1. Introduction
Keratin-associated proteins (KAPs) are components of wool and hair fibres. Typically, a wool or hair fibre comprises the cuticle and cortex, and occasionally they have a medulla, a centrally positioned structure composed of hollow cells within the cortex [
1]. Keratin intermediate filaments are embedded in a matrix of the KAPs [
2], and consequently the KAPs are thought to have a function in determining the characteristics of wool and hair fibres.
The KAP proteins are compact in size yet they are abundant in number. Although only 31 KAP genes (designated
KRTAPs) have been identified in sheep to date [
3,
4], the human genome contains a larger repertoire, with 89 known
KRTAPs assigned into 25 KAP families: KAP1 to KAP13, KAP15 to KAP17, and KAP19 to KAP27 [
5,
6,
7]. Among these, the KAP11 and KAP13 families represent the earliest expressed families in human hair follicles, commencing their expression concurrently with the hair keratins and well before the emergence of the first high glycine–tyrosine KAP [
5,
7,
8,
9]. The human KAP11 family contains a single member, KAP11-1, while KAP13 comprises four family members.
In human hair follicles, only three KAP13 genes (
KRTAP13-1,
KRTAP13-2, and
KRTAP13-3) demonstrate observable expression, with
KRTAP13-2 displaying strong expression in the cuticle [
9]. There is no obvious expression of
KRTAP13-1 in mouse hair follicles, but instead with embryonic mice it is localised in the periderm, the filiform tongue papillae, and the parakeratotic tail epidermis of adult mice [
10]. These findings highlight diversity within the KAP13 family and suggest that different family members may serve different functions or exhibit differential behaviours.
To date, only one member of the KAP13 gene family,
KRTAP13-3, has been described in sheep [
11]. The purpose of this study is to search for additional family members and, if found, ascertain whether these genes are polymorphic. This study also aims to investigate whether any variation detected in the genes is associated with fibre traits in Chinese Tan sheep. This indigenous Chinese breed is recognised for growing wool with a distinguishing ‘spring-like’ crimp that is notable up to approximately 35 days of age, a period traditionally referred to as ‘Er-mao’. The pelts of the lambs are important in the fur trade in the colder regions of Northern and Western China. The wool comprises two types of fibres: a ‘fine’ fibre, which is typically non-medulated, and a heterotypic and ‘coarser’ hair fibre that can be medulated.
2. Materials and Methods
The collection of sheep blood samples adhered to ethical guidelines outlined in the Animal Welfare Act 1999 (NZ Government) and specifically followed Section 7.5 Animal Identification of the Animal Welfare (Sheep and Beef Cattle) Code of Welfare 2010, which covers the collection of blood drops by nicking sheep ears.
2.1. Sheep Investigated and Wool Trait Measurement
In this study, two groups of sheep were investigated. The first group comprised 116 sheep selected from various farms. They were chosen to be unrelated and to represent breeds that have been selected historically for meat, wool, and dual-purpose production systems. These sheep were chosen to create a diverse base to ascertain the likely extent of DNA sequence variation in the KRTAP13-n genes. The sheep comprised 10 breeds from eight unrelated farms and were numbered as follows: South Suffolk (6), Poll Dorset (12), Israeli Assaf (15), Corriedale (10), Romney (20), Texel (10), Merino (20), Coopworth (6), White Dorper (10), and Black Dorper (7). This group was solely used for screening for variation in ovine KRTAP13-n. Because no wool samples were collected from these sheep, they were not subjected to gene association analyses.
The second group comprised 246 Chinese Tan lambs. These were the progeny of ten lambs. Most of the lambs were born as singles, but six of them (three pairs) were born as twins. It was decided to remove the twins from the association analyses, as they might confound the results. Consequently, 240 single-born lambs were used in the general linear modeling approach to ascertain if there was a relationship between wool trait variation and variation in the KRTAP13-n genes studied.
Wool samples were collected from the mid-side region (above the flank over the 13th rib) of the Tan lambs at Er-mao. For each sample, the larger heterotypic hair fibres and the fine wool fibres were separated based on their difference in fibre diameter and length. To do this, the sample was spread out on a flannel-covered board, and then using an index figure to press the base of all the fibres against the board, the other hand was used to grip the top of the longer heterotypic fibres and pull them out of the fibre sample. The fine wool fibres remained on the flannel-covered board. It was repeated several times to ensure that all the heterotypic hair fibres were separated from the fine wool.
The fine and heterotypic fibres were then measured for mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), coefficient of variation of fibre diameter (CVFD), and mean fibre curvature (MFC). The measurements on the heterotypic fibres were undertaken by the New Zealand Wool Testing Authority, Napier, New Zealand, using IWTO sanctioned methods (IWTO-12-2012), while the measurement of the fine wool samples was undertaken by Pastoral Measurements Limited, Timaru, New Zealand.
A sample of venous blood was collected from each sheep and dotted onto TFN blotting paper (Munktell Filter AB, Falun, Sweden). The DNA in the white blood cells was purified from 1.2 mm punches taken from the dried blood on the TFN paper, using a two-step washing process that was described for ovine
KRTAP19-5 [
12]. This method involves incubating the blood card punches in a 20 mM NaOH solution for 30 min at room temperature, aspiration of the NaOH solution, and a subsequent single wash with 1 × TE
−1 buffer (10 mM Tris–HCl, 0.1 mM EDTA, pH 8.0). The prepared samples were air dried and stored until needed.
2.2. Search for KRTAP13-n in the Sheep Genome
The coding sequence of ovine
KRTAP13-3 (JN377429) was used for a BLASTN search of the Sheep Genome Assembly ARS-UI_Ramb_v2.0 (GCA_016772045.1). The genomic sequences that contained open reading frames (ORFs) with high sequence similarity to this coding sequence were presumed to be
KRTAP13-n family members. The homologous genomic sequences identified were then used to design three sets of PCR primers (
Table 1) for amplifying the three separate
KRTAP13-n identified from the sheep genomic DNA purified as described above.
2.3. PCR Amplification and Single Strand Conformation Polymorphism Analysis of KRTAP13-n
PCR primers were synthesised by Integrated DNA Technologies (Coralville, IA, USA). Amplification for all three genes was carried out in 15 μL reactions that contained one prepared punch of the TFN paper, 150 μM of each dNTP (Bioline, London, UK), 0.25 μM of each primer, 2.5 mM Mg
2+, 0.5 U of Taq DNA polymerase (Qiagen, Hilden, Germany), and 1× the reaction buffer supplied with the enzyme. The thermal profile for amplification of the three genes studied consisted of an initial denaturation step at 94 °C for 2 min, followed by 35 cycles of 30 s at 94 °C, 30 s at the annealing temperature shown in
Table 1, and 30 s at 72 °C, and a final extension step for 5 min at 72 °C. The thermal cycling was undertaken in S1000 PCR machines (Bio-Rad, Hercules, CA, USA).
The PCR amplicons were analysed using a single strand conformation polymorphism (SSCP) approach. For each amplicon, a 0.7-μL aliquot of each was mixed with 7 μL of gel loading dye (0.025% bromophenol blue, 0.025% xylene–cyanol, 98% formamide, 10 mM EDTA). After denaturation at 95 °C for 5 min, the DNA samples were cooled on wet ice and loaded on 16 cm × 18 cm, 14% acrylamide/bisacrylamide (37.5:1) (Bio-Rad) gels. Electrophoresis was carried out using the conditions described in
Table 1 and using 0.5× TBE buffer in Protean II xi cells (Bio-Rad).
Upon completion of the electrophoretic separation, the SSCP gels were fixed and stained for 10 min in a solution containing 10% ethanol, 0.5% acetic acid, and 0.2% silver nitrate. Next, the gels were rinsed with distilled water and the banding patterns were revealed by developing the gels in a solution of 3% NaOH and 0.1% HCOH. Development was undertaken until dark-staining bands appeared on the yellow background of the gel. At that time, gel development was halted by removing the developer solution and by the addition of a 10% ethanol and 0.5% acetic acid aqueous solution.
2.4. DNA Sequencing and Sequence Analysis
The amplicons that were selected for sequencing were identified using the PCR–SSCP approach described above. Specifically, amplicons representing different SSCP banding patterns from sheep that appeared to be homozygous for the amplicon sequence were sequenced three times in both directions at the Lincoln University DNA sequencing facility (Lincoln University, Lincoln, New Zealand) using a Sanger dideoxy approach.
For rare alleles at all three loci, which were exclusively identified in heterozygous sheep, a previously described alternative approach was employed. This was described previously [
13]. In this approach, a gel slice corresponding to a SSCP band of the rarer allele is excised from the SSCP gel, macerated, and then used as a template for re-amplification with the original primers. This effectively enables allele-specific amplification, and the resulting second amplicon can be matched to the banding patterns observed on the SSCP gels under the original conditions and then sequenced directly.
Once the sequencing outputs from both directions of read were obtained in triplicate, they were aligned, translated, and subjected to phylogenetic analysis using DNAMAN XL (version 10, Lynnon BioSoft, Vaudreuil, QC, Canada).
2.5. Statistical Analyses
Genetic heterogeneity and polymorphism information content (PIC) were calculated using an online calculator (
https://www.genecalculators.net/pq-chwe-polypicker.html; accessed on 17 July 2024). Association analyses were performed using Minitab version 16 (Minitab Inc., PA, USA). General Linear Models (GLMs) were used to assess the effect of the absence or presence of the
KRTAP13-2 and the
KRTAP13-4 alleles on the various wool traits that were measured. The models incorporated sire and lamb sex because sire was identified to have an influence on all the wool traits, while sex was identified as a factor impacting certain wool traits. The model employed was: Y
jkl = µ + V
j + G
k + S
l + e
jkl; where Y
jkl is the phenotypic value of the trait, µ is the group raw mean for that particular trait, V
j is the effect of the
jth allele (presence or absence), G
k is the effect of lamb sex, S
l is the effect of the
lth sire (10 sires in total), and e
jkl is the random residual effect.
4. Discussion
This study identifies three new
KRTAPs in sheep. The high sequence homology between these
KRTAPs and ovine
KRTAP13-3, their similar chromosomal location, and the close phylogenetic relatedness to the human
KRTAP13-n genes suggest that these new
KRTAPs represent the other three members of the KAP13 gene family,
KRTAP13-1,
KRTAP13-2, and
KRTAP13-4. With the identification of these genes, the number of
KRTAPs identified in sheep increases from 31 [
3,
4] to 34, which possibly only represents one-third of the total number of
KRTAPs present in this species when compared to humans.
An interesting finding is that the
KRTAP13-n genes were more closely related to each other within species than to their orthologs from another species for the coding region, but this relationship changed in the immediate flanking regions where the orthologs from the sheep and humans were more closely related. This suggests that the coding and flanking regions of
KRTAP13-n are subject to distinct selective pressures and/or have evolved through different mechanisms. The coding region appears to have undergone concerted evolution, while the flanking regions show signs of divergent evolution. This kind of evolutionary pattern has been previously reported for the KAP1 family [
15], and it may lead to sequence and structure homogeneity within the
KAP13-n proteins while allowing for regulatory diversity among individual family members.
While the KAP13 family in sheep shares similarities with humans in terms of family members and chromosomal locations, differences exist between these two species. A notable difference is the position of
KRTAP13-2. It is located at one end of the
KRTAP13-n cluster in sheep, but it is situated at the other end of the
KRTAP13-n cluster in humans (
Figure 2). Research in sheep suggests a prevalence of long intergenic non-coding RNA (lincRNA) genes near these
KRTAP13-n genes [
3], and in humans, lincRNAs are known to contain a higher proportion of transposable element-derived sequences [
16]. It is unknown whether these lincRNAs might have had a role in the movement of
KRTAP13-2 in the chromosome.
The association of
KRTAP13-2 with FDSD and CVFD but not with MFD suggests that variation in this gene primarily influences fibre diameter uniformity and not the average fibre diameter for the sample. That is, regardless of the average fibre diameter, fibre diameter variation measured as either a standard deviation of the mean or as CVFD (=FDSD/MFD × 100) is associated with variation in
KRTAP13-2. The mechanism underpinning this association remains unclear, but one proposition might be that
KRTAP13-2 affects wool fibre ellipticity. Wool fibres typically exhibit an elliptical rather than circular cross-sectional shape, and it has been suggested that increased FDSD and CVFD are correlated with increased ellipticity [
17]. In goats, the ellipticity of cashmere fibres has been linked to cuticle thickness, with thicker cuticles associated with greater fibre ellipticity [
18].
In humans, although
KRTAP13-2 expression is detected in the cortex, it exhibits stronger expression in the cuticle [
9]. If the expression pattern of the
KRTAP13-2 ortholog in sheep mirrors this, strong cuticular expression would suggest a potential role in regulating cuticle thickness or rigidity. This could, in turn, influence wool fibre ellipticity and subsequently impact the FDSD and CVFD.
Coarse wool fibres, with their larger surface area, are inherently more susceptible to deformation from external forces compared to fine wool fibres. This is consistent with the wool trait data obtained in this study, where the fine wool had lower FDSD and CVFD than the heterotypic hair fibres. The impact of cuticle thickness or rigidity on ellipticity is expected to be less pronounced in fine wool than in coarse wool, which may explain why the effect was only detected for the heterotypic hair fibres and not for the fine wool.
Ovine
KRTAP13-2 is in proximity to
KRTAP15-1 on sheep chromosome 1, with approximately 3.3 kb between them. Variation in
KRTAP15-1 has previously been reported to affect wool yield, but it is also associated with variation in FDSD in Merino × Southdown-cross sheep [
19]. This is notable because in this study with Chinese Tan sheep,
KRTAP13-2 was found to be associated with variation in FDSD and CVFD. As these studies used sheep from different breeds and farmed in different environments, caution is however required when comparing the results of the two studies.
In ovine
KRTAP13-2, allele
B differs from allele
A at SNPs c.240A/G and c450T/C, and from allele
C at SNPs c.122A/G and c.240A/G. The detection of wool trait associations with allele
B suggests that some of these three SNPs may have a functional effect. However, as alleles
A and
C differ in two of these SNPs (i.e., c.122A/G and c.450T/C), the lack of association detected for alleles
A and
C suggests the functional effect, if any, is possibly because of SNP c.240A/G. This SNP is synonymous and would not change the protein sequence. However, research has suggested that synonymous SNPs can affect mRNA stability, regulate gene expression, and/or alter protein structures [
20,
21]. Alternatively, this SNP may be linked to variation in other regions of the
KRTAP13-2 gene or nearby genes that have a functional effect. Further investigation into the upstream and downstream regions of this gene or other nearby genes is therefore required. Regardless, the variation reported here for
KRTAP13-2 appears to have the potential to be developed as a gene-marker to improve wool fibre uniformity.
The lack of association detected between KRTAP13-4 variation and wool traits in the Tan sheep may be due to the imbalanced frequencies of the alleles. Although two alleles (A and D) had frequencies above 5% and were included in the association study, allele A was very common at 86.9%, while allele D occurred at a frequency of 8.4%. This imbalance may have resulted in an insufficient number of animals carrying allele D in the small population group studied, reducing the likelihood of associations being detectable. Alternatively, variation in this gene may be associated with other wool traits that were not measured in this study, a possibility that cannot be ruled out.
Interestingly, while KRTAP13-2 and KRTAP13-4 are closely placed on the sheep chromosome, they appear to exhibit differences in both the amount of genetic variation and their possible effect on wool traits. This would suggest the mechanisms underlying the variation and the associations detected here require further investigation.
An interesting phenomenon observed in this study is the difference in diversity levels among the members of the KAP13 family. While KRTAP13-2 and KRTAP13-4 exhibit similar levels of diversity, KRTAP13-1 appeared to be conserved in the Tan sheep but polymorphic in the population of sheep of diverse breeds. This raises questions about the factors that influence genetic diversity within the KAP13 family.
Considering that the sheep used for variation screening were randomly selected from diverse breeds farmed for different purposes, it would seem less likely that selective breeding has influenced the diversity observed. Natural selection would also seem unlikely to be a significant factor if the protein encoded primarily functions as a structural component among many others in the wool and hair fibres. However, it is plausible that
KRTAP13-1 is expressed in other tissues or involved in functions related to fitness or survival, despite such functions having not been reported yet. In this respect, in mice,
KRTAP13-1 does not appear to be expressed in hair follicles, but it is found in the periderm of embryonic mice and in the filiform tongue papillae and parakeratotic tail epidermis of adult mice [
10]. This would suggest that further investigation into the expression patterns of the
KRTAP13-n genes, and especially the roles of
KRTAP13-1 in sheep, is warranted.
The allele sequences of ovine
KRTAP13-4 required two different electrophoresis conditions for resolution. In this respect, we explored a range of electrophoresis temperatures (from 4 °C to 35 °C), voltages (from 200 V to 400 V), gel concentrations (from 8% to 12%), and the addition of glycerol to gels. Despite these efforts, no single condition could resolve all the allele sequences of ovine
KRTAP13-4. While optimisation of electrophoresis conditions typically resolves all gene alleles with one set of conditions, the inability to do so with a single gel system has been reported previously for the porcine leptin gene [
22]. This highlights a potential limitation in the detection power of PCR-SSCP analyses for resolving all sequence variation in the genes.