Detection of New Silent Mutation at 348 bp Position in a CD18 Gene in Holstein Cattle Normal and Heterozygous for Bovine Leukocyte Adhesion Deficiency Syndrome

Abstract

In India, Holstein and its crosses are being used extensively in breeding programmes and all these breeding bulls are screened for autosomal recessive genes. Blood samples are collected in ethylenediaminetetraacetic acid (EDTA) coated tubes and DNA was isolated by using phenol-chloroform method. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) wereperformed by using bovine leukocyte adhesion deficiency (BLAD) specific primersand <em>TaqI </em>restriction enzyme for diagnosis of bovine leukocyte adhesion deficiency (BLAD) syndrome. The bull was found to be heterozygous for BLAD allele. The PCR product was sequenced by automated sequencer using the ABI big dye Ver 3.1 for detection of mutation at position 383 bp (A/G). Sequence analysis comparison was performed using the codon code aligner 4.0.4 software. The sequence of carrier animal confirmed polymorphism at 383 bp position. The sequence was also compared with sequence of normal Holstein as a control and the sequence available with NCBI (accession No. NC-007299). The comparison of sequences revealed a heterozygous polymorphism at 348 position (T>C) in a carrier animal whereas in homozygous in a control Holstein which was normal for BLAD. The new polymorphism at 348 position was found to be silent as it does not change amino acid (asparagine, AAT>AAC) within exon 4 of CD18 gene. The partial sequence of new polymorphism / silent mutation has already been submitted to NCBI (accession No. KF840683). Further studies have to be carried out to elucidate the possible association of the CD18 silent point mutation at 348 bp position as a potential molecular marker for milk production traits.