Characterisation of keratin genes associated with wool traits

In the textile industries, wool has to compete with cotton and synthetic fibres. In order to improve the competitiveness of wool fibre, the sheep industry is working to find ways to improve the raw product through the selective breeding of sheep. The use of gene-markers is one approach to selectively breeding genetically superior stock, and this method allows for the early selection of superior livestock, and without having to wait for the animal to grow to maturity. The α-keratins are structural proteins in the cortex of wool fibres, and are assembled in an organized fashion into the keratin intermediate filaments (KIFs). These are enveloped by an inter-filamentous matrix consisting of keratin-associated proteins (KAPs). Variation in the keratins affects wool structure and fibre characteristics, and thus their genes make ideal candidates for the development of gene-markers. Currently 17 human hair keratins have been characterized: eleven of which are type I and six of which are type II human hair keratins, and ten sheep type I and seven sheep type II proteins have been identified. This study identified variation in 13 regions of four keratin genes using polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) analysis. Three regions of KRT83 were investigated, including a portion of the promoter, all of exon 2 (including part of intron 1) and a region encompassing exon 3 - 4 (including all of exon 3, intron 3, exon 4 and part of intron 4). In 300 New Zealand Romney, Merino and White Dorper sheep obtained from 26 farms in New Zealand, one, two and four PCR-SSCP banding patterns were observed for these three regions respectively. The exon 2 region contained two single nucleotide polymorphisms (SNPs) and the exon 3 - 4 region contained five SNPs. Three regions of KRT85 were investigated, including two parts of the promoter ( called promoter 1 and promoter 2) and an exon 3 - 4 (including all of exon 3, intron 3, exon 4 and part of intron 4) region. One, three and two PCR-SSCP banding patterns were observed for these regions respectively. Six SNPs were detected in promoter 2 and two in the exon 3 - 4 region. Four regions of KRT31 were investigated, including a portion of the promoter, all of exon 1 (including part of intron 1), all of exon 3 (including part of intron 2 and intron 3) and exon 7 (including part of intron 6), three, two, two and two PCR-SSCP banding patterns were observed for these regions respectively. The promoter region, the exon 1 region and the exon 3 region contained two SNPs, and the exon 7 regions contained one SNP. Three regions of KRT34 were investigated, including three parts of the promoter (called promoter 1, promoter 2 and promoter 3). Only the promoter 1 region showed polymorphism upon SSCP anslysis, with two unique SSCP patterns being observed. A total of three SNPs were identified in the three promoter 1 variants. Investigation of the effect of variation in the KRT83 exon 3 - 4 region was subsequently undertaken in 489 Merino × Southdown-cross sheep from seven sire-lines. The four variants identified in the original 300 sheep (designated A-D) and a new variant (E containing a new SNP) were observed with a frequency of 64.6%, 15.4%, 6.6%, 10.1% and 3.3%, respectively. General linear mixed-effects models (GLMMs) were used to investigate associations between the presence or absence of the variants and wool traits, with a second set of models testing associations between common genotypes and those traits. The presence of A was associated with a decrease in fibre diameter standard deviation (FDSD) and coefficient of variation of fibre diameter (CVFD). The presence of C was associated with an increase in mean fibre diameter (MFD), mean fibre curvature (MFC) and prickle factor (PF), and a decrease in wool yield [clean fleece weight (CFW)/greasy fleece weight (GFW) ×100] (YIELD). A trend for association between the presence of C and increased FDSD was also detected. The presence of D was associated with an increase in MFD and PF, and a decrease in YIELD. The presence of E was associated with a decrease in CVFD. Genotype AD had a higher GFW and a lower YIELD than AA, and AC and AD tended to have increased MFD compared to AA and AB. The effect of variation in the KRT85 promoter 2 region and its association with wool traits was investigated. The three variants identified in the original 300 sheep (A - C) exhibited unique PCR-SSCP banding patterns with frequencies of 56%, 29% and 15%. In the single-variant GLMMs, the presence of B was associated with an increase in FDSD and PF, and this association persisted for PF, but was lost for FDSD in the multi-variant GLMMs. Although no associations were detected with A and C in the single-variant GLMMs, the presence of A tended to be associated with an increase in YIELD, and the presence of C tended to be associated with a decrease in MFD. Genotype AA had a higher mean YIELD, lower mean FDSD and lower mean PF. The impact of variation in the KRT85 exon 3 - 4 region on wool traits was investigated. Two unique variants (a and b) were observed in 463 of the Merino × Southdown-cross lambs. The frequencies of the two variants detected in these sheep were 48.7% and 51.3% respectively. Three different genotypes were observed with frequencies of 22.68%, 52.05% and 25.27% for aa, ab and bb respectively. The presence of a tended to be associated with an increase in GFW and CFW, and the presence of b was associated with an increased PF. Sheep with genotype ab had a higher GFW, CFW, MSL and PF, than those of genotypes aa and bb. Variation in the KRT31 promoter region of 485 Merino × Southdown-cross, revealed the three variants identified in the original 300 sheep (named A, B and C). These were observed with a frequency of 56%, 29% and 15%, respectively. The presence of A and B had no significant effect on wool traits, but the presence of C was found to be associated with an increase in GFW, CFW and MSL. There was an effect of genotype with BC sheep producing wool of higher CFW and MSL, than AA, AB, AC and BB sheep. The impact of variation in KRT34 promoter 1 region on wool traits was investigated in 460 Merino × Southdown-cross lambs. Their frequencies were 41% and 59% for A and B respectively. Three different genotypes were observed, and the frequencies were AA (10%); AB (57.39%) and BB (32.61%). The presence of A was found to be associated with a decrease in MFD, FDSD and MSL. With the three genotypes tested, sheep with genotype BB had higher MFD and FDSD, than those of genotype AA and AB. This study confirms the potential for KRTs to serve as gene-markers of wool traits