MATERIALS AND METHODS

Animals and experimental samples: In this study, a total of 100 Friesian bull calves were used. The samples were stored at -20°C until needed for DNA extraction. Based on the farm record, the selected animals were weaned at 90 kg b.wt., have their birth weight ranged from 29-38 kg and their weaning age ranged from 75-100 days. Blood samples were collected into anticoagulant disodium EDTA containing tubes. The genomic DNA was extracted using extraction kit (Jena Bioscience, Germany). This study protocol was approved by the animal welfare and ethics committee, Faculty of Veterinary Medicine, Damnhour University.

MYF5-TaqI polymorphism detection: The PCR was done for amplification of a fragment of MYF5 gene with expected amplicon size of 1190 bp using available sequence information from bovine MYF5 published (GenBank accession No. M95684)¹³.

Forward: 5’-GATAGCTGGCTGTGAATGAT-3’
Reverse: 5’-CTGGCAACTGGGGAGAGAGAAG-3’

The polymerase chain reaction mixture was done in a 25 μL consisted of: 2 μL DNA, 9.5 μL H₂O (dd H₂O), 12.5 μL PCR master mix (Jena Bioscience, Germany), 0.5 μL of each primer. The final reaction mixture was achieved in a thermal cycler and the PCR temperature schedule program was carried out by 94°C for 4 min as initial denaturation succeeded by 34 cycles of 94°C for 1 min for denaturation, primer hybridization temperature at 58°C for 1 min, primer extension at 72°C for 1°C and the final elongation at 72°C lasts for 10 min.

The amplified DNA fragments of MYF5 gene were digested with fast digest TaqI (Thermo Scientific, #FD0674) at 65°C for 5 min. The reaction volume was done in 30 μL consisted of: 10 μL PCR product, 17 μL H₂O (dd H₂O), 2 μL 10x fast digest green buffer, 1 μL restriction enzyme. The obtained cleaved fragments were explored by agarose gel electrophoresis then their patterns were visualized under U.V using gel documentation system.

Statistical analysis
Adjustment or correction of non-genetic factors:
Based on farm records, the calf weaning weight was adjusted to 205 days of age by linear interpolation from birth weight, weaning weight and age. Adjustment was carried out using the following equation¹⁴.

where, A is for 205 days weight (kg), B is for the weaning weight (kg), C is for the birth weight (kg) and D is for the weaning age (days).

Association analysis: Statistical analysis was performed using Graphpad statistical software program (Graphpad prism for windows version 5.1, Graphpad software, Inc, Sandiego, CA, USA). General Linear Model (GLM) practice of the statistical analysis system package¹⁵ was used for data analysis to determine association of MYF5 genotypes and body weight.

Gene and genotypic frequencies in MYF5 locus: Based on the electrophoresis results, gene and genotypic frequencies were calculated by allele simple counting¹⁶. Chi-square was carried out to test Hardy-Weinberg equilibrium and show genotype distribution in the cattle population.

RESULTS

One pair of specific primers was used to amplify specific DNA fragments 1190 bp of MYF5 gene (Fig. 1). Restriction analysis of 1190 bp PCR products digested with TaqI revealed that, two fragments (983 and 207 bp) for genotype BB, three fragments (1190, 983 and 207 bp) for genotype AB and undigested fragment (1190 bp) for genotype AA (Fig. 2).

Using PCR-RFLP method, the population of 100 Friesian bull calves was genetically described. Where, the incidence and frequency of MYF5 genotypes and alleles were calculated. In 100 Friesian bull calves, the genotypic frequencies 0.20, 0.46 and 0.34 were for AA, AB and BB genotypes, respectively and the allelic frequencies 0.43 and 0.57 were for A and B alleles.

Fig. 1:	Representative PCR results of MYF5 gene, lane M: DNA marker and lanes 1-7: 1190 bp amplified fragment of MYF5 gene

Fig. 2:	Representative TaqI restriction fragment pattern of MYF5 gene (1190 bp). BB: Restriction fragment of 983 and 207 bp, AB: Restriction fragment of 1190, 983 and 207 bp, AA: Restriction fragment of 1190 bp and M: DNA ladder

Table 1:	Frequency of genotypes and alleles in the MYF5 locus
Chi-square calculated (χ2) = 0.379-No significant differences, Chi square tabulated (χ2) at DF = 1 and p<0.05 = 3.84

Table 2:	Associations of MYF5 genotypes with corrected body weight (LSM±SE)
Means of different levels within the same column having different superscripts are significantly different (p<0.01)

The χ²-test showed that the genotype distributions in the cattle population were in Hardy-Weinberg equilibrium (p<0.05) (Table 1).

Statistical analysis indicated that there was highly significant association (p<0.01) between MYF5 genotypes and body weight. The AB genotype was higher in body weight than both BB and AA genotypes. However, there was no significant variation between AB and BB genotypes in the body weight (Table 2).

DISCUSSION

In this study, PCR amplification of a fragment of MYF5 gene yielded specific PCR product of desirable size (1190 bp). The following DNA restriction fragments were obtained for MYF5-TaqI digestion: Digested (983 and 207 bp) fragments for genotype BB, three fragments (1190, 983 and 207 bp) for genotype AB and undigested fragment (1190 bp) for genotype AA. For a population of 100 Friesian bull calves, the genotypic frequencies 0.20, 0.46 and 0.34 were for AA, AB and BB genotypes, respectively and the allelic frequencies 0.43 and 0.57 were for A and B alleles. The χ²-test showed that the genotype distributions in the cattle population were in Hardy-Weinberg equilibrium (p<0.05). Absence of the significant difference between observed and the expected values for genotype counts indicated the population balanced and follow Hardy-Weinberg equilibrium. This balance may originate from the higher number of the heterozygous genotype (AB) than those homozygous genotypes (AA and BB) which keep the balanced allelic frequencies in the population¹⁷. The association between RFLP-TaqI of the MYF5 gene and body weight was studied. The MYF5 genotypes were highly significant (p<0.01) associated with body weight. The AB genotype was higher in body weight than both BB and AA genotypes. However, there was no significant variation between AB and BB genotypes in the body weight.

Several polymorphisms in MYF5 gene were indicated in different cattle breeds by several previous studies. However, most of association analyses reported opposing results or failed to demonstrate any relationship to growth traits^9,18-23. The association of the SNP in MYF5 with the body weight traits of Nanyang cattle has been investigated⁹. The researchers indicated that there was significant association between the identified SNP in MYF5 gene and all body weight traits except birth weight. A significant effect of SNP on the average daily gain of cattle was also reported¹⁰.

In another study on RFLP-TaqI of the MYF5 gene and growth traits, association between polymorphism of MYF5 Gene and body weight in Jiaxian, Nanyang and Qinchuan breeds of cattle was studied¹¹. According to results, the DNA restriction fragments were obtained for MYF5-Taq/digestion were similar to that denoted in our study. Allele B at MYF5 locus was dominant in these three populations. The frequency of allele B at MYF5 locus in the three Chinese breeds was 0.8275/0.7581/0.7523, respectively. No statistically significant variations in body weight traits were observed between the genotypes of the Jiaxian and Nanyang breeds at MYF5 locus. However, there were statistically significant differences between the genotypes of the Qinchuan breed. The MYF5 gene polymorphisms and their relatedness with body weight was also reported in Hanwoo (Korean cattle)²⁴. Statistical analysis indicated that there was significant association (0.05) between MYF5 gene polymorphisms and live body weight at 6 months of age (LW6).

CONCLUSION

This study supports the significance effect of MYF5 gene as plausible candidate for body weight in cattle. Moreover, the effectiveness of RFLP as a molecular genetic marker contains great genetic potential resource to improve such trait results in effective selection.

ACKNOWLEDGMENT

The authors thank all members of department of Animal Husbandry and Animal Wealth Development, Faculty of Veterinary Medicine, Damanhour University, Egypt for their kind cooperation.