Spatiotemporal Expression and Haplotypes Identification of KRT84 Gene and Their Association with Wool Traits in Gansu Alpine Fine-Wool Sheep

Abstract

Keratin (K) is a major protein component of hair and is involved in hair growth and development. In this study, we analysed the expression, localization, and polymorphism of the K84 gene (KRT84) in Gansu Alpine Fine-wool sheep using immunofluorescence, RT-qPCR, and PARMS (penta-primer amplification refractory mutation system). Haplotypes of KRT84 were also constructed and their relationship with wool traits analysed. It was revealed that KRT84 was highly expressed in hair follicles, including the inner root sheath, outer root sheath, and hair medulla and at all six lamb ages investigated from 1 to 270 days of age. Three SNPs were detected in KRT84 exon 1, and they formed three haplotypes (named H1, H2, and H3) and six genotypes. Analyses revealed an association between haplotype combinations (diplotypes) and the mean fibre curvature, mean staple length, mean staple strength, mean fibre diameter, the coefficient of variation of fibre diameter, and comfort factor for these sheep. These results suggest that KRT84 is of importance in determining several key traits in Gansu Alpine Fine-wool sheep and that the gene could possibly be used as a genetic marker for wool trait selection in these sheep.

Keywords: Gansu Alpine Fine-wool sheep; SNPs; keratin; spatiotemporal expression.

Figure 1

Immunofluorescence staining for K84 in the skin of Gansu Alpine Fine-wool sheep at different times. Immunofluorescence staining of K84 (5×). (A–C) 1d; (D–F) 30 d; (G–I) 60 d; (J–L) 90 d; (M–O) 180 d; (P–R) 270 d. The blue tissue in the figure is the DAPI-labelled nuclear fluorescence staining and the red tissue is the fluorescence staining of K84. C: corneum; DP: dermal papilla; SG: sebaceous gland; ORS: outer root sheath; IRS: inner root sheath; HM: hair matrix.

Figure 2

Levels of K84 in the skin of Gansu Alpine Fine-wool sheep at different times. The data are expressed as means ± S.E. Different letters indicate significant differences between lambs of differing age (p < 0.05).

Figure 3

PCR amplification and sequencing of KRT84 in Gansu Alpine Fine-wool sheep. (A) c.97A/G, (B) c.162C/T, (C) c.286G/A (noting that the overlapping peaks indicated by arrows in A, B, and C are SNPs and that the numbering on the electropherograms reflects the location in the sequencing reaction and not the position of the SNP in the gene). Sequences in A and B are from the forward strand whereas the sequence in C is from the reverse strand. (D) D illustrates the PCR amplification of KRT84.

Figure 4

Genotyping of KRT84 in Gansu Alpine Fine-wool sheep using PARMS analysis. (A) The red, green, and blue dots indicate GA, GG, and AA, respectively; (B) The red, green, and blue indicate CT, TT, and CC, respectively; (C) The red, green, and blue dots indicate GA, GG, and AA, respectively.

Figure 5

Linkage disequilibrium analysis of the KRT84 SNPs in Gansu Alpine Fine-wool sheep. The red square in the figure has an R² value of 1, suggesting a strongly interlocking state.

Figure 6

The mRNA levels of KRT84 in the skin of Gansu Alpine Fine-wool sheep. The β-actin gene (ACTB) was used as a reference gene to calibrate the level of gene expression. The genotype expression levels in skin are presented as means ± S.E. and significant differences (p < 0.05) are denoted by different letters.