Genetic markers for skatole metabolism

Abstract

Novel metabolites and enzymes involved in skatole metabolism are disclosed. The novel metabolites are 3-OH-3-methylindolenine (HMI); 3-methyloxindole (3MOI); indole-3-carbinol (I-3C); and 2-aminoacetophenone (2-AM). Measuring levels of these metabolites in a pig may be useful in identifying the pig's ability to metabolize skatole and its susceptibility to boar taint. The novel enzymes involved in skatole metabolism are aldehyde oxidase and CYP2A6. Enhancing the activity of these enzymes may be useful in enhancing skatole metabolism and reducing boar taint. The identification of the enzyme also allows the development of screening assays for substances that interact with these enzymes and skatole metabolism or for genetic screening to identify pigs on the basis of their skatole metabolism. Pigs having high levels of these enzymes may be selected and bred to produce pigs that have a lower incidence of boar taint.

1 . A method of genetically typing animals to determine those with desired boar taint characteristics, comprising: 
 obtaining a sample of genetic material from said animal; and    assaying for the presence of a genotype in said animal which is associated with improved boar taint, said genotype characterized by the following:    a) a polymorphism in the CYP2A6 gene, said polymorphism being one which is associated with improved boar taint characteristics.    
     
     
         2 . The method of  claim 1  wherein said polymorphism is a t/c polymorphism at nucleotide position 124 of SEQ ID NO:3.  
     
     
         3 . The method of  claim 1  wherein-said polymorphism is a deletion of guanine at nucleotide position 422 of SEQ ID NO:3.  
     
     
         4 . The method of  claim 1  wherein said polymorphism results in a loss of function mutation of CYP2A6.  
     
     
         5 . The method of  claim 1  wherein said polymorphism results in a Phe to Leu change at position 42 of SEQ ID NO:4.  
     
     
         6 . The method of  claim 1  wherein said polymorphism results in a truncated CYP2A6 protein of SEQ ID NO:8.  
     
     
         7 . The method of  claim 1  wherein said step of assaying is selected from the group consisting of: restriction fragment length polymorphism (RFLP) analysis, minisequencing, MALD-TOF, SINE, heteroduplex analysis, one base extension methods, single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE).  
     
     
         8 . A method of genetically typing animals according to skatole metabolism comprising: 
 obtaining a sample of genetic material from said animal;    assaying for the presence of an allele characterized by a polymorphism in a CYP2A6 gene present in said sample, and    correlating said allele with skatole metabolism and concomitant boar taint.    
     
     
         9 . The method of  claim 8  wherein said polymorphism results in a deletion of guanine at position 422 of SEQ ID NO:3, or a c/t transition at position 124 of SEQ ID NO:3.  
     
     
         10 . The method of  claim 8  wherein said step of assaying is selected from the group consisting of: restriction fragment length polymorphism (RFLP) analysis, minisequencing, MALD-TOF, SINE, heteroduplex analysis, one base extension methods, single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE).  
     
     
         11 . The method of  claim 9  further comprising the step of amplifying the amount of CYP2A1 gene or a portion thereof which contains said polymorphism.  
     
     
         12 . A method of determining genetic variability in animals which is linked to skatole metabolism comprising: 
 obtaining a biological sample from a group, line, population or family of animals, said sample comprising a nucleotide sequence encoding an enzyme associated with cytochrome P450 metabolism;    comparing said sequence to a reference sequence to identify a polymorphism;    correlating said polymorphism with variability in skatole metabolism.    
     
     
         13 . A method of screening animals to determine those with desired boar taint characteristics, comprising: 
 obtaining a sample of genetic material from said animal; and    assaying for the presence of a genotype in said animal which is associated with improved boar taint, said genotype characterized by the following:    a) a polymorphism in a cytochrome CYP450 gene, said polymorphism being one which is associated with improved boar taint characteristics.    
     
     
         14 . A nucleotide sequence which encodes a truncated CYP2A6 protein, having an deletion of the guanine at position 422 of SEQ ID NO:3 or its equivalent as determined by BLAST, said nucleotide sequence comprising one or more of the following: 
 (a) SEQ ID NO: 3, or SEQ ID NO:7,    (b) a sequence which will hybridize under conditions of high stringency to the sequences in    (a); or    (c) a sequence with at least about 90% sequence identity to the sequences in (a).    
     
     
         15 . A truncated CYP2A6 protein according to  claim 14 .  
     
     
         16 . A nucleotide sequence which encodes a CYP2A6 protein, having an LEU 42 PHE mutation of SEQ ID NO:3 or its equivalent as determined by BLAST said nucleotide sequence comprising one of the following: 
 (a) SEQ ID NO: 1 or SEQ ID NO:5,    (b) a sequence which will hybridize under conditions of high stringency to the sequences in    (a); or    (c) a sequence with at least about 90% sequence identity to the sequences in (a).    
     
     
         17 . A CYP2A6 skeletal muscle protein, said protein comprising an amino acid sequence comprising one of the following: 
 (a) SEQ ID NO: 2, 4, 6, or 8    (b) conservatively modified variant of (a), or    (c) a sequence with at least about 80% homology to a sequence in (a)    
     
     
         18 . A nucleotide sequence encoding the protein of  claim 17 .  
     
     
         19 . A porcine CYP2A6 protein, comprising the following: 
 a) SEQ ID NO:4    b) conservatively modified variants of SEQ ID NO:4    c) a sequence with 80% homology to SEQ ID NO:4    
     
     
         20 . A nucleotide sequence encoding a CYP2A6 protein comprising: 
 a) a sequence encoding a protein of  claim 19     b) SEQ ID NO: 3    c) a sequence with 90% sequence identity to SEQ ID NO:3    d) a sequence which will hybridize under conditions of high stringency to the complement of SEQ ID NO:3    e) the complement of any of a-d.