Elsevier

Small Ruminant Research

Volume 175, June 2019, Pages 149-154
Small Ruminant Research

Identification of llama KRTAP7-1 and KRTAP8-1 fiber genes and polymorphism screening

https://doi.org/10.1016/j.smallrumres.2019.04.016Get rights and content

Highlights

  • We characterized KRTAP7-1 and KRTAP8-1 genes in llamas.

  • Two non-synonymous substitutions (c.185 G > A and c.237 C > G) were found in KRTAP7-1.

  • All SNP identified in KRTAP8-1 coding region were non synonymous.

  • One of them was a double nucleotide polymorphism that affected two consecutive bases.

  • The c.1-5A > G was located in the regulatory region of KRTAP8-1.

Abstract

Keratin-associated proteins (KAP) are one of the main structural components of hair fiber. Within this protein group, high glycine tyrosine (HGT)-KAP play a crucial role in the definition of their physical-mechanical properties. Polymorphisms in HGT genes have been associated with the variation of different wool traits in sheeps and goats. The Argentine llama is a fiber-producing animal which is valued by the textile industry. However, the genes encoding for fiber proteins have not yet been identified in this species. Here, we focus on studying the HGT-KRTAP7-1 and KRTAP8-1 genes and their variation using the High Resolution Melting (HRM) technique in a sample of 117 llamas. Four single nucleotide polymorphisms (SNPs) were detected in KRTAP7-1, two of which were non-synonymous substitutions leading to amino acid changes in the protein. Of the 5 polymorphisms identified in KRTAP8-1, c.1-5A > G was located in the Kozak sequence, known to regulate protein synthesis level. The other four, two SNPs and one double nucleotide polymorphism (DNP), were found in the coding region and produced three amino acid replacement: c.43 T > C and c.45C > A (p.Y15Q), c.46 G > T (p.G16W) and c.173 A > G (p.Y58C). In summary, most of the polymorphisms found in both KRTAP7-1 and KRTAP8-1 genes produce non-conservative amino acid changes involving tyrosine and glycine residues, which are essential to maintain HGT protein properties. Therefore, these mutations as well as the regulatory SNP here identified could modify the fiber characteristics. We discuss the possible impact of these polymorphisms on KAP7-1 and KAP8-1 structure and/or interaction with other fiber proteins.

Introduction

Hair fiber is a highly organized structure formed by the cuticle, the cortex, and the medulla (Gong et al., 2012; Plowman et al., 2009). Most of the fiber (90%) is composed of the cortex, which in turn consists of keratin intermediate filaments (KIF) embedded in a matrix of keratin-associated proteins (KAP) (Kuczek and Rogers, 1987). KAP are a heterogeneous group encoded by a large number of multigene families, which according to their amino acid composition and molecular size can be classified into three major classes: high sulfur content (HS, ≤30 mol% cysteine), very high sulfur content (UHS, >30 mol% cysteine), and high content of glycine-tyrosine (HGT) (Powell et al., 1997). HGT-KAP comprises the KAP6, KAP7 and KAP8 families. While KAP6 is encoded by a multigene family of at least three members, KAP7-1 and KAP8-1 are encoded by unique genes, KRTAP7-1 and KRTAP8-1, in most species (Rogers et al., 2002). However, a new member of the KAP8 family, KRTAP8-2, has been identified in goats (Jin et al., 2011) and more recently in sheeps (Gong et al., 2014). These proteins play an essential role in determining the shape and mechanical properties of the fiber such as strength and rigidity (Matsunaga et al., 2013; Parry and Steinert, 1992; Plowman et al., 2009; Powell and Beltrame, 1994).

In vitro studies showed that interactions between HGT and the KIF occur mainly through KAP8-1 that binds specifically to the head domain of the intermediate filament protein K85 (Matsunaga et al., 2013). That bridging keeps the intermediate filaments together resulting in a helical organization within the orthocortex, which is distinctive of curly hair in humans (Nagase and Tsuchiya, 2008) and sheeps (Plowman et al., 2007). Crimped fibers such as those observed in Merino sheeps show a well-defined bilateral arrangement of orthocortical and paracortical cells, with the orthocortex on the convex side of the crimp and the paracortex on the inner side of the curvature (Rogers, 2006). The organization of keratin proteins is also different in both cell types; HGT-KAP proteins are relatively more abundant in the orthocortex than in the paracortex, whereas HS-KAPs are more abundant in the paracortex than in the orthocortex. It has been proposed that this bilateral segmentation plays a role in the determination of wool bending (Gong et al., 2016). This is also supported by the finding that the Felting Lustre (FL) mutant in the Merino sheeps, which is characterized by shinny crimpless wool, shows reduced levels of KRTAP 6, 7, and 8 gene expression in the orthocortex (Li et al., 2009). Probably driven by an economic interest, the variation of these genes has been investigated mainly in fiber producing species (Zhao et al., 2009; Gong et al., 2012; Liu et al., 2014). Different studies have reported association between polymorphisms in HGT-KRTAP and wool traits. Parsons et al. (1994) analyzed the association between genomic variation of KRTAP6 and KRTAP8 and production traits in Merino sheeps, finding evidence of linkage between these loci and the mean fiber diameter. In another study in Southdown x Merino cross lambs, a 57 bp deletion in KRTAP6-1 was associated with various fiber characteristics including diameter and prickling factor (Zhou et al., 2015). In Cashmere goats, two single nucleotide polymorphisms (SNPs) in KRTAP8-1 were found to be related with the weight and length of the fiber (Liu et al., 2011).

The llama (Lama glama) and the alpaca (Vicugna pacos) are domestic camelid species, native to South America. Traditionally raised as a multipurpose animal, the Argentine llama has an excellent fiber production capacity with a quality similar to that of alpacas from Bolivia or Peru (Hick et al., 2009). Studies on morphological attributes and fiber traits showed high variation in these characters in Argentine llama herds, therefore presenting great possibilities for improvement through breeding programs (Frank et al., 2006). With the advance of molecular biology techniques, it is possible to obtain genotype information and use it for selective breeding of animals with desirable traits. Nevertheless, very little is known about the genes that control the growth and development of fiber in camelids. Just two reports on this topic have been published. They characterized the Fibroblast Growth Factor 5 (FGF5) gene and analyzed its association with fiber length in the llama (Daverio et al., 2017) and alpaca (Pallotti et al., 2018). On the other hand, genes that encode the structural components of the fiber have not yet been studied in domestic camelids. In the present work, we identified HGT genes KRTAP7-1 and KRTAP8-1 and their genetic variation using the HRM (High Resolution Melting) technique in Argentine llamas. We found sequence variants in both genes that might be useful as markers in association studies with different fiber traits.

Section snippets

Samples

DNA from one individual was initially used for the characterization of the KRTAP7-1 and KRTAP8-1 genes. For polymorphism identification, 117 unrelated llama samples from 4 herds from Catamarca (N = 33), 3 from Jujuy (N = 36), 2 from Buenos Aires (N = 16) and 1 herd from Tucumán (N = 1), Salta (N = 2), La Pampa (N = 12), San Luis (N = 9) and Entre Ríos (N = 8) provinces of Argentina were analyzed. The lack of relationship between the sampled animals was verified by consulting breeder records.

KRTAP7-1 gene

The coding region of the llama KRTAP7-1 gene consisted of a single exon of 264 bp. The sequence also included two fragments of 37 bp and 266 bp corresponding to the 5′ and 3′-untranslated regions (UTR). The complete sequence of the gene and its untranslated regions were deposited in the GenBank database under accession number MH253571.

KRTAP7-1 encoded a putative protein of 87 amino acids, two amino acids longer than that of sheeps and goats. This protein was 100% identical to that of alpaca and

Characterization and genetic variability analysis of the KRTAP7-1 and KRTAP8-1 genes

Llama KRTAP7-1 and KRTAP8-1 genes are both small and intronless, a feature that is common to other KRTAP genes (Liu et al., 2011; McLaren et al., 1997).

According to studies carried out in mammal KRTAP subgenomes, the KAP8-1 protein presents an average amino acid composition of 22.47% Glycine and 19.15% Tyrosine (Khan et al., 2014). Compared to these values, KAP8-1 of llama had more Glycine (25.8%) and less Tyrosine (16.1%). KAP7-1 amino acid composition was similar to other species, although

Conclusion

The KRTAP7-1 and KRTAP8-1 genes are polymorphic in llamas and each of them is predicted to encode 4 protein variants. Moreover, KRTAP8-1 has a variant in the regulatory region that could affect the levels of transcription and translation of the protein. Hence, the polymorphisms identified constitute good markers for association studies and could provide an opportunity for the implementation of molecular information for selective breeding of animals with desirable fiber traits.

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Acknowledgements

The authors thank M. Silbestro for her technical assistance and all llama breeders who allowed us to take samples, especially Lilian Brodtmann. This work was supported by funds from the Scientific Research Commission from the Province of Buenos Aires (CICPBA), Argentina. Di Rocco F is researcher of CICPBA.

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