Genetic polymorphism detection of Casein genes in Albanian local sheep breeds

Vilson Bozgo, Anila Hoda, Lorena Hysi and Lumturi Papa

Department of Animal Science, Agricultural University of Tirana, Albania
ahoda@ubt.edu.al

Abstract

Local sheep breeds play an important role for the Albanian farmers as the mains source of milk, meat and wool. The aim of this study was to detect the genetic polymorphism of exon 17 in alpha -casein gene and exon 4, in kappa -casein gene in three Albanian local sheep breeds: Bardhoka, Ruda and Shkodrane, by the use of PCR-SSCP and PCR-RFLP respectively. The sequence analysis showed a single nucleotide polymorphism (SNP) at position 137 (C>T) and at 168 (T>C) of exon 17 CSN1S1 and exon 4 CSN3, respectively. Analysis of molecular variance (AMOVA) showed that 4% of differentiation was between populations. Genetic differentiation among populations (F_ST) is low, only 4.2%. Gene flow between populations was 5.97.

Keywords: genetic polymorphism, gene flow, AMOVA

Introduction

Local sheep breeds are the main livestock species reared by Albanian farmers, which are managed in extensive and semi extensive conditions and are the main source of milk, meat and wool mainly for family consumption (Hoda and Marsan 2012).

Milk proteins include caseins and whey proteins (Farrell Jr et al 2004). Caseins are a family of phosphoproteins synthesized in the mammary gland in response to lactogenic hormones and secreted as micelles (Feligini et al 2005). Casein genetic polymorphisms are important and well known due to their effects on quantitative traits and technological properties of milk (Ceriotti et al 2004).

The genetic polymorphism of casein genes is studied in different Egyptian breeds (Othman et al 2013a,b), Czech (Sztankoova et al 2012), Italian (Sacchi et al 2005) etc. Feligini et al (2005) have studied SNP in kappa casein coding gene of Pag, Sarda and Pramenka sheep breeds. Genetic polymorphism of CSN1S1 casein gene is studied also in Bulgarian sheep breeds. (Hristova at al 2011, Gencheva and Georgieva 2019).

Previously different markers are used to characterize the local sheep breeds in Albania like microsatellite (Hoda and Marsan 2012, Hoda et al 2010b), AFLP (Hoda et al 2010a), mtDNA (Hoda et al 2014), SNP (Hoda et al 2011), which have shown that these breeds face a high level of admixture. Milk protein polymorphisms have been considered as potential tools for selection of dairy sheep breeds (Ceriotti et al 2004).

The aim of this study was to investigate allele frequencies at casein loci of milk protein genes (exon 17 CSN1S1and exon 4 CSN3) in three Albanian local sheep breeds, Bardhoka, Ruda and Shkodrane.

Material and methods

A total of 93 individuals from three local sheep breeds: Bardhoka, Ruda and Shkodrane were included in the analysis. A description of the breed is provided previously (Hoda and Marsan 2012). Blood samples were collected from 31 unrelated individuals, per each breed, according to the recommendations of ISAG/FAO. DNA was isolated from the whole blood using the standard procedure of phenol-chloroform extraction and Proteinase K digestion. The exon 17 from ovine CSN1S1 gene was analyzed using PCR - SSCP method, meanwhile exon 4 of CSN3 gene was determined by PCR- RFLP analysis as described by Pariset et al (2006).

GenAlEx 6.5 program (Peakall and Smouse 2012) was used to compute the allele frequency per locus, observed and expected heterozygosity, Information index. ARLEQUIN software (Excoffier et al 2005) was used for the analysis of molecular variance (AMOVA). GENEPOP software (Rousset 2008) was used to calculate fixation index F (Weir and Cockerham 1984).

Result and discussion

Sequence analysis of CSN1S1exon 17 in three local sheep breeds from Albania revealed transition C to T, at position 137. The C SNP has the highest frequency at Bardhoka breed (78.6%; Figure 1). The analysis of Exon 4 of CSN3gene detected a SNP at position 168 (T>C).The highest frequency of T was found at Bardhoka breed (92.9%; Figure 1). Ruda breed displayed the highest values of genetic diversity at each locus, while Bardhoka showed the lowest value for the CSN3 locus.

Figure 1. Allelic frequencies per locus and breed

According to Takezaki and Nei (1996), a marker can be used in the selection program if the average heterozygosity in a breed is in the range of 0.3 to 0.8. Based on the results obtained here, both loci cannot be considered suitable for selection, however, they could be used for estimation of breed genetic diversity.

F statistics and gene flow (Nm), per each locus, are shown in Table 2. The data show that the global heterozygote deficit across populations (F_IT) is 21.3%. The heterozygote deficit due to inbreeding within populations (F_IS) is 17.9%. Genetic differentiation among populations (F_ST) is low, only 4.2%. The low level of genetic differentiation is supported by gene flow across breeds over two loci, which is 5.97.

The analysis of molecular variance (AMOVA) displayed 3.98% differentiation among populations, similar as explained by F_ST values(Table 3). AMOVA shows that only 4% of total variation is found among the population.

The substitution of nucleotide between variant C to T at position 137 of exon 17 change the aminoacid Thr137 with Ile137. At exon 4 of CSN3 gene, thymine was substituted for a cytosine while the corresponding amino acid remains a tyrosine. The same finding has shown Feligini et al (2005), for Pag, Pramenka and Sarda breeds, from Croatia, Serbia and Italy, respectively.

The use of genetic polymorphism of casein genes in molecular marker-assisted selection (MAS) is of great interest to improve milk productivity in farm animals (Kumar et al 2006). The genetic polymorphisms of milk proteins are often associated with quantitative and qualitative parameters in milk being potential candidate markers that should be included in breeding strategies (Selvaggi et al 2014). Identification of polymorphisms in ovine milk is more important because sheep milk is commonly used solely for cheesemaking (Feligini et al 2005). Therefore the results obtained here can be used also for an association between genetic variants and ovine milk traits, as previously done by Corral et al (2010) and to consider the molecular markers in selection schemes for milk production in sheep breeds.

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Received 19 February 2020; Accepted 30 March 2020; Published 1 May 2020

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