Genetic testing for improved cattle fertility

Abstract

Arrays of nucleic acid molecules, kits, methods of genotyping and marker assisted bovine breeding methods based on novel SNPs on genes of the bovine transforming growth factor-β (TGF-β) signaling pathway for improved bovine fertilization rate. The methods and compositions of the present invention are related to SNPs in the DNA-binding protein inhibitor 3 (ID3) gene, and in the bone morphogenetic protein 4 (BMP4) gene corresponding to position 2702 of SEQ ID NO: 2. Also disclosed are methods for determining viability of developing bovine embryos by measuring the expression level of one or more target genes in the TGF- signaling pathway, and selecting for implantation only embryos whose target gene expression level is not up-regulated.

What is claimed is: 
     
         1 . An isolated oligonucleotide molecule consisting of position 1213 of SEQ ID NO: 1 and between 11 and 150 contiguous nucleotides adjacent to position 1213 of SEQ ID NO: 1, wherein position 1213 is cytosine or thymine; or an isolated oligonucleotide molecule consisting of position 2702 of SEQ ID NO: 2 and between 11 and 150 contiguous nucleotides adjacent to position 2702 of SEQ ID NO: 2, wherein position 2702 is cytosine or adenine. 
     
     
         2 . The oligonucleotide molecule according to  claim 1 , which comprises at least about 15 contiguous nucleotides adjacent to position 1213 of SEQ ID NO:1, or to position 2702 of SEQ ID NO: 2. 
     
     
         3 . The oligonucleotide molecule according to  claim 2 , which comprises at least about 20 contiguous nucleotides adjacent to position 1213 of SEQ ID NO:1, or position 2702 of SEQ ID NO: 2. 
     
     
         4 . The oligonucleotide molecule according to  claim 1 , which consists of not more than about 100 nucleotides. 
     
     
         5 . The oligonucleotide molecule according to  claim 1 , which consists of not more than about 50 nucleotides. 
     
     
         6 . The oligonucleotide molecule according to  claim 1 , wherein position 1213 of SEQ ID NO: 1 or position 2702 of SEQ ID NO: 2 is within 4 nucleotides of the center of the oligonucleotide molecule. 
     
     
         7 . The oligonucleotide molecule according to  claim 6 , wherein position 1213 of SEQ ID NO: 1 or position 2702 of SEQ ID NO: 2 is at the center of the oligonucleotide molecule. 
     
     
         8 . The oligonucleotide molecule according to  claim 1 , wherein position 1213 of SEQ ID NO: 1 or position 2702 of SEQ ID NO: 2 is at the 3′-end of the oligonucleotide molecule. 
     
     
         9 . An array of nucleic acid molecules comprising the isolated oligonucleotide molecule according to  claim 1  supported on a substrate. 
     
     
         10 . A kit comprising an isolated oligonucleotide molecule of  claim 1 , and a suitable container. 
     
     
         11 . A method for detecting single nucleotide polymorphism (SNP) in a gene encoding a DNA binding protein inhibitor 3 (ID3 gene), in a bovine cell, the method comprising determining the identity of a nucleotide on the ID3 gene of the cell at a position corresponding to position 1213 of SEQ ID NO: 1, and comparing the identity to the nucleotide identity at a corresponding position of in SEQ ID NO: 1; or a method for detecting single nucleotide polymorphism (SNP) in a gene encoding a bone morphogenetic protein 4 (BMP4 gene) in a bovine cell, the method comprising determining the identity of a nucleotide on the BMP4 gene of the cell at a position corresponding to position 2702 of SEQ ID NO: 2, and comparing the identity to the nucleotide identity at a corresponding position of in SEQ ID NO: 2. 
     
     
         12 . The method according to  claim 11 , wherein the bovine cell is an adult cell, an embryo cell, a sperm, an egg, a fertilized egg, or a zygote. 
     
     
         13 . A method according to  claim 11 , wherein the identity of the nucleotide is determined by sequencing a nucleic acid molecule comprising a suitable portion of the ID3 or BMP4 gene of the cell comprising respectively a position corresponding to position 1213 of SEQ ID NO: 1 or position 2702 of SEQ ID NO: 2. 
     
     
         14 . A method according to  claim 13 , wherein the nucleic acid molecule is isolated from the cell via amplification by the polymerase chain reaction (PCR) of genomic DNA of the cell, or by RT-PCR of the mRNA of the cell. 
     
     
         15 . A method according to  claim 11 , wherein the identity of the nucleotide is determined by hybridizing a suitable probe to a nucleic acid preparation from the cell. 
     
     
         16 . A method according to  claim 15 , wherein the probe is labeled with a detectable label. 
     
     
         17 . A method according to  claim 11 , wherein the identity of the nucleotide of both copies of the ID3 gene or of the BMP4 gene of the cell is determined 
     
     
         18 . A method according to  claim 17 , wherein the identity of the nucleotide of both copies of the sequence is determined based on genotypes of the parent of the cell, genotypes of the daughter of the cell, or both. 
     
     
         19 . A method for determining whether an individual bovine animal is suitable as a gamete donor for natural mating, artificial insemination or in vitro fertilization, the method comprising detecting the SNP according to  claim 11 , and excluding as gamete donor an individual whose genotype is TT at a position in the ID3 gene corresponding to position 1213 of SEQ ID NO: 1, or selecting as a gamete donor an individual whose genotype is TT at a position in the BMP4 gene corresponding to position 2702 of SEQ ID NO: 2. 
     
     
         20 . A method of selecting a bovine embryo for planting in a uterus, the method comprising genotyping the embryo according to  claim 11  while preserving the viability of the embryo, and excluding from planting an embryo whose genotype is TT at a position in the ID3 gene corresponding to position 1213 of SEQ ID NO: 1, or selecting for planting an embryo whose genotype is TT at a position in the BMP4 gene corresponding to position 2702 of SEQ ID NO: 2. 
     
     
         21 . A method for selectively breeding of cattle using a multiple ovulation and embryo transfer procedure (MOET), the method comprising superovulating a female animal, collecting eggs from said superovulated female, in vitro fertilizing said eggs from a suitable male animal, implanting said fertilized eggs into other females allowing for an embryo to develop, and genotyping said developing embryo according to  claim 11 , and terminating pregnancy if the genotype of the developing embryo is TT at a position in the ID3 gene corresponding to position 1213 of SEQ ID NO: 1, or allowing pregnancy to proceed if the genotype of the embryo is TT at a position in the BMP4 gene corresponding to position 2702 of SEQ ID NO: 2. 
     
     
         22 . A method of selecting a bovine embryo for planting in a uterus for further development, the method comprising
 1) Obtaining at least one blastomere from a developing embryo while maintaining the viability of the developing embryo,     2 ) Determining in the blastomere the level of expression of at least a Target Gene selected from the group of genes consisting of DNA-binding protein inhibitor 3 (ID3), thrombospondin-2 (THBS2), bone morphogenetic protein 4 (BMP4), growth differentiation factor-9 (GDF9), BMP binding endothelial regulator (BMPER), and decorin (DCN), SMAD family member 2 (SMAD2), thrombospondin-4 (THBS4), protein phosphatase 2, regulatory subunit A, alpha (PPP2R1A), and ribosomal protein S6 kinase, 70 kDa, polypeptide 2 (RPS6KB2), and   3) Excluding from planting an embryo whose level of expression of at least one of the genes is up-regulated.   
     
     
         23 . A method according to  claim 22 , wherein an embryo is excluded from planting whose level of expression of at least two of the genes is up-regulated. 
     
     
         24 . The method according to  claim 22 , wherein the level of expression of the gene is determined by obtaining cDNA from mRNA extracted from the blastomere, and performing real time qPCR on the cDNA. 
     
     
         25 . The method according to  claim 24 , wherein at least one or more housekeeping gene is used as internal control. 
     
     
         26 . The method according to  claim 22 , wherein a sample of at least 10 embryos are tested simultaneously, and the embryos in the sample are divided into a first group of high Target Gene expression and a second group of low Target Gene expression, and the embryos in the first group are excluded from implanting.