Methods and compositions for genetically detecting improved milk production traits in cattle

Abstract

An isolated nucleic acid molecule comprising a polymorphic site selected from the group consisting of positions 164, 269, 284, 407 and 989 of SEQ ID NO: 1, an array or a kit comprising the same. Also provided are a method for detecting single nucleotide polymorphism (SNP) in bovine proteinase inhibitor (PI) gene, a method for haplotyping a bovine cell, a method for progeny testing of cattle based on said haplotyping, a method for selectively breeding of cattle based on haplotyping a parent animal. The present invention further provides a method for testing a dairy cattle for its milk production trait, comprising haplotyping its cells, wherein a cattle having haplotypes 1, 3, 4 or 5 indicates that the cattle has desirable milk production trait. Haplotype 1 indicates that the cattle has the most desirable milk production trait.

1 . An isolated nucleic acid molecule comprising a polymorphic site selected from the group consisting of positions 164, 269, 284, 407 and 989 of SEQ ID NO: 1 and at least 17 contiguous bases of SEQ ID NO: 1 adjacent to the polymorphic site, wherein the nucleic acid molecule comprises 
 i) an adenine base at position 164 of SEQ ID NO: 1;    ii) a guanine base at position 164 of SEQ ID NO: 1;    iii) a cytosine base at position 269 of SEQ ID NO: 1;    iv) a thymine base at position 269 of SEQ ID NO: 1;    v) a guanine base at position 284 of SEQ ID NO: 1;    vi) a thymine base at position 284 of SEQ ID NO: 1;    vii) a guanine base at position 407 of SEQ ID NO: 1;    viii) a cytosine base at position 407 of SEQ ID NO: 1;    ix) a cytosine base at position 989 of SEQ ID NO: 1; or    x) a thymine base at position 989 of SEQ ID NO: 1;    or a nucleic acid molecule that is fully complementary to a nucleic acid sequence of (i)-(x), provided that the a nucleic acid molecule is not one consisting of SEQ ID NO: 1.    
     
     
         2 . A nucleic acid molecule according to  claim 1 , which comprises at least 15 contiguous bases of SEQ ID NO: 1 adjacent to the polymorphic site.  
     
     
         3 . A nucleic acid molecule according to  claim 1 , which comprises at least 20 contiguous bases of SEQ ID NO: 1 adjacent to the polymorphic site.  
     
     
         4 . An isolated nucleic acid molecule according to  claim 1 , which comprises not more than 150 nt.  
     
     
         5 . An isolated nucleic acid molecule according to  claim 1 , which comprises not more than 100 nt.  
     
     
         6 . An isolated nucleic acid molecule according to  claim 1 , which comprises not more than 50 nt.  
     
     
         7 . A nucleic acid molecule according to  claim 1 , wherein the polymorphic site is within 4 nucleotides of the center of the nucleic acid molecule.  
     
     
         8 . A nucleic acid molecule according to  claim 7 , wherein the polymorphic site is at the center of the nucleic acid molecule.  
     
     
         9 . A nucleic acid molecule according to  claim 1 , wherein the polymorphic site is at the 3′-end of the nucleic acid molecule.  
     
     
         10 . An array of nucleic acid molecules comprising at least two nucleic acid molecules according to  claim 8 .  
     
     
         11 . A kit comprising a nucleic acid molecule of  claim 1 , and a suitable container.  
     
     
         12 . A method for detecting single nucleotide polymorphism (SNP) in bovine proteinase inhibitor (PI) gene, wherein the PI gene have a nucleic acid sequence of SEQ ID NO: 1, the method comprising determining the identity of a nucleotide at position 164, 269, 284, 407 or 989, and comparing the identity to the nucleotide identity at a corresponding position of SEQ ID NO: 1.  
     
     
         13 . A method according to  claim 12 , wherein the identity of at least two positions of positions 164, 269, 284, 407 and 989 are determined.  
     
     
         14 . A method according to  claim 12 , wherein the identity of all of positions 164, 269, 284, 407 and 989 are determined.  
     
     
         15 . A method for haplotyping a bovine cell, comprising determining the identity of the nucleotides of at least two positions of 164, 269, 284, 407 and 989 of bovine PI gene having a sequence of SEQ ID NO: 1, and comparing the identities at the respective positions to that shown in the table below:  
       
         
           
                 
                 
                 
                 
                 
                 
               
                     
                 
                     
                 
                   POSITION 
                   164 
                   269 
                   284 
                   407 
                   989 
                 
                     
                 
                   Wild type 
                   G 
                   C 
                   G 
                   G 
                   C 
                 
                   Haplotype 1 (ACGCT) 
                   A 
                   C 
                   G 
                   C 
                   T 
                 
                   Haplotype 2 (GTTGT) 
                   G 
                   T 
                   T 
                   G 
                   T 
                 
                   Haplotype 3 (GCGGT) 
                   G 
                   C 
                   G 
                   G 
                   T 
                 
                   Haplotype 4 (GTTGC) 
                   G 
                   T 
                   T 
                   G 
                   C 
                 
                   Haplotype 5 (GCGGC) 
                   G 
                   C 
                   G 
                   G 
                   C 
                 
                   Haplotype 6 (ACGCC) 
                   A 
                   C 
                   G 
                   C 
                   C 
                 
                     
                 
                     
                 
             
                
                
                
                
               
               
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       thereby determining the haplotype.  
     
     
         16 . A method according to  claim 15 , wherein the bovine cell is an adult cell, an embryo cell, a sperm, an egg, a fertilized egg, or a zygote.  
     
     
         17 . A method according to  claim 15 , wherein the identity of the nucleotide is determined by sequencing the PI gene, or a relevant fragment thereof, isolated from the cell.  
     
     
         18 . A method according to  claim 17 , wherein the PI gene or a relevant fragment thereof 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.  
     
     
         19 . A method according to  claim 17 , wherein the PCR or RT-PCR is conducted with a pair of primers selected from the group consisting of (1) SEQ ID NO: 2 and SEQ ID NO: 3; and (2) SEQ ID NO: 4 and SEQ ID NO: 5.  
     
     
         20 . A method according to  claim 17 , wherein both copies of the PI gene in the cell are haplotyped.  
     
     
         21 . A method for progeny testing of cattle, the method comprising collecting a nucleic acid sample from said progeny, and haplotyping said nucleic sample according to  claim 15 .  
     
     
         22 . 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 haplotyping said developing embryo according to  claim 15 , and terminating pregnancy if said developing embryo is not haplotype 1, 3, 4 or 5.  
     
     
         23 . A method according to  claim 22 , wherein pregnancy is terminated is said embryo is not haplotype 1.  
     
     
         24 . A method for selectively breeding dairy cattles, comprising selecting a bull that is homozygously haplotype 1 and using its semen for fertilizing a female animal.  
     
     
         25 . A method according to  claim 24 , wherein the female animal is in vitro fertilized.  
     
     
         26 . A method according to  claim 24 , wherein MOET procedure is used.  
     
     
         27 . A method according to  claim 24 , wherein said female animal is also homozygously haplotype 1.  
     
     
         28 . A method for testing a dairy cattle for its milk production trait, comprising haplotyping its cells according to  claim 15 , wherein a cattle having haplotype 1,3, 4 or 5 indicates that the cattle has desirable milk production trait.  
     
     
         29 . A method according to  claim 28 , wherein a cattle having haplotype 1 indicates that the cattle has a desirable milk production, health or reproduction trait.