A nucleotide substitution in the ovine KAP20-2 gene leads to a premature stop codon that affects wool fibre curvature.

Abstract

The keratin-associated proteins (KAPs) form a matrix that cross-links the main component of the wool fibre, the keratin intermediate filaments. They are therefore believed to play an important role in defining the physical and mechanical properties of the fibre. Although over 80 functional KAP genes (KRTAPs) have been identified in humans, only 29 have been found in sheep,1-3 All of these are polymorphic, and variation in some of these ovine KRTAPs has been associated with wool traits.1-6 Variation in the gene encoding the high glycine-tyrosine KAP20-2 protein (KRTAP20-2) has been reported to affect cashmere fibre weight traits.7 A blast search in the Ovine Genome Assembly Oar_v4.0 with the caprine KRTAP20-2 coding sequence (MF973462) identified on chromosome 1 an orthologous open reading frame (NC_019458.2:12334029_123134217), which has seven nucleotide substitutions relative to the caprine sequence. The sequence was located in a region that contains 13 other known KRTAPs (Fig. S1). A comparison of the sheep KRTAP20-2 with the human and caprine homologues and with other ovine HGT-KAP genes is shown in Fig. S2. Analysis of a 273-bp PCR fragment containing this open reading frame by single-stranded conformational polymorphism (SSCP) revealed two nucleotide sequence variants (Fig. S3), corresponding to a single nucleotide polymorphism (SNP) c.160A>T. This is the only difference between these sequences and the sheep v4.0 sequence, with the A nucleotide being found in that genome assembly sequence. Both sequences have been submitted to GenBank with accession nos. MH071391 and MH071392, for the A- and T-containing sequences respectively. This introduces a premature stop codon that removes the nine C-terminal amino acid residues. In spite of a high frequency of SNPs and insertion/deletions in KRTAPs,1 a nonsense mutation has not been reported to date. The PCR-SSCP analysis of 250 Merino × Southdown-cross sheep (from three sire lines) for which wool trait data were available, revealed that the A and T variants occur at a frequency of 71.8% and 28.2% respectively. Analyses using general linear models (GLMs) in minitab version 16 indicate that the wool fibre from homozygous AA sheep have a higher mean fibre curvature (MFC) than does the wool fibre from sheep carrying the TT genotype (Table 1). Theoretically, this effect may also reflect linkage between KRTAP20-2 and other genes in the same chromosome region. However, the effect observed for KRTAP20-2 differs from the associations observed for three nearby KRTAPs (KRTAP6-1, KRTAP22-1 and KRTAP6-3), which are at a distance of approximately 73 kb, 80 kb and 87 kb respectively from KRATP20-2 (Fig. S1). Specifically, previous research on Merino-cross sheep has revealed that KRTAP22-1 appears to affect wool yield,3 whereas KRTAP6-1 and KRTAP6-3 appear to affect mean fibre diameter (MFD) associated traits, but none of these genes were associated with the curvature.4, 8 Our data also suggest an association between variation in KRTAP20-2 and MFD (P = 0.091), but this trait in these Merino-cross sheep correlated with MFC (r = 0.490; P < 0.001) (Table S1). This study was financially supported by Ningxia Agricultural Breeding Program and the Lincoln University Gene-Marker Laboratory. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.