Identification of novel single nucleotide polymorphisms in candidate genes for mutton quality in Indian sheep  

Reena Arora1 , Harikesh Singh Yadav2 , Dinesh Kumar Yadav3
1. Animal Biotechnology Division, National Bureau of Animal Genetic Resources, Karnal-132001, Haryana, India
2. Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar-243122, Bareilly, Uttar Pradesh, India
3. Livestock Information Management Section, National Bureau of Animal Genetic Resources, Karnal-132001, Haryana, India
Author    Correspondence author
Animal Molecular Breeding, 2014, Vol. 4, No. 1   doi: 10.5376/amb.2014.04.0001
Received: 10 Oct., 2014    Accepted: 06 Nov., 2014    Published: 24 Nov., 2014
© 2014 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Arora et al., 2014, Identification of novel single nucleotide polymorphisms in candidate genes for mutton quality in Indian sheep, Animal Molecular Breeding, Vol.4, No.1, 1-5 (doi: 10.5376/amb.2014.04.0001)


The study reports the identification of novel SNPs in candidate genes for mutton quality across a panel of eleven phenotypically and geographically diverse Indian sheep breeds. The investigated candidate genes were CAPN4, CAST, FABP3 and DGAT1 which are known to be polymorphic. Nine novel SNPs could be identified across the studied gene loci which were in the heterozygous condition. One SNP in the exon 1 of DGAT1 was non-synonymous. The average expected heterozygosity determined in the eleven Indian sheep breeds at these loci was 0.328. The allele and genotype frequencies for all the identified SNPs were determined. The average haplotype and nucleotide diversity across all loci were 0.545 and 0.206 respectively. The information generated provides preliminary indication of the functional diversity present in Indian sheep. Future studies will be required to establish the effect of the reported SNPs in the Indian sheep populations.

Genes; Polymorphism; Indian sheep; Mutton; SNP

The Indian mutton industry is largely dependant on regional demands and a preference for particular meat among some communities in specific geographical areas.The variability of mutton quality that can be seen within and between different sheep breeds may be the result of genetic variation found in genes linked to these traits. The existence of major genes associated with mutton quality provides excellent opportunities for improving its quality. The major constraint for improving mutton quality in Indian sheep is the lack of knowledge on physical as well as genetic attributes of mutton quality from different sheep breeds/populations. The candidate gene approach involving genes with known expression of certain proteins provides genomic information that may be used in genetic improvement of sheep in the absence of resource flocks in the country. Some candidate genes like calpain (CAPN4), calpastatin(CAST), diacylglycerol acyltransferase 1 (DGAT1) and fatty acid binding protein 3 (FABP3) have been implicated in muscle development and fat metabolism (Goll et al. 1992; Calvo et al. 2004; Scata et al. 2009). The CAPN4 is acytosolic endopeptidase that requires calcium for catalytic activity, whereas CAST is a specific endogenous inhibitor that acts on calpain (Ma et al. 1993). These genes are involved in muscle development, growth (Nonneman and Koohmaraie, 1999) as well as in eating quality (tenderness) of the mutton (Byun et al. 2009). The DGAT1 and FABP3 are involved in fatty acid metabolism and associated with milk fat content and marbling (Scata et al. 2009; Xu et al. 2009). It is imperative that knowledge be acquired of the available Indian sheep populations which are as yet untouched reservoirs of potential genes defining mutton quality. The present study was therefore, undertaken with the aim to screen Indian sheep for previously reported polymorphic loci in candidate genes possibly associated with mutton quality traits.

1 Results
Nine novel SNPs were identified in the four gene loci for mutton quality traits, across a panel of 11 diverse Indian sheep breeds. These SNPs were named as per their position in the available reference sequence (Table 1). All the SNPs were observed to be novel in Indian sheep and were in the heterozygous condition. Six transitions and three transversions were observed. Two of the SNPs DGAT1 g.355G > T and FABP3 g.4993A > G were in the coding region whereas the remaining SNPs were observed in the introns. One of the novel SNPs (DGAT1 g.355G > T) in the exon 1 region was nonsynonymous leading to a change in the amino acid (Asp to Tyr).

Table 1 Estimates of observed heterozygosity (Ho), total expected heterozygosity (Ht), allele and genotype frequencies across the identified SNPs in Indian sheep

The heterozygosity as well as allele and genotype frequencies for all SNPs are given in Table 1. The observed heterozygosity values varied from 0.071 to 0.832, while the expected heterozygosity ranged from 0.069 to 0.569. The minor allele frequency was > 0.033 across all SNPs. The major allele was near fixation at DGAT1 g.434C > G SNP. The homozygous genotypes AA in CAST g.326A > G, TT in DGAT1 g.355G > T and GG in DGAT1 g.434C > G were not observed in the Indian sheep investigated.
The number of haplotypes per locus (Hap.No.), haplotype diversity (Hd) and nucleotide diversity (Pi) for all the investigated loci are given in Table 2. The CAST locus was observed to be the most diverse with maximum number of haplotypes (10), while CAPN4 showed two haplotypes only. The average haplotype and nucleotide diversity across all loci were 0.545 and 0.206 respectively.

Table 2 Haplotype diversity across candidate genes for mutton quality traits in Indian sheep

FST statistic (Weir 1996), which measures the genetic differentiation between populations (by estimating the genetic variance among populations divided by total genetic variance of the entire population) revealed low differentiation (0.174) among the sheep populations investigated. The average gene flow between the breeds was 1.29.
2 Discussion
Differences in mutton quality traits in sheep have been attributed to genetic variations in relevant genes (Kijas et al. 2007; Hickford et al. 2010). In this study, detection of novel nucleotide variations in Indian sheep, at candidate genes for mutton quality, known to be polymorphic, reveal availability of greater genetic diversity at these loci.
Polymorphism in the CAPN4 gene has also been reported in Karakul sheep breed by SSCP analysis (Shahroudi et al. 2006), but these have not been associated with any production trait. The detection of four new SNPs in the CAST gene locus, in our study, reflects that this locus habours substantial diversity. These results are supported by previous studies where two alleles were reported in the CAST gene using the RFLP technique (Palmer et al. 1998; Shahroudi et al. 2006). Additionally, four alleles have been identified in the intron 12 region in New Zealand crossbred sheep by SSCP (Byun et al. 2009). The FABP3 and DGAT1 genes have been associated with marbling or intramuscular fat in pigs (Li et al. 2010) and cattle (Cho et al. 2008). Some variations in these genes are associated with milk fat content in sheep (Calvo et al. 2004; Scata et al. 2009). Xu et al. (2009) have reported association of an SNP in exon 17 with marbling score in Chinese sheep. The non-synonymous SNP identified in exon 1 of DGAT1 gene (g.355G > T), in this study, may influence the gene expression. However, this needs to be substantiated by further studies.
The average expected heterozygosity at these loci was 0.328. The results of this study suggest that sufficient level of heterozygosity exists in these sheep breeds at the investigated gene loci. The gene diversity observed at these loci was slightly less as compared to that reported previously for wool keratin intermediate filament (0.420, Arora et al. 2008) and β - lactoglobulin (0.422, Arora et al. 2010) gene loci in Indian sheep.
Identification of SNPs associated with mutton traits may not only help in improvement of the production but also provide economic gains to the rearers. Two SNPs in the GDF8 gene known to be associated with mutton quality and carcass traits in Texel (Kijas et al. 2007) and New Zealand Romney sheep (Hickford et al. 2010) have also been identified in Indian sheep (Arora et al. 2013). Although the sample size is small the information generated provides preliminary indication of functional diversity present in Indian sheep.
The diversity available in Indian sheep breeds at candidate genes for mutton quality, revealed from this study, presents the opportunity to exploit the use of either allele/genotype, once the association is validated in the Indian sheep population.In order to substantiate these observations future studies will need to establish the effect of reported SNPs in the Indian sheep populations.
3 Material and methods
3.1 Samples
A panel of eleven Indian sheep breeds/populations representing wide phenotypic and geographic diversity was used for identification of SNPs. Ten unrelated genomic DNA samples from each sheep population namely Bandur (also known as Mandya and Bannur), Chokla, Deccani, Ganjam, Garole, Madgyal, Magra, Malpura, Muzaffarnagri, Nali and Nellore were included in the panel. These breeds are distributed in three agroecological zones and are phenotypically diverse. The utility, distribution, 6-month body weight and dressing percentage of the breeds/populations are presented in Table 1. Chokla, Magra, Nali, Malpura and Muzzafarnagri belong to North-western arid and semi arid region. Deccani, Madgyal, Nellore and Bandur are from the Southern Peninsular region and Ganjam and Garole are from the Eastern region of the country. As per utility patterns these breeds have been classified as carpet wool (Chokla, Magra, Nali), mutton carpet-wool (Ganjam, Malpura and Muzaffarnagri) and mutton (Bandur, Deccani, Garole, Madgyal, Nellore) breeds (Acharya et al. 1982) (Table 3).

Table 3 Distribution zone, utility, 6-month body weight and dressing percentage of breeds/populations investigated

3.2 PCR amplification and sequencing
The primers for CAPN4 (exon 4, 5 and intron 4), CAST (exon 12, intron 12 and partial exon 13), DGAT1 (5’UTR, exon1 and intron1) and FABP3 (exon 2 and intron 2) gene were taken from published literature (Shahroudi et al. 2006; Byun et al. 2009; Scata et al. 2009; Calvo et al. 2004). Each locus was amplified by PCR after standardizing the annealing temperature. The amplified products were checked on agarose gel. Fifty microlitres of the PCR amplified product was purified using QIAquick PCR Purification Kit (QIAGEN). The purified PCR amplicons were sequenced on ABI 3100 sequencer in both forward and reverse direction.
3.3 Data Analysis
Multiple alignments of the sequences were performed with MEGA 4.1 (Tamura et al. 2007) and DNAMAN ver 5.2.10 (Lynnon BioSoft, Vaudreuil, Quebec, Canada).All positions containing gaps and missing data were eliminated from the dataset. Insertions and deletions were excluded from all estimates. The sequences were analysed for polymorphic sites, nucleotide diversity (π) and haplotype diversity (h), genetic differentiation (FST) and gene flow (Nm) using DNASPv5 (Librado et al. 2009). Allele and genotype frequencies were estimated for the identified SNPs.
The authors acknowledge the help rendered by officials of State Animal Husbandry Departments of different States and the sheep farmers for their help in collection of blood samples. We are grateful to Director, National Bureau of Animal Genetic Resources (NBAGR, Karnal), Indian Council of Agricultural Research (ICAR, New Delhi) for providing necessary facilities and Mr. Rakesh Kumar (Senior Technical Assistant, NBAGR Karnal) for technical assistance.
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