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Genetic polymorphism of casein alpha-S1 gene in Tunisian local Goat


a a

Borni Jemmali*, Mounir Kamoun, a Marwa Haddar, a Abderrahmene Ben Gara, a Houcine Selmi, a Moncef Hammami, a Marouene Amraoui, a Hamadi Rouissi, a Rekik Boulbaba.
a

Department of Animal Production, Higher Institute of Agriculture of Mateur, Tunisia

ARTICLE INFO
Article history: Received 15 Septembers 2012 Revised 26 September 2012 Accepted 08 October 2012 Available online 02 November 2012

ABSTRACT The genetic polymorphism of the casein alpha-S1 locus was investigated in Tunisian goat. Blood samples were collected from local goat breed. Genomic DNA samples were obtained from leukocytes of 75 dairy goats and regions of interest in the gene were amplified through Polymerase Chain Reaction (PCR), then evaluated in agarose gels. For better characterization of the single nucleotide polymorphism, if exist, a PCR-Restriction Fragment Length Polymorphism study was performed employing the endonuclease XmnI. DNA amplification using primers produced fragments with sizes of 457 bp. The PCR products of primer (223 bp) digested by restriction enzyme Xmn1 produced four fragments at 223 bp, 212bp, 161bp and 150-bp. The results showed that local goat breed had different genotypes A/A, B/C, C/C and DD. Our results revealed that the CSN1S1 allelic variants in tested breed showed different genotypes, three of them were homozygous 12.5%, 60.5% and 12.8% respectively for A/A, C/C and D/D and the other was heterozygous B/C (14.2%). Identification of different variants of the casein alpha-S1 can be used to improve milk quality of local goat breed.
crossbreeding trials. In 2011, Ammar et al1. reported that goat population in Tunisia is around 1.5 million concentrated in the arid regions, mainly in the South. The local genotype (Arbi) is the most prevalent and kid meat is the main product from this breeding. Two breeding systems were identified: pastoral and agro-pastoral systems. The averages of fertility, fecundity and prolificacy were generally low (genetic characteristic of the breed) and varied between 86-91%, 8992% and 130-140% respectively. The lowest values were recorded for herds managed under pastoral systems. Kiddings are concentrated in autumn-winter. Goats are always associated with poor quality lands, with difficult access. Thus mortality of kids is often greater than 12% and the abortion rate usually exceeds 10%. The common feeding systems are grazing during the day and housing at night where supplementation with concentrate feeds is provided for lactating does. Nutrient productivity of pastoral rangelands varied between 30 and 60 UF/ha/year during favourable years (rainfall around 100 mm/year) and 10-20 UF/ha/year during dry years (rainfall < 50 mm/year). Goat production in Tunisia is generally confined to marginal areas in mixed flocks with sheep. This dominant
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Keywords: Goat, polymorphism, milk genes, PCR-RFLP

Introduction:
Djemali (2000) reported that goat population is approximately 1 million and 300 thousand heads of which 750,000 breeding females. The majority is located in southern regions of Tunisia. About 95% of this population is native with black coat, horns and long drooping ears. Average adult weights are 40 and 60 kg for females and males respectively. They are hardy animals with a good ability to walk long distances and a good tolerance for salt in water (3.5 g/l). Native goat breed is known as "ARBI" breed .
Corresponding author: Dr. Jemmali Borni *, E-mail address: bornijemali@yahoo.fr Citation: Dr. Jemmali Borni * (Genetic polymorphism of casein alpha-S1 gene in Tunisian local Goat) BIOMIRROR: 1-5/bm- 1526080112 Copyright: Dr. Jemmali Borni *. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Imported breeds like Maltaise, Alpine, Boer, Saanen, Murciana, Damasquine and others have been used in various

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production system has rendered performance recording and monitoring very difficult to carry out except for a reduced number of experimental flocks. Very few references are therefore available on the genetic ability of the local goat and its genetic improvement was mainly undertaken through crossing with imported exotic breeds. Most crossbreeding programmes whether in the north or in the south had an integrated, system-adapted approach (Rekik anr Ben Hammouda, 2000). Under Tunisian conditions, goats are critical to the development of sustainable and environmentally sound production systems. Efforts should be intensified to improve productive and reproductive performances of these animals using genetic methods such as selection assisted by molecular markers. Qualitative characters, such as meat quality, enzymes, milk protein types, are among those being investigated for the possibilities which they provide of improving the accuracy of estimating genetic merit o f sires and cows and practicing selection at an earlier age. The development of molecular techniques has generated methods for the identification of DNA markers (RFLP, RAPID, AFLP, SNP, etc.). These markers have been used to find regions of DNA closely linked to them with a quantitative effect on traits, the so-called Quantitative Trait Loci (QTL). Molecular techniques have also allowed finding major genes responsible for an important part of the genetic variance of some traits. Genotypes of animals (both males and females) for these QTL, or major genes, can be easily identified early in their lives. This has been proposed (in some cases they are already being used) as an aid to increase annual genetic gain through selection; what has been called Marker-Assisted Selection (MAS). Casein gene polymorphism in goats has been extensively investigated due to the less allergenicity of goats milk than that of cows (Haenlein, 2004)7. Therefore, a considerable amount of data on the structure and diversity of casein genes in goat has accumulated in the literature (Cosenza et al., 2008; Ramunno et al., 2005)14. The structure of the CSN1S1 gene coding for the alfa-S1-Casein (s1-Cn) has been studied in detail by several researchers (Jansa-Perez et al., 1994; Ramunno et al., 2005)15. To date, at least 17 alleles of CSN1S1 gene have been detected. The alleles are characterized by single nucleotide substitutions, and insertions or deletions (Marletta et al., 2007)11. Ramunno et al. (2005)14 have studied the structure of the CSN1S1 gene in goat and detected a simple tandem repeat (STR) of (GT)1112 motive at the 18th intron of this gene. The authors have reported that, the alleles A and N are linked to (GT)11 and the allele F, is linked to (GT)12 motive. Microsatellites or simple tandem repeats (STR) are noncoding DNA fragments with short tandem repeat sequences. Due to their high mutation rates, microsatellites show high polymerphism (Schltterer, 2000). Microsatellites have been widely used for the examining of population structure (Luikart et al., 199910; Bozkaya et al., 2007)3, gene and quantitative trait loci (QTL) mapping (19Vaiman et al. 1996; 12Minvielle et al., 2006), and forensic purposes (Nizamlioglu et al., 2006) in different animal species. No study has been found on the variability of the microsatellite locus in the CSN1S1 gene in Tunisian goat populations. Therefore, the purpose of this study was to investigate the genetic polymorphism of CSN1S1 in local goat population.
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Material and Methods:


Sample collection and DNA extraction: A total of 52 dairy cattle were randomly selected from private farm. Blood samples were collected in vacutainer tubes containing EDTA (1 mg mL-1). Genomic DNA was extracted using standard protocol (Easy-DNA Kit Invitrgen) and stored at -20C until used in assay. The concentration of DNA samples was estimated using UVvisible range spectrophotometer and diluted to 50 ng/L before PCR amplification. All the DNA samples had 260/280 OD ratios in the range of 1.8 to 2, indicating high purity. DNA was also examined by loading samples on 0.8 % agarose gel and visualizing the band under UV light with a Gel Doc 1000 system (BioRad) after ethidium bromide staining. PCR amplification The PCR was performed in a final volume of 50 L containing 100 ng of template DNA, 50 pmole of each primer,5l of 10PCR buffer (20 mM TrisHCl pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 0.2 mM of dNTPs, and 1 U of Taq DNA polymerase (SibEnzyme Ltd) (Ramunno et al., 2000): Forward: 5'- TTCTAAAAGTCTCAGAGGCAG -3' Reverse: 5'- GGGTTGATAGCCTTGTATGT -3' This solution was initially denatured at 94 C for 5 min. followed by 39 cycles of denaturation (95 C for 30 s), annealing (62 C for 45 s), and elongation (72 C for 1 min) and a final extension at 72 C for 10 min. The PCR products were electrophoresed on 1.5% agarose gels in order to check the quality and specificity of DNA fragment amplification. PCR-RFLP condition For PCR-RFLP analysis, the 223 bp PCR products were digested with Xmn1 (BioLabs). Restriction fragments were separated by electrophoresis in a 2.5 % agarose gel and their sizes were estimated using the molecular markers. The results were taken into account when the sum of all the restriction fragments for Xmn1 enzyme was in the range of 223 bp (Ahmed and Othman, 2009). 20 l of PCR products was digested for 4h at 37C with 10 units of restriction enzyme. Digested products were separated by electrophoresis on a 1.5 % agarose gel and visualized with Ethidium bromide under UV light with a Gel Doc 1000 system (BioRad) after ethidium bromide staining. Table1. PCR and PCR-RFLP expected alleles Gene Enzyme Xmn1 PCR 223 bp PCR-RFLP 223; 212; 161 and 150

Result and Discussion: Quality and quantity of extracted DNA from analyzed samples was tested by electrophoresis on agarose gel (Figure 1). Electrophoresis showed a height DNA quality. DNA quantity was estimated using the molecular markers quantity for each band.

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Figure 1. Quality uality of extracted genomic DNA from different goat individuals (lanes 1 1 18).

From all analyzed individuals of goat, DNA fragments containing the CSN1S1 gene were amplied ed for nucleotide sequencing via the polymerase chain reaction (PCR) using

adequate primer pairs. As expected, the size of PCR product of CSN1S1 gene was 223 bp (Figure 2). This result is similar to that found by Ahmed and Othman (2009), who studied the genetic polymorphism of casein alpha S1.

Figure 2. Amplification of genomic DNA from different cattle individuals of local goat population. M: 1kb Molecular weight marker (invitrogen DNA Ladder).

The PCR amplied ed product was observed as 223 bp (Figure 2). Four different variant groups of (150-150) 150) bp, (161 (161-223) bp, (223-223) bp and (212-212) 212) bp were obtained (Figure 3) after restriction digestion with XmnI. Genotyping at the DNA level showed that the CC genotype had the highest frequency in analysed goats (60.5 %). The PCR products of primer (223 bp) digested by restriction triction enzyme Xmn1 produced four

fragments at 223 bp, 212bp, 161bp and 150-bp. 150 The results showed that local goat breed had different genotypes A/A, B/C, C/C and DD. Our results revealed that the CSN1S1 allelic variants in tested breed showed different genotypes, three of them were homozygous 12.5%, 60.5% and 12.8% respectively for A/A, C/C and D/D and the other was heterozygous B/C (14.2%).

Figure 3. The electrophoretic pattern obtained after digestion of PCR amplified goat CSN1S1 pr products oducts with XmnI.

This is the first study in Tunisia that contributes with results about the polymorphism of the CSN1S1 locus of goats. The

goat calcium-sensitive sensitive caseins (alphas1, beta and alphas2) represent, over many years, an excellent model for

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ISSN 0976 9080 BM, an open access journal

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demonstrating that the major part of the variability observed in the content of these proteins in goat milk is mostly due to the presence of autosomal alleles at single structural loci (CSN1S1, CSN2 and CSN1S2 respectively) clustered on a 200 kb segment of chromosome 6; furthermore, CSN1S1 and CSN2 are convergently transcribed and are only 12 kb apart (Cosenza et al., 2005). CSN1S1 is characterized by 19 exons ranging in size from 24 (exons 5, 6, 7, 8, 10, 13, 16) to 17.5 kb. The goat CSN1S1 locus has been characterized by at least 13 alleles, which have been associated with different levels of protein synthesis. The amount of total casein in goat milk was positively correlated with the presence of as1 casein allele and was highest in case of A, B and C alleles (Kumar et al., 2007). Gene substitution effect, alpha, for a SNP is the average change of genotypic value that results when one allele is replaced by the other allele of same locus. Estimated additive (al) and dominance (dl) effects of SNP were collected and gene substitution effects (al) were calculated (a1 = a1+(1-2 pi) di); where pl is the frequency of the major allele at 1st SNP position (Dagnachew, 2011).
8. Jansa-Perez M, Leroux C, Sanchez A Martin P (1994) Occurrence of a LINE sequence in the 3 UTR of the goat s1-casein E- encoding allele associated with reduced protein synthesis level. Gene, 147: 179-187. Kumar A, Rout PK, Mandal A, Roy R (2007) Identication of the CSN1S1 allele in Indian goats by the PCR-RFLP method. Animal (2007), 1:8, pp 10991104. Luikart G, Buji-Duval MP, Ertugrul O, Zagdsuren Y, Maudet C, Taberlet P (1999). Power of 22 microsatellite markers in fluorescent multiplexes for parentage testing in goats (Capra hircus). Anim. Genet. 30: 431-438. Marletta D, Criscione A, Bordonaro S, Guastella AM, DUrso G (2007) Casein polymorphism in goats milk. Lait, 87: 491-504. Minvielle F, Kayang BB, Inoue-Murayama M, Miwa M, Vignal A, Gourichon D, Neau A, Monvoisin JL, Ito S (2006) Search for QTL affecting the shape of the egg laying curve of the Japanese quail. BMC Genet. 7: p. 26. Nizamlioglu M, Kurar E, Bulut Z, Inal S, Erzurum F (2006) Microsatellite analysis of some horse breeds in Turkey: usefulness for parentage testing. FEBS J. 273: 365-366. Ramunno L, Cosenza G, Rando A, Pauciullo A, Illario R, Gallo D, Berardino D, Masina P (2005) Comparative analysis of gene sequence of goat CSN1S1 F and N allele and characterization of CSN1S1 transcript variants in mammary gland. Gene, 345: 289-299. Ramunno L, Cosenza G, Pappalardo M (2000) Identification of the goat CSN1S1F allele by means of PCRRFLP method. Animal Genetics, v.31, p.333-346, 2000. Rekik M, Ben Hammouda M (2000) A steering frame for the genetic improvement of sheep and goats in Tunisia. Dans : Options Mditerranennes, Series A, 43, p. 129136. Sahar A, Othman E (2009) Genotyping Analysis of Milk Protein Genes in Different Goat Breeds Reared in Egypt. JGEB Vol. 7, No. 2, pp 33-39. Schltterer C (2000) Evolutionary dynamics of microsatellite DNA. Chromosoma, 109: 365-371. Vaiman D, Schibler L, Bourgeois F, Oustry A, Amiguest Y, Cribiu EP (1996) A genetic linkage map of the male goat genome. Genetics, 144: 279-305..

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Conclusion :
Our results revealed that the CSN1S1 allelic variants in tested breed showed different genotypes, three of them were homozygous 12.5%, 60.5% and 12.8% respectively for A/A, C/C and D/D and the other was heterozygous B/C (14.2%). Identification of different variants of the casein alpha-S1 can be used to improve milk quality of local goat breed. Further studie of CSN1S1 caseins could be utilized in breeding schemes aiming at the improvement of the quality of processed milk of Tunisian local goat breeds.
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References:
1. Ammar H, Boda R, Ben Younes M, Lpez S (2011) Goat breeding systems in the South of Tunisia (Tataouine). Options Mditerranennes. Economic, social and environmental sustainability in sheep and goat production systems. A no. 100, pp 283-288. Binyam S, Dagnachew GT, Sigbjorn L, Tormod A (2011) Casein SNP in Norwegian goats: additive and dominance effects on milk composition and quality. Genetics Selection Evolution 2011, 43:31 Bozkaya F, Kuss AW, Geldermann H (2007) DNA variants of the MHC show location-specific convergence between sheep, goat and cattle. Small Rumin. Res. 70: 174-182. Cosenza G, Paciullo A, Colimono L, DAvino A, Mancusi A, Ramunno L (2008) Genotyping at the CSN1S1 locus by PCR-RFLP and AS-PCR in a Neapolitan goat population. Small Rumin. Res. 74: 84-90. Cosenza G, Pauciullo A, Gallo D, Berardino DD, Ramunno L (2005) A Ssp I PCR-RFLP detecting a silent allele at the goat CSN2 locus. J Dairy Res. 2005 Nov;72(4):456-9. Djemali M (2000) Genetic improvement objectives of sheep and goats in Tunisia. Lessons learned. In: Analysis and definition of the objectives in genetic improvement programmes in sheep and goats. Options Mediterraneennes. Serie A: Seminaires Mediterraneens (France), no. 43. Gabina, D. Animal Production and Health Div (2000), p. 121-127. Haenlein GFW (2004) Goat milk in human nutrition. Small Rumin. Res. 51: 155-163.

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