Yuzhu Luo

Wool Keratin-Associated Protein Genes in Sheep—A Review

The importance of sheep’s wool in making textiles has inspired extensive research into its structure and the underlying genetics since the 1960s. Wool keratin-associated proteins (KAPs) are a key structural component of the wool fibre. The characterisation of the genes encoding these proteins has progressed rapidly with advances in the nucleotide and protein sequencing. This review describes our knowledge of ovine KAPs, their categorisation into families, polymorphism in the proteins and genes, the clustering and chromosomal location of the genes, some characteristics of gene expression and some potential effects of the KAPs on wool traits. The extent and nature of genetic variation in wool KAP genes and its association with fibre characteristics, provides an opportunity for the development of gene-markers for selective breeding of sheep to produce better wool with properties highly matched to specific end-uses.

A 57-bp deletion in the ovine KAP6-1 gene affects wool fibre diameter.

High glycine-tyrosine keratin-associated proteins (HGT-KAPs) are predominantly present in the orthocortex of wool fibres. They vary in abundance in different wools and have been implicated in regulating wool fibre properties, but little is known about the functional roles of these proteins in the fibre matrix. In this study, we used polymerase chain reaction--single-strand conformational polymorphism (PCR-SSCP) analysis to screen for variation in a gene encoding the ovine HGT-KAP6-1 protein. We identified three gene variants (A, B and C). Variants A and B were similar to each other, with only three nucleotide differences occurring downstream of the coding sequence. However, variant C had a 57-bp deletion that would notionally result in a loss of 19 amino acids in the protein. The presence of C was found to be associated with an increase in mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), coefficient of variation of fibre diameter (CVFD) and prickle factor (percentage of fibres over 30 microns; PF). Sheep of genotype BC produced wool of greater MFD, FDSD and PF than sheep of genotypes AA, AB and BB. The CVFD was greater in the BC sheep than the AB sheep. The results suggest that variation in ovine KRTAP6-1 affects wool fibre diameter-associated traits and that the 57-bp deletion in this gene would lead to coarser wool with greater FDSD, CVFD and PF.

Identification and sequence analysis of the keratin-associated protein 24-1 (KAP24-1) gene homologue in sheep.

Keratin-associated proteins (KAPs) are major structural components of hair and wool fibres, and play a critical role in determining the properties of the fibre. While over 100 KAP genes that have been grouped into 27 KAP families have been identified in mammals, most homologues remain unidentified in sheep. A BLAST search of the Ovine Genome Assembly v2.0 using a human KRTAP24-1 coding sequence (NM_001085455), identified a putative ovine KAP24-1 gene clustered with six other known KAP genes on chromosome 1. The KAP24-1 gene was amplified from the genomic DNA of 260 New Zealand Romney-cross sheep and stem-loop conformational polymorphism (SLCP) analysis of the amplicons revealed four unique banding-patterns, representing four different DNA sequences. These sequences were not closely homologous with any known ovine KRTAP and the highest similarity was with KRTAP24-1 sequences from humans, cattle, dog, pig, Sumatran orangutan and northern white-cheeked gibbon. This suggests that the sequences were allelic variants of ovine KRTAP24-1. Among these four sequences, seven nucleotide substitutions in the coding region were identified and four of the substitutions were non-synonymous. The putative ovine KAP24-1 polypeptide consisted of 252 amino acids. While probably belonging to the high-sulphur KAP group, the polypeptide had a moderate level of cysteine, but a high content of serine and tyrosine. The polypeptide possesses two putative N-glycosylation sites and a number of residues that may be O-glycosylated and/or phosphorylated.

Identification of the ovine keratin-associated protein 22-1 (KAP22-1) gene and its effect on wool traits

Keratin-associated proteins (KAPs) are structural components of wool and hair fibers. To date, eight high glycine/tyrosine KAP (HGT-KAP) families have been identified in humans, but only three have been identified in sheep. In this study, the putative ovine homolog of the human KAP22-1 gene (KRTAP22-1) was amplified using primers designed based on a human KRTAP22-1 sequence. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) was used to screen for variation in KRTAP22-1 in 390 Merino × Southdown-cross lambs and 75 New Zealand (NZ) Romney sheep. Three PCR-SSCP banding patterns were detected and DNA sequencing revealed that the banding patterns represented three different nucleotide sequences (A–C). Two single nucleotide polymorphisms (SNPs) were identified in these sequences. Variant B was most common with a frequency of 81.3% in NZ Romney sheep, while in the Merino × Southdown-cross lambs, A was more common with a frequency of 51.8%. The presence of B was found to be associated with increased wool yield and decreased mean fiber curvature (MFC). Sheep of genotype BB or AB had a higher wool yield than those of genotype AA. These results suggest that ovine KRTAP22-1 variation may be useful when developing breeding programs based on increasing wool yield, or decreasing wool curvature.

Identification of four new gene members of the KAP6 gene family in sheep.

KAP6 is a high glycine-tyrosine keratin-associated protein (HGT-KAP) family. This family is thought to contain multiple genes. In this study, we used a KRTAP6 coding sequence to search the Ovine Genome (v3.1) and identified five homologous regions (R1–R5). All these regions contained an open reading frame, and they were either identical to, or highly similar to, sheep skin Expressed Sequence Tags (ESTs). Phylogenetic analysis revealed that R1–R5 were clustered with KAP6 sequences from different species and formed a group distinct to other HGT-KAPs. R1 was very similar to the characterised KRTAP6-1 sequence, but the remaining genes appeared to be new. PCR primers were designed to amplify and confirm the presence of these new genes. Amplicons were obtained for all of the 96 sheep investigated. Six, five, three and six PCR-SSCP patterns representing six, five, three and six DNA sequences were observed for KRTAP6-2 to KRTAP6-5 respectively. KRTAP6-2 and KRTAP6-4 had five and three SNPs respectively. Three SNPs and a 45-bp insertion/deletion were detected for KRTAP6-3, and five SNPs and an 18-bp insertion/deletion were identified for KRTAP6-5. Allele frequencies for these KAP6 genes differed between Merino and Romney sheep.

Haplotyping using a combination of polymerase chain reaction-single-strand conformational polymorphism analysis and haplotype-specific PCR amplification.

A single nucleotide polymorphism (SNP) may have an impact on phenotype, but it may also be influenced by multiple SNPs within a gene; hence, the haplotype or phase of multiple SNPs needs to be known. Various methods for haplotyping SNPs have been proposed, but a simple and cost-effective method is currently unavailable. Here we describe a haplotyping approach using two simple techniques: polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and haplotype-specific PCR. In this approach, individual regions of a gene are analyzed by PCR-SSCP to identify variation that defines sub-haplotypes, and then extended haplotypes are assembled from the sub-haplotypes either directly or with the additional use of haplotype-specific PCR amplification. We demonstrate the utility of this approach by haplotyping ovine FABP4 across two variable regions that contain seven SNPs and one indel. The simplicity of this approach makes it suitable for large-scale studies and/or diagnostic screening.

Polymorphism of the bovine ADRB3 gene

The β3-adrenergic receptor (ADRB3) is predominantly expressed in white and brown adipose tissue and mediates the lipolytic and thermogenic effects of high catecholamine concentrations. Variation in the ADRB3 gene (ADRB3) has been associated with obesity and the earlier onset of non-insulin-dependent diabetes mellitus in some ethnic groups, as well as some production traits of sheep, but to date variation of bovine ADRB3 has not been reported. In this study, variation in the promoter region of bovine ADRB3 was investigated in 737 cattle by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis. Six PCR-SSCP patterns representing six allelic variations and containing four single nucleotide polymorphisms (SNPs) and three nucleotide deletions/insertions were observed. Allele A was the most common allele (93.83%), whereas alleles C, D, E and F were rare (0.07, 1.09, 0.41, and 0.34%, respectively). The variation identified here might have an impact on both the function and level of expression of bovine ADRB3.

Variation in the ovine C-type lectin dectin-1 gene (CLEC7A)

Dectin-1 is a pattern-recognition receptor that predominantly recognizes beta-glucans from fungi, plants and bacteria. It is therefore implicated in immunological defenses against pathogens that have beta-glucans. In this study, variation in exons 4-6 of the ovine dectin-1 gene (CLEC7A) was investigated. These exons encode the putative domain for beta-glucan recognition. Three, three and four unique PCR-single-strand conformational polymorphism (PCR-SSCP) patterns were detected in exons 4, 5 and 6, respectively. DNA sequencing also revealed three, three and four different nucleotide sequences for the respective exons. All of these sequences were unique, but shared high sequence homology with the CLEC7A cDNA sequences from sheep, cattle and pigs. Either one, or a combination of two sequences was found for each exon in each sheep. This suggests that these sequences represent allelic variants of the ovine CLEC7A gene, although only defined within the exons investigated. The sequence data confirmed four single nucleotide polymorphisms (SNPs) in exon 4, two in exon 5 (with one at the intronic boundary) and three in exon 6. Of these SNPs, the two in exon 4 and two in exon 6 would result in amino acid substitutions. The variation detected here may have an impact on the recognition of beta-glucans and therefore affect the ovine immune response.

Extensive Diversity of the ADRB3 Gene in Chinese Sheep Identified by PCR-SSCP

The β3-adrenergic receptor (ADRB3) is the main mediator of the lipolytic and thermogenic effects of high catecholamine concentrations. Polymorphism in the ovine ADRB3 gene has been reported to be associated with birth weight, growth rate, carcass composition, and cold survival. Eight alleles have been identified in New Zealand sheep, but to date, breeds from other countries have not been studied in detail. In this study, we employed PCR-SSCP to detect ADRB3 polymorphism in the Hu, Tan, small-tailed Han, and Tibet breeds of Chinese sheep. Thirteen SSCP patterns were observed, including the eight previously reported and five new patterns. The newly identified SSCP patterns represent five novel sequences, increasing the reported number of alleles of ADRB3 from 8 to 13. In these Chinese sheep, the most common allele was the previously reported allele C (49.0%); the A allele was the next most common (22.0%). These results suggest that Chinese sheep have more diversity in ADRB3 than New Zealand sheep and therefore have possibly been selected either naturally or via breeding for different traits.

Variation in the ovine KAP6-3 gene (KRTAP6-3) is associated with variation in mean fibre diameter-associated wool traits

Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis was used to investigate variation in the ovine KAP6-3 gene (KRTAP6-3) in 383 Merino × Southdown-cross lambs from four sire-lines, and to determine whether this variation affects wool traits. Five PCR-SSCP banding patterns, representing five different nucleotide sequences, were detected, including four previously identified (named A, B, C, and F) variants and one newly identified (named G) variant. A new non-synonymous single nucleotide polymorphism (SNP) and a 45-bp deletion were detected in variant G. Of the three common genotypes (AA, AB, and AG) identified in these sheep, wool from sheep that were AG, on average, had a lower mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), and prickle factor (PF) than wool from AA sheep, whereas wool from AB sheep, on average, had a higher MFD, FDSD, and PF than wool from AA sheep. This suggests that variation in ovine KRTAP6-3 affect MFD, FDSD, and PF, and that this gene may have potential for use as a gene-maker for improving fibre diameter-associated wool traits.