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The Polymorphism of a Novel Mutation of KAP13.1 Gene and its Associations with Cashmere Traits on Xinjiang Local Goat Breed in China



Y. Fang, W.J. Liu, F.Q. Zhang, Y.G. Shao and S.G. Yu
 
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ABSTRACT

Genetic variations of KAP13.1 gene have been studied in 816 animals of Xinjiang goat breeds in China. The genotypes and allele frequencies of KAP13.1 gene were detected by PCR-RFLP techniquies. At the same time, parts of our samples were sequenced and analyzed with cashmere production traits data. The results showed that TT genotype significantly higher of body weight after combed trait than GT genotype (p<0.05) and down cashmere thickness trait showed genotype GT with significant higher than GG (p<0.05) in Nanjiang cashmere goat. In Xinjiang goat, the fiber diameter showed significant differences (p<0.05) among the age two, three and four years old. The fiber diameter trait in one year old cashmere goat had significant differences (p<0.05) compared with two, three and four years old. The frequencies of the KAP13.1-T allele in Xinjiang goat (n = 220), Nanjiang cashmere goat (n = 310) and Bogeda cashmere goat breeds (n = 286) were 0.996, 0.568 and 0.969, 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. The mutation in our animals at KAP13.1 locus was recorded for the first time. In conclusion, The results possibly revealed that the polymorphism existed in the three Xinjiang local goat breed and a missense mutation was possibly caused by variations in the number of the decapeptide repeat structures. Further analysis of results leads us to believe that the polymorphism of KAP13.1 gene might be relevant to fiber diameter and other cashmere traits. Thus, molecular genetic study of KAP13.1 gene represented valuable results for genetic conservation purposes and economic production of cashmere traits.

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Y. Fang, W.J. Liu, F.Q. Zhang, Y.G. Shao and S.G. Yu, 2010. The Polymorphism of a Novel Mutation of KAP13.1 Gene and its Associations with Cashmere Traits on Xinjiang Local Goat Breed in China. Asian Journal of Animal and Veterinary Advances, 5: 34-42.

DOI: 10.3923/ajava.2010.34.42

URL: https://scialert.net/abstract/?doi=ajava.2010.34.42

INTRODUCTION

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 are the main structure of all hairs and wool (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). Human hair is very resistant to external stimuli and high stability due to keratins (Barba et al., 2009). So, the keratins and Keratin-Associated Proteins (KAPs) play an essential role in hairs and wool.

The keratins and Keratin-Associated Proteins (KAPs) are one of the largest gene families in mammalian genomes encode, which is a heterogeneous group of proteins that make up about 90% of the cashmere fiber (Rogers et al., 2002). The 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 sulfur KAPs (<30 mol% cysteine content), the ultra-high sulfur KAPs (>30 mol% cysteine content) and the high tyrosine/glycine KAPs (Powell and Rogers, 1996). 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 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).

KAP13.1 is one of high sulfur KAPs which are important for the hair structure, localized in the hair cortex, showed matrix, cuticular expression and might play an important role in the hair forming compartment (Rogers et al., 2002). Apparent molecular weights of the high sulfur proteins were 26.5-43.0 kd, but it is probably higher than the real values 75-150% (Marshall, 1983). The high sulfur proteins predominantly found in the cuticle and some also found in the cortex (Irvine, 2005).

China has a centuries-old history of breeding cashmere goat and abundant cashmere breeds resources and Xinjiang is one of the biggest cashmere-producer in China. The Xinjiang goat is a native indigenous breed. The breed of Nanjiang cashmere goat is hybrid offspring of Liaoning cashmere goat, which is famous for its high cashmere yield, strong adaptability and stable heritage. Nanjiang goat is hybrid offspring of Liaoning cashmere goat breed and Xinjiang indigenous goat and was named by Ministry of livestock of the People’s Republic of Xinjiang (Xinjiang Autonomous Region) in 1997 of China.

To date, no polymorphisms of KAP13.1 gene have been reported for Xinjiang local cashmere goat. Therefore, the aim of our study is analyzed the genetic variations of KAP13.1 gene in 816 individuals of Xinjiang goat breeds in China by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) and DNA sequencing methods.

MATERIALS AND METHODS

Animal Source
Eight hundred and sixteen blood samples from unrelated animals (Xinjiang goat, n = 220; Nanjiang cashmere goat, n = 310; BoGeDa cashmere goat, n = 286) were collected and used in this study in June of 2008. 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 Nanjiang goat were from Urumuqi of XinJiang in China. Many records of cashmere traits and body weight were used for statistical analysis.

DNA 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 KAP13.1 (GenBank accession number AY510115), one pair of PCR primers was designed with Primer5.0, as follow:

Forward: 5'- TCCCATCGTAACTCACAT -3',
Reverse: 5'- AGGTCGGTTGGAAAGAAA -3'

They were used to amplify 501 bp PCR products, containing the CDS region of KAP13.1 gene.

PCR Amplification
One pair of PCR primers was designed using Primer 5.0 software to amplify the whole CDS region of Capra KAP13.1 gene (high sulfur KAPs) (GenBank accession number AY510115), the size of the PCR products was 501 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 5 min 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.

PCR-RFLP and DNA Sequencing
Aliquots of 10 μL PCR products of KAP13.1 were incubated with 6 units restriction enzyme PvuII (Promega) for 6 hours at 37°C, then electrophoresed on 2% agarose gels with 1xTBE buffer (89 mM Tris, 89 mM boric acid, 2 mM Na2EDTA), containing 200 ng mL-1 ethidium bromide. A 7 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 (100v) for 0.4-0.6 h.

The PCR fragments from different patterns in the three breeds were amplified by the pair of primers were sequenced in both directions by ABI PRIZM 501bp DNA sequencer (PerkinElmer) and the sequences were analyzed with BioXM software (version 2.6).

Statistical Methods and Analysis
Based on the genotypes of KAP13.1 locus in Xinjiang local goat breeds, the genotypic frequencies, the allelic frequencies and Hardy-Weinberg equilibriums were directly calculated. Differences in genotypic frequencies at KAP13.1 locus among Xinjiang indigenous goat and cashmere goat populations in China were analyzed using a χ2-test, which were performed by SPSS software (version 16.0) (Norusis, 2008). 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.

Image for - The Polymorphism of a Novel Mutation of KAP13.1 Gene and its 
        Associations with Cashmere Traits on Xinjiang Local Goat Breed in China

Furthermore, statistical analysis was performed for the records of cashmere traits in Xinjiang goat (XJG, n = 220) and Nanjiang cashmere goat (NJG, n = 310). All analyses were done in two steps, first using a full animal model and then using a reduced animal model. The full animal model included fixed effects of marker genotype, age of ram, ewe, sex, farm, body weight, after combed cashmere fineness, down cashmere thickness, cashmere yield and random effects of animal. The reduced model was used in the final analysis (Boldman et al., 1993; Zhao et al., 2004). The software SPSS (version 16.0) was used to analyze the relationship between the genotypes and cashmere traits in goat. The reduced linear model with fixed effects was established and included effects of ewe, ram within ewe, age and genotype, as well as interaction between ram and genotype was involved. Reduced linear model:

Yijklm = u+Si+Dij+Ak+Gl+(SG) il+Eijklm

where, Yijklm was the trait measured on each of the ijklmth animal, u was the overall population mean, Si was the fixed effect associated with the ith ram, Dij was the fixed effect associated with jth ewe with ram i, Ak was fixed effect due to the kth age, Gl was the fixed effect associated with lth genotype (KAP13.1/TT, GTand GG genotype), (SG)il was interaction between the ith ewe and the lth genotype and Ejiklm was the random error.

An effect associated with farm, sex were not matched in the linear model, as the preliminary statistical analyses indicated that these effect did not have a significant influence on variability of traits in analyzed populations. The Least Square Means Estimates (LSM) with standard errors for three genotypes of KAP13.1 gene and growth traits (Zhao et al., 2004).

RESULTS

In this study, the CDS region of KAP13.1 gene demonstrated polymorphic patterns in three breeds populations by PCR-RFLP method and DNA sequencing (Fig. 1), the result revealled a mutation T>G (CAGCTG>CAGCGG) and three genotypes (named genotype TT, GT and GG (Fig. 2). The mutation was confirmed by restriction digestion (PvuII-CAG/CTG), which resulted an important missense mutation in KAP13.1 protein, namely, CTG (Leu) > CGG (Arg). In detail, the T>G mutation was located in (g.186 T>G) nucleotide position of GenBank Accession No. AY510115 at KAP13.1 gene locus.

The result showed that one mutation located in the CDS region of KAP13.1 gene in Xinjiang local goat breeds, which leads to an important deletion restriction-reaction polymerase locus. Three genotypes (TT, GT and GG) were firstly detected by restriction enzyme PuvII. Genotypic and allele frequencies of KAP13.1 gene in the three breeds were showed in Table 1 and 2. The frequencies of alleles T and G were 0.568, 0.432 in Nanjiang cashmere goat, 0.969, 0.031 in BoGeDa cashmere goat and they were 0.996, 0.004 in Xinjiang goat, respectively.

Table 1: Genotype distribution and allelic frequencies at the KAP13.1 gene locus
Image for - The Polymorphism of a Novel Mutation of KAP13.1 Gene and its 
        Associations with Cashmere Traits on Xinjiang Local Goat Breed in China
Bold values: Genotype frequencies at the KAP13.1 gene locus. χ2 (HWE): Hardy-Weinberg equilibrium χ2 value, NJG: NanJiang cashmere goat, BGDG: BoGeDa cashmere goat, XJG: XinJiang cashmere goat

Image for - The Polymorphism of a Novel Mutation of KAP13.1 Gene and its 
        Associations with Cashmere Traits on Xinjiang Local Goat Breed in China
Fig. 1: Sequencing maps from different genotypes in cashmere goat KAP13.1 gene

Image for - The Polymorphism of a Novel Mutation of KAP13.1 Gene and its 
        Associations with Cashmere Traits on Xinjiang Local Goat Breed in China
Fig. 2: PCR-RFLP patterns of the KAP13.1 gene in cashmere goat, M: 100 bp ladder marker, lane 1, 2: Homozygous TT genotype, lane 3: Heterozygous GT genotype, lane 4, 5, 6: Homozygous GG genotype

Table 2: Genetic index in three Xinjiang local goat in China
Image for - The Polymorphism of a Novel Mutation of KAP13.1 Gene and its 
        Associations with Cashmere Traits on Xinjiang Local Goat Breed in China
Ho: Gene homozygosities, He: Gene heterozygosities, Ne: Effective allele number, PIC: Polymorphic information content

T allele and TT genotype were predominant in three breeds. The genotypic frequencies at KAP13.1 locus were significantly different between three breeds. The χ2-test showed that the genotype distributions of KAP13.1 gene were not in agreement with Hardy-Weinberg equilibrium in three breeds (Table 1). Based on χ2-test, genotypic frequencies of the various polymorphism at KAP13.1 gene were found to be significantly different in the three breeds (χ2 = 247.337, df = 4, p<0.01).

Gene homozygosity, gene heterozygosity, effective allele numbers and PIC of KAP13.1 gene in the three breeds were shown in 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), Xinjiang goat population and Bogeda cashmere goat population were with low polymorphic level, Nanjiang cashmere goat population was with median polymorphic level. Gene heterozygosity, effective allele numbers and PIC of KAP13.1 gene in Nanjiang cashmere goat population showed higher than that of Xinjiang goat and Bogeda cashmere goat population.

In this study, we also revealed that the polymorphism of KAP13.1 gene associated with body weight after combed, after combed cashmere fineness (fiber diameter), down cashmere thickness and cashmere yield (cashmere wool fiber weight) (Table 3, 4). Further analysis suggested that the animals with TT genotype significantly higher body weight after combed than GT (p<0.05) in Nanjiang cashmere goat. The down cashmere thickness trait showed GT was significantly higher than GG (p<0.05) in Nanjiang cashmere goat. No significant differences were observed between the TT, GT and GG genotypes in the Nanjiang cashmere goat related to cashmere production traits (p>0.05) (data not show). In Xinjiang goat, the fiber diameter was significant differences (p<0.05) among the age of two, three and four years old. The fiber diameter trait in one year old cashmere goat recorded significant differences (p<0.05) compared with two, three and four years old cashmere goat (Table 3).

Table 3: Association of genotypes at the KAP13.1 gene with growth traits in XinJiang goat
Image for - The Polymorphism of a Novel Mutation of KAP13.1 Gene and its 
        Associations with Cashmere Traits on Xinjiang Local Goat Breed in China
BWC: Body weight after combed, CD: Cashmere fineness, DCT: Down cashmere thickness, CY: Cashmere yield. *Values with different superscripts within the same line differ significantly at p<0.05 (a, b, c). SE: Standard error of means

Table 4: Association of genotypes at the KAP13.1 gene with growth traits in XinJiang cashmere goat
Image for - The Polymorphism of a Novel Mutation of KAP13.1 Gene and its 
        Associations with Cashmere Traits on Xinjiang Local Goat Breed in China
Data are expressed as Mean±SE. BWC: Body weight after combed, CD: Cashmere fineness; DCT: Down cashmere thickness, CY: Cashmere yield

DISCUSSION

Many studies reported genetic polymorphisms in the human high sulfur hair keratin-associated proteins, 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). It is known that the fiber diameter, cashmere yeild and down cashmere thickness were important cashmere production traits in cashmere goat. Present study is considered as the firstly detected novel mutation in the coding region of KAP13.1 gene in three cashmere goat breeds in China.

The study of SNP in our results was not a casual mutation, it showed missense mutation in KAP13.1 protein leucine to arginine, which is essential for optimal growth in infancy and childhood and for nitrogen equilibrium in adults (Jmaes John, http://searchwarp.com/swa51880.htm). The genome is highly redundant in terms of tRNA species for each amino acids but enigmatically under-represents a number of specific codons (Shah et al., 2008), that may be linked to cashmere goat in the coding or regulatory regions of the gene which is not be a causal mutation for the cashmere goat growth traits. The polymorphic level of Xinjiang goat population and Bogeda cashmere goat population were lower than that of Nanjiang cashmere goat population.

The χ2-test results obtained in our study 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.

Despite all the available information concerning KAP13.1 gene in human and mouse, there is no information known for cashmere goat or other livestock. Present results showed that the mutations of KAP13.1 were associations with fiber traits and it was firstly detected in Xinjiang local goat breeds of China. Therefore, KAP13.1 gene seems to be promising as it plays an important role in fiber diameter traits. Considering the economic importance of the cashmere production traits to the livestock industry, it appears clearly that essential further researches on KAP13.1 gene in the livestock should be done.

CONCLUSION

In conclusion, the present study revealed a novel SNP in CDS region of KAP13.1 gene. The SNP is significantly associated with fiber diameter and down cashmere thickness trait. The G allele was a recessive in the three goat breeds and it should be rejected 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.

ACKNOWLEDGMENTS

This study was supported by GEF Applied Research Project (No. GEF052456 CHA).

Y. Fang and W.J. Liu developed the concept and desigened the experiment. Y. Fang, W.J. Liu, F.Q. Zhang, S.G. Yu and Y.G. Shao isolated the genomic DNAs from goat blood. Some of goat blood samples collection were supported by researcher F.Q. Zhang. The pair of primer design and the mutation of gene detection was done by Y. Fang, He also carried out the experiment and some bioinformatics analysis. W.J. Liu and Y.G. Shao conducted some bioinformatics analysis.

We thanks for anonymous reviewers for their helpful suggestions on the quality improvement of the present study, also.

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