Volume 16, Issue 3 (9-2025)
Res Anim Prod 2025, 16(3): 24-44 |
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Moradgholi K, Dashab G R, Rokouei M, Faraji Arough H. (2025). Bioinformatic Analysis of the FEZL Gene sequence in Sistani, Holstein, and their Crossbreds. Res Anim Prod. 16(3), 24-44. doi:10.61882/rap.2025.1485
URL: http://rap.sanru.ac.ir/article-1-1485-en.html
URL: http://rap.sanru.ac.ir/article-1-1485-en.html
1- Animal Science Department, College of Agriculture, University of Zabol, Zabol, Iran
2- Department of Ostrich, Special Domestic Animals Institute, Research Institute of Zabol, Zabol, Iran
2- Department of Ostrich, Special Domestic Animals Institute, Research Institute of Zabol, Zabol, Iran
Abstract: (1197 Views)
Extended Abstract
Background: FEZL is one of the candidate genes related to performance, quality traits of milk, and resistance to mastitis disease in dairy cows. Mastitis is one of the common diseases in the dairy cattle population, which imposes a lot of economic losses on the farmer. Studies on genetic variation in the FEZL gene locus and their relationship with the milk somatic cell count (SCC) showed a significant relationship between genetic variants in the mentioned locus and the SCC, which is indicative of mastitis. Moreover, conflicting results have been reported about the mentioned gene variants' effects on milk's quantitative and qualitative traits. Therefore, this research aims to investigate the amount of different deletion, addition, and mutation polymorphisms in the FEZL locus, to identify important variants related to milk composition, and to investigate the difference between pure breeds and their crossbreds in Sistani and Holstein cows.
Methods: This research was conducted on 20 pure breeds of Sistani and Holstein cows (10 cows from each group) and their crossbreds of Sistani and Holstein (7 heads) from the Research Center for Sistani Cattle and Native Herds of the Sistan region. Blood was randomly selected and taken from the jugular vein. Quantitative and qualitative traits of milk were measured from morning and evening milking within three months. Total DNA from blood samples was extracted by the salt-optimized method. Then, the extracted DNA was electrophoresed on a 0.5% agarose gel, and samples of good quality were used for the PCR amplification reaction. A 229 bp fragment from the FEZL gene locus was amplified using a specific primer pair with temperature cycling in a thermocycler. Sequencing was done with Chromas software in FASTA format, followed by removing low-quality sequences. In the next step, the production areas of a single thread were removed from the back-and-forth sequence, and cantig production was done using CAP3 software. Alignment of 27 samples was performed with MEGA software in pairs and multiples. Four samples were removed due to unfavorable alignment, and 23 samples were saved for further analysis. The polymorphic regions caused by mutations, deletions, and additions, as well as haplotyping, were analyzed with MEGA6 and DNASP5 software. Finally, the effect of each marker on milk quality traits was estimated using the single marker model and the GLM procedure of SAS9 software. The type 1 error in multiple tests was reduced using the Benferroni test at the 5% probability level. Finally, the means of the groups were compared with the Tukey-Kramer method at the probability level of 5%.
Results: A total of 86 polymorphic regions were identified in exon 1 of the FEZL gene among pure and crossbred populations. In all samples, the percentages of transitional and transversional substitutions were 32.56% and 67.44%, respectively. The percentages of transitional and transversional substitutions were estimated at 50.6 and 49.4% in the Holstein breed, 28.08 and 71.92% in the Sistani breed, and 33.55 and 66.45% in crossbreds. By comparison, the lowest and the highest percentages of transitional substitution were observed in Sistani and Holstein breeds, respectively, but Holstein and Sistani breeds showed the lowest and the highest percentages of transversional substitution, respectively. The highest and the lowest coefficients of transition substitution ratio to transversional, which is an indicator of evolution during the past periods, belonged to Holstein (0.75) and Sistani (0.30) breeds, respectively. In all populations, thymine and Cytosine were respectively the most and least frequent bases in the nucleotide sequence of the exon 1 region of the FEZL gene. The highest diversity within breeds belonged to Holstein and Sistani crossbreds (0.15), and the lowest diversity was recorded for the Sistani breed (0.044). Among different breeds, the highest degree of divergence was between crossbreds with Holstein (0.126) and the lowest between Sistani and crossbreds (0.012). Of the 77 polymorphic loci (SNPs), 33 cases were two-variant, 9 cases were three-variant, and two were four-variant. In the milk composition, the Sistani and Holstein breeds contained the lowest and the highest percentages of fat, protein, lactose, and fat-free dry matter, respectively, while middle percentages were found in the crossbreds. The effect of breeds was significant on all milk traits, except for protein percentage (P < 0.01). Among the identified markers, the marker at the position of 97 bp of the exon 1 of the FEZL gene had the highest effect on the fat, lactose, protein, and milk dry matter percentage traits (P < 0.01).
Conclusion: Despite the existence of different polymorphisms in the exon 1 of the FEZL gene and the amount of genetic diversity existing between and within the Holstein and Sistani breeds, it can be considered a useful marker in the breeding of native livestock, and the combinability strength of variants in crossbreds can be used to improve the economic traits of milk. Furthermore, polymorphism in the mentioned area was still used as an indicator to improve somatic cell counts in milk as an indicator of mastitis control in dairy cows, which was not possible to measure in this research and is recommended in future studies.
Background: FEZL is one of the candidate genes related to performance, quality traits of milk, and resistance to mastitis disease in dairy cows. Mastitis is one of the common diseases in the dairy cattle population, which imposes a lot of economic losses on the farmer. Studies on genetic variation in the FEZL gene locus and their relationship with the milk somatic cell count (SCC) showed a significant relationship between genetic variants in the mentioned locus and the SCC, which is indicative of mastitis. Moreover, conflicting results have been reported about the mentioned gene variants' effects on milk's quantitative and qualitative traits. Therefore, this research aims to investigate the amount of different deletion, addition, and mutation polymorphisms in the FEZL locus, to identify important variants related to milk composition, and to investigate the difference between pure breeds and their crossbreds in Sistani and Holstein cows.
Methods: This research was conducted on 20 pure breeds of Sistani and Holstein cows (10 cows from each group) and their crossbreds of Sistani and Holstein (7 heads) from the Research Center for Sistani Cattle and Native Herds of the Sistan region. Blood was randomly selected and taken from the jugular vein. Quantitative and qualitative traits of milk were measured from morning and evening milking within three months. Total DNA from blood samples was extracted by the salt-optimized method. Then, the extracted DNA was electrophoresed on a 0.5% agarose gel, and samples of good quality were used for the PCR amplification reaction. A 229 bp fragment from the FEZL gene locus was amplified using a specific primer pair with temperature cycling in a thermocycler. Sequencing was done with Chromas software in FASTA format, followed by removing low-quality sequences. In the next step, the production areas of a single thread were removed from the back-and-forth sequence, and cantig production was done using CAP3 software. Alignment of 27 samples was performed with MEGA software in pairs and multiples. Four samples were removed due to unfavorable alignment, and 23 samples were saved for further analysis. The polymorphic regions caused by mutations, deletions, and additions, as well as haplotyping, were analyzed with MEGA6 and DNASP5 software. Finally, the effect of each marker on milk quality traits was estimated using the single marker model and the GLM procedure of SAS9 software. The type 1 error in multiple tests was reduced using the Benferroni test at the 5% probability level. Finally, the means of the groups were compared with the Tukey-Kramer method at the probability level of 5%.
Results: A total of 86 polymorphic regions were identified in exon 1 of the FEZL gene among pure and crossbred populations. In all samples, the percentages of transitional and transversional substitutions were 32.56% and 67.44%, respectively. The percentages of transitional and transversional substitutions were estimated at 50.6 and 49.4% in the Holstein breed, 28.08 and 71.92% in the Sistani breed, and 33.55 and 66.45% in crossbreds. By comparison, the lowest and the highest percentages of transitional substitution were observed in Sistani and Holstein breeds, respectively, but Holstein and Sistani breeds showed the lowest and the highest percentages of transversional substitution, respectively. The highest and the lowest coefficients of transition substitution ratio to transversional, which is an indicator of evolution during the past periods, belonged to Holstein (0.75) and Sistani (0.30) breeds, respectively. In all populations, thymine and Cytosine were respectively the most and least frequent bases in the nucleotide sequence of the exon 1 region of the FEZL gene. The highest diversity within breeds belonged to Holstein and Sistani crossbreds (0.15), and the lowest diversity was recorded for the Sistani breed (0.044). Among different breeds, the highest degree of divergence was between crossbreds with Holstein (0.126) and the lowest between Sistani and crossbreds (0.012). Of the 77 polymorphic loci (SNPs), 33 cases were two-variant, 9 cases were three-variant, and two were four-variant. In the milk composition, the Sistani and Holstein breeds contained the lowest and the highest percentages of fat, protein, lactose, and fat-free dry matter, respectively, while middle percentages were found in the crossbreds. The effect of breeds was significant on all milk traits, except for protein percentage (P < 0.01). Among the identified markers, the marker at the position of 97 bp of the exon 1 of the FEZL gene had the highest effect on the fat, lactose, protein, and milk dry matter percentage traits (P < 0.01).
Conclusion: Despite the existence of different polymorphisms in the exon 1 of the FEZL gene and the amount of genetic diversity existing between and within the Holstein and Sistani breeds, it can be considered a useful marker in the breeding of native livestock, and the combinability strength of variants in crossbreds can be used to improve the economic traits of milk. Furthermore, polymorphism in the mentioned area was still used as an indicator to improve somatic cell counts in milk as an indicator of mastitis control in dairy cows, which was not possible to measure in this research and is recommended in future studies.
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