 cycles of 94.degree. C. for 45 s, touchdown
annealing from 65-50.degree. C. for 45 s (<2.degree. C./cycle),
72.degree. C. for 45 s, and a final extension at 72.degree. C. for
7 min. For individual genotyping, primers STAT7 and STAT8 were used
to amplify 50 ng genomic DNA and the PCR products were digested
with the restriction enzyme BstII that distinguishes alleles C and
G of the SNP. The digestion products were electrophoresed on a 1.5%
agarose gel.
[0078] Statistical Analysis: Sires and their daughters are
genotyped, and phenotypic data was available for the daughters. The
linear model used was
Y.sub.ijk=.mu.+genotype.sub.i+sire.sub.j+Map+e.sub.ijk where
Y.sub.ijk is the yield deviation (milk, fat, protein) of daughter
k, .mu. is the mean, genotypei is the effect of genotype i,
sire.sub.j is the sire j effect, Map is M paratuberculosis
infection status (noninfected=0, infected=1), and e.sub.ijk is the
residual.
[0079] The results are shown in Table 4 below: TABLE-US-00004 TABLE
4 Estimates of the effects of CG genotypes, standard errors (SE)
and P-values for milk production traits (kg) as a deviation from
the effect of the genotype (CC) in the UW resource population Milk
yield .+-. Fat yield .+-. Protein yield .+-. Genotype SE (P) SE (P)
SE (P) CG 445.9 .+-. 179.4 14.53 .+-. 7.00 9.65 .+-. 4.86 (0.0133)
(0.0386) (0.073)
[0080] Table 4 shows the estimates of the genotype effects for milk
yield, protein and fat yield in the UW resource population.
Compared to genotype CC, genotype CG was associated with a
significant increase in milk yield (445.9 Kg), fat yield (14.53 Kg)
and protein yield (9.65 Kg).
[0081] Accordingly, in another embodiment, the present invention
provides a method of improving dairy cattle breeding. Specifically,
because an ideal genotype for a milk-producing cow is CG, for both
survivability and production traits, a natural mating between two
CC parents is not desirable. Because a GG parent does not exist,
CC.times.GG (which would produce 100% CG progeny) is not possible.
Thus, parents should be selected for CG.times.CG matings or
CC.times.CG matings. Both give 50% CG progeny, and 25% CC.
CC.times.CG matings are particularly preferred because it is more
desirable for the remaining 25% of the resultant embryos to have a
CC genotype, than 25% GG for them to have genotype which will die
at very early stages of the embryo development, which will occur in
a CG.times.CG mating. In addition, CC.times.CG mating is preferred
because milk production traits may be otherwise compensated by
additional genetic traits.
[0082] In situations where there are more control, for example in
in vitro fertilization and embryo transfer, the present invention
further provides a method of selecting parents (CG), such that
fertilization occurs between CC.times.CG or CG.times.CG parents, in
combination with a step to de-select CC embryos (poor production
traits) and GG embryos (early embryo death), so that only CG
embryos are planted.
[0083] The foregoing description and examples have been set forth
merely to illustrate the invention and are not intended to be
limiting. Since modifications of the disclosed embodiments
incorporating the spirit and substance of the invention may occur
to persons skilled in the art, the invention should be construed
broadly to include all variations falling within the scope of the
appended claims and equivalents thereof. All references cited
hereinabove and/or listed below are hereby expressly incorporated
by reference.
Sequence CWU 1
1
3 1 21 DNA artificial PCR PRIMER 1 gagaagttgg cggagattat c 21 2 20
DNA artificial PCR PRIMER 2 gtgttctcgt tcttgagcag 20 3 1800 DNA Bos
taurus 3 accatcatcc tggatgacga gctgatccag tggaagcggc ggcagcagct
ggcggggaac 60 ggagggcccc ccgagggcag cctggatgtg ctacagtcct
ggtaccaggg gtggggggcg 120 gggaggggca ggcagcagag tggtgctgcc
agctgctgtt tgcgcccacg tctacatgag 180 cagctggctc cctctgtctg
ggcgcgggtc ttatcccacc agtggtgtgt ttggtgctga 240 caccggtgtc
cctttctgtg ccccctcccc tgggaggatg ctggggtggg gccaggtggc 300
aaagtggcgc tcaggctggt tggaccccag tcagtgtcgc tcctcctggg tgtttctctg
360 gtttttttgg aaggcagggc atctctgctg tgcccagtgc acaggcgagg
tggctcgggc 420 accaggcctt cctgggggtg gagctgggtg tgggccttgt
ccccgcctgg gcgcytgcca 480 gcttctggcc tggaggacgg gggtgaagcc
cgtgtccttc ccttgggccc tggggctcgg 540 gttcaggtgt gagaagttgg
cggagattat ctggcagaac cggcagcaga tccgcagagc 600 cgagcacctc
tgccagcagc tgcccatccc cggccccgtg gaggagatgc tggctgaggt 660
caacgccacc atcactgaca tcatctcagc cctggtgacc aggtgactcc tggccacgcc
720 ccgctcccat ctggttgccc tgggttgggg gcagcagggt ctttgcagat
ggggagctct 780 ggcttaaatc cttcagtttc tgcctcacac cctcctccca
tccctctcca tcccctgttg 840 ctatggcctc ttgctgtcga cctcacccag
tatttctcgt ggacactaca cgggcatttg 900 tctcctgcaa ctcctttcag
ctgctgagtt ccttttactg cctcccttcc cgccagctcc 960 cctgactcac
agtggcccca gggagggtgg actgtccgca aaccctccct tcacctgctc 1020
agcctggtgc aaggcaagcc tccccacgtg gaaggtgggg ccagagtcct gtcccctgaa
1080 gtgctcctgt cccttgtgtc tccgcagcac cttcatcatc gagaagcagc
cccctcaggt 1140 cctgaagacc cagaccaagt tcgcggccac cgtgcgcctg
ctggtgggtg ggaagctgaa 1200 cgtgcacatg aacccccccc aggtgaaggc
caccatcatc agcgagcagc aggccaagtc 1260 actgctcaag aacgagaaca
cccgcaagta tgctgcccgc tccttcatct gccctccccc 1320 agctcagcct
ctgctctgta gctggggtcc caggtgatga ggacacacgg ggcctcccac 1380
tctttgtcta gcattgcaat gacagatgac tctgtctgtc tgggggtatt cctcgcacac
1440 agagcaaatc aaggacttca ctattagggt gtcccaggat ctgtgctgag
cactggcaca 1500 gtgctgggat ccacaccaaa cttggctcca tcacggccca
acctttaggc tagcaggcag 1560 cagacgtgag aattgattac ttgctggcat
gtgaaaagag agacactggg gcaccatgaa 1620 agtgggtcat ggagggggtg
ggacagaatg acagctagtt tcaaggctgc ggggtgacta 1680 ggaggagatg
gtgaacgggc gtggagaggg cagacgttcg ggcagagaga acacgggcct 1740
gtctggaaga ggagcagaca aagggcgtgg tggatggaga acccggggtg aggtcctggg
1800
* * * * *