WO2015093988A1

WO2015093988A1 - Genetic markers of offspring mortality and uses therefor

Info

Publication number: WO2015093988A1
Application number: PCT/NZ2014/050023
Authority: WO
Inventors: Richard John Spelman; Mathew Douglas LITTLEJOHN; Michel Alphonse Jumien Georges; Carole Yvone Louise Chislaine CHARLIER
Original assignee: Livestock Improvement Corporation Limited
Priority date: 2013-12-18
Filing date: 2014-12-18
Publication date: 2015-06-25

Abstract

The present invention relates to methods of determining whether or not an animal carries alteration or variation in the OBFCl gene, being a biological marker linked to offspring mortality, and particularly, but not exclusively, to methods for selecting or rejecting animals, one or more cells or embryos, animal evaluation, breeding animals, and herd formation, using this marker.

Description

GENETIC MARKERS OF OFFSPRING MORTALITY AND USES THEREFOR

FIELD

The present invention relates to methods of determining whether or not an animal carries a biological marker linked to offspring mortality, and particularly, but not exclusively, to methods for selecting or rejecting animals, one or more cells or embryos, animal evaluation, breeding animals, and herd formation.

BACKGROUND

In farming, offspring mortality can result in significant economic losses. In the New Zealand dairy cow population, for example, the death of calves and young livestock causes losses of up to 1000 to 2000 animals per annum.

There are many factors which cause or influence offspring mortality in animals. These include, for example, farming and environmental conditions, disease, and genetic mutation. The estimated economic loss due to offspring mortality, particularly abortion or prenatal deaths and perinatal mortality, is 1.5 million to 3 million dollars per annum.

Identification of animals carrying genetic alterations which may result in offspring mortality and implementing appropriate selection and breeding programmes may help reduce the loss of animals and increase productivity in farming.

OBJECT

It is an object of the present invention to provide a method of determining whether or not an animal carries a biological marker linked to offspring mortality, a method for estimating the worth of an animal, a method for selecting or rejecting an animal, a method for breeding animals, a method of forming a herd, a method for cloning an animal, a nucleic acid, a peptide, a kit and/or at least to provide the public with a useful choice.

STATEMENT OF INVENTION

The inventors contemplate that an alteration or variation in the OBFCI gene which disrupts the gene can be lethal to an animal which is homozygous for that alteration or variation. Methods involving the analysis of such alterations or variations can be used to determine whether or not an animal is a carrier for a lethal alteration or variation and risks passing this to their offspring. Such methods may be used for selecting, screening and breeding animals, farm management, and for esthnating an animal's worth to a particular industry, for example. In a first aspect, the invention provides a method for determining whether or not an animal carries a genetic marker linked to offspring mortality, the method comprising at least the step of analysing a nucleic acid from said animal to determine whether or not it includes one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith, wherein where the nucleic acid includes one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith, it is determined to carry a genetic marker linked to offspring mortality.

In one embodiment, the method may be used to determine whether or not an animal is more likely than not to be a carrier for offspring mortality. In one embodiment, the method may be used to determine whether or not an animal is a carrier for offspring mortality.

In a second aspect, the invention provides a method for determining whether an animal is or is more likely than not a carrier for offspring mortality, the method comprising at least the step of analysing a nucleic acid from said animal to determine whether or not it includes one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith, wherein where the nucleic acid includes one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith, it is determined to be a carrier or to be more likely than not to be a carrier for offspring mortality.

In one embodiment of the first or second aspects, where the animal is heterozygous for one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith, the animal is identified as more likely than not to be a carrier for offspring mortality. In one embodiment, it is identified as being a carrier for offspring mortality.

In one embodiment of the first or second aspects, the invention provides a method for determining whether or not an animal is or is more likely than not to be a carrier for offspring mortality, the method comprising at least the step of analysing a nucleic acid from said animal to determine whether or not it includes the genetic marker 24720155TTdel on chromosome 26 of Bos taurus and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, the presence of the genetic marker 24720155TTdel and/or the presence of one or more genetic marker in linkage disequilibrium therewith infers that the animal is more likely than not a carrier for offspring mortality. In one embodiment, it is inferred to be a carrier for offspring mortality. In a third aspect the invention provides a method for selecting or rejecting an animal, the method comprising at least the step of analysing a nucleic acid from said animal to determine whether or not it includes one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith, wherein the presence of one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith infers that the animal is more likely than not a carrier for offspring mortality. In one embodiment, in the presence of one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith the animal is inferred to be a carrier for offspring mortality.

In one embodiment of the third aspect, an animal is selected if it does not have one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, an animal is rejected if it has one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment of the third aspect, where the animal is heterozygous for one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith, it is inferred that the animal is more likely than not a carrier for offspring mortality. In one embodiment, where the animal is heterozygous for one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith the animal is inferred to be a carrier for offspring mortality.

In one embodiment of the third aspect, the invention provides a method for selecting or rejecting an animal, the method comprising at least the step of analysing a nucleic acid from said animal to determine whether or not it includes the genetic marker 24720155TTdel on chromosome 26 of Bos taurus and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the method comprises at least the steps of:

a) analysing a nucleic acid from said animal to determine whether or not it includes the genetic marker 24720155TTdel on chromosome 26 of Bos taurus and/or one or more genetic marker in linkage disequilibrium therewith; and,

b) selecting or rejecting an animal based on the presence of the genetic marker 24720155TTdel on cliromosome 26 of Bos taurus and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, where the genetic marker 24720155TTdel and/or one or more genetic marker in linkage disequilibrium therewith is present it is inferred that the animal is more likely than not a carrier for offspring mortality and the animal may be rejected. In one embodiment, where the genetic marker 24720155TTdel and/or one or more genetic marker in linkage disequilibrium therewith is present it is inferred that the animal is a carrier for offspring mortality and the animal may be rejected.

In one embodiment of the third aspect, the method is performed for the purpose of selecting or rejecting an animal for milking purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting an animal for beef farming. In another embodiment, the method is performed for the purpose of selecting or rejecting an animal for breeding purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting an animal for inclusion in a herd.

In a fourth aspect, the invention provides a method for estimating the worth of an animal and/or its offspring, the method comprising at least the step of analysing a nucleic acid from said animal to determine whether or not it includes one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, when one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith is present it is inferred that the animal is more likely than not to be a carrier for offspring mortality. In one embodiment, where one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith is present the animal is inferred to be a carrier for offspring mortality.

In one embodiment of the fourth aspect, where the animal is heterozygous for one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith, it is inferred that the animal is more likely than not to be a carrier for offspring mortality. In one embodiment, where the animal is heterozygous for one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith, it is inferred that the animal is a carrier for offspring mortality

In one embodiment of the fourth aspect, the invention provides a method for estimating the worth of an animal and/or its offspring, the method comprising at least the step of analysing a nucleic acid from the animal to determine whether or not it includes the genetic marker 24720155TTdel on chromosome 26 of Bos taurus and/or one or more genetic marker in linkage disequilibrium thereof. In one embodiment, where the genetic marker 24720155TTdel and/or one or more genetic marker in linkage disequilibrium therewith is present it is inferred that the animal is more likely than not to be a carrier for offspring mortality. In one embodiment, it is inferred that the animal is a carrier for offspring mortality.

In a fifth broad aspect, the invention provides a method for breeding animals which comprises selecting at least a first animal that does not include one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith and mating said first animal with a second animal.

In one embodiment of the fifth aspect, the method further comprises selecting the second animal on the basis that it does not have a genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment of the fifth aspect, the invention provides a method for breeding animals which comprises selecting at least a first animal that does not have the genetic marker 24720155TTdel and/or one or more genetic marker in linkage disequilibrium therewith and mating said first animal with a second animal.

In one embodiment, the method further comprises the step of selecting the second animal on the basis that it does not have the genetic marker 24720155TTdel and/or one or more genetic marker in linkage disequilibrium therewith.

In a sixth aspect, the invention provides a method for selecting or rejecting one or more cell or embryo, the method comprising at least the step of analysing a nucleic acid from said one or more cell or embryo, or an animal from which the one or more cell or embryo is derived, to determine whether or not it includes one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment of the sixth aspect, one or more cell or embryo is selected if it, or an animal from which it is derived, does not have one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, one or more cell or embryo is rejected if it, or an animal from which it is derived, has one or more genetic variation winch disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment of the sixth aspect, the invention provides a method for selecting or rejecting one or more cell or embryo, the method comprising at least the step of analysing a nucleic acid from said one or more cell or embryo, or an animal from which it is derived, to determine whether or not it includes the genetic marker 2472015 STTdel on chromosome 26 of Bos taurus and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the method comprises at least the steps of:

a) analysing a nucleic acid from said one or more cell or embryo, or an animal from which it is derived, to determine whether or not it includes the genetic marker 24720155TTdel and/or one or more genetic marker in linkage disequilibrium therewith; and, b) selecting or rejecting one or more cell or embryo based on the presence or absence of the genetic marker 24720155TTdel and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, where the genetic marker 24720155TTdel and/or one or more genetic marker in linkage disequilibrium therewith is present the one or more cell or embryo is rejected. In one embodiment, where the genetic marker 2472015 STTdel and/or one or more genetic marker in linkage disequilibrium therewith is absent the one or more cell or embryo is selected.

In one embodiment of the sixth aspect, the method is performed for the purpose of selecting or rejecting one or more cell or embryo for use in cloning an animal and/or breeding an animal. In one embodiment, breeding an animal may involve IVF.

In a seventh aspect, the invention provides a method for breeding animals the method comprising at least the step of selecting a first gamete that does not include one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith and fusing said first gamete with a second gamete to form a zygote.

In one embodiment of the seventh aspect, the method further comprising selecting the second gamete on the basis that it does not include one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith.

In an eighth aspect, the invention provides a method of breeding an animal, the method comprising at least the step of selecting an embryo that does not include one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith. In a ninth aspect, the invention provides a method of cloning an animal, the method comprising at least the step of selecting one or more cell that does not include one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment of the first to ninth aspects, the methods of the invention involve analysing a nucleic acid to determine whether or not it includes one or more genetic variation which disrupts the OBFC1 gene alone or in combination with one or more genetic marker in linkage disequilibrium therewith. In one embodiment, the methods of the invention involve analysing only one or more genetic marker in linkage disequilibrium with one or more genetic variation which disrupts the OBFC1 gene.

In one embodiment of the first to ninth aspects, the methods of the invention involve analysing a nucleic acid to determine whether or not it includes the genetic marker 24720 55TTdel on chromosome 26 of Bos taurus alone or in combination with one or more genetic marker in linkage disequilibrium therewith. In one embodiment, the methods of the invention involve analysing only one or more genetic marker in linkage disequilibrium with the genetic marker 24720155TTdel on chromosome 26 of Bos taurus.

In a tenth aspect, the invention provides a method for determining whether or not an animal carries a biological marker linked to offspring mortality, the method comprising at least the step of observing the level and/or activity of one or more of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof.

In one embodiment of the tenth aspect, the method comprises comparing the level and/or activity of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof against one or more standard.

In one embodiment of the tenth aspect, the method may be used to determine whether or not an animal is more likely than not to be a carrier for offspring mortality. In one embodiment, the method may be used to determine whether or not an animal is a carrier for offspring mortality.

In an eleventh aspect, the invention provides a method for determining whether an animal is or is more likely than not a carrier for offspring mortality, the method comprising at least the step of observing the level and/or activity of one or more of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof.

In one embodiment of the eleventh aspect, the method comprises comparing the level and/or activity of OBFCl, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof against one or more standard.

In a twelfth aspect the invention provides a method for selecting or rejecting an animal the method comprising at least the step of observing the level and/or activity of one or more of OBFCl, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof.

In one embodiment of the twelfth aspect, the method comprises comparing the level and/or activity of OBFCl , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof against one or more standard.

In one embodiment, an animal is selected if the level and/or activity of OBFCl, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof is indicative of an animal who is not a carrier for offspring mortality. In one embodiment, an animal is rejected if the level and/or activity of OBFCl , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof is indicative of an animal who is a carrier for offspring mortality.

In one embodiment of the twelfth aspect, the method is performed for the purpose of selecting or rejecting an animal for milking purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting an animal for beef farming. In another embodiment, the method is performed for the purpose of selecting or rejecting an animal for breeding purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting an animal for inclusion in a herd.

In a thirteenth aspect, the invention provides a method for estimating the worth of an animal and/or its offspring, the method comprising at least the step of observing the level and/or activity of one or more of OBFCl , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof. In one embodiment of the thirteenth aspect, the method comprises comparing the level and/or activity of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof against one or more standard.

In a fourteenth aspect, the invention provides a method for breeding animals, the method comprising at least the step of selecting a first animal if it has a level and/or activity of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof which is indicative of an animal who is not a carrier for offspring mortality and mating said first animal with a second animal.

In one embodiment of the fourteenth aspect, the method further comprises selecting a second animal if it has a level and/or activity of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof which is indicative of an animal who is not a carrier for offspring mortality.

In one embodiment of the fourteenth aspect, the method comprises observing the level and/or activity of one or more of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof in the first and/or second animal. In one embodiment, the method comprises comparing the level and/or activity of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof against one or more standard.

In a fifteenth aspect, the invention provides a method for selecting or rejecting one or more cell or embryo, the method comprising at least the step of observing the level and/or activity of one or more of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof.

In one embodiment of the fifteenth aspect, the method comprises comparing the level and/or activity of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof against one or more standard.

In one embodiment of the fifteenth aspect, one or more cell or embryo is selected if the level and/or activity of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof from said one or more cell or embryo, or an animal from which it is derived, is indicative of an animal who is not a carrier for offspring mortality. In one embodiment, one or more cell or embryo is rejected if the level and/or activity of OBFC 1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof from said one or more cell or embryo, or an animal from which it is derived, is indicative of an animal who is a carrier for offspring mortality.

In one embodiment of the fifteenth aspect, the method is performed for the purpose of selecting or rejecting one or more cell or embryo for use in cloning an animal and/or breeding an animal. In one embodiment, breeding an animal may involve IVF.

In a sixteenth aspect, the invention provides a method for breeding animals, the method comprising at least the step of selecting a first gamete if the level and/or activity of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof is indicative of an animal who is not a carrier for offspring mortality and fusing said first gamete with a second gamete to form a zygote.

In one embodiment of the sixteenth aspect, the method further comprises selecting the second gamete if the level and/or activity of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof is indicative of an animal who is not a carrier for offspring mortality.

In a seventeenth aspect, the invention provides a method of breeding an animal, the method comprising at least the step of selecting an embryo if the level and/or activity of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof is indicative of an animal who is not a carrier for offspring mortality.

In an eighteenth aspect, the invention provides a method of cloning an animal the method comprising at least the step of selecting one or more cell where the level and/or activity of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof is indicative of an animal who is not a carrier for offspring mortality.

In one embodiment of the tenth to eighteenth aspects, the methods comprise at least the steps of: a) detecting one or more of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding one or more thereof in a one or more cell, embryo or sample from an animal; and, π

c) comparing the level and/or activity of the one or more of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding one or more thereof against one or more standard.

In one embodiment of the tenth to eighteenth aspects, the one or more standard comprises a level and/or level of activity of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof which is associated with an animal or animals which is/are known not to be a carrier/carriers for offspring mortality. In one embodiment, a lower level and/or level of activity of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof compared to the standard infers that an animal is more likely than not a carrier for offspring mortality. In one embodiment, a lower level and/or level of activity compared to the standard infers that an animal is a carrier for offspring mortality. In one embodiment of the twelfth or fifteenth aspects, an animal, one or more cell or embryo is rejected if it has a lower level and/or level of activity of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof compared to the standard.

In one embodiment of the tenth to eighteenth aspects, the one or more standard comprises a level and/or level of activity of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof which is associated with an animal or animals which is/are known to be a carrier/carriers for offspring mortality. In one embodiment, a higher level and/or level of activity of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof compared to the standard infers that an animal is more likely than not, not to be a carrier for offspring mortality. In one embodiment, a higher level and/or level of activity compared to the standard infers that an animal is not a carrier for offspring mortality. In one embodiment, substantially the same or a lower level and/or level of activity of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof compared to the standard infers that an animal is more likely than not to be a carrier for offspring mortality. In one embodiment, a substantially similar, substantially the same or a lower level and/or level of activity compared to a standard infers that an animal is a carrier for offspring mortality. In one embodiment of the twelfth or fifteenth aspects, an animal, one or more cell or embryo is selected if it has a higher level and/or level of activity of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof compared to the standard. In one embodiment of the twelfth or fifteenth aspects, an animal, one or more cell or embryo is rejected if it has substantially similar, substantially the same or a lower level and/or level of activity of OBFCl , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof compared to the standard.

In one embodiment of the tenth to eighteenth aspects, the level of OBFCl, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof is the level of expression thereof. In another embodiment, the method involves observing the level of activity of OBFCl, one or more precursor thereof, one or more isoform thereof, and/or one or more fragment thereof.

In a nineteenth aspect, the invention provides a method for determining whether or not an animal carries a biological marker linked to offspring mortality, the method comprising at least the step of analysing the amino acid sequence of one or more OBFCl, one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof from said animal, wherein where it includes one or more variation in the amino acid sequence which disrupts OBFCl, it is determined to carry a biological marker linked to offspring mortality.

In a twentieth aspect, the invention provides a method for determining whether an animal is or is more likely than not a carrier for offspring mortality, the method comprising at least the step of analysing the amino acid sequence of one or more OBFCl , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof from said animal to determine whether or not it includes one or more variation which disrupts OBFCl , wherein where it includes one or more variation in the amino acid sequence which disrupts OBFCl , it is determined to be a carrier or to be more likely than not to be a carrier for offspring mortality.

In one embodiment of the nineteenth or twentieth aspects, where the amino acid sequence of one or more OBFCl , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof is truncated it is inferred that the animal is more likely than not a carrier for offspring mortality. In one embodiment, it is inferred that the animal is a carrier for offspring mortality. In one embodiment of the nineteenth or twentieth aspects, where the one or more OBFC1, one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof comprises the amino acid sequence of SEQ ID No. 4 or a part thereof the animal is more likely than not a carrier for offspring mortality. In one embodiment, it is inferred that the animal is a carrier for offspring mortality.

In a twenty first aspect, the invention provides a method for selecting or rejecting an animal, the method comprising at least the step of analysing the amino acid sequence of one or more OBFC1, one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof to determine whether or not it includes one or more variation in the amino acid sequence which disrupts OBFC1 , wherein the presence of one or more variation which disrupts OBFC1 infers that the animal is more likely than not a carrier for offspring mortality. In one embodiment, the presence of one or more variation which disrupts OBFC1 infers that the animal is a carrier for offspring mortality.

In one embodiment of the twenty first aspect, an animal is selected if it does not have one or more variation which disrupts OBFC1. In one embodiment, an animal is rejected if it does have one or more variation which disrupts OBFC1.

In one embodiment of the twenty first aspect, where the amino acid sequence of one or more OBFC1 , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof is truncated it is inferred that the animal is more likely than not a carrier for offspring mortality and the animal may be rejected. In one embodiment, it is inferred that the animal is a carrier for offspring mortality and the animal may be rejected.

In one embodiment, where the one or more OBFC1 , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof comprises the amino acid sequence of SEQ ID No, 4 or a part thereof the animal is more likely than not a carrier for offspring mortality and the animal may be rejected. In one embodiment, it is inferred that the animal is a carrier for offspring mortality and the animal may be rejected.

In one embodiment of the twenty first aspect, the method is performed for the purpose of selecting or rejecting an animal for milking purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting an animal for beef farming. In another embodiment, the method is performed for the purpose of selecting or rejecting an animal for breeding purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting an animal for inclusion in a herd. In a twenty second aspect, the invention provides a method for estimating the worth of an animal and/or its offspring, the method comprising at least the step of analysing the amino acid sequence of one or more OBFCI , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof from said animal to determine whether or not it includes one or more variation which disrupts OBFCI, wherein the presence of one or more variation which disrupts OBFCI infers that the animal is more likely than not to be a carrier for offspring mortality. In one embodiment, the presence of one or more variation which disrupts OBFCI infers that the animal is a carrier for offspring mortality

In one embodiment of the twenty second aspect, where the amino acid sequence of one or more OBFCI , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof is truncated it is inferred that the animal is more likely than not a carrier for offspring mortality. In one embodiment, it is inferred that the animal is a carrier for offspring mortality.

In one embodiment, where the one or more OBFCI, one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof comprises the amino acid sequence SEQ ID No. 4 or a part thereof the animal is more likely than not a carrier for offspring mortality. In one embodiment, it is inferred that the animal is a carrier for offspring mortality.

In a twenty third aspect, the invention provides a method for breeding animals which comprises selecting at least a first animal that does not have one or more amino acid variation which disrupts OBFCI and mating said first animal with a second animal.

In one embodiment of the twenty third aspect, the method further comprises the step of selecting the second animal on the basis that it does not have one or more variation which disrupts OBFCI .

In one embodiment of the twenty third aspect, where the amino acid sequence of one or more OBFCI , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof is truncated it is inferred that the animal is more likely than not a carrier for offspring mortality and the animal is not selected. In one embodiment, it is inferred that the animal is a carrier for offspring mortality and the animal is not selected.

In one embodiment, where the one or more OBFCI, one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof comprises the amino acid sequence SEQ ID No. 4 or a part thereof the animal is more likely than not a carrier for offspring mortality and the animal is not selected. In one embodiment, it is inferred that the animal is a carrier for offspring mortality and is not selected.

In another embodiment, if the animal has the wild-type amino acid sequence of OBFCl, at least in the region of the alteration described herein, the animal may be selected.

In a twenty fourth aspect, the invention provides a method for selecting or rejecting one or more cell or embryo, the method comprising at least the step of analysing the amino acid sequence of one or more OBFCl , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof from said one or more cell or embryo, or an animal from which the one or more cell or embryo is derived, to determine whether or not it includes one or more variation which disrupts OBFCl .

In one embodiment of the twenty fourth aspect, the absence of one or more variation which disrupts OBFCl infers that the one or more cell or embryo is suitable for use (or at least more suitable for use than one or more cell or embryo having one or more variation which disrupts OBFCl) in a method for breeding or cloning an animal which is more likely than not, not to be a carrier for offspring mortality. In one embodiment, the absence of one or more variation which disrupts OBFCl infers that the one or more cell or embryo is suitable for use (or at least more suitable for use than one or more cell or embryo having one or more variation which disrupts OBFCl) in a method for breeding or cloning an animal which is not a carrier for offspring mortality.

In one embodiment of the twenty fourth aspect, one or more cell or embryo is selected if it, or an animal from which it is derived, does not have one or more variation which disrupts OBFCl . In one embodiment, one or more cell or embryo is rejected if it, or an animal from which it is derived, has one or more variation which disrupts OBFCl .

In one embodiment of the twenty fourth aspect, the invention provides a method for selecting or rejecting one or more cell or embryo, the method comprising at least the step of analysing the amino acid sequence of one or more OBFCl , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof from said one or more cell or embryo, or an animal from which the one or more cell or embryo is derived, to determine whether or not OBFCl is truncated. In one embodiment, the one or more cell or embryo is rejected if OBFCl is truncated. In one embodiment, one or more cell or embryo is rejected where one or more OBFCl , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof comprises the amino acid sequence of SEQ ID No. 4 or a part thereof.

In one embodiment of the twenty fourth aspect, the method is performed for the purpose of selecting or rejecting one or more cell or embryo for use in cloning an animal and/or breeding an animal. In one embodiment, breeding an animal may involve IVF.

In a twenty fifth aspect, the invention provides a method for breeding animals, the method comprising at least the step of selecting a first gamete that does not have one or more amino acid variation which disrupts OBFCl and fusing said first gamete with a second gamete.

In one embodiment of the twenty Fifth aspect, the method further comprises selecting the second gamete where it does not have one or more amino acid variation which disrupts OBFCl .

In a twenty sixth aspect, the invention provides a method of breeding an animal, the method comprising at least the step of selecting an embryo that does not have one or more amino acid variation which disrupts OBCF1.

In a twenty seventh aspect, the invention provides a method of cloning an animal, the method comprising at least the step of selecting one or more cell that does not have one or more amino acid variation which disrupts OBFCl .

In one embodiment of the nineteenth to twenty seventh aspects, the methods of the invention involve analysing the amino acid sequence of one or more OBFCl , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof from said animal to determine whether or not it includes one or more variation which disrupts OBFCl alone or in combination with one or more other biological marker.

In a twenty eighth aspect, the invention provides a method of forming a herd, the method comprising at least the steps of:

a. performing a method of any one or more of the first, second, third, fourth, sixth, tenth, eleventh, twelfth, thirteenth, fifteenth, nineteenth, twentieth, twenty first, twenty second, twenty fourth aspects of the invention;

b. selecting or rejecting an animal based on the results of step a.;

c. forming a herd of selected animals. In one embodiment of the twenty eighth aspect, an animal is rejected if it is inferred to be a carrier or to be more likely than not to be a carrier for offspring mortality. In one embodiment, an animal is selected if it is inferred not to be a carrier or to be more likely than not, not a carrier for offspring mortality.

In certain embodiments of the twenty eighth aspect, an animal is rejected if it is includes one or more of:

one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith;

one or more amino acid variation which disrupts OBFC 1 ; and,

a level or activity of OBFCl , one or more a precursor thereof, one or more isoform thereof, one or more fragment thereof and/or one or more nucleic acid encoding any one or more thereof is indicative of an animal or animals who is or is more likely than not to be a carrier for offspring mortality.

In certain embodiments of the twenty eighth aspect, an animal is selected if it:

does not have one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith; and,

does not have one or more amino acid variation which disrupts OBFCl ; and/or,

has a level or activity of OBFCl , one or more a precursor thereof, one or more isoform thereof, one or more fragment thereof and/or one or more nucleic acid encoding any one or more thereof which is indicative of an animal or animals who is or is more likely than not, not to be a carrier for offspring mortality.

In a twenty ninth aspect, the invention provides a method for identifying one or more genetic variation in the OBFCl gene which infers that an animal is or is more likely than not to be a carrier of offspring mortality. In one embodiment, the method comprises identifying one or more genetic variation in the OBFCl gene which disrupts the gene. In one embodiment, where the one or more alteration results in a decrease in the level and/or activity of OBFCl it is inferred that the animal is more likely than not to be a carrier of offspring mortality. In one embodiment, where the one or more alteration results in a decrease in the level and/or activity of OBFCl it is inferred that the animal is a carrier of offspring mortality.

In a thirtieth aspect, the invention provides a method for identifying whether or not an animal (and/or its offspring), one or more cell or embryo has or may have one or more genetic alteration which is linked to offspring mortality the method comprising observing a nucleic acid sequence to identify whether or not it includes one or more genetic variation in the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, where the nucleic acid is identified to have one or more genetic variation in the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith, the animal, cell or embryo is identified as having one or more genetic alteration that is linked to offspring mortality. In one embodiment, the one or more genetic alteration disrupts the OBFCl gene.

In a thirty first aspect, the invention provides a method for identifying one or more variation in one or more OBFCl, one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof which infers that an animal is or is more likely than not to be a carrier of offspring mortality. In one embodiment, the method comprises identifying one or more variation which disrupts OBFCl . In one embodiment, where the one or more variation results in a decrease in the level and/or activity of OBFCl it is inferred that the animal is more likely than not to be a carrier of offspring mortality. In one embodiment, where the one or more variation results in a decrease in the level and/or activity of OBFCl the animal is inferred to be a carrier of offspring mortality.

In a thirty second aspect, the invention provides a method for identifying whether or not an animal (and/or its offspring), one or more cell or embryo has or may have one or more amino variation which is linked to offspring mortality the method comprising observing the amino acid sequence of one or more OBFCl, one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof to determine whether or not it includes one or more variation. In one embodiment, where the one or more OBFCl , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof is identified to have one or more variation the animal, cell or embryo is identified as having one or more variation that is linked to offspring mortality. In one embodiment, the one or more amino acid variation disrupts OBFCl .

In one embodiment of the methods of the first to thirty second aspects of the invention the animal is, or the one or more cell or embryo is from, a mammal. In one particular embodiment, the animal, one or more cell or embryo is from the Bovidae family. In one embodiment, the animal, one or more cell or embryo is bovine. In a particular embodiment the bovine is Bos taurus or Bos indicus. In a particular embodiment, the bovine is chosen from the group consisting Jersey, Holstein-Friesian or crossbred dairy cattle. In one embodiment, the methods of the first to thirty second aspects of the invention may further involve analysing one or more additional biological markers. In one embodiment, the one or more biological markers is one or more genetic markers.

In one embodiment, the methods of the first to thirty second aspects of the invention may further involve taking a sample from the animal.

In another aspect, the invention provides an isolated nucleic acid encompassing the genetic marker 24720155TTdel on chromosome 26 of Bos taurus and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, the invention provides a nucleic acid comprising or consisting the sequence of SEQ ID No. 2 or a functionally equivalent variant thereof.

In one embodiment, the nucleic acid is chosen from the group comprising DNA, cDNA and mRNA.

In another aspect, the invention provides other isolated nucleic acids and/or peptides and/or proteins as may be described herein.

In another aspect, the invention provides a peptide comprising or consisting the sequence of SEQ ID No. 4. In one embodiment, the invention provides a fragment, isoform or precursor of the peptide comprising or consisting the sequence of SEQ ID No. 4 or a part of any one or more thereof or a functionally equivalent variant thereof.

In other aspects, the invention provides:

An animal selected by a method of any one or more of the third, twelfth and twenty first aspects;

One or more cell or embryo selected by a method of any one or more of the sixth, fifteenth or twenty fourth aspects;

Offspring or an animal produced by a method of any one or more of the fifth, seventh, eighth, ninth, fourteenth, sixteenth, seventeenth, eighteenth, twenty third, twenty fifth, twenty sixth and twenty seventh aspects.

In aspects of the invention involving selection or rejection of an animal, one or more cell, embryo, or gamete and/or breeding and/or cloning, the absence of one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith infers that the animal, one or more cell, gamete or embryo is suitable for use (or at least more suitable for use than an animal, one or more cell, gamete or embryo that has one of more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith) in a method for breeding or cloning an animal which is more likely than not, not to be a carrier for offspring mortality. In one embodiment, the absence of one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith infers that the animal, one or more cell, gamete or embryo is suitable for use (or at least more suitable for use than an animal, one or more cell, gamete or embryo that has one of more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith) in a method for breeding or cloning an animal which is not a carrier for offspring mortality.

In aspects of the invention involving the selection or rejection of an animal, one or more cell, gamete or embryo, and/or breeding and/or cloning, the presence of one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith infers that the animal, one or more cell, gamete or embryo is not suitable for use (or at least less suitable for use than an animal, one or more cell, gamete or embryo that does not have one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith) in a method for breeding or cloning an animal which is more likely than not, not to be a carrier for offspring mortality. In one embodiment, the presence of one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith infers that the animal, one or more cell, gamete or embryo is not suitable for use (or at least less suitable for use than an animal, one or more cell, gamete or embryo that does not have one or more genetic variation which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith) in a method for breeding or cloning an animal which is not a carrier for offspring mortality.

The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all

combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

FIGURES

These and other aspects of the present invention, which should be considered in all its novel aspects, will become apparent from the following description, which is given by way of example only, with reference to the accompanying figures, in which:

Figure 1 : shows SEQ ID No. 1 - as described further herein after. Figure 2: shows SEQ ID No. 2 - as described further herein after.

Figure 3: shows SEQ ID No. 3 - as described further herein after.

Figure 4: shows SEQ ID No. 4 - as described further herein after.

PREFERRED EMBODIMENT(S)

The following is a description of the present invention, including preferred embodiments thereof, given in general terms. The invention is further elucidated from the disclosure given under the section "Examples" which provides, inter alia, data supporting the invention.

The inventors have identified a genetic variation in the OBFCl (oligonucleotide/oligosaccharide- binding fold containing 1) gene which they believe is embryonic lethal (when two copies are in an animal, it dies in utero or dies at birth). The inventors contemplate that methods involving the analysis of nucleic acid to determine whether or not it includes such an alternation can be used, inter alia, to determine whether or not an animal is or is at least likely to be a carrier for offspring mortality. The inventors also contemplate that the analysis of one or more genetic marker which is in linkage disequilibrium with the specific marker they have identified may also be used for the same purpose. In addition, haplotypes including the genetic marker of the invention and/or one or more marker in linkage disequilibrium therewith may also be used for this purpose.

The specific genetic variation identified by the inventors' results in a frame-shift mutation which alters the amino acid sequence of OBFCl . This alteration is predicted to result in the introduction of a new stop codon 28 amino acids downstream, resulting in a truncated protein. Accordingly, methods of the invention may comprise the analysis of the amino acid sequence of OBFCl, fragments, isoforms, and/or precursors thereof, and/or of the levels (including amount, expression and activity levels) of OBFCl and observing the size of OBFCl , fragments, isoforms, precursors thereof and/or a nucleic acid encoding any one or more thereof.

This is the first time that alterations in OBFCl have been associated with offspring mortality, and thus the first time a link between OBFCl and offspring mortality has been made. Accordingly, the inventors contemplate that any genetic alteration which disrupts this gene may be lethal to an animal which is homozygous for it. As such, the invention should be taken to encompass methods involving the analysis of nucleic acid from an animal to determine whether or not it includes any such alteration or variation in the gene, analysis of the amino acid sequence of OBFCl (including reference to fragments, isoforms, and/or precursors thereof) to determine whether or not it includes any alteration that disrupts OBFCl, and/or the level (including the amount and level of expression, for example) and/or activity of OBFCl to determine whether or not an animal is or is at least likely to be a carrier for offspring mortality. While the description which follows may focus on the analysis of the nucleotide sequence or amino acid sequence at a particular position, it should be understood to extend to the analysis of the sequence at any other position within the gene or OBFC1 protein.

Analysis of one or more biological marker of OBFC1 in accordance with the invention may also be used for selecting or rejecting one or more cell or embryo. Such analysis may aid in methods for breeding or cloning animals, for example.

Analysis of one or more biological marker in accordance with the invention may assist in: predicting phentotypic performance, including use in production management systems known as Marker Assisted Selection; the selection or rejection of animals for breeding and/or other purposes; managing animals in order to maximise their individual potential performance and value; estimating the worth or economic value of an animal; improving profits related to selling animals and/or products produced from the animals; improving the genetics of a population of animals by selecting and breeding desirable animals; generating and maintaining herds of animals; cloning animals likely to have or not have a specific trait; predicting the suitability of an animal and/or its offspring to use in different industries and/or breeding programmes or cloning. It should be appreciated that animals may be screened any time during their life, including early at birth or as embryos or foetuses, to predict life time performance and segregated or managed to suit their genotype and therefore predicted phenotype. Animals may be tested or screened at any time during their life, including but not limited to early at birth, as gametes, zygotes, embryos, foetuses.

Definitions

The term "biological marker(s)" as used herein should be taken broadly and includes, for example, genetic markers, the level of one or more protein (including reference to one or more fragments thereof, one or more precursors thereof, one or more isoforms thereof) or a nucleic acid encoding one or more protein (including reference to one or more fragments thereof, one or more precursors thereof, one or more isoforms thereof), the level of expression of one or more gene or protein (including reference to one or more fragments thereof, one or more precursors thereof, one or more isoforms thereof), the level of activity of one or more protein (including reference to one or more fragments thereof, one or more precursors thereof, one or more isoforms thereof) and/or variation in the amino acid sequence of a protein (including reference to one or more fragments thereof, one or more precursors thereof, one or more isoforms thereof) which may include observation of the size of a protein (including reference to one or more fragments thereof, one or more precursors thereof, one or more isoforms thereof) or nucleic acid. The term "genetic marker" as used herein refers to nucleic acids or specific genetic loci (including specific nucleotide positions) that are polymorphic or contain sequence alterations or variations within a population, the alleles of which can be detected and distinguished by one or more analytic methods. The term "genetic marker" further includes within its scope a plurality of genetic markers co- segregating, in the form of a "haplotype". In this context, the term "haplotype" refers to a plurality of genetic markers that are generally inherited together. Typically, genetic markers within a haplotype are in linkage disequilibrium. Reference herein to analysing a nucleic acid to determine the nucleotide sequence of a "genetic marker" or at a particular genetic position should be taken to include analysing and determining the nucleotide sequence on either strand of the nucleic acid. Further, reference to nucleotide or nucleic acid "sequence" should be taken broadly to include both a single nucleotide as well as two or more nucleotides.

The term "single nucleotide polymorphism" (SNP) refers to nucleic acid sequence variations that occur when a single nucleotide in the genome sequence is altered. A single nucleotide polymorphism may also be a single nucleotide insertion or deletion. The different nucleotides within a SNP are referred to as an allele.

The term "genotype" as used herein means the genetic constitution or nucleotide sequence at one or more genetic locus, in particular the nucleotide sequence of an allele of a genetic locus.

"Linkage disequilibrium" should be taken broadly to refer to the tendency of the presence of an allele at one genetic locus to predict the presence of an allele at one or more other genetic loci (for example a distinct genetic marker). The genetic loci need not necessarily be on the same chromosome.

However, in a preferred embodiment, the genetic loci are located on the same chromosome.

One measure of linkage disequilibrium is DELTA², which is calculated using the formula described by Devlin et al (Genomics 29 (2):311-22 (1995)), and is a measure of how well an allele X at a first genetic loci predicts the occurrence of an allele Y at a second genetic loci. A DELTA² value of 1.0 indicates the prediction is perfect (for example, if Y is present then X is present). It should be appreciated that reference to linkage disequilibrium herein should not be taken to imply a DELTA² value of 1.0. In particular embodiments, the linkage disequilibrium between an allele at one genetic locus and an allele at a second genetic locus, has a DELTA² value of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and most preferably 1.0. Skilled persons will readily appreciate methods for determining whether any two alleles are in linkage disequilibrium. However, by way of example, see Genetic Data Analysis II, Weir, Sinauer

Associates, Inc. Publishers, Sunderland, Mass., 1996.

A "variation in the amino acid sequence" of OBFCl, isoform, fragment and/or precursor thereof should be considered broadly to include any change in the amino acid sequence. By way of example only, it should be taken to include subsitution of any one or more amino acid, addition of one or more amino acid and/or deletion of one or more amino acid.

A "genetic variation" in the OBFCl gene should be considered broadly to include any change in the nucleotide sequence. By way of example only, it should be taken to include subsitution of any one or more nucleotide, addition of one or more nucleotide and/or deletion of one or more nucleotide.

Where the invention is described in terms of identifying or determining whether or not a nucleic acid includes one or more genetic variation or a peptide or protein includes one or more variation in the amino acid sequence, it should be appreciated that such variation is a difference in the nucleic acid or amino acid sequence compared to the nucleic acid sequence associated with one or more animal which is not a known carrier for offspring mortality. Typically, the variation will be determined in relation to a "reference sequence". In certain embodiments, the reference sequence is a nucleic acid encoding part or all of a OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith or the amino acid sequence of OBFCl , an isoform, a precursor or a fragment thereof, which is associated with an animal that is not a known carrier for offspring mortality. In one embodiment, the nucleic acid reference sequence is SEQ ID No. 1 or a part thereof. In one embodiment, the amino acid reference sequence is SEQ ID No. 3 or a part thereof. In other embodiments, the reference sequence could be a nucleic acid or peptide or protein having a sequence which is known to be associated with an animal or animals who is/are a carrier(s) for offspring mortality. Accordingly, reference to "variation" should not be restricted to mean that the nucleic acid, peptide or protein being analysed is different from the reference sequence.

Certain embodiments of the invention may be described herein with reference to "a part" of OBFCl , a fragment, precursor or isoform thereof. In one embodiment, such a "part" comprises or consists at least 4 consecutive amino acids of SEQ ID No. 4, or at least 10, 20, 30, 40, 50, 60, 70, 80 or more consecutive amino acids of SEQ ID No. 4. In one embodiment, such a "part" comprises or consists at least 4 consecutive amino acids of SEQ ID No. 4, or at least 10, 20, 30, 40, 50, 60, 70, 80 or more consecutive amino acids of SEQ ID No. 4 which includes one or more amino acid variation which disrupts OBFCl . In certain embodiments, "a part" of OBFCl, a fragment, precursor or isoform thereof comprises or consists at least the amino acids from position 127 to at least 1 4 of SEQ ID No. 4.

Certain embodiments of the invention may be described herein with reference to "a region" of the OBFCl gene. In one embodiment, the region will encompass a part of a OBFCl gene in which one or more genetic alteration which disrupts the OBFCl gene resides. In one particular embodiment, the region will encompass a part of a OBFCl gene in which the genetic marker 24720155TTdel and/or one or more marker in linkage disequilibrium therewith resides. In one embodiment, such a region comprises or consists at least 4 consecutive nucleotides of SEQ ID No. 2, or at least 10, 20, 30, 40, 50, 60, 70 80 or more consecutive nucleotides of SEQ ID No. 2. In one embodiment, a "region" of the gene comprises or consists of the nucleotides encoding amino acids from position 127 to 154 of SEQ ID No. 4.

A genetic alteration or variation which "disrupts" the OBFCl gene, may be any genetic change which has an affect on the level, expression or activity of the OBFCl gene product (including reference to isoforms, fragments and/or precursors thereof). By way of example, it may decrease the level of expression or alter the structure or function of the gene product. Similarly, a variation or alteration in the amino acid sequence which disrupts OBFCl may be any change in the amino acid sequence of OBFCl which has an effect on the level or level of activity of OBFCl (including reference to isoforms, fragments and/or precursors thereof). The term "disrupts" should not be taken to imply that there is substantially no OBCF1 or activity, although this may be preferred, but rather taken to encompass any change in the level or actvity, in one embodiment a decreased level or level of activity.

One can readily determine whether a variation disrupts OBFCl or the OBFCl gene using standard assays known in the art. However, by way of example, expression levels can be measured using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), northern blotting, microarray analysis, RNA sequencing for measuring cDNA levels and western blotting, PAGE, mass

spectrometry, and immunoprecipitation for measuring protein levels.

In general, where reference is made to an "increase" or "decrease" in the level or activity of OBFCl , an isoform thereof, a fragment thereof, a precursor thereof, and/or a nucleic acid encoding any one or more thereof it should be taken broadly to include any increase or decrease in said level compared to a reference animal or animals or a standard. In one particular embodiment, the reference animal or animals are those having wild-type OBFCl . In another embodiment, the reference animal or animals are those having one or more genetic or amino acid variation which is associated with offspring mortality. Reference may also be made herein to a "higher" or a "lower" level or activity of OBFC1, an isoform thereof, a fragment thereof, a precursor thereof, and/or a nucleic acid encoding any one or more thereof compared to a reference animal or animals or a standard. This should not be taken to imply a particular level or activity of OBFC1 , an isoform thereof, a fragment thereof, a precursor thereof, and/or a nucleic acid encoding any one or more thereof. One can readily determine whether a variation results in an increase or decrease in the level or activity of OBFClor a higher or lower level or activity of OBFC1 compared to a standard using standard assays known in the art, including those techniques exemplified herein after.

In one embodiment the inventors contemplate that the level or level of activity of OBFC1 in a cell or animal having one or more variation which disrupts OBFC1 and/or the OBFC1 gene will be at least approximately 20% lower than an animal or animals which do not have one or more genetic variation which disrupts the OBFC1 gene (including reference to the same animal if it it did not have one or more such variation). In one particular embodiment, the inventors contemplate that the level would be significantly lower.

Reference to the "OBFC1 gene" should be taken to include reference to the coding and non-coding regions of the gene, including upstream and downstream regulatory elements.

While the inventors have identified the biological markers of the invention in bovine animals, they contemplate that it is equally applicable to a variety of different mammals. Various mammals can suffer from "offspring mortality". Accordingly, the term "animal" is used herein primarily in reference to mammals. In one particular embodiment, the mammal is one within within the Bovidae family. In particular embodiments, the animal is a bovine animal. More particularly the animal is Bos taurus or Bos indicus. In one particular embodiment the animal is a beef or dairy breed. By way of further example, the animal may be chosen from the group of animals including, but not limited to, Jersey, Holstein-Friesian, Ayrshire, crossbred dairy cattle, Angus, Hereford, Simrnental and crossbred beef cattle.

"Offspring mortality" should be taken broadly to mean the death of an animal prior to or after birth, including for example, abortion or prenatal death, perinatal death, and neonatal death. In certain preferred embodiments, it relates to abortion or prenatal death. In another embodiment, it relates to perinatal death (for example, for a bovine animal, stillborn after 270 days of gestation up until approximately 24 hours after birth). In another embodiment, it encompasses neonatal death (for example, death between 1 an 28 days of age). In another embodiment, it relates to death between 1 and 6 months. A "functionally equivalent variant" of any particular nucleic acid, protein or peptide referred to herein should be taken broadly to encompass any nucleic acid, peptide or protein whose sequence may vary from the specific sequence provided but which nucleic acid, peptide or protein retains substantially the same function; for example, in the case of an oligonucleotide used to detect a genetic marker of the invention, the ability to bind to a particular target nucleic acid or prime a particular reaction with the desired specificity). The phrase "functionally equivalent" should not be taken to imply that the variant has the same level of activity as the nucleic acid, peptide or protein of which it is a variant, although this may be desired. In one embodiment, "functionally equivalent variants" of any particular nucleic acid, peptide or protein will have at least approximately 80%, approximately 90%, approximately 95%, or approximately 99% sequence homology or similarity to the sequence of which they are a variant. In one particular embodiment, the "functionally equivalent variants" of any particular nucleic acid, peptide or protein will have at least approximately 80%, approximately 90%, approximately 95%, or approximately 99% sequence identity to the sequence of which they are a variant.

As used herein the "worth" of an animal refers to an index used to evaluate the value of an animal, for breeding purposes, inclusion in a herd, herd management, for example. The "worth" is the sum of the estimated value of one or more characteristics which may be associated with the animal, typically weighted by an economic value. Exemplary characteristics include milk fat, protein, milk volume, liveweight, fertility, and milk somatic cells. Additional characteristics may include, for example, disease susceptibility, size, mortality rates and health. The term "worth" should be taken to encompass "breeding worth" and other known indexes used to assess the value of an animal. Persons skilled in the art to which the invention relates will readily appreciate methods and formulae suitable for estimating breeding worth on the basis of any number of different characteristics. Results, data and/or information generated by a method of the invention may be used in calculations for estimating "worth".

It should be appreciated that where methods of the invention relate to breeding animals, any appropriate breeding methods may be utilised including for example natural insemination, artificial insemination and in vitro fertilisation. Accordingly, the word "mating" should be construed broadly and not limited to the physical pairing of two animals.

As noted herein previously herein, the methods of the invention may be used to identify animals suitable for cloning. They may also be used during cloning processes, to determine for example whether or not one or more cell, embryo or cloned animal has one or more genetic variation in the OBFC1 gene. Any appropriate cloning method could be used. However, by way of example, such cloning techniques include somatic cell nuclear transfer, chromatin transfer, and embryo splitting. Persons of general skill in the art will readily appreciate appropriate somatic cell nuclear transfer and chromatin transfer methodologies. However, by way of example, the methods described in the following publications may be used: Bovine somatic cell nuclear transfer, Ross PJ and Cibelli, JB 2010. Methods in Molecular Biology 636: 155-177; and, Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattle. Mesquita FS, Machado SA, Drnevich J, Borowicz P, Wang Z, Nowak RA. Anim Reprod Sci. 2013 Jan 30; 136(4):231-44. doi:

10.1016/j .anireprosci.2012.10.030. Epub 2012 Nov 8.

Where IVF is employed in the context of the invention, any appropriate IVF methodology may be used, as will be apparent to persons of general skill in the art to which the invention relates. However, by way of example, appropriate methods are described, for example, in: Imai K, Tagawa M, Yoshioka H, Matoba S, Narita M, et al. (2006) The efficiency of embryo production by ovum pick-up and in vitro fertilization in cattle. J Reprod Dev 52: 19-29.

Methods of the invention may involve taking a sample from an animal to be tested. The sample may be any appropriate tissue or body fluid sample. In one embodiment, the sample is one or more cell, blood, muscle, bone, somatic cell(s), saliva, or semen. Such samples can be taken from the animal using standard techniques known in the art. It should be appreciated that a sample may be taken from an animal at any stage of life, including prior to birth; by way of non-limiting example, a zygote, an embryo, a feotus. Individual gametes could also be tested using the methods of the invention. This may assist in breeding and/or cloning programmes. Accordingly, "sample" should be taken to include a zygote, embryonic tissue, foetal tissue and gametes. A sample may also be taken after the death of an animal. The samples are analysed using techniques which allow for the observation or analysis of one or more biological marker, as will be described further herein after.

In addition, it should be appreciated that where analysis or observation of one or more biolocial marker in an animal is conducted during gestation, the analysis or observation could be conducted by analysing protein, peptide, nucleic acid or one or more cell of that animal that may be present in the maternal blood supply, placenta, amniotic fluid or any other maternal tissue or fluid prior to birth of the animal. Accordingly, reference to analysing a nucleic acid from an animal, analysing OBFC1 , a precursor, an isoform and/or fragment thereof from an animal, observing the level or activity of one or more OBFC1 , a precursor, an isoform and/or fragment thereof in an animal, and the like, should be taken to include reference to analysing and/or observing one or more of these from that animal that may be present in a maternal tissue or fluid. "Embryo" should be taken broadly to include an organism from the first division of the zygote. In certain embodiments, an embryo is an organism between the first division of the zygote until the time it becomes a foetus. Reference to an "embryo" should be taken to include reference to an organism at different developmental stages, including a blastula, blastocyst, gastrula, and morula for example.

In one aspect, the invention provides methods for the selection or rejection of one or more cells. In certain embodiments, such "cells" may include a gamete (for example, sperm or ovum) or zygote. Selection of such cells may be of use in an IVF program, for example. In other embodiments, such "cells" may be somatic cells, embryonic cells, embryonic stem cells, cells in a cell line, one or more cells of use in cloning, for example. Selection of these cells may be of use in cloning procedures, or preparing cell lines for use in cloning and other procedures, for example.

Certain aspects and embodiments of the invention may be described herein with reference to "fusing a first and second gamete" to form a zygote. This phrase should be taken broadly to include fertilisation processes, such as may be used in in vitro fertilisation processes. Skilled persons will readily appreciate standard means of "fusing" gametes to form a zygote.

For ease of reference, the methods of the invention may be described herein after in terms of analysing a biological marker (such as a nucleic acid sequence, amino acid sequence, level of a protein and peptide or activity level of a protein or peptide) in or from an "animal" or to determine whether or not an "animal" has a particular marker linked to offspring mortality or determining the genotype of an "animal", and the like. It should be appreciated that the methods of the invention are also applicable to analysing and determining whether or not individual cells, including gametes, and embryos may have relevant biological markers. Accordingly, reference to "animals" should also be taken to include reference to one or more cell or embryo, unless the context requires otherwise.

The invention may be described herein with reference to an amino acid or nucleic acid sequence, or a level or level of activity of OBFCl (including reference to one or more isoform, precursor or fragment thereof) being "indicative" of an animal (or animals) who is or is more likely than not to be a carrier for offspring mortality or being "indicative" of an animal who is not or is more likely than not, not to be a carrier for offspring mortality. This can be taken to mean that a nucleic acid or amino acid sequence includes one or more variation (or genetic marker in linkage disequilibrium therewith) which disrupts OBFCl or the OBFCl gene and is known to be associated with such animal or animals. In the case of the level or level of activity of OBFCl (including reference to one or more isoform, precursor or fragment thereof), "indicative" should not be taken to mean that the level is exactly the same as a level associated with such animal or animals. However, in one embodiment the level or level of activity is substantially similar or substantially the same as that associated with such animal or animals. Reference to "animal or animals" should be taken to mean that a particular reference or standard is based on a value from a single animal or pooled or averaged from a group of animals.

Genetic marker(s)

In one aspect, a method of the invention involves the analysis of a nucleic acid (including one or more nucleic acid) from an animal to determine whether or not it includes one or more genetic marker as herein before described (for example, one or more genetic alteration which disrupts the OBCF1 gene and/or a genetic marker in linkage disequilibrium therewith). In one embodiment, the one or more genetic marker is any alteration that results in a frameshift of the protein coding sequence. In one embodiment, the one or more genetic marker is any alteration that results in truncation of the protein which it encodes. In one embodiment, the one or more genetic marker is located in a region encoding amino acids from position 127 to 370 of OBFC1.

The specific marker identified by the inventors is a 2 base pair deletion of TT in the TTT polynucleotide tract located from position 24720155-24720157 within the OBFC1 gene on chromosome 26 of Bos taurus. The two alleles of this marker may be referred to herein as 24720155TTdel (the 2bp deletion mutant), and 24720155TTwt (the wild type or reference allele). However, it is noted that different nucleotide numbering systems can be used to describe insertion/deletion polymorphisms. For the avoidance of ambiguity, the polymorphism could also be referred to as 24720154CTT>C. The Entrez Gene ID for the OBFC1 gene is 514213 (http://www.ncbi.nlm. nm.gov/gene/?term=STNl_BOVIN). The sequence and position is based on the genomic sequence of chromosome 26 in bovine build UMD3.1 (AC_000183.1 in the GenBank database http://www.ncbi.nlm.nih.gov/). Further sequence information is provided in SEQ ID No. 1 (wild-type) and SEQ ID No, 2 (mutant) herein after.

It will be appreciated that the precise location of a particular genetic marker of the invention may vary slightly from genome to genome; for example, in a different species of animal, or different breed of animal, the location of the marker may vary. However, persons of skill in the art to which the invention relate will be able to readily identify a particular marker in different genomes through routine sequence alignment and with knowledge that it resides in the OBFC1 gene. To account for this variation in the location of a genetic marker across genomes reference to a position of a particular marker herein should be taken to mean a position "corresponding to" a particular position of chromosome 26 of Bos taurus" in the UMD3.1 genome build. For example, reference to the genetic marker 24720155TTdel on chromosome 26 of Bos Taurus should be taken to mean that the genetic marker is at a position corresponding to position 24720155 on chromosome 26.

While the inventors have observed that the genetic marker 24720155TTdel is indicative of offspring mortality, they contemplate that any variation in the nucleotide sequence at this genetic position may be indicative of an animal being a carrier for offspring mortality. The invention should be interpreted accordingly.

In one embodiment, the methods involve the analysis of a nucleic acid (including one or more nucleic acid) to determine whether or not it includes the genetic marker 24720155TTdel on chromosome 26 of Bos taurus. Alternatively, or in addition, the methods may involve analysing the nucleotide sequence of a nucleic acid to determine the nucleotide sequence of one or more genetic marker in linkage disequilibrium with this genetic marker. In one particular embodiment, the methods of the invention may involve analysing the nucleotide sequence of a nucleic acid to determine the haplotype. In one embodiment, the haplotype includes the genetic marker 24720155TTdel on chromosome 26 of Bos taurus.

In one embodiment, where the genetic marker 247201 5TTdel and/or one or more genetic marker in linkage disequilibrium therewith is present it is inferred that an animal is more likely than not a carrier for offspring mortality. In one embodiment, it is inferred that an animal is a carrier for offspring mortality. In such cases, the animal may be rejected for use in a herd or breeding programme, for example. In another embodiment, if the animal has the wild-type allele of the genetic marker, or the nucleic acid sequence encoding OBFCl , at least in the region of the specific alteration described herein, the animal may be selected for use in a herd or breeding programme, for example. The "region of the specific alteration" is intended to mean the nucleic acid sequence encoding amino acids from position 127 to 370 of a wild-type OBFCl (SEQ ED No. 3, for example).

The genetic marker identified by the inventors results in a frame shift in the region encompassing amino acids 127 to 1 4 of OBFCl . Accordingly, the methods of the invention could involve the analysis of any one or more nucleotides in the region encoding amino acids 127 to 154, wherein one or more variation in this region suggests the presence of one or more genetic marker which disrupts the OBFCl gene. In one embodiment, the methods of the invention could involve analysis of one or more nucleotides in the region encoding amino acids 127 to 370, wherein the presence of one or more variation in this region suggests the presence of one or more genetic marker which disrupts OBFC 1 As a genetic alteration which disrupts the OBFC1 gene may involve insertion and/or deletion of one or more nucleotide (for example the TT deletion of the specific marker identified by the inventors), it should be appreciated that the methods of the invention may involve analysis of the size of the OBFC1 gene or a part or region thereof.

It should also be appreciated that one could analyse the nucleic acid sequence of either strand of the nucleic acid to identify the sequence at a particular genetic locus or osition; for example, instead of analysing the strand associated with the sequence variant listed above, the nucleotide sequence of the opposite or complementary strand of DNA could be analysed. Persons of skill in the art will readily appreciate nucleic acid sequence variations on such opposite strand which correlate with the genotypes mentioned above, having regard to the information contained herein and nucleic acid base pairing principles (ie, A pairs with T and C pairs with G). SEQ ID No. 1 and 2 herein after reflect the minus strand of the OBFC1 gene. Skilled persons will readily appreciate the sequence of the plus strand. Accordingly, while the invention may be described herein in terms of determining whether or not a nucleic includes a specific genetic marker (such as the 2472015 STTdel allele) it should be read to encompass the step of determining the genetic marker by observing the sequence on either strand.

As noted above, the invention also encompasses use of one or more genetic markers which are in linkage disequilibrium with a marker of the invention. Such markers may be analysed instead of or in addition to a genetic marker of the invention.

Nucleic acids can be analysed to determine the genotype/sequence of the genetic markers described herein according to any appropriate technique. Such techniques include for example polymerase chain reaction (PCR), including allele-specific PCR, gel electrophoresis, the use of oligonucleotide probe hybridisation, Southern blotting, direct sequencing, restriction digestion, restriction fragment length polymorphism (RFLP), single-strand confirmation polymorphism (SSCP), LCR (ligase chain reaction), denaturing gradient gel electrophoresis (DGGE), the use of allele-specific oligonucleotides (ASOs), the use of proteins which recognize nucleic acid mismatches, such as Exoli mutS protein, RNAse protection assays, oligonucleotide array hybridisation (for example microarray), denaturing HPLC (dHPLC), fluorescence quenching PCR (TaqMan™ Applied Biosystems, CA 94404, USA), High Resolution Melting (HRM), and matrix-assisted laser desorption/ionisation time-of-flight mass spectroscopy (MALDI-TOF MS). Combinations of two or more of such techniques may be used. Such combination may increase the sensitivity of the analysis being conducted.

The technique(s) used will depend on the nature of the marker to be detected as will be appreciated by skilled persons. For example, single nucleotide polymorphisms (SNPs), may be analysed using those techniques capable of resolving a single nucleotide difference between sequences; for example, direct sequencing or LCR, allele-specific PCR, RFLP, SSCP, DGGE, using allele-specific oligonucleotides (ASOs), or proteins which recognize nucleic acid mismatches, oligonucleotide array hybridisation, dHPLC, fluorescence quenching PCR and matrix MALDI-TOF MS,

Any one or more of the techniques mentioned hereinbefore (including for example, SSCP, RFLP, DGGE, dHPLC and direct sequencing) may be used to analyse genetic markers which may include insertion or deletion of one or more nucleotide.

It should be appreciated that certain of the techniques of use in analysing a genetic marker in accordance with the invention will utilise one or more oligonucleotides which hybridise to a genetic region encompassing the marker, adjacent to the marker, or flanking the marker. Such oligonucleotides may be DNA, RNA or derivatised forms thereof and include nucleic acid primers, such as PCR and LCR primers, and nucleic acid probes.

Persons of ordinary skill in the art to which the invention relates will readily appreciate appropriate oligonucleotides of use in the invention having regard to one or more of the nucleic acid sequence of chromosome 26, particularly in the genetic regions proximal to the genetic marker, the nature of the genetic marker to be analysed, and the general principles of nucleic acid hybridisation. The nucleic acids will be capable of hybridising in a specific manner to a target nucleic acid and in the case of primers they will be capable of priming a PCR or like reaction. While such nucleic acids will preferably have 100% complementarity to their target region of the mRNA or cDNA of the protein of interest, they may contain one or more non-complementary nucleotides at a particular position while still substantially retaining specificity for the target nucleic acid to which they are designed to bind. By way of example, the nucleic acids may have approximately 80%, approximately 90%, approximately 95%, or approximately 99% complementarity or homology to its target. By way of further example, in certain cases, the oligonucleotides may be designed such that a mismatch at a particular nucleotide position is indicative of the nature of the genetic marker being analysed (for example, a SNP). By way of example, a mismatch in the nucleotide present at the 3' end of an LCR primer will inhibit the reaction providing an indication of the nature of the nucleotide at that position. Mismatches may similarly be utilised in techniques including RNAse protection assays and allele- specific PCR, as well as in fluorescence quenching PCR, for example. Typically, the nucleic acids will hybridise to their target nucleic acid under stringent hybridisation conditions (see for example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The oligonucleotide probes or primers may be of any length as is appropriate for a particular application, having regard to the sequence of the genetic region to which they are designed to bind. A probe or primer will typically be capable of forming a stable hybrid with the complementary sequence to which it is designed to hybridise. Accordingly, the length is dependent on the nucleic acid composition and percent homology between the oligonucleotide and its complementary sequence, as well as the hybridisation conditions which are utilised (for example, temperature and salt concentrations). Such hybridisation factors are well known in the art to which the invention relates. By way of example, oligonucleotides of use in the present invention may be from 2 to 500 nucleotides in length. In one embodiment, particularly where they are used as primers, the oligonucleotides may be of approximately 15 nucleotides to 30 nucleotides in length.

Oligonucleotide probes and primers of use in the invention may be prepared by any number of conventional DNA synthesis methods including recombinant techniques and chemical synthesis, or they may be purchased commercially. It will be appreciated that the usefulness of any probe or primer may be evaluated, at least notionally, using appropriate software and sequence information for the nucleic acid encoding the protein of interest. For example, software packages such as Primer3 (http://primer3.sourceforge.net/), PC OligoS (National Bioscience Inc), Amplify (University of Wisconsin), and the PrimerSelect program (DNAStar Inc) may be used to design and evaluate primers.

Where amplification techniques (for example PCR) are used in methods of the invention amplification may be conducted according to conventional procedures in the art to which this invention relates, such as described in US Patent No 4,683,202. By way of example PCR reactions will generally include 0.1 μΜ-1 μΜ of each primer, 200μΜ each dNTP, 3-7mM MgCl₂, and 1U Taq DNA polymerase. Further, exemplary PCR cycling conditions include: denaturation at a temperature of approximately 94°C for 30 to 60 seconds, annealing at a temperature calculated on the basis of the sequence and length of the primer (as herein after discussed) for 30 to 60 seconds, and extension at a temperature of approximately 70°C to 72°C for 30 to 60 seconds. By way of example, between 25 and 45 cycles are run.

It will be appreciated by those of ordinary skill in the art that any amplification conditions provided herein are merely exemplary and may be varied so as to optimise conditions where, for example, alternative PCR cyclers or DNA polymerases are used, where the quality of the template DNA differs, or where variations of the primers not specifically exemplified herein are used, without departing from the scope of the present invention. The PCR conditions may be altered or optimised by changing the concentration of the various constituents within the reaction and/or changing the constituents of the reaction, altering the number of amplification cycles, the denaturation, annealing or extension times or temperatures, or the quantity of template DNA, for example. Those of skill in the art will appreciate there are a number of other ways in which PCR conditions may be optimised to overcome variability between reactions.

It will be understood that whilst not specifically exemplified herein, appropriate annealing temperatures for any primer within the scope of the present invention may be derived from the calculated melting temperature of that primer. Such melting temperatures may be calculated using standard formulas, such as that described in Sambrook and Russell, 2001. As will be understood by those of ordinary skill in the art to which this invention relates annealing temperatures may be above or below the melting temperature but generally an annealing temperature of approximately 5°C below the calculated melting temperature of the primer is suitable.

Oligonucleotides used for detection and/or analysis of genetic markers in accordance with the invention may be modified to facilitate such detection. Similarly, nucleic acid products obtained using techniques such as PCR may be modified to facilitate detection and/or analysis. For example, the nucleic acid molecules may be labelled to facilitate visual identification using techniques standard in the art. By way of example nucleic acids may be radio-labelled using P³² as may be described in Sambrook and Russell, 2001. Further, nucleic acids may be appropriately labelled for use in colorigenic, fluorogenic or chemiluminescence procedures.

It will be appreciated that the methods of the invention may employ one or more control samples. Such control samples may be positive or negative controls for a particular genetic marker. The type of control samples used may vary depending on such factors as the nature of the genetic marker being analysed and the specific technique being used for such detection and analysis. Positive controls may include samples having known nucleic acid sequences (including nucleic acids of a known size), for example. Negative controls may include samples having no nucleic acid present. By way of general example, positive control samples could include one or more nucleic acids known to have a particular nucleotide sequence at one or more relevant position. In one embodiment, the method may utilise a control sample having a sequence which is associated with an animal being a carrier for offspring mortality. In another embodiment, the method may utilise a control sample having a sequence which is associated with an animal known not to be a carrier for offspring mortality.

The methods of this embodiment of the invention may involve comparing the sequence of a nucleic acid being tested to one or more reference sequences, as herein before described. Typically, the methods of the invention will involve taking a sample from an animal to be tested, as noted herein before.

In order to facilitate detection of a genetic marker in accordance with the invention, a sample may be processed prior to analysis. For example, the sample may be processed to isolate nucleic acid from the sample to be analysed or to amplify a specific genetic region to be analysed.

In one embodiment, nucleic acid is isolated or extracted from the sample prior to analysis. In one embodiment, genomic DNA is isolated or extracted from the sample. In an alternative embodiment, mRNA may be isolated or extracted from the sample. In such a case, the mRNA may be converted to cDNA using reverse transcription techniques known in the art. Techniques for isolating nucleic acids from samples will be readily appreciated by skilled persons. By way of Example, methods of use in isolating nucleic acids are described in Sambrook and Russell, 2001.

In an alternative form of this embodiment of the invention analysis of the nucleic acid may occur in situ obviating the need to extract nucleic acid from the sample. This may be done using PCR for example. Skilled persons will readily appreciate appropriate techniques and methodology to this end (see for example, Sambrook and Russell, 2001).

The methods of the invention may be combined with one or more other methods of use in assessing genotype, predicting phenotype, selecting an animal based on certain characteristics, estimating breeding values or estimating worth and the like. Accordingly, the methods of the invention may include, in addition to analysis of a genetic marker identified herein, analysis of additional genetic markers, and/or the level of expression of certain genes/proteins, and/or one or more phenotypic traits, for example.

Peptide and Protein Markers

In another aspect, the methods of the invention comprise analysing one or more of OBFC1 , one or more precursor thereof, one or more isoform thereof, and/or one or more fragment thereof to determine whether or not it includes one or more variation in the amino acid sequence which disrupts OBFC1, in one particular embodiment, one which results in a decrease in the activity of OBFC1. In one particular embodiment, the variation in the amino acid sequence is a deletion of one or more amino acid. In one embodiment, the amino acid sequence of OBFC1 is truncated. Accordingly, it should be appreciated that the methods of the invention may involve analysis of the size of OBFC1 , one or more precursor thereof, one or more isoform thereof, and/or one or more fragment thereof. In one particular embodiment, the presence of an OBFC1 which is smaller than 370 amino acids infers that an animal, cell or embryo carries a biological marker linked to offspring mortality. In one embodiment, where OBFCl is observed to be 154 amino acids in length, it infers that an animal, cell or embryo carries a biological marker linked to offspring mortality.

In one embodiment, the one or more variation in the amino acid sequence which disrupts OBFCl is located in the region encoding amino acids from position 127 to 370 of OBFCL

In one embodiment, the one or more variation results in a frame shift mutation. In one embodiment, the one or more variation results in a frame shift mutation resulting in a truncated protein. In one embodiment, the one or more variation results in a frame shift from amino acid 127 of OBFCl . In one embodiment, the one or more variation results in truncation from an amino acid at position 154 of OBFCl . In one embodiment, the one or more variation results in a frame shift from amino acid 127 and truncation from an amino acid at position 154. In one embodiment, the one or more variation results in a protein or peptide comprising or consisting the amino acid sequence of SEQ ID No. 4.

As the specific variation in OBFCl observed by the inventors results in frame shift from amino acid 127 and truncation from amino acid 154, the methods of the invention could involve analysis of one or more amino acids in this region, wherein one or more variation in this region suggests the presence of one or more amino acid variation which disrupts the OBFCl gene.

In one embodiment, where the amino acid sequence of one or more OBFCl , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof is truncated it is inferred that an animal is more likely than not a carrier for offspring mortality. In one embodiment, it is inferred that an animal is a carrier for offspring mortality. In one embodiment, where the one or more OBFCl , one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof comprises or consists the amino acid sequence SEQ ID No. 4 or a part thereof the animal is more likely than not a carrier for offspring mortality. In one embodiment, it is inferred that an animal is a carrier for offspring mortality. In such cases, the animal may be rejected for use in a herd or breeding programme, for example. In another embodiment, if an animal has the wild-type amino acid sequence, at least in the region of the alteration described herein, the animal may be selected for used in a herd or breeding programme. The "region of the alteration" is intended to mean the amino acid sequence from position 127 to position 370 of a wild-type OBFCl {SEQ ID No. 3, for example).

OBFCl , isoforms thereof, precursors thereof, and/or fragments thereof may be analysed using standard techniques known in the art. However, by way of example, peptide sequencing methods, mass spectrometry, Western blotting and ELISA could be used. The methods may employ one or more control samples, such as positive and/or negative controls for a particular amino acid sequence variation. The type of control samples used may vary depending on such factors as the type of variation being analysed and the specific technique being used for detection and analysis. Positive controls may include samples having known amino acid sequences (including proteins and peptides of a known size), for example. Negative controls may include samples having no peptide present. In one embodiment, the method may utilise a control sample having a sequence which is associated with an animal being a carrier for offspring mortality. In another embodiment, the method may utilise a control sample having a sequence which is associated with an animal known not to be a carrier for offspring mortality.

The methods of this embodiment of the invention may involve comparing the sequence of a peptide or protein being tested to one or more reference sequences, as herein before described.

The methods of the invention may involve taking a sample for an animal to be tested. In order to facilitate analysis of a peptide in accordance with the invention, a sample may be processed prior to analysis according to any of a number of known methods. For example, the sample may be processed to remove one or more one or more high abundance proteins that might make it difficult to analyse OBFC1 , an isoform, fragment or precursor thereof. Exemplary techniques which may be employed to process a sample prior to analysis of OBFC1, an isoform, fragment or precursor thereof are described elsewhere herein.

Level of Peptides and Proteins

In another aspect, the methods of the invention involve observing the level of one or more of OBFC1 (including reference to any one or more isoform of OBFC1, any one or more precursor of OBFC1 , any one or more fragment of OBFC1), and/or any one or more nucleic acid encoding one or more of the foregoing.

The inventors contemplate that a decrease in the level (for example, a decrease in the level of expression) of OBFC1 (including reference to one or more isoform, precursor or fragment thereof) and/or a nucleic acid encoding same infers an animal will more likely than not be a carrier of offspring mortality. In one embodiment, the inventors contemplate that any decrease in the level may be considered to infer that an animal is a carrier for offspring mortality. In one embodiment, at least a 20% decrease in the level may be considered to infer that an animal is or is more likely than not a carrier for offspring mortality compared to an animal or animals thai do not carry a biological marker linked to offspring mortality (including reference to the same animal if it did not have such biological marker).

The methods of this embodiment of the invention will typically involve taking a sample from an animal, observing the level (in one embodiment the level of expression) of OBFC1 (including reference to one or more isoform, precursor and/or fragment thereof) or nucleic acids encoding same, and comparing the level against one or more standard. In one embodiment, a difference in the level between the sample and the one or more standard infers whether the animal will more likely than not be a carrier or is a carrier of offspring mortality, or the animal is determined to carry a biological marker linked to offspring mortality.

In one embodiment, a standard comprises a level of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof which is associated with an animal or animals which is/are known not to be a carrier/carriers for offspring mortality. In one embodiment, a lower level of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof from the animal compared to the standard infers that the animal is more likely than not a carrier for offspring mortality, or the animal is determined to carry a biological marker linked to offspring mortality. In one embodiment, a lower level of OBFC1 compared to the standard infers that the animal is a carrier for offspring mortality.

In one embodiment, a standard comprises a level of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof which is associated with an animal or animals which is/are known to be a carrier/carriers for offspring mortality. In one embodiment, substantially similar, substantially the same or a higher level of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof from the animal compared to the standard infers that the animal is more likely than not, not to be a carrier for offspring mortality. In one embodiment, substantially similar, substantially the same or a higher level compared to a standard infers that the animal is not a carrier for offspring mortality. In one embodiment, substantially similar, substantially the same or a lower level of OBFC1, one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof from the animal compared to the standard infers that the animal is more likely than not to be a carrier for offspring mortality. In one embodiment, substantially similar, substantially the same or a lower level compared to a standard infers that the animal is a carrier for offspring mortality. In practise, the method of this aspect of the invention may comprise using two or more standards. For example, it may comprise measuring the level of one or more of OBFC1 , one or more precursor thereof, one or more isoform thereof, one or more fragment thereof, and/or one or more nucleic acid encoding any one or more thereof across both one or a pool of wild-type animals and one or a pool of animals known to carry an alteration which disrupts OBFCL The samples from the animals to be tested would then be compared with the pooled values to determine whether they are indicative of an animal carrying one or more alteration or indicative of a wild-type animal.

Where it is inferred that an animal is or is more likely than not, not to be a carrier for offspring mortality, the animal may be selected for inclusion in a herd or breeding programme, for example. Where it is inferred that an animal is or is more likely than not to be a carrier for offspring mortality, the animal may be rejected for inclusion in a herd or breeding programme, for example.

OBFC1 (including reference to one or more precursors, fragments and/or isoforms thereof) and nucleic acids encoding same may be detected and the levels thereof compared to a standard using any one or a combination of techniques which are of use in identifying, quantifying and/or highlighting differential levels or expression of one or more proteins. Such techniques will be readily appreciated by persons of ordinary skill in the art to which the invention relates. However, by way of example, the levels of OBFC1 (including reference to one or more precursors, fragments and/or isoforms thereof) may be measured using protein purification methods, immunological techniques, separation of proteins based on characteristics such as molecular weight and isoelectric point including gel electrophoresis (for example, PAGE, including 2D PAGE) and microfluidics-based technologies as for example in gel-free protein separation techniques, and mass spectrometry (MS) utilizing isobaric label based MS such as iTRAQ or label-free approaches such as multiple reaction monitoring (MRM).

Appropriate immunological techniques include enzyme linked immunosorbent assay (ELISA) (sandwich ELISA, double sandwich ELISA, direct ELISA, microparticle ELISA), radioimmunoassay (RIA), immunoprecipitation, Western blotting, immunohistochemical staining, antibody arrays, or agglutination assays. Protocols for carrying out such techniques are readily available; for example, see "Antibodies a Laboratory Manual", Cold Spring Harbor Laboratory Press (1988).

Antibodies of use in such immunological techniques may be purchased commercially or produced according to standard methodology in the art having regard to the nature of the proteins to be tested. For example, polyclonal antibodies and monoclonal antibodies may be produced in accordance with the procedures described in the text "Antibodies a Laboratory Manual" (Cold Spring Harbor Laboratory Press, 1988) using one or more of the proteins or a fragment thereof as antigen.

Preferably monoclonal antibodies are used.

Nucleic acid-based techniques of use in determining the level of a nucleic acid (for example cDNA levels) may include differential display procedures, Northern Blotting, competitive PCR, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), microarray analysis, and RNA sequencing. Persons skilled in the art to which the invention relates will readily appreciate methodology for performing these techniques.

Nucleic acids, such as oligonucleotide probes and primers, of use in detecting expression levels of proteins in accordance with the invention (for example using Northern blotting or competitive PCR) will be readily appreciated by skilled persons having regard to the information contained herein and any published amino acid and/or nucleic acid sequence information for OBFCl . The nucleic acids will be capable of hybridising in a specific manner to an mRNA or cDNA associated with OBFCl and in the case of primers they will be capable of priming a PCR or like reaction.

Mass spectroscopy techniques of use in the invention are described for example in "Proteins and proteomics-A laboratory manual" (RJ Simpson, Cold Spring Harbour Laboratory Press (2002).

The difference in the levels of OBFCl (including reference to one or more fragment, precursor and/or isoform) or nucleic acids encoding same in a sample versus a standard may be compared using standard technology having regard to the method employed to detect the protein or nucleic acid. For example, colorimetric and fluorometric techniques may be used in which a detection molecule (such as an antibody or nucleic acid probe or primer) is labelled with a molecule which can be visualised by the naked eye or otherwise detected using a spectrophotometer, or fluorometer for example.

Alternatively, detection molecules could be labelled with radio-isotopes. Incorporating labels into nucleic acids during PCR amplification where it is employed (as opposed to labelling a detection molecule such as a probe or primer), is also contemplated.

Methods for labelling molecules and subsequently measuring the intensity of signals generated will be known to those of skill in the art to which the invention relates.

It should be appreciated that in addition to analysing samples and standards, the methods of the invention may include the testing of one or more positive or negative control samples to ensure the integrity of the results. For example, one could include a sample containing no protein/nucleic acid and one or more samples containing a known level of protein/nucleic acid so that results can be calibrated across different runs of the method. The sample may be processed prior to analysing OBFCl (including reference to one or more isoform, precursor and/or fragment) and/or a nucleic acid encoding same to facilitate analysis of the proteins or nucleic acids. Skilled persons will readily appreciate appropriate processing steps and techniques suitable for performing them.

In one embodiment, high abundance proteins which have the potential to make it difficult to analyse, such as detect and/or measure the level of OBFCl (including reference to one or more isoform, precursor and or fragment) may be removed from the sample. For example, Top6 or Top7 depletion may be used. The sample may also be subject to proteolytic digestion. As such detection of a protein or isoform in accordance with the invention should be taken to include detection of any one or more fragments thereof. Fragments should be of a length sufficient to ensure specificity to OBFCl . Such fragments will for example be at least 8 amino acids in length, more preferably at least 10, 15 or 20 amino acids in length.

Processing steps for preparing the sample for analysis of nucleic acids encoding OBFCl (including reference to one or more isoform, precursor and/or fragment) may include lysing cells, isolating mR A, and generating cDNA using standard procedures such as reverse transcription-PCR as will be known in the art to which the invention relates. In one embodiment, mRNA may be observed in situ.

Skilled persons may readily appreciate other means by which the sample may be processed for use in the invention.

Activity of Proteins and Peptides

In another aspect, methods of the invention may involve observing the level of activity of OBFCl (including reference to one or more precursor, isoform and/or fragment thereof).

The inventors contemplate that a decrease in the level of activity of OBFCl (including reference to one or more isoform, precursor or fragment thereof) infers an animal will more likely than not be a carrier for offspring mortality. In one embodiment, the inventors contemplate that any decrease in the level of activity may be considered to infer that an animal is a carrier of offspring mortality. In one embodiment, at least a 20% decrease in the level of activity may be considered to infer that an animal is or is more likely than not a carrier for offspring mortality compared to an animal or animals that do not carry a biological marker linked to offspring mortality (including reference to the same animal if it did not have such biological marker). The methods of this embodiment of the invention may involve taking a sample from an animal, observing the level of activity of OBFCl (including reference to one or more isoform, precursor and/or fragment thereof), and comparing the level of activity against one or more standard. In one embodiment, a difference in the level of activity between the sample and the one or more standard infers whether the animal will more likely than not be a carrier or is a carrier for offspring mortality, or an animal is determined to carry a biological marker linked to offspring mortality.

In one embodiment, a standard comprises a level of activity of OBFCl , one or more precursor thereof, one or more isoform thereof, and/or one or more fragment thereof which is associated with an animal or animals which is/are known not to be a carrier/carriers for offspring mortality. In one embodiment, a lower level of activity of OBFCl , one or more precursor thereof, one or more isoform thereof, and/or one or more fragment thereof from the animal compared to the standard infers that the animal is more likely than not a carrier for offspring mortality, or an animal is determined to carry a biological marker linked to offspring mortality. In one embodiment, a lower level of activity compared to a standard infers that the animal is a carrier for offspring mortality.

In one embodiment, a standard comprises a level of activity of OBFCl , one or more precursor thereof, one or more isoform thereof, and/or one or more fragment thereof which is associated with an animal or animals which is/are known to be a carrier/carriers for offspring mortality. In one embodiment, a higher level of activity of OBFCl, one or more precursor thereof, one or more isoform thereof, and/or one or more fragment thereof from the animal compared to the standard infers that the animal is more likely than not, not to be a carrier for offspring mortality. In one embodiment, a higher level of activity compared to a standard infers that the animal is not a carrier for offspring mortality. In one embodiment, substantially similar, substantially the same or a lower level of activity of OBFCl , one or more precursor thereof, one or more isoform thereof, and/or one or more fragment thereof from the animal compared to the standard infers that the animal is more likely than not to be a carrier for offspring mortality. In one embodiment, substantially similar, substantially the same or a lower level of activity compared to a standard infers that the animal is a carrier for offspring mortality.

In practise, the method of this aspect of the invention may comprise using two or more standards. For example, it may comprise measuring the activity of one or more of OBFCl , one or more precursor thereof, one or more isoform thereof, and/or one or more fragment thereof, across both one or a pool of wild-type animals and one or a pool of animals known to carry an alteration which disrupts OBFCl . The samples from the animals to be tested would then be compared with the pooled values to determine whether they are indicative of an animal carrying one or more alteration or indicative of a wild-type animal. Where it is inferred that an animal is or is more likely than not, not to be a carrier for offspring mortality, the animal may be selected for inclusion in a herd or breeding programme, for example. Where it is inferred that an animal is or is more likely than not to be a carrier for offspring mortality, the animal may be rejected for inclusion in a herd or breeding programme, for example.

The level of activity of OBFCl (including reference one or more isoforms, fragments and/or precursors) may be measured using standard methodology as known in the art, having regard to the function of OBFCl . By way of example, the methods used may involve one or more of the following techniques: Immunoprecipitation, Western blotting, ELISA, mass spectrometry, surface plasmon resonance, isothermal titration calorimetry, luciferase assays and reporter gene assays.

The difference in the level of OBFCl (including reference to one or more fragment, precursor and/or isoform) activity in a sample versus a standard may be compared using standard technology having regard to the method employed to detect the activity. For example, colorimetric and fluorometric techniques may be used in which a detection molecule (such as an antibody or nucleic acid probe or primer) is labelled with a molecule which can be visualised by the naked eye or otherwise detected using a spectrophotometer, or fluorometer for example. Alternatively, detection molecules could be labelled with radio-isotopes. However, by way of example the methodology described in Perrot- Applanat et al, 1997 Mol Endo 11 (8) may be used.

It should be appreciated that in addition to analysing samples and standards, the methods of the invention may include the testing of one or more positive or negative control samples to ensure the integrity of the results. For example, one could include a sample containing no protein and one or more samples containing a protein with a known level of activity so that results can be calibrated across different runs of the method.

The sample may be processed prior to analysing OBFCl (including reference to one or more isoform, precursor and/or fragment) activity. Skilled persons will readily appreciate appropriate processing steps and techniques suitable for performing them.

In one embodiment, high abundance proteins which have the potential to make it difficult to analyse, such as detect and/or measure the level of activity of OBFCl (including reference to one or more isoform, precursor and/or fragment) may be removed from the sample. For example, Top6 or Top7 depletion may be used. By way of further example, immunoprecipitation of the protein of interest could also be used. Processing steps for preparing the sample for analysis of OBFC1 (including reference to one or more isoform, precursor and/or fragment) activity may include cell lysis, immunoprecipitation and preparation of cell membranes, for example. Persons skilled in the art will readily appreciate other useful techniques that may be used.

Breeding and cloning:

As mentioned herein before, methods of the invention may be used in breeding programmes and for selecting animals (including selecting their gametes, for example) for such purposes. Such methods may comprise identifying at least one first animal that is inferred not to be a carrier or not to be more likely than not to be a carrier of offspring mortality (using one or more method as described herein) and mating said animal with a second animal. In one embodiment, the method may comprise further identifying at least one second animal that is inferred not to be a carrier or not to be more likely than not to be a carrier of offspring mortality (using one or more method as described herein). For example, a method of the invention may comprise selecting a first animal (and optionally also a second animal) identified not to have one or more biological marker linked to offspring mortality. In a preferred embodiment, the mating will produce one or more offspring.

The invention also encompasses breeding methods which comprise: 1) selecting a first gamete and/or a second gamete using a method of the invention and fusing said first gamete with said second gamete to form a zygote; 2) selecting an embryo using a method of the invention. The invention also provides a method of cloning an animal comprising selecting one or more cell using a method of the invention. One or more methods of the invention (such as those for selecting or rejecting one or more cell or embryo) may be used to identify and select appropriate gametes and embryos for these breeding methods. For example, a method of the invention may comprise selecting a gamete, embryo or cell identified not to have one or more biological marker linked to offspring mortality.

In the breeding methods of the invention, animals may be mated using any appropriate methods including naturally, artificial insemination or IVF. In such cases, individual gametes may be selected for use in the process. Such gametes may be selected using a method of the invention; for example, a method of the invention may be used to identify animals that are inferred not to be a carrier or not to be more likely than not to be a carrier of offspring mortality and gametes from those animals selected for use in a breeding program or process or gametes may be tested in accordance with the invention and then selected for use in a breeding program or process. In one particular embodiment, a method of selecting or rejecting one or more animal (according to the third, twelfth and/or twenty first aspects of the invention described herein before, for example) could be used to select the first and/or second animal and their gametes used in IVF. In another embodiment, a method of selecting or rejecting one or more cells (according to the sixth, fifteenth and/or twenty fourth aspects of the invention described herein before, for example) could be used to select a first and/or second gamete and selected gametes used in IVF. Following selection of male and female gametes, the female gamete is fertilised in vitro. At the relevant time, one or more embryo is transferred to a gestational carrier.

In one embodiment, in vitro fertilisation of a female gamete may occur and then a method of the invention used to determine whether or not an embryo has a desired genotype/phenotype and should be selected or rejected for further use in a breeding programme. This might occur where individual gametes, or animals from which they have been obtained or derived, have not been tested to determine if they are likely not to be a carrier for offspring mortality prior to fertilisation (accordingly, the invention should be taken to include methods of breeding where the first and/or second animal and/or gametes are not selected on the basis of such a test, but a resulting embryo or offspring is tested and selected). Alternatively, a method of the invention could be used where the individual gametes or animals from which they have been obtained or derived have been tested and selected on the basis of having a desirable genotype/phenotype, for quality control purposes or to double check that the resulting embryos have the same desirable genotype/phenotype.

Optionally, following mating of the animals, one or more method of the invention may be used to determine whether or not any offspring has or may be inferred to have the desired characteristics. Such testing may occur at any time during the life of the offspring, including before birth; by way of example only, testing an embryo, a foetus, amniotic fluid, placenta, maternal blood, at birth.

In certain cases, cloning may be used to generate an animal. In such cases, the method may comprise identifying at least one first animal that is inferred not to be a carrier or not to be more likely than not to be a carrier of offspring mortality (using one or more method as described herein) and using the nucleus or chromatin from one or more cell of that animal in a cloning procedure (such as somatic cell nuclear transfer, chromatin transfer techniques, and embryo splitting). Such cloning methods are described, for example, in Bovine somatic cell nuclear transfer Ross PJ and Cibelli, JB 2010. Methods in Molecular Biology 636: 155-177. At the relevant time during the cloning procedure, one or more embryo will be transferred to a gestational carrier. In certain embodiments, a cloning procedure may utilise a cell derived from a cell line and a method of the invention may be used to select such a cell which is, or cell line whose cells are, capable of being used to generate an animal which is not or is more likely than not to be a carrier of offspring mortality. In one embodiment, the cell line may be an embryonic cell line.

One or more cell of use in cloning may be selected using a method of the invention. Following selection of one or more cells a cloning procedure can be conducted. For example, a method of the invention may be used to identify animals that are inferred not to be a carrier or not to be more likely than not to be a carrier of offspring mortality and cells from those animals selected for use in a cloning process. Similarly, a method of the invention may be used to identify cells from a cell line which comprise or do not comprise one or more genetic or amino acid alteration as described herein and may or may not be of use in generating an animal that is more likely than not or is not a carrier of offspring mortality. Methods of the sixth, fifteenth and/or twenty fourth aspects of the invention may be used for such purposes. Methods of the invention could also be used to identify animals whose cells could be used to generate cell lines for cloning purposes.

In one particular embodiment, a method of selecting or rejecting one or more animal (according to the third, twelfth and/or twenty first aspects of the invention described herein before, for example} could be used to select an animal for cloning. In another embodiment, a method of selecting or rejecting one or more cells (according to the sixth, fifteenth and/or twenty fourth aspects of the invention described herein before, for example) could be used to select one or more cells of use in cloning.

Optionally, at various stages during the cloning procedure, one or more method of the invention may be used to determine whether or not any cloned animal may be inferred to be or to be more likely than not a carrier of offspring mortality. Such testing may occur at any time during the life of the cloned animal. By way of example only, testing of a blastocyst, an embryo, a foetus, amniotic fluid, placenta, maternal blood, at birth.

In addition, a cloning method of the invention may involve selecting desirable cells without testing those cells or the animals or cell line from which they came for the presence or absence of a biological marker associated with offspring mortality. The cloning procedure can be initiated and then a method of the invention used to determine whether an embryo, foetus or animal resulting from the cloning procedure has a relevant biological marker in accordance with the invention and an embryo, foetus or animal selected where it has a desirable genotype/phenotype. The breeding and cloning methods of the invention may involve subjecting one or more cell, zygote, embryo and/or feotus, for example, to any one of a number of standard growth and/or gestation methods.

Forming a Herd

The invention also provides methods for forming a herd of animals. Such methods may comprise determining whether or not an animal carries a biological marker linked to offspring mortality according to the first, tenth, nineteenth, thirty first and/or thirty second aspect of the invention described herein, determing whether or not an animal (and/or its offspring) is more likely than not to be a carrier for offspring mortality according to the second, eleventh and/or twentieth aspects of the invention described herein, selecting or rejecting an animal according to the third, twelfth and/or twenty first aspects of the invention as described herein, and/or estimating the worth of an animal according to the fourth, thirteenth and/or twenty second aspects of the invention as described herein, for example. In certain embodiments, methods of the invention which involve selecting or rejecting one or more cells may also be used to select one or more animals for inclusion in a herd. Animals may be selected or rejected for inclusion in the herd based on the results of one or more of the aforementioned methods of the invention. In certain embodiments, where an animal is identified to have one or more biological marker in accordance with the invention or is inferred to be or to be more likely than not to be a carrier of offspring mortality, and/or not to be a desirable worth, it may be rejected and not selected for inclusion in the herd. Where an animal is identified not to have one or more biological marker in accordance with the invention or is inferred not to be or to be more likely than not, not to be a carrier of offspring mortality and/or to have a desirable "worth", it may be selected for inclusion in the herd.

Accordingly, in certain embodiments methods of this aspect of the invention involve testing one or more animals or cells in accordance with a method of any one or more of the first, tenth, nineteenth, second, eleventh, twentieth, third, twelfth, twenty first, fourth, thirteenth, twenty second, sixth, fifteenth, twenty fourth, thirty first and thirty second aspects of the invention, selecting animals having a desirable genotype/phenotype or inferred to have one or more desired characteristic or worth and forming a herd with the selected animals.

The invention should also be taken to include a herd formed by the methods described herein.

The herd of animals may be formed for any desirable reason. However, by way of example only, it may desirable to form a herd for: beef farming; milk production. Nucleic Acids, Peptides, Antibodies

The invention also provides nucleic acids carrying one or more genetic marker of the invention. For example, isolated nucleic acids ecompassing a region of a OBFC1 gene in which the genetic marker 24720155TTdel and/or one or more marker in linkage disequilibrium therewith reside are encompassed by the invention. In one embodiment, the nucleic acid comprises or consists of the sequence of SEQ ID No, 2, or is a functionally equivalent variant thereof.

The invention also encompasses nucleic acids which can hydridise, preferably under stringent conditions (as herein before described), to a region of a OBFC1 gene in which the genetic marker 24720155TTdel resides. Such nucleic acids may be used as probe or primers or otherwise in analysis of genetic markers of the invention, as herein before described.

Nucleic acids of the invention may have 100% sequence identity, homology or complementarity to the relevant region of a OBFC1 gene, but may also have some sequence variation. For example, nucleic acids of the invention may have approximately 80%, approximately 90%, approximately 95% or approximately 99% sequence identity, homology or complementarity.

The nucleic acids may be of any appropriate length. In one embodiment, they are at least 4 nucleotides in length, or at least 10, 20, 30, 40, 50, 60, 70, 80 or more nucleotides in length.

By way of example only, nucleic acids of the invention include DNA, mRNA and cDNA.

The invention also provides peptides or proteins encompassing an amino acid variation associated with one or more genetic marker of the invention. These may be of any appropriate length. In one embodiment, they are at least 4 amino acids in length, or at least 10, 20, 30, 40, 50, 60, 70, 80 or more amino acids in length. In one embodiment, the peptide is 154 amino acids in length. In one embodiment, a peptide of the invention comprises or consists the amino acid sequence of SEQ ID No. 4 or is a functionally equivalent variant thereof. In another embodiment, the invention provides an isoform, precursor or fragment of a peptide comprising or consisting the amino acid sequence of SEQ ID No. 4 or a part thereof.

In another embodiment, the invention provides one or more antibodies which bind to a peptide or protein encompassing the amino acid variation associated with one or more genetic marker of the invention. In one embodiment, the one or more antibodies are monoclonal antibodies. In another embodiment, the one or more antibodies are polyclonal antibodies. Kits

The invention also relates to kits which are of use in a method of the invention.

In one embodiment, the kit comprises at least one or more reagents suitable for analysis of one or more biological marker in accordance with the invention. Reagents suitable for analysis of one or more of the markers include one or more nucleic acid probes and/or primers and one or more antibodies, as herein before described. Skilled persons will readily appreciate other appropriate reagents suitable for detecting or observing the biological markers and having regard to the various techniques mentioned herein before.

Reagents of use in processing samples for analysis may also be contained in the kits of the invention. The kits may also comprise one or more standard and/or other controls including nucleic acids, peptides or proteins whose sequence at a particular position is known. Further, kits of the invention can also comprise instructions for the use the components of the kit as well as printed charts or the like that could be used as standards against which results obtained from test samples could be compared. Reagents may be held in any suitable container.

EXAMPLES

The inventors have identified a genetic variation on chromosome 26. The name of the gene that this variation is in is called OBFCl (oligonucleotide/oligosaccharide-binding fold containing 1) and denoted with locus tag of BOS_233S2. fhttp://www.ncbi.nlm.nih.gov/gene/?tenn=STNl BQVEN).

The variation is a 2 base pair deletion of TT (referred to herein as the genetic marker 24720154CTT>C or 24720155TTdel on chromosome 26 of Bos taurus). The data generated and observations made by the inventors indicates that the variation is embryonic lethal; when two copies are in a calf, it dies in utero or dies at birth.

Sequence information for the OBFCl gene is provided herein after:

SEQ ID NO. 1: >bosTau6_refGene_NM_001076849 range=chr26:24699353-24738868.

Genomic sequence (minus strand) indicating the reference form of the OBFCl gene. Exons are provided in uppercase, introns and intergenic sequence is provided in lowercase. TTT tract which is site of TT deletion in mutant OBFCl is highlighted. Bases indicated are 'A's since sequence represents minus strand.

SEQ Π) No. 2: >bosTau6_refGene_NM_001076849 range=chr26:24699353-24738868.

Genomic sequence (minus strand) indicating the OBFCl gene bearing the 24720155TTdel mutation. Exons are provided in uppercase, introns and

intergenic sequence is provided in lowercase. Site of deletion highlighted.

Missing bases are 'AA' since sequence illustrated represents the minus strand.

SEQ Π⁾ No. 3: Protein sequence indicating the reference (wild type) form of OBFC1.

NCBI Reference Sequence: NP_001070317.1

>gi|l 16003915 [ref] P_001070317.11 CST complex subunit STN1 [Bos taurus].

SEQ ID No. 4: Protein sequence indicating the predicted mutant form of OBFC 1. The

highlighted part of the protein indicates the predicted amino acid sequence change resulting from the 24720155TTdel variant, with a new translation frame beginning at amino acid 127. All amino acids downstream of this residue can be expected to change as a result of this new reading frame. The introduction of a predicted new stop codon after amino acid 154 should be noted. This is predicted to result in a large truncation of amino acids relative to the OBFC1 reference sequence (154 amino acids versus 370 for the wild-type form).

The variation has not been seen in the homozygous state in a New Zealand bovine population with over 11 ,000 animals genotyped and 95,000 animals having genotypes imputed. Mating data where carrier sires are mated to daughters of carrier sires have a higher proportion of animals (3.66%) not having a calf in the following season. This is confirmed by 3.15% more animals being recorded as being empty for carrier sire matings to daughters of carrier sires. The inventors also note a 2% lower rate of females being reared when they result from carrier sire matings to daughters of carrier sires. Data generated from 100 sires and dams that are heterozygous for the variation showed no progeny that are homozygous for the variation, when you would expect to see 25 such progeny.

The inventors have observed the variation primarily in the Jersey population, but also see it in Holstein-Friesian and the crossbred populations.

Research and Results

The inventors tested the hypothesis that an animal that has two copies of the variation does not survive. The research programme followed three different strategies to identify if the variation of interest affects fertility and/or calf survival: 1. Over 10,000 animals were genotyped to identify if there were any animals that have two copies of the variation of interest.

2. Sires in the population were genotyped or imputed for the variation of interest. Mating and survival data where carrier sires were mated to daughters of carrier sires was compared to when the carrier sires were mated to daughters of non-carrier sires. The inventors investigated to see if there were more empty cows and lower survival rates of calves.

3. Over 90,000 animals have been genotyped in the population. A number of the animals have both their sire and dam also genotyped. This is termed a trio. The inventors tested to see whether or not, in the situation where both sire and dam are heterozygous (have one copy of the variation), the expected ¼ of animals having two copies of the variation were present in the population.

Genotyping

Genotyping over 1 1,000 animals did not identified any animals that have two copies of the variation (Table 1). It would be expected, given the frequency of the variation, that there would be 12 animals that would have two copies of the variation. To see zero is highly significant with a p-value of 0.0004. The allele frequency is 3%.

Table 1 : Genotype results for the two variations of interest

Chr26:24720154 10841; 0 0.0004

Sires in the population were genotyped or imputed for the variation of interest. Mating data for four seasons (2008-1 1) in the NZ dairy population was investigated. Mating data for carrier sires was extracted and the data divided into two groups:

1. Where carrier sires have been mated to daughters of carrier sires; and,

2. Where carrier sires been mated to daughters of non-carrier sires.

For Group 1 it would be expected that 50% of the daughters would be carriers. When a carrier sire is mated to a carrier female it is expected that 25% of the progeny would be homozygous for the variation. Given 50% of the daughters will be carriers, then 12.5% of the progeny from these matings should be homozygous for the variation. If the variation affects the ability of the embryo/foetus to survive full term then one would expect more of the animals in this group not to have a parturition/calving the next season. If the calf does reach full term then one may see a lower survival rate for this group of calves.

Whereas for Group 2, given that the sire of the cow is not a carrier then the cow can only be a carrier from inheriting it from her dam. The allele frequency for this variation is 3% therefore only 1.5% of these cows would be carriers of the variation and less than half a percent of the resultant progeny would have two copies of the variation.

It is expected, given the allele frequency of the variation, that there would be a 3-4% difference in number of cows having a calving in the following season.

There were 296999 matings from carrier sires over the 2008-11 seasons and 18316 of these matings were to daughters of a carrier sire. The difference between Group 1 (CC) and Group 2 (CN) is 3.66% and is highly statistically significant with a p-value less than 0.0001 (Table 2).

Table 2: Mating data for carrier sires mated to daughters of carrier sires (CC) and daughters of non- carriers (CN)

A number of the animals were culled before the next season. The proportion of the animals in the CC group that were culled due to the animals being empty is 41.03% compared to 37.88% for the CN group.

The fate of calves born is recorded by farmers and has been investigated. The percent of female calves that were reared is 2.2% lower for the calves generated from the matings of carrier sires to daughters of carrier sires compared to matings to daughters of non-carrier sires (Table 3).

Table 3: Survival data for carrier sires mated to daughters of carrier sires (CC) and daughters of non- carriers (CN)

GG 5573 86.7

CN 88937 88.9

p-val <0.0001

Trios

Over 90,000 animals have been genotyped in the population studied. A number of the animals have both their sire and dam also genotyped. This is termed a trio. The inventors tested to see whether or not, in the situation where both sire and dam are heterozygous (have one copy of the variation), the expected ½ of animals having two copies of the variation were present in the population.

For the variation there were 100 trios where the sire and the dam are both heterozygous and one would expect to have 25 progeny that are homozygous for the variation. There are 61 heterozygous 39 homozygous common progeny and no animals homozygous for the variation.

The invention has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation. However, a person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to a certain extent or substituted for known equivalents without departing from the scope of the invention. It should be appreciated that such modifications and equivalents are herein incorporated as if individually set forth. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. Furthermore, titles, headings, or the like are provided to enhance the reader's comprehension of this document, and should not be read as limiting the scope of the present invention.

The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference. However, the reference to any applications, patents and publications in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Throughout this specification and any claims which follow, unless the context requires otherwise, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of "including, but not limited to".

Claims

CLAIMS:

1. A method for determining whether or not an animal carries a genetic marker linked to offspring mortality, the method comprising at least the step of analysing a nucleic acid from said animal to determine whether or not it includes one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith, wherein where the nucleic acid includes one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith, it is determined to carry a genetic marker linked to offspring mortality.

2. A method for determining whether an animal is or is more likely than not a carrier for offspring mortality, the method comprising at least the step of analysing a nucleic acid from said animal to determine whether or not it includes one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith, wherein where the nucleic acid includes one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith, it is determined to be a carrier or to be more likely than not to be a carrier for offspring mortality.

3. A method for selecting or rejecting an animal, one or more cell or embryo, the method comprising at least the step of analysing a nucleic acid from an animal, one or more cell or embryo to determine whether or not it includes one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith.

4. A method as claimed in claim 3 wherein an animal, one or more cell or embryo is selected if it, or an animal from which the one or more cell or embryo is derived, does not have one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith and an animal, one or more cell or embryo is rejected if it, or an animal from which the one or more cell or embryo is derived, has one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith.

5. A method for estimating the worth of an animal and/or its offspring, the method comprising at least the step of analysing a nucleic acid from said animal to determine whether or not it includes one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith, wherein when one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith is present it is inferred that the animal is or is more likely than not to be a carrier for offspring mortality.

6. A method for determining whether or not an animal carries a biological marker linked to offspring mortality, the method comprising at least the step of analysing the amino acid sequence of one or more OBFC1, one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof from said animal to determine whether or not it includes one or more variation in the amino acid sequence which disrupts OBFC1 , wherein where it includes one or more variation in the amino acid sequence which disrupts OBFC1, it is determined to carry a biological marker linked to offspring mortality.

7. A method for determining whether an animal is or is more likely than not a carrier for offspring mortality, the method comprising at least the step of analysing the amino acid sequence of one or more OBFC1, one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof from said animal, wherein where it includes one or more variation in the amino acid sequence which disrupts OBFC1, it is determined to be a carrier or to be more likely than not to be a carrier for offspring mortality.

8. A method for selecting or rejecting an animal, one or more cell or embryo, the method comprising at least the step of analysing the amino acid sequence of one or more OBFC1, one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof from an animal, one or more cell or embryo to determine whether or not it includes one or more variation which disrupts OBFC1.

9. A method as claimed in claim 8 wherein an animal, one or more cell or embryo is selected if it, or an animal from which the one or more cell or embryo is derived, does not have one or more variation which disrupts OBFC1 and an animal, one or more cell or embryo is rejected if it, or an animal from which the one or more cell or embryo is derived, does have one or more variation which disrupts OBFC1.

10. A method for estimating the worth of an animal and/or its offspring, the method comprising at least the step of analysing the amino acid sequence of one or more OBFC1, one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof from said animal to determine whether or not it includes one or more variation which disrupts OBFC1, wherein the presence of one or more variation which disrupts OBFC1 infers that the animal is or is more likely than not to be a carrier for offspring mortality.

11. A method for breeding animals which comprises selecting at least a first animal that does not include one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith and/or one or more amino acid variation which disrupts OBFC1, and mating said first animal with a second animal.

12. A method for breeding animals, the method comprising at least the step of selecting a first gamete that does not have one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith and/or one or more amino acid variation which disrupts OBFC1, and fusing said first gamete with a second gamete to form a zygote.

13. A method of claim 11 or 12, the method further comprising selecting the second animal or second gamete on the basis that it does not include one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith and/or one or more amino acid variation which disrupts OBFC1.

14. A method of breeding an animal, the method comprising at least the step of selecting an

embryo that does not have one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith and/or one or more amino acid variation which disrupts OBFC1.

15. A method of cloning an animal, the method comprising at least the step of selecting one or more cell that does not have one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith and/or one or more amino acid variation which disrupts OBFC1.

16. A method as claimed in any one of claims 1 to 5 and 11 to 15 wherein the one or more genetic variation comprises 24720155TTdel at a position corresponding to position 24720155 on chromosome 26 of Bos taurus and/or one or more genetic marker in linkage disequilibrium therewith.

17. A method as claimed in any one of claims 6 to 16 wherein the one or more amino acid variation results in truncation of OBFC1.

18. A method of forming a herd, the method comprising at least the steps of:

a. performing a method of any one or more of claims 1 to 10;

b. selecting or rejecting an animal based on the results of step a.; and,

c. forming a herd of selected animals.

19. A method as claimed in claim 18 wherein an animal is rejected if it is includes one or more of:

o one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith;

o one or more amino acid variation which disrupts OBFC1 ; and,

o a level or activity of OBFC1, one or more a precursor thereof, one or more isoform thereof, one or more fragment thereof and/or one or more nucleic acid encoding any one or more thereof is indicative of an animal who is a carrier for offspring mortality; and,

an animal is selected if it:

o does not have one or more genetic variation which disrupts the OBFC1 gene and/or one or more genetic marker in linkage disequilibrium therewith;

o does not have one or more amino acid variation which disrupts OBFC1 ; and/or, o has a level or activity of OBFCl, one or more a precursor thereof, one or more isoform thereof, one or more fragment thereof and/or one or more nucleic acid encoding any one or more thereof which is indicative of an animal who is not a carrier for offspring mortality.

20. A method for identifying one or more genetic variation in the OBFCl gene and/or one or more amino acid variation in one or more OBFCl, one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof which infers that an animal is or is more likely than not to be a carrier of offspring mortality, the method comprising identifying one or more genetic variation which disrupts the OBFCl gene and/or one or more amino acid variation which disrupts OBFCl.

21. A method as claimed in claim 20 wherein where the one or more variation results in a decrease in the level and/or activity of OBFCl it is identified as a variation which can infer that an animal is or is more likely than not to be a carrier of offspring mortality.

22. A method for identifying whether or not an animal (and/or its offspring), one or more cell or embryo has or may have one or more genetic alteration and/or one or more amino acid variation which is linked to offspring mortality the method comprising observing a nucleic acid sequence and/or the amino acid sequence of one or more OBFCl, one or more precursor thereof, one or more isoform thereof and/or one or more fragment thereof, to identify whether or not it includes one or more amino acid variation which disrupts OBFCl and/or one or more genetic alteration which disrupts the OBFCl gene and/or one or more genetic marker in linkage disequilibrium therewith.

23. A method as claimed in claim 22 wherein where the one or more variation results in a decrease in the level and/or activity of OBFCl it is identified as a variation which can infer that an animal is or is more likely than not to be a carrier of offspring mortality.

24. An animal selected by a method of any one of claims 3, 4, 8 and 9.

25. One or more cell or embryo selected by a method of any one of claims 3, 4, 8 and 9.

26. Offspring or an animal produced by a method of any one of claims 11 to 17.

27. A herd formed by a method of claim 18 or 19.

28. An isolated nucleic acid encompassing the genetic marker 24720155TTdel which is at a position corresponding to position 24720155 on chromosome 26 of Bos taurus and/or one or more genetic marker in linkage disequilibrium therewith.

29. An isolated nucleic acid as claimed in claim 28 wherein the nucleic acid comprises or consists of the sequence of SEQ ID No. 2 or is a functionally equivalent variant thereof.

30. An isolated peptide comprising or consisting the sequence of SEQ ID No. 4 or a functionally equivalent variant thereof.