The Study On RAPD Markers Related To Holstein Cattle Milk Yield Trait And The Analysis Of These Markers In Bioinformatics

To determine genes or associated DNA markers underlying the milk yield trait, 68 Chinese Holstein cattles were scanned by use of RAPD (random amplified polymorphic DNA) technique.In this experiment, we grouped 36 Chinese Holstein cattles as the high- milk-yield subgroup, in which the minimum individual milk yield was 8500Kg and 32 Chinese Holstein cattles as the low-milk-yield subgroup, in which the maximum individual milk yield was 5600Kg. Firstly, two bovine genomic DNA pools were generated from the two subgroups. After the establishment of the RAPD-PCR optimal conditions by the method of orthogonal design, the above two pools were scanned with 320 arbitrary 10 mer primers. Alltogether 2956 DNA bands were amplified, i.e.nearly 3000 loci were screened. 24 primers were found to be polymorphic and used for the following analysis.By testing individually we found 5 RAPD markers named S139-921, S409-414, S217-1227, S198-258 and S417-J were associated with milk yield trait. Of them 4 RAPD markers have been successfully sequenced. The RAPD marker S139-921 was further transformed into a SCAR marker and named as yield-S139.In order to further dig the bioinformation of these 4 RAPD fragments, we analyzed them by bioinformatics methods. The results were:⑴ Four RAPD marker fragments were homology analyzed, one of which, RAPD marker S139-921, had high homology with some bovine genes. We found these genes had a common region--Bov-B LINE/BovB, and the region just contains the homogoly region. ⑵By searching the Bos_Taurus_WGS_Trace database with BLASTN program, we got many Contigs for RAPD marker S139-921. Using DNAMAN 5.0 software, we jointed these Contigs and extended the RAPD fragment S139-921 from 921 bp to 2141 bp in two orientations. The left end was extended 562 bp, and the right end was extended 658 bp.⑶ By searching the cow repeats database by use of RepeatMasker Server, we found the extended sequence is a LINE, including a part of Bov-B LINE/BovB, a part of L1MC/D LINE/L1, and a part of SINE. That indicated the RAPD marker S139-921 was a part of LINE DNA fragment.⑷ Based on above results, we drew a conclusion: RAPD marker S139-921 was a part of LINE DNA fragment, and speculated that some QTLs or genes associated with milk yield trait may lie near this LINE region, and the existence of this LINE region may cause the difference of the milk yield.