Understanding functional implication of β-casein gene variants in four cattle breeds characterized using AmpliSeq approach

Abstract

Here, we designed a custom panel targeting whole β-casein gene SNPs of zebu and taurine cattle breeds to identify variants and applicability in dairy cattle genotyping. We sequenced two libraries consisting of different pools of primer sets from 95 individuals on the Illumina MiSeq. Consequently, over 92% target regions were amplified and 71 SNPs were available after quality filtering. Only three intronic variants were novel while majority of the identified variants were catalogued in dbSNP as known variants. Identified missense SNPs lead to variant A1/A2, B, F and A3, located in exon 7 only. For confirmation, A1/A2 locus was genotyped using PCR-RFLP. Variant B was observed in all animals, either in homozygous or in heterozygous form. Variants A1, F and A3 predicted to have a deleterious effect on protein function by decreasing the structural stability. Additionally, SIFT score revealed that the A1 variant might affect the protein function.

Keywords: AmpliSeq; Cattle; Next generation sequencing; Single nucleotide polymorphism; β-Casein.

Fig. 1

Coverage of each amplicon covering β-casein gene across 95 samples calculated using BAMSTATS v0.3.2; Amplicon on X- axis and average coverage on Y-axis

Fig. 2

The overlap of identified SNPs by β-casein gene sequencing using Illumina custom amplicon panel in four cattle breeds

Fig. 3

Sample wise distribution of SNPs. In mosaic plot, yellow color indicates heterozygous genotype, red color indicates homozygous altered genotype, white color indicates homozygous wild type genotype and blue color indicates the nucleotide position not sequenced. Green, grey, peach and violet color in bar graph on top X-axis of mosaic plot indicates number of SNPs reported in four breeds viz., Gir, Kankrej, HF crossbred and HF, respectively

Fig. 4

Representative image of PCR–RFLP of β-casein gene resolved on 2.0% agarose gel; well M is loaded with step-up 100 bp ladder; wells numbered 1–9 in top lane are PCR product of β-casein exon-7 fragment; wells numbered with 1–9 in lower lane are PCR product digested with DdeI. Digestion patterns were produced: A1A1: 121 bp; A2A2: 86 bp and 35 bp; A1A2: 121 bp, 86 bp and 35 bp