Genetic test for the identification of carriers of complex vertebral malformations in cattle

Abstract

Genetic markets for identifying bovine carriers of complex vertebral malformation (CVM) disease gene are described. The genetic markers, including the microsatellite markers, BM4129, INRAA003, BMS2790, ILSTS029, INRA123, BM220, HUJ246, BMS862, BMS937, BL1048, BMS2095 and BMS1266 and the bovine SLC35A3 gene, are located on bovine chromosome BTA3. The G/T polymorphism at position 559 of the bovine SLC35A3 gene is identified as being causative and diagnostic for CVM in cattle.

1 . A method for detecting bovine complex vertebral malformation (CVM) in a Holstein-Friesian subject comprising a bovine SLC35A3 gene comprising detecting the presence or absence of at least one genetic marker located on bovine chromosome BTA3 in the region flanked by and including the polymorphic microsatellite markers BM4129 and BMS 1266, wherein said at least one genetic marker is linked to the bovine SLC35A comprising a coding sequence which encodes either 
 i) SEQ ID NO.:17, or    ii) A polypeptide which differs from SEQ ID NO: 17 solely in that there is a phenylalanine at position 180,    wherein the presence of a genetic marker linked to the coding sequence encoding phenylalanine at position 180 of SEQ ID NO: 17 is indicative of CVM.    
     
     
         2 . The method according to  claim 1 , wherein the at least one genetic marker is located in the region flanked by and including the polymorphic microsatellite markers INRAA003 and BMS937.  
     
     
         3 . The method according to  claim 1 , wherein the at least one genetic marker is located in the region flanked by and including the polymorphic microsatellite markers INRAA003 and ILSTS029.  
     
     
         4 . The method according to  claim 1 , wherein the at least one genetic marker is the microsatellite marker INRAA003  
     
     
         5 . The method according to  claim 1 , wherein the at least one genetic marker is the microsatellite marker BMS2790.  
     
     
         6 . The method according to  claim 1 , wherein the at least one genetic marker is the microsatellite marker ILSTS029.  
     
     
         7 . The method according to  claim 1 , wherein the at least one genetic marker is the microsatellite marker INRA123.  
     
     
         8 . The method according to  claim 1 , wherein the at least one genetic marker i the microsatellite marker BM220.  
     
     
         9 . The method according to  claim 1 , wherein the at least one genetic marker is the microsatellite marker HUJ246.  
     
     
         10 . The method according to  claim 1 , wherein the at least one genetic marker is the microsatellite marker BMS862.  
     
     
         11 . The method according to  claim 1 , wherein the at least one genetic marker is the microsatellite marker BMS937.  
     
     
         12 . The method according to  claim 1 , wherein SEQ ID NO: 17 encodes the bovine SLC35A3 protein encoded by the bovine SLC35A3 gene.  
     
     
         13 . The method according to  claim 12 , wherein the bovine SLC35A3 protein is encoded by a cDNA sequence comprising the nucleotide sequence of SEQ ID NO:18.  
     
     
         14 . The method according to  claim 1 , wherein the detection of the presence or absence of the coding sequence encoding polypeptide ii) is performed by a technique selected from the group consisting of allele-specific PCR, minisequencing, primer-extension, pyro-sequencing, PCR-RFLP, allele-specific rolling circle amplification and primer-extension followed by MALDI-TOF mass-spectrometry.  
     
     
         15 . The method according to  claim 1  wherein said at least one genetic marker is genetically linked to a gene or disease trait causing bovine complex vertebral malformation disease at a lod score of at least 3.0, such as at least 4.0, including at least 5.0, such as at least 6.0, including at least 7.0, such as at least 8.0, including at least 9.0 such as at least 10.0, including at least 11.0, such as at least 12.0.  
     
     
         16 . The method according to  claim 1 , wherein the at least one marker is a combination of genetic markers.  
     
     
         17 . The method according to  claim 4 , wherein the primer pair for amplifying the microsatellite marker INRAA003 is SEQ ID NO:1 and SEQ ID NO:2.  
     
     
         18 . The method according to  claim 5 , wherein the primer pair for amplifying the microsatellite marker BMS2790 is SEQ ID NO:3 and SEQ ID NO:4.  
     
     
         19 . The method according to  claim 6 , wherein the primer pair for amplifying the microsatellite marker ILSTS029 is SEQ ID NO:5 and SEQ ID NO:6.  
     
     
         20 . The method according to  claim 7 , wherein the primer pair for amplifying the microsatellite marker INRA123 is SEQ ID NO:07 and SEQ ID NO:8.  
     
     
         21 . The method according to  claim 8 , wherein the primer pair for amplifying the microsatellite marker BM220 is SEQ ID NO:9 and SEQ ID NO:10.  
     
     
         22 . The method according to  claim 9 , wherein the primer pair for amplifying the microsatellite marker HUJ246 is SEQ ID NO:11 and SEQ ID NO:12.  
     
     
         23 . The method according to  claim 10 , wherein the primer pair for amplifying the microsatellite marker BMS862 is SEQ ID NO:13 and SEQ ID NO:14.  
     
     
         24 . The method according to  claim 11 , wherein the primer pair for amplifying the microsatellite marker BMS937 is SEQ ID NO:15 and SEQ ID NO:16.  
     
     
         25 . A diagnostic kit for use in detecting the presence in a bovine subject of at least one genetic marker associated with bovine complex vertebral malformation (CVM), comprising at least one oligonucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, and combinations thereof.  
     
     
         26 . The method according to  claim 1 , wherein the at least one genetic marker is located in the region flanked by and including the polymorphic microsatellite markers INRA003 and BMS 862.  
     
     
         27 . The method according to  claim 1 , wherein the at least one genetic marker is located in the region flanked by and including the polymorphic microsatellite markers INRA003 and HUJ246.  
     
     
         28 . The method according to  claim 1 , wherein the at least one genetic marker is located in the region flanked by and including the polymorphic microsatellite markers INRA 003 and BM220.  
     
     
         29 . The method according to  claim 1 , wherein the at least one genetic marker is located in the region flanked by and including the polymorphic microsatellite markers INRA003 and INRA 123.  
     
     
         30 . The method according to  claim 1 , wherein the at least one genetic marker is located in the region flanked by and including the polymorphic microsatellite markers INRA 003 and BMS2790.  
     
     
         31 . The method according to  claim 1 , wherein the at least one genetic marker is located in the region flanked by and including the polymorphic microsatellite markers INRA003 and BMS 862.  
     
     
         32 . The method according to  claim 1 , wherein the at least one genetic marker is located in the region flanked by and including the polymorphic microsatellite markers INRA003 and HUJ246.  
     
     
         33 . The method according to  claim 1 , wherein the at least one genetic marker is located in the region flanked by and including the polymorphic microsatellite markers INRA 003 and BM220.  
     
     
         34 . The method according to  claim 1 , wherein the at least one genetic marker is located in the region flanked by and including the polymorphic microsatellite markers INRA003 and INRA 123.  
     
     
         35 . The method according to  claim 1 , wherein the at least one genetic marker is located in the region flanked by and including the polymorphic microsatellite markers INRA 003 and BMS2790.