Polymorphisms of the PRLR Gene and Their Association with Milk Production Traits in Egyptian Buffaloes

Abstract

Prolactin (PRL) and its receptor (PRLR) were considered as potential genetic markers for milk production and quality traits in cattle. However, little information is available regarding PRLR genetic diversity and association studies with milk traits in Egyptian water buffaloes. Therefore, the present study was conducted to search for mutations in PRLR and determine their associations with milk performance in these animals. Exon3 (E3) and E10 of PRLR were screened for polymorphisms using single strand conformation polymorphism (SSCP) and sequencing in 400 buffaloes. The associations between haplotypes and milk production (fat%, protein%, lactose%, and solid%) traits as well as mRNA and protein levels of PRL and PRLR were studied. Two single nucleotide polymorphisms (SNPs) in E10 were detected: g.11685G>A (p.Ala494Thr) and g.11773T>C (p.Val523Aal). The G and T alleles were wild (ancestral) alleles, while the A and C alleles were mutant alleles. These SNPs resulted in four haplotypes; AC, AT, GC, and GT. Buffaloes with wild GT haplotypes showed significantly higher milk yield, fat% and protein%, mRNA and protein levels of PRL and PRLR in milk somatic cells than other animals. Animals carrying mutant AC haplotype had inferior milk traits and lowest levels of associated mRNAs and proteins. With these results, we could conclude that the selection of buffaloes with wild GT haplotypes for g.11685G>A and g.11773T>C SNPs of the PRLR gene might improve the milk production traits of Egyptian water buffaloes.

Keywords: Egyptian buffalo; milk performance; mutations; prolactin receptor.

Figure 1

Identification of SNPs in buffalo PRLR(L2). (A) Agarose gel of PRLR(L2) amplified fragments (305 bp) from six different samples (lanes 1-6). (B) PCR-SSCP banding patterns show five different patterns (P1-P5) in five different samples. (C) A representative sequence chromatogram from one sample shows the sites of the two SNPs (two orange boxes), primers (forward (F) and reverse (R), the two green arrows), and amino acid sequences (colored letters). (D,E) Sequences chromatogram spanning the site of g.11685G>A (D) and g.11773T>C (E) SNPs (arrowheads) and the altered amino acids (p.Ala494Thr and p.Val523Ala). Ala, alanine; M; 1Kb ladder; Thr, threonine; Val, valine.

Figure 2

Real-time PCR graphical presentation showing expression of PRLR and PRL genes in milk SCs in animals carrying AC, AT, GC, and GT haplotypes. Data are expressed as fold change mean ± SEM. Number of samples per each haplotype was nine. The mutant AC haplotype was considered as the control. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 3

(A) Western blot bands showing expression of PRL and PRLR proteins in milk SCs in animals carrying AC, AT, GC, and GT haplotypes. (B) Band quantification of PRL and PRLR. Data are expressed as fold change mean ± SEM. Number of samples per each haplotype was nine. The mutant AC haplotype considered as the control. * p < 0.05, *** p < 0.001, and **** p < 0.0001.