Alpha-lactalbumin gene polymorphism: a preliminary study on two breeds of the river buffalo (Bubalus bubalis)

Abstract

An experiment was carried out to investigate Single Nucleotide Polymorphisms (SNPs) in the coding regions of α-LA gene in two Indian buffalo breeds vis. Murrah (the best milk yielder) and South Kanara (used in agriculture operations). SNPs in α-LA potentially alter the gene expression and may be associated with differences in milk yield and quality. High molecular weight genomic DNA was extracted from 16 peripheral blood samples. Based on the 3090 bp sequences for bovine gene for alpha-lactalbumin (NCBI GenBank X06366) four primers were designed and used for amplification. The PCR products were electrophoresed, visualized and documented. Single Stranded Conformational Polymorphisms (SSCP) technique was used to screen for SNPs using the DCode Universal Mutation Detection System with 6% acrylamide gel. The individuals (samples) showing polymorphisms and also two samples per breed for each exon were further probed by DNA sequencing. DNA was eluted from the gel using a gel elution Kit. For sequencing, PCR reactions contained 5ul of eluted DNA and 10 pmoles of primer and 4ul of reaction mix. The sequencing products were analysed with the ABI Prism 377 DNA automated sequencer. In order to detect possible polymorphisms, the buffalo α-LA gene sequences observed were compared with the sequence for the same gene in NCBI gene bank AF194373. Sequence of exon 1 of all the animals were determined by direct sequencing. All the 8 buffaloes belonging to both the breeds showed a substitution at position 864 (C→T) and one South Kanara buffalo showed substitution at position 1264 (A→G). The polymorphism at position 864 (C→T) results in substitution of an amino acid P→S while the observed polymorphism at 1264 was a silent mutation in the coding region of the gene. No SNPs were observed in other three exons. The substitution observed in all the buffaloes concerned reveals Indian buffaloes which are riverine breeds differing from the buffaloes of China.