Genotype Identification And Editing For Polled Of Mongolia Cattle

Horns are a distinctive feature of the Bovidae family and its basic function is fighting and self-defense.In the modern cattle industry,however,these appendages have become undesirable due to their dangerous for both breeders and animals and economic losses.Under the feeding conditions,horns are usually dehorned within a week after birth by physical or chemical methods.The man-made dehorning process can go against with the animal welfare and make harmful to animals.Naturally,there exist natural hornless cattle,which result from gene mutations as P_202ID,P_80kbID and P_219ID.In the present study,total of 38 cattle including 22 polled,12 horned Mongolian cattle and 4 crosses between Mongolian cattle and Angus origin were selected to genotype the polled Mongolian cattle.The genotyping was analyzed by polymerase chain reaction?PCR?,agarose gel electrophoresis and sequence.The result showed that the Mongolian cattle POLLED genotype belongs to P_219ID mutation,which is in consistent with the polled genetype of the hornless Turano Mongolian cattle.Additionally,we sequenced the mitochondrial DNA and found that Mongolian cattle are origins of Bos taurus.Furtherly,it confirmed that Mongolian cattle originated to Mongolian Turano system.In order to produce hornless Mongolian cattle,the genome editing technique CRISPR/Cas9 systern was used to mediate P_202ID genotype knockin at POLLED locus in horned Mongolian cattle.Two vectors were constructed and electroporated co-transferred into fetal fibroblasts of Mongolian cattle.The flow cytometry slected monoclonal cells were then subjected to genomic extraction,PCR amplification and sequencing to identify positive monoclonal cells.Finally,4 strains of heterologous recombinant monoclonal cell lines were obtained.In conclusion,the genetype of the hornless Mongolian cattle is P_219ID mutation and is in consistent with the polled Turano Mongolian cattle;the polled genetype P_219ID mutation was edited by CRISPR/Cas9 system and positive transgenic cells were obtained.