Variation in the caprine keratin-associated protein 15-1 (KAP15-1) gene affects cashmere fibre diameter

Open Access Archives Animal Breeding Variation in the caprine keratin-associated protein 15-1 (KAP15-1) gene affects cashmere fibre diameter Mengli Zhao1,2 , Huitong Zhou1,2,3 , Jon G. H. Hickford1,2,3 , Hua Gong2,3 , Jiqing Wang1,2 , Jiang Hu1,2 , Xiu Liu1,2 , Shaobin Li1,2 , Zhiyun Hao1,2 , and Yuzhu Luo1,2 1 Gansu Key Laboratory of Herbivorous Animal Biotechnology, Faculty of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China 2 International Wool Research Institute, Faculty of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China 3 Gene-Marker Laboratory, Faculty of Agriculture and Life Sciences, Lincoln University, Lincoln 7647, New Zealand Correspondence: Jiqing Wang () and Yuzhu Luo () Received: 5 December 2018 – Revised: 4 March 2019 – Accepted: 5 March 2019 – Published: 26 March 2019 Abstract. Keratin-associated proteins (KAPs) are a structural component of cashmere fibre, and variation in some KAP genes (KRTAPs) has been associated with a number of caprine fibre traits. In this study, we report the identification of KRTAP15-1 in goats. Sequence variation in the gene was detected using the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) technique in 250 Longdong goats, and six variants (named A to F) containing eight single nucleotide polymorphisms (SNPs) were identified. Five of the SNPs were non-synonymous and would lead to putative amino acid changes. Reverse-transcription polymerase chain reaction (RT-PCR) analysis revealed that KRTAP15-1 was expressed in secondary hair follicles but not in heart tissue, liver tissue, lung tissue, kidney tissue or the longissimus dorsi muscle. Despite being rich in cysteine, the caprine KAP15-1 protein possesses a high content of serine and moderate content of glycine and phenylalanine. Association analyses revealed that KRTAP15-1 variant A was associated with decreased mean fibre diameter (MFD), and this effect appeared to be dominant; while variant C was found to be associated with increased MFD, the effect being recessive. The findings suggest that caprine KRTAP15-1 is highly polymorphic and that variation in this gene affects cashmere MFD. 1 Introduction Hair and cashmere are produced by the primary and the secondary hair follicles, respectively, of cashmere goats. As a consequence of the characteristic of being softer, lighter and stronger, with better insulating properties, cashmere fibres are sometimes called “soft gold”, this reflecting how highly the fibre is valued. The price of cashmere fibres is comparatively much higher than wool and mohair, and cashmere fleece weight, mean fibre diameter (MFD) and unstraightened fibre length are the most important traits that determine the economic return for the production of cashmere. The main components of cashmere fibres are keratins (Ks), which form keratin intermediate filaments and keratinassociated proteins (KAPs), which form a matrix cross- linking the keratin intermediate filaments. The Ks and KAPs are therefore believed to play an important role in determining the characteristics of the fibre. KAPs have a high content of either cysteine or glycine and tyrosine, and based on this content, the proteins can be divided into three broad groups: the high-sulfur (HS) group, which contains less than 30 mol % cysteine; the ultra-highsulfur (UHS) group, which contains more than 30 mol % cysteine, and the high glycine and tyrosine (HGT) group, which has 35 mol %–60 mol % glycine and tyrosine (Gong et al., 2016). Within these groups, the KAPs can be further subdivided into families based on their sequence similarity, and over 100 KAP genes (called KRTAPs) belonging to 27 fam- Published by Copernicus Publications on behalf of the Leibniz Institute for Farm Animal Biology (FBN). Original study Arch. Anim. Breed., 62, 125–133, 2019 https://doi.org/10.5194/aab-62-125-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. 126 M. Zhao et al.: Variation in the caprine keratin-associated protein 15-1 (KAP15-1) gene ilies have been identified to date across mammalian species (Gong et al., 2010). KAP15-1 is a single gene-member family belonging to the HS-KAP group. The KAP15-1 gene (KRTAP15-1) has been described in mice and humans (Pruett et al., 2004; Rogers et al., 2002) and recently in sheep (Li et al., 2018). In sheep, variation in KRTAP15-1 has been reported to affect wool yield (Li et al., 2018). Despite a caprine KRTAP15-1 sequence having been deposited in the NCBI GenBank database (accession number AY510116.1), little is known about KRTAP15-1 variation and its effect on fibre traits in this species. In this study, we attempted to identify KRTAP15-1 in goats, to search for potential variation in the gene and to investigate its effect on cashmere fleece traits. 2 2.1 Materials and methods Goats and DNA samples Two hundred and fifty-three Longdong cashmere goats fed at the Yu sheng Cashmere Goat Breeding Company in Huan County of Gansu Province were investigated. At 1 year of age, cashmere fibres were collected by combing the goats, and the weight of fibre collected per goat was measured. A sample of fibre from the mid-side region was collected from each goat and sent for the measurement of mean crimped fibre length and MFD at the Inner Mongolia Agricultural University, Inner Mongolia, China. Blood samples from these goats were collected directly onto FTA cards (Whatman BioScience, Middlesex, UK). A two-step washing procedure was used to purify the goat genomic DNA for polymerase chain reaction (PCR) amplification from 1.2 mm punches of the dried blood spots, using the protocol described in Zhou et al. (2006). 2.2 Animal tissues Three 3-year-old Longdong cashmere goats were slaughtered, and tissue from the skin, heart, liver, lungs, kidneys and longissimus dorsi muscle was collected and rapidly frozen for storage in liquid nitrogen. The separation of primary and secondary hair follicles from the skin tissue used the method described by Jin et al. (2011). Briefly, the skin sample was cut into a strip of a width of 1.0 cm. Next, the hair follicle bulbs were exposed by removing the subcutaneous fat using a dissecting needle and the follicle tissues separated from the surrounding tissues. Once isolated, the hair follicles were sorted under a microscope into primary and secondary follicles, this being based on whether they had sweat glands or not. 2.3 Polymerase chain reaction amplification A comparison of the caprine KRTAP15-1 sequence (accession number AY510116.1) and the ovine Arch. Anim. Breed., 62, 125–133, 2019 KRTAP15-1 sequences (accession numbers MH742372 – MH742375) suggested that the primers designed for ovine KRTAP15-1 (Li et al., 2018), would also amplify a 495 bp fragment covering the entire coding sequence of caprine KRTAP15-1. The sequences of these primers were 5’-GAACTCAGAACTCCCAACAG-3’ and 5’-TAACCATGAGGTGACT (...truncated)