摘要Background:Complex vertebral malformation (CVM) and bovine leukocyte adhesion deficiency (BLAD) are two autosomal recessive lethal genetic defects frequently occurring in Holstein cattle,identifiable by single nucleotide polymorphisms.The objective of this study is to develop a rapid and reliable genotyping assay to screen the active Holstein sires and determine the carrier frequency of CVM and BLAD in Chinese dairy cattle population.Results:We developed real-time PCR-based assays for discrimination of wild-type and defective alleles,so that carriers can be detected.Only one step was required after the DNA extraction from the sample and time consumption was about 2 hours.A total of 587 Chinese Holstein bulls were assayed,and fifty-six CVM-carriers and eight BLAD-carriers were identified,corresponding to heterozygote carrier frequencies of 9.54％ and 1.36％,respectively.The pedigree analysis showed that most of the carriers could be traced back to the common ancestry,Osborndale lvanhoe for BLAD and Pennstate lvanhoe Star for CVM.Conclusions:These results demonstrate that real-time PCR is a simple,rapid and reliable assay for BLAD and CVM defective allele detection.The high frequency of the CVM allele suggests that implementing a routine testing system is necessary to gradually eradicate the deleterious gene from the Chinese Holstein population.