Asian Journal of Animal and Veterinary Advances

Year: 2010 | Volume: 5 | Issue: 6 | Page No.: 402-409
DOI: 10.3923/ajava.2010.402.409

A Novel 15 bp Deletion Mutation at KAP16.5 Locus in Cashmere Goat of China

Liu Wu Jun, Fang Yi, Shao Yong-gang, Fang Guang Xin, Tian Ke Chuan, Huang Xi Xia, Chen Hong and Zhang Fu Quan

Abstract: In this study, a novel deletion mutation in the whole CDS region at cashmere goat KAP16.5 gene locus is reported, which result showed the deletion of KAP16.5 gene in 816 cashmere goat samples was firstly detected in China. At the same time, parts of these samples sequenced. However, there are not many studies of goat KAP16.5 gene and its polymorphisms in literary sources. The polymorphism was studied in three Xinjiang local goat breeds of China. The results showed that there was 15 bp-deletion mutation in this gene. The frequencies of the KAP16.5-Y allele in Xinjiang goat (n = 220), Nanjiang cashmere goat (n = 310) and BoGeDa cashmere goat breeds (n = 286) were 0.9591, 0.8694 and 0.8846, respectively. The χ²-test showed that the genotype distributions in these three cashmere goat breeds were not in agreement with Hardy-Weinberg equilibrium. According to the classification of PIC, Nanjiang cashmere goat was more polymorphic at this locus. Moreover, the 15 bp-deletion mutation was described at KAP16.5 locus for the first time and not have described at KAP16.5 locus. Then, the deletion polymorphisms within the KAP16.5 have been associated with economic production traits data in Nanjiang cashmere goat breed shown no significant (p>0.05).

Keywords: deletion mutation, Cashmere goat, goat, cashmere traits, breed and KAP16.5 gene

INTRODUCTION

The hair keratin-associated proteins are a major component of the matrix between the hair keratins form intermediate filaments and are thought to form the rigid hair shaft through a cross-linked network with the hair keratins form intermediate filaments (Powell and Rogers, 1994). Mammalian skin consists of three major compartments: epithelium, dermis and hypodermis. It is important that epidermis is a derivative of the surface ectoderm, as a protective barrier and specific appendages including hair, nails and different eccrine glands (Jonker et al., 2004; Bond et al., 1996). The cuticle, the cortex and the medulla is a main structure of all hairs and wool (Bond et al., 1996; Langbein et al., 1999, 2001; Hawkins and Ragnarsdottir, 2009; Feughelman, 2002). The internal cortical cells are long polyhedral spindle-shaped structures (Jones, 2001), which mechanically, are the most important component of any α-keratin fiber (Feughelman, 2002). The flattened overlapping, cuticle cells surround the cortex and forms the external layer of the fiber (Marshall et al., 1991). The structure of the cashmere fiber largely involves the expression of hair keratins and their keratins-associated proteins (Langbein et al., 1999, 2001). The keratins and Keratin-Associated Proteins (KAPs) are a large heterogeneous group of proteins that make up about 90% of the wool fiber (Powell and Rogers, 1994). The human hair is very resistant to external stimuli and high stability is due to keratin (Barba et al., 2009). The keratins and Keratin-Associated Proteins (KAPs) plays an essential role in hairs and wool.

KAPs are encoded by a large number of multigene families. Furthermore, the KAP genes are small in size less than 1 kb, generally contain a single exon. The KAPs have been divided into three categories, the high sulphur KAPs (<30 mol% cysteine content), the ultra-high sulphur KAPs (>30 mol% cysteine content) and the high tyrosine/glycine KAPs (Barba et al., 2009). The hair keratins represent the type I (acidic) and type II (basic) two multigene families. They form the 8-10 nm intermediate filaments (KIF) of trichocytes by co-polymerization of type I and type II members, which are differentially expressed during hair fiber development (Langbein et al., 1999, 2001). Previous studies have introduced these KAPs are using the abbreviations KAP1.n through KAP23.n for these members known at that time with n referring to a number identifying individual members, also subdivided into 23 distinct families (Rogers and Powell, 1993) and more than 100 KAP genes have been isolated from human and other mammalian species. Genetic markers for the keratin and keratin-associated protein genes have been associated with variation in fiber diameter and staple strength (McLaren et al., 1997).

The KAP16.5 gene is one of high sulphur KAPs, which are important for the hair structure. Apparent molecular weights of the high sulphur proteins were 26.5-43.0 kd, but it is probably higher than the real values 75-150% (Marshall, 1983). The high sulphur proteins predominantly found in the cuticle with some also found in the cortex (Irvine, 2005). All KAP16 gene exhibit strong expression in a narrowly defined pattern restricted to the lower and middle cortical region of the hair shaft in both developing and cycling hair (Pruett et al., 2004).

To date, no polymorphisms of KAP16.5 gene have been reported for China cashmere goat. Therefore, the aim of our study is analyzed the genetic variations of KAP16.5 gene in 816 goat individual in China by electrophoresed on agarose gels, PAGE and DNA sequencing methods.

MATERIALS AND METHODS

Animal Source
Eight hundred and sixteen all unrelated animals (Xinjiang goat, n = 220; Nanjiang cashmere goat, n = 310; Bogeda cashmere goat, n = 286) were collected and used in this study in March to December of 2009. The Xinjiang goat were from the breeding centre of KuErLe of XinJiang in China, the Nanjiang cashmere goat were from AkeSu Goat Research Center of XinJiang and Bogeda cashmere goat were from Urumuqi of XinJiang in China. Many records of cashmere traits and body weight were used for statistical analysis.

3DNA Preparation and Primer Design
Genomic DNA of 816 cashmere goat were isolated from 2% heparin-treated blood samples and stored at -80°C, following standard procedures (Sambrook and Russell, 2001). According to the sequence of KAP16.5 (GenBank accession number AY510117), one pair of PCR primers was designed with Primer5.0, as follow:

It was used to amplify 272 bp PCR products for cashmere goat KAP16.5 gene of the whole CDS region.

PCR Amplification
One pair of PCR primer were designed using Primer 5.0 software to amplify the whole CDS region of Capra KAP16.5 gene (high sulphur KAPs) (GenBank accession number AY510117), the size of the PCR products was 272 bp. The 25 μL volume contained: 50 ng genomic DNA, 0.5 μM of each primer, 1xBuffer (including 1.5 mM MgCl₂), 200 μM dNTPs and 0.625 units of Taq DNA polymerase (MBI). The PCR was performed using the following program: 94°C for 5min followed by 33 cycles of 94°C for 40 sec, annealing for 35 sec and 72°C for 35 sec and a final extension at 72°C for 10 min.

Deletion Mutation Polymorphism in the KAP16.5 Gene and DNA Sequencing
PCR products were electrophoresed on 4% agarose gels with 1xTBE buffer (89 mM Tris, 89 mM boric acid and 2 mM Na₂EDTA), containing 200 ng mL^-1 ethidium bromide. A 6.5 μL aliquot of PCR products was added to 1.5 μL of loading dye (0.025% bromophenol blue, 0.025% xylene cyanol, 40%, w/v sucrose) and the gels were run at a constant voltage (100 V) for 0.5-1.0 h.

Aliquots of 5 μL PCR products were loaded on 10% PAGE gel (80x73x0.75 mm) in 1xTBE buffer and constant voltage 110 V for 3.5 h. The gel was stained with 0.1% silver nitrate solution. The two methods show equal data results: genotype YY, YZ and ZZ.

The PCR fragments from different patterns in the three breeds were amplified by the pair of primer, then sequenced in both directions by ABI PRIZM 272 DNA sequencer (PerkinElmer) analyze the sequences with BioXM software (version 2.6).

Statistical Methods and Analysis
In these Chinese goat breeds, base on the genotypes of KAP16.5 locus, genotypic frequencies, allelic frequencies and Hardy-Weinberg equilibriums were directly. Differences in genotypic frequencies at Capra hircus KAP16.5 locus among Xinjiang indigenous goat and cashmere goat populations in China were analyzed using a χ²-test, which were performed by SPSS software (1999). Population genetic indexes, such as He (gene heterozygosity), Ho (gene homozygosity), Ne (effective allele numbers) and PIC (Polymorphism Information Content) were calculated according to Nei and Roychoudhury (1974) and Nei and Li (1979), respectively.

RESULTS

Few studies related to polymorphisms of KAP16.5 gene in cashmere goat had been reported. In this study, the entire CDS region and its flanking region of cashmere KAP16.5 gene demonstrated polymorphic patterns in three cashmere goat populations of China. However, the deletion polymorphisms (named genotype YY, YZ and ZZ) were firstly detected at KAP16.5 locus (Fig. 1a, b).

Frequencies of allele KAP16.5-Y allele in the analyzed populations were 0.8694, 0.8846 and 0.9591 for Nanjang cashmere goat (NJG), BoGeDa cashmere goat (BGD) and Xinjiang goat (XJG), respectively. The genotypic frequencies at KAP16.5 locus were significantly different among Xinjiang goat, Nanjiang cashmere goat and Bogeda cashmere goat based on a χ²-test (χ² = 34.588, df = 4, p<0.001), as well as allelic frequencies among the three populations (χ² = 8.721, df = 2, p<0.05). The χ²-test showed that the genotype distributions of NJG, BGD and XJG breeds were not in agreement with Hardy-Weinberg equilibrium (Table 3). In present populations, the population genetic parameters (namely, homozygosity, heterozygosity, effective allele numbers (Ne) and PIC (Polymorphism Information Content)) were calculated (Table 2). According to the classification of PIC (high polymorphism if PIC value>0.5, median polymorphism if 0.25<PIC value<0.5 and low polymorphism if PIC value<0.25) (Botstein et al., 1980). Genotypic and allele frequencies of KAP16.5 gene in the three breeds were shown in Table 1 and 2. Value of homozygosty estimate varied from 0.773 (NJG) to 0.922 (XJG) and Ne ranged from 1.085 (XJG) to 1.294 (NJG). PIC values varied from 0.075 (XJG) to 0.201 (NJG). The three goat populations were at low polymorphic level.

Taking nucleotide sequence AY510117 (GenBank Accession Number AY510117) as criterion, nucleotide sequences from 24 individuals with 3 different genotypes were aligned with the multiple sequence alignment program ClustalW (http://www.ebi.ac.uk/clustalw). A novel 15-bp deletion was detected among them (Fig. 2, 3; GenBank Accession No. AY510117). According to the mutation nomenclature and proposals (Den Dunnen and Antonarakis, 2000), the 15 bp deletion mutation was described as r.115-129 deletion (-GGCCTGGGCTTTGGC-) mutation (Fig. 2, 3). Observed significant differences of genotypic frequencies for YY, YZ and ZZ indicate that the breeds are different according to content of allele Z-with deletion by χ²-test (χ² = 8.721, df = 2, p<0.05) (Table 3). Taking in consideration, that the breeds studied represent different capara utility type (economic production of cashmere traits), the genotypic distribution possibly effect utility type of the breeds. Allele KAP16.5 gene was detected in all the cashmere goat breeds. Probably, deletion was not spread in the breed discussed.

The association of novel 15 bp deletion in KAP16.5 gene with cashmere production traits (body weight after combed, cashmere fiber diameter, down cashmere thickness, cashmere yield) in Nanjang cashmere goat were analyzed (Table 3).

The cashmere production traits in the records had no significant associated with genotypes studied (p>0.05).

DISCUSSION

Many previous studies reported genetic polymorphisms in human high sulfur hair keratin-associated protein, which also play an important role in determining various wool traits as well as the variation in fiber diameter (McLaren et al., 1997; Parsons et al., 1994). Few studies have been reported related to KAP16.5 gene in human and mouse, which also not have been reported in goat. It is known that the fiber diameter, cashmere yield and down cashmere thickness were important cashmere production traits in cashmere goat. Present study is considered as the firstly detected a deletion mutation in the coding region of KAP16.5 gene in these cashmere goat breeds in China.

Notably, all KAP16 gene exhibit strong expression in a narrowly defined pattern restricted to the lower and middle cortical region of the hair shaft in both developing and cycling hair. The study of 15-bp deletion mutation in our results was not a casual mutation, which is essential for KAP16.5 gene CDS region, it showed 15-bp deletion mutation in KAP16.5 protein Cys, Ala, Cys, Cys and Ala, which may be linked to cashmere goat in the coding or regulatory regions of the gene. The result of polymorphic level of Xinjiang goat population was lower than that of Nanjiang and Bogeda cashmere goat population. In order to better understand the detailed genetic variation of KAP16.5 gene, we analyzed it with cashmere production traits in the records. Moreover, the research results revealed no significant impact for genotypes studied (p>0.05) by artificial selection and breeding. So, the objective of this study was to detect the polymorphism within goat KAP16.5 gene in Chinese animals, which may possibly contributed to conducting association analysis and evaluating them as genetic markers in cashmere production and other performance for animal breeding and genetics. In order to get more genetic variation information we would do pay more attention to further research in the future.

The χ²-test results obtained in present study that may be caused by nonrandom mating and among the three Xinjiang local goat breed selected some of the excellent individuals could violate the Hardy-Weinberg equilibrium.

For all the available information concerning KAP16.5 gene in human and mouse, there is no information known for cashmere goat. Our study is considered as the firstly detected the deletion novel mutation in the coding region of KAP16.5 gene in three cashmere goat breeds and associated with cashmere production traits of China. Therefore, the presence of 15 bp deletion in KAP16.5 gene might straddling influence the body weight after combed, cashmere fiber diameter, down cashmere thickness, cashmere yield. The KAP16.5 gene seems to be promising as it plays an important role in fiber diameter traits. It is important that cashmere production traits to the livestock industry, it appears clearly that essential further researches on KAP16.5 gene in the livestock should be done.

CONCLUSION

In this study, we revealed a novel 15-bp deletion polymorphism in the whole CDS region of KAP16.5 gene. The deletion mutation is not significantly associated with body weight after combed, cashmere fiber diameter, down cashmere thickness and cashmere yield trait. The Z allele was a straddling influence in the three goat breeds and it should be pay attention from the breeding schemes of goat. Therefore, this study considered as a preliminary important research for improving the Xinjiang local goat breeds and the breeding of genuine cashmere in China. Also, the present study is one of initial studies performed on China goat cashmere traits and provides preliminary information about alleles and genotypes of KAP16.5 gene in three goat breeds of China.

ACKNOWLEDGMENTS

This study was supported by The project of key Science and Research Program of Xinjiang High School SandR Plan (No. 20090608115118343), GEF Applied Research Project (No. GEF052456 CHA). The project of National Key Technology RandD Program in the 11th Five-year Plan of China (2008BADB2B05).

REFERENCES