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Short Communication · Volume 49, Issue 4, 30 July 2017 · Open Access

Molecular Effects of Polymorphism in the 3'UTR of Osteopontin Gene in Riverine Buffalo

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Affiliations & Notes ⌄
  1. 1Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan 2Virtual University of Pakistan, Lahore, Pakistan , * Corresponding author: asifnadeem@uvas.edu.pk
Article Info ⌄
Journal:
Pakistan Journal of Zoology
Published:
30 July 2017
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From the article

  • Once a reliable correlation between DNA polymorphisms and economically important traits has determined, such markers or polymorphisms may be incorporated in marker-assisted selection (MAS) breeding programs.
  • Alligned sequence showed an SNP in 3’ UTR of SPP1 gene in riverine buffalo (Table I).
  • The identified SNP 37421762 (C>A) in the 3′UTR of the gene was found non-significant in all analyzed milk production traits.
  • However, the effects of genes used in marker-assisted selection (MAS) program should be known and consistent in different environments and genetic backgrounds to increase the selection efficacy of dairy animals.

Selected from the article's own text.

Abstract

Nucleotide sequencing of bovine osteopontin gene showed a novel DNA polymorphism 37421762 (C>A) in the 3’-untranslated region (3’-UTR) of the gene. Association test was performed to analyze its relation with milk composition but it was found non-significant in all analyzed traits. So, it was inferred that it might be due to environmental or epigenetic effect, sampling number or a breed difference. To know the functional consequences of the identified polymorphism, RNA secondary structure analysis was performed and SNP was found changing the termination loop shape in mRNA. However, to increase the selection efficacy of dairy animals, the effects of candidate polymorphism should further be screened in different environments and genetic backgrounds.

Materials and methods

In order to analyze the Osteopontin polymorphism, fifty blood samples of Nili Ravi (Riverine buffalo) were collected from different livestock farms and processed for genomic DNA extraction .

Specific primer set

5’ -TATAGACTTTGGTGTGGAAG- 3’ (forward),

5’ -CCCTGCTTTAATGTATCCTT- 3’ (reverse) was designed to amplify the 3’UTR of SPP1 gene in riverine buffalo. A total of 25 μL reaction mixture containing 1 µL DNA (50 ng/µL), 2.5 µL PCR buffer (2mM), 2.5 µL dNTPs (25mM), 2.5 µL MgCl2, 1 µL of each forward and reverse primers (10 pmol), 0.75 μL Taq DNA polymerase (5U / μL), and 13.75 µL of deionized water was used for PCR amplification. Then the amplicons were precipitated and sequenced by Sanger chain termination method using AB 3130 XL genetic analyzer (Applied Bio systems Inc., Foster City, CA).

Sequences were aligned by using ClustalW and analyzed for genetic variation. p.37421762 (C/A) was found in 3’ UTR of Osteopontin gene. Polymorphism was further analyzed for Chi2 testing, Hardy Weinberg Equilibrium (HWE) and association analysis by One Way ANOVA (Mean±SE) (Tables 1, II). SNP was found obeying HWE. Further p.37421762 was analyzed for RNA secondary structure (Supplementary Fig. 1) and there was

Table I.- Allelic and genotypic frequencies and HWE of identified polymorphism in 3’ UTR of SPP1 gene.

SNP ID

Allele frequency

Genotype frequency

 HWE P< 0.05

A

B

AA

AB

BB
p.37421762 C/A

0.6829

0.3171

0.2861

0.1651

0.5383

0.0945

Table II.- Association analysis of identified SNP in 3’ UTR of SPP1 gene with milk composition traits.

Genetic variation Milk composition

AA (Mean ± SE)

AB (Mean ± SE)

BB (Mean ± SE)

P-value (0.05)

n=17

n=29

n=33
p.37421762 C/A Milk fat

6.16± 0.6361

6.44±1.5665

6.31±0.5129

0.620834
SNF

7.22±0.3552

7.48 ± 0.3487

7.22 ± 0.1447

0.883244
Lactose

3.28±1.7022

3.67±0.8933

3.44±0.7762

0.727452
Protein

2.98±0.3824

3.53±0.2377

3.33±0.3552

0.829834
Ash

0.73±0.6338

0.76±0.4751

0.88±0.2851

0.744613

significant difference in the termination loop of mRNA that might hinder the termination and Poly-A tailing of the mRNA molecule effecting functionality of the gene.

Results and Discussion

Conventionally, phenotypic and biochemical markers have been used to identify a potential animal for economically important traits in dairy breeds. Now, the selection efficiency of complex quantitative traits depends on the identification of candidate genes as well as DNA polymorphism in them. Once a reliable correlation between DNA polymorphisms and economically important traits has determined, such markers or polymorphisms may be incorporated in marker-assisted selection (MAS) breeding programs. Thus, the use of validated markers can substanially increase selection efficacy of animals . Analysis of animal databases has revealed that about 344 quantitative trait loci (QTL) are associated with milk production traits and 71 with mastitis related traits ; . Association between DNA polymorphism and milk production traits have been reported for a number of genes including: including diacylglycerol acyltransferase 1 (DGAT1), fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), CSN1S1, ATP-binding cassette, subfamily G, member 2 (ABCG2), growth hormone receptor (GHR), oxidised low-density lipoprotein receptor 1 (OLR1), prolactin (PRL), signal transducer activator of transcription 5A (STAT5A), osteopontin (SPP1), leptin, alpha-lactalbumin (LALBA), Transcription factor Pit 1 (POU1F1), CYP11B1 and PPARGC1A ; ; ; ; ; ; ; .

The SPP1 gene was selected for association test with milk composition traits as several reported markers has showed its effects on milk production . Alligned sequence showed an SNP in 3’ UTR of SPP1 gene in riverine buffalo (Table I). DNA mutations in 3’ UTR and intronic portion of the genes may alter the mRNA stability and splice sites of pre-mRNA, consequently leading to the assemblage of truncated proteins . The identified SNP 37421762 (C>A) in the 3′UTR of the gene was found non-significant in all analyzed milk production traits. The SPP1 gene has been reported for g.8514C>T polymorphism associated with milk composition traits ; . However, the current study was found contradictory to them and no SNP was identified in this region of the gene. Cohen et al. (2004) reported that SPP1 is a candidate gene affecting milk protein percentage, but the identified SNP in 3′UTR did not show any significant relationship with milk protein content. It may be due to environmental or epigenetic effect, sampling number or a breed difference. However, the effects of genes used in marker-assisted selection (MAS) program should be known and consistent in different environments and genetic backgrounds to increase the selection efficacy of dairy animals.

Supplementary material

There is supplementary material associated with this article. Access the material online at: http://dx.doi.org/10.17582/journal.pjz/2017.49.4.sc8

Statement of conflict of interest

Authors have declared no conflict of interest.

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