Identification of Polymorphisms and Genetic Diversity Studies of Interferon Regulatory Factor 3(IRF3) Gene in Muturu,White Fulani and N’Dama Cattle

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The study was carried out to genetically characterize the IRF3 gene in the, N’Dama, Muturu and White Fulani cattle. DNA was extracted from the blood samples using the Zymo-spin extraction kit. ARLEQUIN 2.0001 software was used to estimate the basic population genetic statistics while DnaSP version 5.10.01 was used to estimate genetic diversity indices and test for deviation from neutrality. A total number of 18 and 29 Single nucleotide polymorphisms (SNPs) were observed after using the software called codon code aligner in exons 1–2 and 5–6 of the IRF3 gene respectively in the three cattle breeds after polymerase chain reaction and sequencing. In exons 1–2, the Muturu (MU) possessed the highest value of SNPs (16) and genetic diversity indices, while the N’Dama (ND) possessed the least (1). In exons 5–6, the highest value of SNPs (14) was observed in the White Fulani and the genetic diversity indices was also high while the Muturu had the least. Analysis of molecular variation (AMOVA) carried out for the loci under consideration revealed a higher level of variation among populations than within populations. It was therefore concluded that the IRF3 gene had many polymorphisms and was highly diversified in Nigerian cattle breeds. Genetic diversity Cattle Interferon Regulatory Factor (IRF3) Polymorphisms INTRODUCTION The interferon regulatory factors (IRF) are made up of developed family with similar transcribed proteins recognized firstly as controllers of the IFN-alpha/beta gene promoters, as well as the IFN-stimulated response element (ISRE) of some IFN-stimulated genes (Hiscott et al ., 1999). It has been reported that in mammals, nine members which belong to the interferon regulatory factor (IRF) family, (IRF-1 to IRF-9) have been identified (Taniguchi et al., 2001 ). The Interferon regulatory factor 3 (IRF3) gene encodes interferon regulatory factor 3, a member of the interferon regulatory transcription factor (IRF) family and is much revealed and is found in the cytoplasm of undiseased cells (Jann et al., 2009 ). Prior to infection, the micro-organism virus triggers phosphorylation of C-terminal serine/threonine residues and consequently amounts to a conformational change in IRF3 with exposure of both the DNA binding domain (DBD) and Interferon associated domains (IAD), which leads to homo- or hetero-dimerization,, association with CBP/p300 coactivators, cytoplasm-to-nucleus transfer, activation of multiple target genes and stimulation of DNA binding to the IFN-stimulated response elements (ISREs). This gene was also reported to function in the stimulation of type I IFNs following virus infection and is an expressed phosphoprotein of 427 amino acids in humans (Au et al., 1995 ). The cattle IRF3 gene consists 8 exons and 7 introns and encodes a 417-amino acid protein. It is a phosphoprotein which is made up of an N-terminal DNA binding domain (DBD domain), a C-terminal IRF-associated domain (IAD) and a transactivation domain, coupled with the importance it plays in the defense of its host and survival of the cells, the activity of IRF3 is strictly controlled. This gene is one of the strongest positional candidate genes implicated in a host of health-related phenotypes such as general disease resistance not only in cattle but in mice and humans as well. The gene was also reported to function in the ability to adapt to infections caused by protozoans in mice and cattle (Jann et al., 2009 ). Cattle populations have several polymorphisms at the IRF3 locus that change single amino acids. Parts of the highest polymorphic regions of this gene in cattle are exons 2, 5 and 6 (Ensembl cow release 92). Considering the importance of this gene, characterization was done in three Nigerian cattle breeds which include; Muturu, White Fulani and N’Dama cattle breeds. This study is therefore aimed to identify polymorphisms in IRF3 gene and evaluate genetic diversity indices in Nigerian, Muturu, N’Dama and White Fulani Cattle breeds. MATERIALS AND METHODS Animals and Sampling An amount of 190 animals were purposively sampled in this study. These comprised 85 White Fulani cattle, 72 Muturu and 33 N’Dama cattle. Samples of White Fulani cattle were collected at four locations; Ajani Farms, Ogbomosho, Odeda Local Government, Ogun State and the Cattle Production Venture of the Federal University of Agriculture, Abeokuta (FUNAAB), all located in Nigeria. The N’Dama breed was sampled at the Institute of Agricultural Research and Training Moor Plantation, Ibadan, off-site ranch at Ilora in Oyo State and the Federal Department of Livestock N’Dama Conservation Programme Ranch at Fashola in Oyo State while the Muturu breed was sampled at Odeda Local Government in Ogun State, Ipokia Local Government in Ogun State and Institute of Food Security, Environmental Resources and Agricultural Research facility at the FUNAAB all located in Nigeria. DNA Extraction Genomic DNA was extracted from the blood samples using Zymo-Spin IIC™ extraction kit. The extraction process was done at the Central Biotechnology Laboratory located found at Federal University of Agriculture, Abeokuta, Ogun State, Nigeria. The manufacturer’s protocol was used in carrying out the extraction after which the quantification of the extracted DNA was done for the determination of the concentration and purity using Nano-drop spectrophotometer in line with with protocol reported by Desjaldins and Conklin, 2010 . After the quantification, the DNA samples were stored at -4 o C for further analyses. Primer design and DNA amplification Bovine exons 1–2 and exons 5–6 IRF3 gene specific primers were designed at Stab vida genetic laboratory located in Caprica-Portugal using Fast Polymerase Chain Reaction (PCR) software. Primer length, Primer sequence, annealing temperatures and the product sizes of the amplicons are presented in Table 1 . For amplification, 10-20ng of genomic DNA was added to the reaction containing 0.4mM of primers forward and reverse, 1mM of each dNTPs, 1.5mM of MgCl2 and 1.5u Taq polymerase and amplified by Magnetic Beads carboxylate cycler at following conditions; one cycle of initial denaturation of 15 minutes at 96 º C, final denaturation of 30 seconds at 95 º C, optimum annealing temperature of 60 º C for 30 seconds, extension at 70 º C for 2 minutes in 35 cycles with one cycle of the final extension performed at 70 º C for 5 minutes. Sequencing of PCR products The amplicons were purified using commercial kit (Magnetic Beads Carboxylate MC Lab, USA). The purified products were sequenced using BigDye® terminator cycle sequencing kit on ABI 3730xl (Applied Biosystems) DNA analyzer at Stab vida genetic laboratory situated in Caprica-Portugal. IRF3 exons 1–2 and exons 5–6 DNA sequence analysis The analysis of exons 1–2 in 62 animals (WF: 22; MT: 23 and ND: 17) out of the three cattle breeds covered a the length of one thousand and twenty five base pairs (1025bp) each after cleaning and trimming of the sequences, while the exons 5–6 in 64 animals (White Fulani:20, Muturu:22 and N’Dama:22) of the three cattle breeds found in Nigeria covered a sequence length of seven hundred base pairs (700bp) each after cleaning and trimming of the sequences with Bioedit and MEGA 5. Multiple sequence alignment was done on all the sequences of nucleotide using CLUSTAL W software (Thompson et al., 1994 ). The Single Nucleotide Polymorphisms in the bovine IRF3 gene exons 1–2 and exons 5–6 of each breed were identified using Codon code aligner software (htpp;// www.codoncode.com/aligner ). The DNA sequence polymorphism programme (DnaSP) version 5.10.01 was used to estimate haplotype frequencies, nucleotide diversity and sequence conservation. Tajima D and Fu’s Fs was also performed to test for deviation from neutrality (Tajima, 1989 ; Fu, 1997 ) using the same software. ARLEQUIN 2.0001 software (Excoffier et al., 1992 ) was used to estimate the basic population genetic statistics such as Analysis of Molecular Variance (AMOVA), population pairwise F st values and Standard genetic distances among the populations. RESULTS Single Nucleotide Polymorphisms(SNPs) identified in exons 1–2 of cattle IRF3 gene A total number of 18 Single Nucleotide Polymorphisms (SNPs) were discovered in exons 1–2 of the IRF 3 gene in all the three cattle breeds used for the study (presented in Table 2 ). Sixteen SNPs were detected in the Muturu breed, four were detected in the White Fulani breed and one was detected in the N’Dama breed. Two SNPs detected were common to the Muturu and White Fulani, while one SNP was shared by the N’Dama and White Fulani. High numbers of the SNPs detected were transversion type mutations. Table 2 contd: Single Nucleotide Polymorphisms Identified in exons 1-2 of Bovine Interferon Regulatory Factor 3 gene Genetic Diversity of exons 1–2 of IRF3 gene of the three cattle breeds Table 3 represents the result of the genetic diversity study of exons 1 and 2 of the IRF 3 gene in the three breeds of cattle. The numbers of sequences used were twenty two for the White Fulani, twenty three for the Muturu and seventeen for the N’Dama cattle respectively. A total number of four polymorphic sites (3 singletons and 1 parsimony informative site) were identified in this region of the IRF 3 gene in the White Fulani cattle. Sixteen polymorphic sites (16 parsimony informative sites) were identified in the Muturu cattle, while only one polymorphic site which was a parsimony informative site was identified in the N’Dama cattle. The highest number of haplotypes were found in the Muturu cattle (4), followed by the White Fulani (3), while the N’Dama had the least. The Muturu cattle had the highest haplotype diversity value of 0.628, followed by the White Fulani (0.382), while the N’Dama had the least value of 0.260. The Muturu cattle had the highest value of 0.0075 for nucleotide diversity while the N’Dama had the lowest value of 0.0004. With respect to the average number of nucleotide differences, the Muturu breed recorded the highest value of 7.570; the White Fulani cattle came second with a value of 0.519, while the N’Dama cattle had the lowest value of 0.382. It could be noticed that there was an enormous sequence conservation cutting across the sequences, with the N’Dama cattle having the highest value of 0.999, the White Fulani was in second place with 0.996, while the Muturu cattle recorded 0.980. Table 3: Genetic Diversity of exons 1-2 of the IRF3 gene in three breeds of Nigerian Cattle. Test for deviation from neutrality Table 4 showed the result of the test of neutrality of the exons 1–2 of the IRF 3 gene in the three breeds of cattle. The Tajima’s D and Fu’s F statistics were both insignificant (p > 0.05) for both White Fulani cattle (-1.479 and − 1.390) and the N’Dama (0.5655 and 0.834), but were both observed to be positive and significant (p < 0.05) for Muturu cattle (2.679 and 11.654). Table 5 revealed the result of the analysis of molecular variance (AMOVA) for exons 1–2 of the IRF 3 gene in the three breeds of cattle. The variation amongst the three breeds was much higher (93.03%) than that of the variation within populations (6.97%). The AMOVA also had an F ST value of 0.93. Table 6 contd: Single Nucleotide Polymorphisms Identified in exons 5-6 of Bovine Interferon Regulatory Factor 3 gene WF-White Fulani; MT- Muturu; ND-N’Dama. Total number of 29 SNPs were observed in Exons 5-6 of the IRF 3 gene in the three cattle breeds used for the study (Table 6). Fourteen Single Nucleotide Polymorphisms (SNPs) were present in White Fulani cattle; thirteen were observed in N’Dama and eight in Muturu. Four SNPs (62G>C, 125C>G, 262C>A and 403A>G) were shared between the N’Dama and White Fulani cattle, one SNP (290C>T) was shared between the Muturu and N’Dama cattle, while one SNP (241T>G) was observed to be shared by the three cattle breeds at the exon 5-6 region of the IRF3 gene. Fifteen of the identified SNPs were transversion mutations while the remaining 14 were transition in nature. WF-White Fulani, MT-Muturu, ND-N’Dama. Table 7 represented the results of the genetic diversity study of exons 5-6 in IRF 3 gene in the breeds(three) of cattle that is being studied. A total number of fourteen polymorphic sites (7 singleton variable sites and 7 parsimony informative site) were identified in this region of the IRF 3 gene found in the White Fulani breed cattle, 8 polymorphic sites (3 parsimony informative sites along with 5 singletons variable sites) were observed in Muturu cattle while 13 polymorphic sites (8 parsimony informative sites along with 5 singleton variable sites) were observed in N’Dama Cattle. The maximum amount of haplotypes were discovered in the White Fulani cattle (12), followed by the N’Dama (11), with the Muturu having the least of six (6) haplotypes in these regions. The White Fulani Cattle breed possessed the maximum haplotype diversity value of 0.93, followed by the N’Dama (0.87), while the Muturu had the lowest value of 0.54. The White Fulani Cattle had the highest value of 0.0049 for nucleotide diversity; N’Dama had 0.0043 while Muturu had the lowest value of 0.0015. With respect to the average number of nucleotide differences, the White Fulani Cattle recorded the maximum value of 3.337; the N’Dama had 2.913 while the Muturu cattle had the lowest value of 1.048. Table 8 showed the result of the test for neutrality of the exons 5–6 of the IRF 3 gene in the three breeds of cattle used for the study. The Tajima’s D and Fu’s F statistics were both not significant (p > 0.05) for the three cattle breeds. The three breeds had negative values (White Fulani − 0.78 and − 6.06; Muturu − 1.72 and − 3.16; N’Dama − 0.65 and − 4.81) for Tajima’s D and Fu’s F statistics respectively. Table 9 revealed the result of the analysis of molecular variation (AMOVA) for exons 5–6 of the IRF 3 gene in the three breeds of cattle. The variation among the three breeds was much higher (98.76%) than that of the variation within populations (1.24%) in this region. The AMOVA had an F st value of 0.99. DISCUSSION This study was done to detect polymorphisms found in exons 1–2 and exons 5–6 of IRF3 gene in the N’Dama, White Fulani and Muturu cattle breeds. These exons were observed to be the most polymorphic regions of the candidate gene in Cattle (Ensembl cow release 92). An overall amount of 18 nucleotide polymorphisms were identified in the exons 1–2 in three cattle breeds, implying the polymorphic nature of the region, most especially in the Muturu and White Fulani breeds both having a higher number of SNPs than the N’Dama which had only one, but generally this region of the gene in the three breeds was quite conserved. According to Dobzhansky ( 1970 ), common in nature are polymorphisms and they are related to genetic variation, biodiversity and adaptation; as a result, they perform an important role in keeping varieties of form in a population inhabiting different environment. The enormous polymorphisms occuring in this region in the Muturu breed also imply the gene’s involvements in different physiological and metabolic activities like response to immune, trypanotolerant nature and susceptibility to heat stress (Adebambo, 2001 ; Udeh et al., 2011 ; Styslinger, 2011 ). A single mutation discovered to be common to both White Fulani and N’Dama breeds suggests a shared common ancestor in their evolutionary development. This particular mutation could also play a role in growth and development, likewise heat tolerance considering the fact that these two breeds are larger animals and have been reported to be more heat tolerant than the Muturu (Behl et al., 2010 ; Udeh et al., 2011 ). Two mutations were also observed to be common to both the White Fulani and the Muturu, which likewise suggests not just a common ancestor but the likelihood that these mutations could also be responsible for immunological activities such as tolerance and resistance to intestinal helminthes, ticks and tick-borne diseases which are some of the characteristics shared by these two breeds (Claxton and Laperre, 1991; Mattioli et al., 2000 ). In exons 5–6 of the IRF3 gene, a total number of 29 polymorphic regions were observed in the three cattle breeds under study, implying that this site of the cattle IRF3 gene also possess large amount of polymorphisms with the Muturu possessing little amount of sites mutated. Carlson ( 2008 ) reported that differences in the Deoxyribonucleicacid (DNA) sequences of beings affect how they influence diseases and react to pathogens, vaccines, drugs, chemicals and other agents. High amount of Single Nucleotide Polymorphisms (SNPs) in exons 5–6 of the White Fulani breed could be due to their transhumance movements and activities which has exposed them to different climatic and weather conditions. A single mutated site was identified which was common to the three breeds; suggesting a common ancestor in their evolutionary development or an ancestral allele which could have arisen due to introgression or crossbreeding. The four mutated sites in exons 5–6 of the IRF3 gene, being shared by the N’Dama and White Fulani still suggested a common ancestor at a point, with these mutations also likely to be involved in growth and heat tolerance activities, considering the fact that the two breeds are larger animals and have been reported to be more heat-tolerant than the Muturu (Behl et al., 2010 ; Styslinger, 2011 ). A single mutated site in exons 5–6 was also identified as being shared by the Muturu and the N’Dama. The two breeds are both Taurines and some level of ancestral relationship would be expected between them. This shared SNP could influence a vital and important role in the immune reaction and response in the two breeds, which have been extensively reported to be the major trypano-tolerant breeds of cattle in Nigeria; making them thrive in areas highly-infested by tsetse flies (Akinwunmi and Ikpi, 1985; Charles,1991; Tawah and Rege, 1996 ; Adebambo, 2001 ). The absence of this particular mutation in the White Fulani cattle which is susceptible to trypanosomiasis reinforces the likely implication of this SNP in resistance to the disease. Analysis of the genetic diversity of IRF3 gene could be a great asset for improvement of various traits being influenced by the gene through marker-assisted selection. A total number of seven haplotypes were identified in exons 1–2 of the IRF3 gene across the three breeds. The highest number of haplotypes (4) was identified in the Muturu, which also had the highest haplotype and nucleotide diversity; this is an indication of high genetic diversity in this region of the gene in Muturu which will respond better to selection when compared with other breeds used for the study. The higher genetic variation in exons 5–6 of the IRF3 gene of White Fulani must have contributed to its adaptability, as genetic variation is important in helping organisms to adapt to an ever changing environment. This higher genetic variation in this breed could also be attributed to the lack of artificial selection pressure when compared to N’Dama and Muturu which are mostly bred in breeding stations and by subsistent farmers. Tajima D and Fu’ Fs test are commonly used test of neutrality in population genetic studies. It summarizes the sequence data into a single value (Tajima, 1989 ). The test of neutrality carried out on exons 1–2 region of the candidate gene in the White Fulani produced negative results which could be interpreted as a signal of population expansion in the absence of selection, the negative Tajima value is also related to the presence of singletons which were detected in the White Fulani cattle. The test of neutrality on exons 1–2 of the Muturu and N’Dama cattle yielded positive results for both Tajima’s D and Fu’s Fs, suggesting a situation of balancing selection and lack of singletons as was observed in these two breeds. The same test of neutrality was carried out on exons 5–6 of the candidate gene in the three breeds, the result yielded negative Tajima D and Fu Fs values suggesting some level of population expansion in the absence of selection with respect to this particular region, an excess amount of singletons were also detected in this region across the three breeds which is also related to the negative values obtained for the test of neutrality (William et al., 1995 ). In recent times, it has been argued that singletons or rare variants located in different genes could play a more important role in disease susceptibility than common variants (Bodmer and Bonilla, 2008 ; Saint Pierre and Génin, 2014 ). These rare genetic variants not initially captured by genome-wide association studies using single nucleotide polymorphism-chips have now become detectable with the advent of next-generation sequencing technologies. Goldstein et al., ( 2013 ) also believed that the process of purifying selection played a major role in maintaining a low frequency of such rare variants capable of strongly predisposing individuals to diseases in populations. The presence of singletons in exons 1–2 and 5–6 of the IRF3 gene in the White Fulani cattle could therefore play a significant role in this particular breeds’ susceptibility to diseases. Singletons were also observed to be present in exons 5–6 of the candidate gene in the Muturu and N’Dama which would also suggest the likelihood of such playing critical roles in disease susceptibility and probably poor heat tolerance. Analysis of molecular variance (AMOVA) is a statistical model for the molecular variation in a single species ( Excoffier et al., 1992 ). It is widely used in population genetics to test the hypothesis that genetic diversity within two populations is not significantly different from that which would result from pooling the two populations (Excoffier et al., 1992 ; Anderson, 2001 ). Analysis of exons 1–2 and 5–6 of the IRF3 gene in N’Dama, Muturu and White Fulani based on AMOVA demonstrated more variation among population compared to within population indicating that gene exchange or crossbreeding among the populations used for the study was low. In conclusion, this study to the best of our knowledge would be the first to report polymorphisms and genetic diversity of the IRF3 gene in the Nigerian White Fulani, Muturu and N’Dama cattle. The results of different analyses carried out in the study revealed the Muturu breed had the highest values for genetic diversity indices for exons 1–2 of the IRF3 gene while the White Fulani cattle breed had the highest genetic diversity indices for exons 5–6 of the IRF3 gene which we believe must have contributed to its adaptability considering the transhumance activities it is exposed to. The study also revealed some of the polymorphisms identified were shared by the different breeds suggesting the possibility of common ancestors and similar functions. The polymorphic nature of the candidate gene in the three cattle breeds would also suggest a form of nature’s response to ability of pathogens to evolve to evade the immune system, considering the gene has been reported to perform various immune functions. The SNPs detected within exons 1–2 and 5–6 of the IRF3 gene can therefore be used as genetic markers for association studies in further researches. Declarations Acknowledgements: Appreciation goes to the Directors and staffers of the Ipokia Local Government in Ogun State, the Federal Department of Livestock N’Dama Conservation Programme Ranch at Fashola in Oyo State, The Institute of Agricultural Research and Training Moor Plantation, Ibadan, off-site ranch at Ilora in Oyo State Odeda Local Government in Ogun State, for their assistance in allowing us to sample their available Cattle. We also thank the directors and staffers of the Central Biotechnology laboratory for allowing us analyse the cattle blood samples, Cattle Production Venture(CPV) and Institute of food Security Environmental Resources and Agricultural Research (IFSERAR) all located at the Federal University of Agriculture, Abeokuta, Ogun State, Nigeria for allowing us use their available cattle for this research work. Funding Statement: The authors declare that the research was not funded by any grants before, during and after the research and during the preparation of this manuscript. Data Availability Statement: Data will be made readily available upon its request from the editorial board. Author’s Contribution: All Authors (Abubakar A Mohammed, Michael O. Ozoje,, John S. De Campos, Christian O. Ikeobi, Talabi, A.O., Samuel O. Durosaro, Moses B. Ilori and Julius A. Aderoju, contributed to the study conception and design. Material preparation, data collection and Analysis were performed by Abubakar A. Mohammed, John S. De Campos, Samuel O. Durosaro, Julius A. Aderoju and Moses B. Ilori. The first draft of the manuscript was written by Abubakar A. Mohammed and all authors commented on the manuscript. Michael O. Ozoje, Christian O. Ikeobi and Talabi, A.O. provided the scientific structured content and all authors (Abubakar A Mohammed, Michael O. Ozoje,, John S. De Campos, Christian O. Ikeobi, Talabi, A.O., Samuel O. Durosaro, Moses B. Ilori and Julius A. Aderoju) read and approved the final manuscript. Compliance with Ethical Standards: This study was approved by the University ethics Research committee with approval project number of FUNAAB/AEWC/2021/0019. Conflict of Interest Statement: The authors have no financial or non-financial interests to disclose. Therefore , the authors declare that they have no conflict of interest and as such personal relationships that could have influenced the final report in this paper. We declare to the editor of this journal that this is our original work and has not been submitted for publication elsewhere. Animal welfare was not compromised anytime during this study and all our research protocols were cleared by the institution prior to embarking on the activities reported here. References Adebambo, A. O. 2001. The Muturu: A rare sacred breed of cattle in Nigeria. Animal Genetic Resource Information 31: 27-36. Akinwumi, J. A. and Ikpi, A. E. 1985. Trypano-tolerant cattle production in southern Nigeria. A report submitted to International Livestock Centre for Africa (ILCA) Adis Ababa – Ethiopia, Pp 2-5. Anderson, M.J. 2001. A new method for non-parametric multivariate analysis of variance. Australian Journal of Ecology 26 : 32–46. Au, W.C., Moore, P.A., Lowther, W., Juang, Y.T. and Pitha, P.M. 1995. Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. Proc. Natl. Acad. Sci. USA 92: 11657–11661. Behl, R., Behl, J. and Joshi, B . 2010 . Heat tolerance mechanisms in cattle—status in zebu cattle: a review. The Indian Journal of Animal Sciences 80(9): 1-12 Bodmer, W. and Bonilla, C. 2008. Common and rare variants in multifactorial susceptibility to common diseases. Nature Genetics . 40(6): 695–701. Carlson, B. 2008. "SNPs — A Shortcut to Personalized Medicine". Genetic Engineering and Biotechnology News (Mary Ann Liebert, Inc.) 28 (12). http://www.genengnews.com/gen-articles/snps-a-shortcut-to-personalized medicine/2507/. Retrieved 2017-07-06. Claxton, J. and Leperre, P. 1991. Parasite burdens and host susceptibility of Zebu and N'Dama cattle in village herds in Gambia. Veterinary Parasitology 40 :293-304. Charles, G. H. 1991. Cattle Genetic Resources . Elsevier Health Sciences.p34 Desjaldins, P. and Conklin, D. 2010. Nanodrop microvolume quantitation of nucleic acid. Journal of Visual Experiment 45: 2565-2570. Dobzhansky, T. 1970. Genetics of the Evolutionary Process . New York: Columbia University Press. Pp505 Ensembl cow release 92. http://m.ensembl.org Excoffier, L., Smouse, P. E. and Quattro, J. M. 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data. Genetics 131: 479-491. Fu, Y. X. 1997. Statistical test of neutrality and Mutation . Genetics 147: 915–925 . Goldstein, D.B., Allen, A., Keebler, J., Margulies, E.H., Petrou, S., Petrovski, S., Sunyaev, S. 2013. Sequencing studies in human genetics: design and interpretation . Nature Reviews Genetics 14(7): 460–70 Hiscott, J., Pitha,P.; Genin, P., Nguyen, H., Heylbroeck, C., Mamane, Y., Algarte, M. and Lin,R.1999. Triggering the Interferon Response: The Role of IRF-3 Transcription Factor. Journal of Interferon & Cytokine Research 19(1): 1-13. Jann ,O.C., King, A., Corrales, N.L., Anderson, S.I., Jensen, K., Ait-Ali, T., Tang, H., Wu, C., Cockett, N.E., Archibald, A.L. and Glass, E.J. 2009. Comparative genomics of toll-like receptor signaling in five species. BMC Genomics 10: 216. Mattioli, R.C., Pandey, V.S., Murray, M. and Fitzpatrick, J.L. 2000. Immunogenetic influences on tick resistance in African cattle with particular reference to trypanotolerant N’Dama ( Bos taurus ) and trypanosusceptible Gobra zebu ( Bos indicus ) cattle. Acta Tropica 75: 263–277. Saint Pierre, A. and Génin, E. 2014. How important are rare variants in common disease? Brief Functional Genomics 13(5): 353-361. Styslinger, M. 2011. N’Dama: Ancient West African cattle. Blogs.worldwatch.org/nourishing the planet. Tajima, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123: 585–595. Taniguchi, T.; Ogasawara,K.; Takaoka,A. and Tanaka,N. 2001. IRF family of transcription factors as regulators of host defense. Annual Review of Immunol ogy 19: 623–655. Tawah, C.L. and Rege, J.E.O. 1996. White Fulani cattle of west and central Africa.Anim. Genet. Review. Info.Bull.17:137-158 Thompson, J.D., Higgins, D.G. and Gibson, T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighing, position-specific gap penalties and weighting matrix choice. Nucleic Acid Research 22: 4673-1377 . Udeh, I., Akporhuarho, P. O. and Onogbe, C. O. 2011. Phenotypic Correlations Among Body Measurements and Physiological Parameters In Muturu and Zebu cattle. Journal of Agricultural & Biological Science 6(4):1 William, J.; Ballard, O.and Kreitman,M. 1995. Is mitochondrial DNA a strictly neutral marker? Trends in Ecology and Evolution 10: 485-488. Cite Share Download PDF Status: Published Journal Publication published 19 Mar, 2025 Read the published version in Tropical Animal Health and Production → Version 1 posted Editorial decision: Accept with minor revision 02 Apr, 2024 Reviewers agreed at journal 21 Feb, 2024 Reviewers invited by journal 15 Feb, 2024 Editor assigned by journal 04 Feb, 2024 First submitted to journal 03 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3917862","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273311567,"identity":"46e00066-e367-484c-a5c3-6c4120becc63","order_by":0,"name":"Abubakar Akinfolarin Mohammed","email":"data:image/png;base64,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","orcid":"https://orcid.org/0009-0005-2071-5155","institution":"Federal College of Education Special","correspondingAuthor":true,"prefix":"","firstName":"Abubakar","middleName":"Akinfolarin","lastName":"Mohammed","suffix":""},{"id":273311568,"identity":"aa2dd404-415f-4cdb-aa72-75ad133ae8c8","order_by":1,"name":"Micheal Ozoje","email":"","orcid":"","institution":"University of Agriculture Abeokuta: Federal University of Agriculture Abeokuta","correspondingAuthor":false,"prefix":"","firstName":"Micheal","middleName":"","lastName":"Ozoje","suffix":""},{"id":273311569,"identity":"2bb1f77a-93fb-4fd9-a697-eaa5b50597ed","order_by":2,"name":"John De Campos","email":"","orcid":"","institution":"University of Agriculture Abeokuta: Federal University of Agriculture Abeokuta","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"De Campos","suffix":""},{"id":273311570,"identity":"2c3a30f2-21f8-4d3f-ad43-d745366ed6d0","order_by":3,"name":"Christian Ikeobi","email":"","orcid":"","institution":"University of Agriculture Abeokuta: Federal University of Agriculture Abeokuta","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Ikeobi","suffix":""},{"id":273311571,"identity":"04e98b9d-4d7c-4300-a8a3-02e1982f28e2","order_by":4,"name":"Adewale Talabi","email":"","orcid":"","institution":"University of Agriculture Abeokuta: Federal University of Agriculture Abeokuta","correspondingAuthor":false,"prefix":"","firstName":"Adewale","middleName":"","lastName":"Talabi","suffix":""},{"id":273311572,"identity":"a7718c85-9d71-4539-9501-cd3de9dfe90e","order_by":5,"name":"Samuel Durosaro","email":"","orcid":"","institution":"University of Agriculture Abeokuta: Federal University of Agriculture Abeokuta","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Durosaro","suffix":""},{"id":273311573,"identity":"0b21a415-e8a3-4405-8d0f-9d52fb92dcae","order_by":6,"name":"Babatunde Ilori","email":"","orcid":"","institution":"University of Agriculture Abeokuta: Federal University of Agriculture Abeokuta","correspondingAuthor":false,"prefix":"","firstName":"Babatunde","middleName":"","lastName":"Ilori","suffix":""},{"id":273311574,"identity":"dfb8216c-169d-4162-80aa-d268bef4e802","order_by":7,"name":"Johnson Aderoju","email":"","orcid":"","institution":"University of Agriculture Abeokuta: Federal University of Agriculture Abeokuta","correspondingAuthor":false,"prefix":"","firstName":"Johnson","middleName":"","lastName":"Aderoju","suffix":""}],"badges":[],"createdAt":"2024-02-01 15:09:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3917862/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3917862/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11250-025-04356-0","type":"published","date":"2025-03-19T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79120453,"identity":"18e787b2-38b2-4405-8f49-90839b5b2d6b","added_by":"auto","created_at":"2025-03-24 16:08:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":784691,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3917862/v1/1f0abe87-80af-45f6-9ec1-d53b0a1464bb.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eIdentification of Polymorphisms and Genetic Diversity Studies of Interferon Regulatory Factor 3(IRF3) Gene in Muturu,White Fulani and N’Dama Cattle\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe interferon regulatory factors (IRF) are made up of developed family with similar transcribed proteins recognized firstly as controllers of the IFN-alpha/beta gene promoters, as well as the IFN-stimulated response element (ISRE) of some IFN-stimulated genes (Hiscott \u003cem\u003eet al\u003c/em\u003e., 1999). It has been reported that in mammals, nine members which belong to the interferon regulatory factor (IRF) family, (IRF-1 to IRF-9) have been identified (Taniguchi et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Interferon regulatory factor 3 (IRF3) gene encodes interferon regulatory factor 3, a member of the interferon regulatory transcription factor (IRF) family and is much revealed and is found in the cytoplasm of undiseased cells (Jann et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Prior to infection, the micro-organism virus triggers phosphorylation of C-terminal serine/threonine residues and consequently amounts to a conformational change in IRF3 with exposure of both the DNA binding domain (DBD) and Interferon associated domains (IAD), which leads to homo- or hetero-dimerization,, association with CBP/p300 coactivators, cytoplasm-to-nucleus transfer, activation of multiple target genes and stimulation of DNA binding to the IFN-stimulated response elements (ISREs). This gene was also reported to function in the stimulation of type I IFNs following virus infection and is an expressed phosphoprotein of 427 amino acids in humans (Au et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The cattle IRF3 gene consists 8 exons and 7 introns and encodes a 417-amino acid protein. It is a phosphoprotein which is made up of an N-terminal DNA binding domain (DBD domain), a C-terminal IRF-associated domain (IAD) and a transactivation domain, coupled with the importance it plays in the defense of its host and survival of the cells, the activity of IRF3 is strictly controlled. This gene is one of the strongest positional candidate genes implicated in a host of health-related phenotypes such as general disease resistance not only in cattle but in mice and humans as well. The gene was also reported to function in the ability to adapt to infections caused by protozoans in mice and cattle (Jann et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCattle populations have several polymorphisms at the IRF3 locus that change single amino acids. Parts of the highest polymorphic regions of this gene in cattle are exons 2, 5 and 6 (Ensembl cow release 92). Considering the importance of this gene, characterization was done in three Nigerian cattle breeds which include; Muturu, White Fulani and N\u0026rsquo;Dama cattle breeds. This study is therefore aimed to identify polymorphisms in IRF3 gene and evaluate genetic diversity indices in Nigerian, Muturu, N\u0026rsquo;Dama and White Fulani Cattle breeds.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eAnimals and Sampling\u003c/h2\u003e\n \u003cp\u003eAn amount of 190 animals were purposively sampled in this study. These comprised 85 White Fulani cattle, 72 Muturu and 33 N\u0026rsquo;Dama cattle. Samples of White Fulani cattle were collected at four locations; Ajani Farms, Ogbomosho, Odeda Local Government, Ogun State and the Cattle Production Venture of the Federal University of Agriculture, Abeokuta (FUNAAB), all located in Nigeria. The N\u0026rsquo;Dama breed was sampled at the Institute of Agricultural Research and Training Moor Plantation, Ibadan, off-site ranch at Ilora in Oyo State and the Federal Department of Livestock N\u0026rsquo;Dama Conservation Programme Ranch at Fashola in Oyo State while the Muturu breed was sampled at Odeda Local Government in Ogun State, Ipokia Local Government in Ogun State and Institute of Food Security, Environmental Resources and Agricultural Research facility at the FUNAAB all located in Nigeria.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eDNA Extraction\u003c/h2\u003e\n \u003cp\u003eGenomic DNA was extracted from the blood samples using Zymo-Spin IIC\u0026trade; extraction kit. The extraction process was done at the Central Biotechnology Laboratory located found at Federal University of Agriculture, Abeokuta, Ogun State, Nigeria. The manufacturer\u0026rsquo;s protocol was used in carrying out the extraction after which the quantification of the extracted DNA was done for the determination of the concentration and purity using Nano-drop spectrophotometer in line with with protocol reported by Desjaldins and Conklin, \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e. After the quantification, the DNA samples were stored at -4\u003csup\u003eo\u003c/sup\u003eC for further analyses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003ePrimer design and DNA amplification\u003c/h2\u003e\n \u003cp\u003eBovine exons 1\u0026ndash;2 and exons 5\u0026ndash;6 IRF3 gene specific primers were designed at Stab vida genetic laboratory located in Caprica-Portugal using Fast Polymerase Chain Reaction (PCR) software. Primer length, Primer sequence, annealing temperatures and the product sizes of the amplicons are presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. For amplification, 10-20ng of genomic DNA was added to the reaction containing 0.4mM of primers forward and reverse, 1mM of each dNTPs, 1.5mM of MgCl2 and 1.5u Taq polymerase and amplified by Magnetic Beads carboxylate cycler at following conditions; one cycle of initial denaturation of 15 minutes at 96\u003csup\u003e\u0026ordm;\u003c/sup\u003eC, final denaturation of 30 seconds at 95\u003csup\u003e\u0026ordm;\u003c/sup\u003eC, optimum annealing temperature of 60\u003csup\u003e\u0026ordm;\u003c/sup\u003eC for 30 seconds, extension at 70\u003csup\u003e\u0026ordm;\u003c/sup\u003eC for 2 minutes in 35 cycles with one cycle of the final extension performed at 70\u003csup\u003e\u0026ordm;\u003c/sup\u003eC for 5 minutes.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1708412409.png\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eSequencing of PCR products\u003c/h2\u003e\n \u003cp\u003eThe amplicons were purified using commercial kit (Magnetic Beads Carboxylate MC Lab, USA). The purified products were sequenced using BigDye\u0026reg; terminator cycle sequencing kit on ABI 3730xl (Applied Biosystems) DNA analyzer at Stab vida genetic laboratory situated in Caprica-Portugal.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eIRF3 exons 1\u0026ndash;2 and exons 5\u0026ndash;6 DNA sequence analysis\u003c/h2\u003e\n \u003cp\u003eThe analysis of exons 1\u0026ndash;2 in 62 animals (WF: 22; MT: 23 and ND: 17) out of the three cattle breeds covered a the length of one thousand and twenty five base pairs (1025bp) each after cleaning and trimming of the sequences, while the exons 5\u0026ndash;6 in 64 animals (White Fulani:20, Muturu:22 and N\u0026rsquo;Dama:22) of the three cattle breeds found in Nigeria covered a sequence length of seven hundred base pairs (700bp) each after cleaning and trimming of the sequences with Bioedit and MEGA 5. Multiple sequence alignment was done on all the sequences of nucleotide using CLUSTAL W software (Thompson et al., \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e). The Single Nucleotide Polymorphisms in the bovine IRF3 gene exons 1\u0026ndash;2 and exons 5\u0026ndash;6 of each breed were identified using Codon code aligner software (htpp;//\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.codoncode.com/aligner\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe DNA sequence polymorphism programme (DnaSP) version 5.10.01 was used to estimate haplotype frequencies, nucleotide diversity and sequence conservation. Tajima D and Fu\u0026rsquo;s Fs was also performed to test for deviation from neutrality (Tajima, \u003cspan class=\"CitationRef\"\u003e1989\u003c/span\u003e; Fu, \u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e) using the same software. ARLEQUIN 2.0001 software (Excoffier et al., \u003cspan class=\"CitationRef\"\u003e1992\u003c/span\u003e) was used to estimate the basic population genetic statistics such as Analysis of Molecular Variance (AMOVA), population pairwise F\u003cem\u003est\u003c/em\u003e values and Standard genetic distances among the populations.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003eSingle Nucleotide Polymorphisms(SNPs) identified in exons 1\u0026ndash;2 of cattle IRF3 gene\u003c/h2\u003e\n \u003cp\u003eA total number of 18 Single Nucleotide Polymorphisms (SNPs) were discovered in exons 1\u0026ndash;2 of the IRF 3 gene in all the three cattle breeds used for the study (presented in Table \u003cspan\u003e2\u003c/span\u003e). Sixteen SNPs were detected in the Muturu breed, four were detected in the White Fulani breed and one was detected in the N\u0026rsquo;Dama breed. Two SNPs detected were common to the Muturu and White Fulani, while one SNP was shared by the N\u0026rsquo;Dama and White Fulani. High numbers of the SNPs detected were transversion type mutations.\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1708412490.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2 contd: Single Nucleotide Polymorphisms Identified in exons 1-2 of Bovine Interferon Regulatory Factor 3 gene\u003c/strong\u003e\u003c/p\u003e\n \u003cdiv\u003e\n \u003cdiv align=\"left\"\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1708412536.png\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003eGenetic Diversity of exons 1\u0026ndash;2 of IRF3 gene of the three cattle breeds\u003c/h2\u003e\n \u003cp\u003eTable \u003cspan\u003e3\u003c/span\u003e represents the result of the genetic diversity study of exons 1 and 2 of the IRF 3 gene in the three breeds of cattle. The numbers of sequences used were twenty two for the White Fulani, twenty three for the Muturu and seventeen for the N\u0026rsquo;Dama cattle respectively. A total number of four polymorphic sites (3 singletons and 1 parsimony informative site) were identified in this region of the IRF 3 gene in the White Fulani cattle.\u003c/p\u003e\n \u003cp\u003eSixteen polymorphic sites (16 parsimony informative sites) were identified in the Muturu cattle, while only one polymorphic site which was a parsimony informative site was identified in the N\u0026rsquo;Dama cattle. The highest number of haplotypes were found in the Muturu cattle (4), followed by the White Fulani (3), while the N\u0026rsquo;Dama had the least. The Muturu cattle had the highest haplotype diversity value of 0.628, followed by the White Fulani (0.382), while the N\u0026rsquo;Dama had the least value of 0.260.\u003c/p\u003e\n \u003cp\u003eThe Muturu cattle had the highest value of 0.0075 for nucleotide diversity while the N\u0026rsquo;Dama had the lowest value of 0.0004. With respect to the average number of nucleotide differences, the Muturu breed recorded the highest value of 7.570; the White Fulani cattle came second with a value of 0.519, while the N\u0026rsquo;Dama cattle had the lowest value of 0.382. It could be noticed that there was an enormous sequence conservation cutting across the sequences, with the N\u0026rsquo;Dama cattle having the highest value of 0.999, the White Fulani was in second place with 0.996, while the Muturu cattle recorded 0.980.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 3: Genetic Diversity of exons 1-2 of the IRF3 gene in three breeds of\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eNigerian Cattle.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1708413061.png\"\u003e\u003c/strong\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eTest for deviation from neutrality\u003c/h2\u003e\n \u003cp\u003eTable \u003cspan\u003e4\u003c/span\u003e showed the result of the test of neutrality of the exons 1\u0026ndash;2 of the IRF 3 gene in the three breeds of cattle. The Tajima\u0026rsquo;s D and Fu\u0026rsquo;s F statistics were both insignificant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) for both White Fulani cattle (-1.479 and \u0026minus;\u0026thinsp;1.390) and the N\u0026rsquo;Dama (0.5655 and 0.834), but were both observed to be positive and significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for Muturu cattle (2.679 and 11.654).\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1708412600.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cdiv\u003e\n \u003cdiv align=\"left\"\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1708412617.png\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cp\u003eTable \u003cspan\u003e5\u003c/span\u003e revealed the result of the analysis of molecular variance (AMOVA) for exons 1\u0026ndash;2 of the IRF 3 gene in the three breeds of cattle. The variation amongst the three breeds was much higher (93.03%) than that of the variation within populations (6.97%). The AMOVA also had an F\u003csub\u003e\u003cem\u003eST\u003c/em\u003e\u003c/sub\u003e value of 0.93.\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1708412671.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cdiv\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eTable 6 contd: Single Nucleotide Polymorphisms Identified in exons 5-6 of Bovine Interferon Regulatory Factor 3 gene\u003c/strong\u003e\u003c/p\u003e\n \u003cdiv\u003e\n \u003cdiv align=\"left\"\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1708412712.png\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eWF-White Fulani; MT- Muturu; ND-N\u0026rsquo;Dama.\u003c/p\u003e\n \u003cp\u003eTotal number of 29 SNPs were observed in Exons 5-6 of the IRF 3 gene in the three cattle breeds used for the study (Table 6). Fourteen Single Nucleotide Polymorphisms (SNPs) were present in White Fulani cattle; thirteen were observed in N\u0026rsquo;Dama and eight in Muturu.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;Four SNPs (62G\u0026gt;C, 125C\u0026gt;G, 262C\u0026gt;A and 403A\u0026gt;G) were shared between the N\u0026rsquo;Dama and White Fulani cattle, one SNP (290C\u0026gt;T) was shared between the Muturu and N\u0026rsquo;Dama cattle, while one SNP (241T\u0026gt;G) was observed to be shared by the three cattle breeds at the exon 5-6 region of the IRF3 gene. Fifteen of the identified SNPs were transversion mutations while the remaining 14 were transition in nature.\u003c/p\u003e\n \u003cdiv\u003e\n \u003cdiv align=\"left\"\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1708412794.png\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; WF-White Fulani, MT-Muturu, ND-N\u0026rsquo;Dama.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTable 7 represented the results of the genetic diversity study of exons 5-6 in IRF 3 gene in the breeds(three) of cattle that is being studied. A total number of fourteen polymorphic sites (7 singleton variable sites and 7 parsimony informative site) were identified in this region of the IRF 3 gene found in the White Fulani breed cattle, 8 polymorphic sites (3 parsimony informative sites along with 5 singletons variable sites) were observed in Muturu cattle while 13 polymorphic sites (8 parsimony informative sites along with 5 singleton variable sites) were observed in \u0026nbsp;N\u0026rsquo;Dama Cattle.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eThe maximum amount of haplotypes were discovered in the White Fulani cattle (12), followed by the N\u0026rsquo;Dama (11), with the Muturu having the least of six (6) haplotypes in these regions. The White Fulani Cattle breed possessed the maximum haplotype diversity value of 0.93, followed by the N\u0026rsquo;Dama (0.87), while the Muturu had the lowest value of 0.54. The White Fulani Cattle had the highest value of 0.0049 for nucleotide diversity; N\u0026rsquo;Dama had 0.0043 while Muturu had the lowest value of 0.0015. With respect to the average number of nucleotide differences, the White Fulani Cattle recorded the maximum value of 3.337; the N\u0026rsquo;Dama had 2.913 while the Muturu cattle had the lowest value of 1.048.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1708412834.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cdiv\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cp\u003eTable \u003cspan\u003e8\u003c/span\u003e showed the result of the test for neutrality of the exons 5\u0026ndash;6 of the IRF 3 gene in the three breeds of cattle used for the study. The Tajima\u0026rsquo;s D and Fu\u0026rsquo;s F statistics were both not significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) for the three cattle breeds. The three breeds had negative values (White Fulani \u0026minus;\u0026thinsp;0.78 and \u0026minus;\u0026thinsp;6.06; Muturu \u0026minus;\u0026thinsp;1.72 and \u0026minus;\u0026thinsp;3.16; N\u0026rsquo;Dama \u0026minus;\u0026thinsp;0.65 and \u0026minus;\u0026thinsp;4.81) for Tajima\u0026rsquo;s D and Fu\u0026rsquo;s F statistics respectively.\u003c/p\u003e\n \u003cdiv\u003e\n \u003cdiv align=\"left\"\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1708412853.png\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eTable \u003cspan\u003e9\u003c/span\u003e revealed the result of the analysis of molecular variation (AMOVA) for exons 5\u0026ndash;6 of the IRF 3 gene in the three breeds of cattle. The variation among the three breeds was much higher (98.76%) than that of the variation within populations (1.24%) in this region. The AMOVA had an F\u003cem\u003est\u003c/em\u003e value of 0.99.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study was done to detect polymorphisms found in exons 1\u0026ndash;2 and exons 5\u0026ndash;6 of IRF3 gene in the N\u0026rsquo;Dama, White Fulani and Muturu cattle breeds. These exons were observed to be the most polymorphic regions of the candidate gene in Cattle (Ensembl cow release 92). An overall amount of 18 nucleotide polymorphisms were identified in the exons 1\u0026ndash;2 in three cattle breeds, implying the polymorphic nature of the region, most especially in the Muturu and White Fulani breeds both having a higher number of SNPs than the N\u0026rsquo;Dama which had only one, but generally this region of the gene in the three breeds was quite conserved. According to Dobzhansky (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1970\u003c/span\u003e), common in nature are polymorphisms and they are related to genetic variation, biodiversity and adaptation; as a result, they perform an important role in keeping varieties of form in a population inhabiting different environment. The enormous polymorphisms occuring in this region in the Muturu breed also imply the gene\u0026rsquo;s involvements in different physiological and metabolic activities like response to immune, trypanotolerant nature and susceptibility to heat stress (Adebambo, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Udeh et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Styslinger, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA single mutation discovered to be common to both White Fulani and N\u0026rsquo;Dama breeds suggests a shared common ancestor in their evolutionary development. This particular mutation could also play a role in growth and development, likewise heat tolerance considering the fact that these two breeds are larger animals and have been reported to be more heat tolerant than the Muturu (Behl et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Udeh et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Two mutations were also observed to be common to both the White Fulani and the Muturu, which likewise suggests not just a common ancestor but the likelihood that these mutations could also be responsible for immunological activities such as tolerance and resistance to intestinal helminthes, ticks and tick-borne diseases which are some of the characteristics shared by these two breeds (Claxton and Laperre, 1991; Mattioli et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn exons 5\u0026ndash;6 of the IRF3 gene, a total number of 29 polymorphic regions were observed in the three cattle breeds under study, implying that this site of the cattle IRF3 gene also possess large amount of polymorphisms with the Muturu possessing little amount of sites mutated. Carlson (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) reported that differences in the Deoxyribonucleicacid (DNA) sequences of beings affect how they influence diseases and react to pathogens, vaccines, drugs, chemicals and other agents. High amount of Single Nucleotide Polymorphisms (SNPs) in exons 5\u0026ndash;6 of the White Fulani breed could be due to their transhumance movements and activities which has exposed them to different climatic and weather conditions. A single mutated site was identified which was common to the three breeds; suggesting a common ancestor in their evolutionary development or an ancestral allele which could have arisen due to introgression or crossbreeding.\u003c/p\u003e \u003cp\u003eThe four mutated sites in exons 5\u0026ndash;6 of the IRF3 gene, being shared by the N\u0026rsquo;Dama and White Fulani still suggested a common ancestor at a point, with these mutations also likely to be involved in growth and heat tolerance activities, considering the fact that the two breeds are larger animals and have been reported to be more heat-tolerant than the Muturu (Behl et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Styslinger, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). A single mutated site in exons 5\u0026ndash;6 was also identified as being shared by the Muturu and the N\u0026rsquo;Dama. The two breeds are both Taurines and some level of ancestral relationship would be expected between them. This shared SNP could influence a vital and important role in the immune reaction and response in the two breeds, which have been extensively reported to be the major trypano-tolerant breeds of cattle in Nigeria; making them thrive in areas highly-infested by tsetse flies (Akinwunmi and Ikpi, 1985; Charles,1991; Tawah and Rege, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Adebambo, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The absence of this particular mutation in the White Fulani cattle which is susceptible to trypanosomiasis reinforces the likely implication of this SNP in resistance to the disease.\u003c/p\u003e \u003cp\u003eAnalysis of the genetic diversity of IRF3 gene could be a great asset for improvement of various traits being influenced by the gene through marker-assisted selection. A total number of seven haplotypes were identified in exons 1\u0026ndash;2 of the IRF3 gene across the three breeds. The highest number of haplotypes (4) was identified in the Muturu, which also had the highest haplotype and nucleotide diversity; this is an indication of high genetic diversity in this region of the gene in Muturu which will respond better to selection when compared with other breeds used for the study. The higher genetic variation in exons 5\u0026ndash;6 of the IRF3 gene of White Fulani must have contributed to its adaptability, as genetic variation is important in helping organisms to adapt to an ever changing environment. This higher genetic variation in this breed could also be attributed to the lack of artificial selection pressure when compared to N\u0026rsquo;Dama and Muturu which are mostly bred in breeding stations and by subsistent farmers.\u003c/p\u003e \u003cp\u003eTajima D and Fu\u0026rsquo; Fs test are commonly used test of neutrality in population genetic studies. It summarizes the sequence data into a single value (Tajima, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). The test of neutrality carried out on exons 1\u0026ndash;2 region of the candidate gene in the White Fulani produced negative results which could be interpreted as a signal of population expansion in the absence of selection, the negative Tajima value is also related to the presence of singletons which were detected in the White Fulani cattle. The test of neutrality on exons 1\u0026ndash;2 of the Muturu and N\u0026rsquo;Dama cattle yielded positive results for both Tajima\u0026rsquo;s D and Fu\u0026rsquo;s Fs, suggesting a situation of balancing selection and lack of singletons as was observed in these two breeds.\u003c/p\u003e \u003cp\u003eThe same test of neutrality was carried out on exons 5\u0026ndash;6 of the candidate gene in the three breeds, the result yielded negative Tajima D and Fu Fs values suggesting some level of population expansion in the absence of selection with respect to this particular region, an excess amount of singletons were also detected in this region across the three breeds which is also related to the negative values obtained for the test of neutrality (William et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). In recent times, it has been argued that singletons or rare variants located in different genes could play a more important role in disease susceptibility than common variants (Bodmer and Bonilla, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Saint Pierre and G\u0026eacute;nin, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These rare genetic variants not initially captured by genome-wide association studies using single nucleotide polymorphism-chips have now become detectable with the advent of next-generation sequencing technologies. Goldstein et al., (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) also believed that the process of purifying selection played a major role in maintaining a low frequency of such rare variants capable of strongly predisposing individuals to diseases in populations. The presence of singletons in exons 1\u0026ndash;2 and 5\u0026ndash;6 of the IRF3 gene in the White Fulani cattle could therefore play a significant role in this particular breeds\u0026rsquo; susceptibility to diseases. Singletons were also observed to be present in exons 5\u0026ndash;6 of the candidate gene in the Muturu and N\u0026rsquo;Dama which would also suggest the likelihood of such playing critical roles in disease susceptibility and probably poor heat tolerance. Analysis of molecular variance (AMOVA) is a statistical model for the molecular variation in a single species \u003cem\u003e(\u003c/em\u003eExcoffier et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). It is widely used in population genetics to test the hypothesis that genetic diversity within two populations is not significantly different from that which would result from pooling the two populations (Excoffier et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Anderson, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Analysis of exons 1\u0026ndash;2 and 5\u0026ndash;6 of the IRF3 gene in N\u0026rsquo;Dama, Muturu and White Fulani based on AMOVA demonstrated more variation among population compared to within population indicating that gene exchange or crossbreeding among the populations used for the study was low.\u003c/p\u003e \u003cp\u003eIn conclusion, this study to the best of our knowledge would be the first to report polymorphisms and genetic diversity of the IRF3 gene in the Nigerian White Fulani, Muturu and N\u0026rsquo;Dama cattle. The results of different analyses carried out in the study revealed the Muturu breed had the highest values for genetic diversity indices for exons 1\u0026ndash;2 of the IRF3 gene while the White Fulani cattle breed had the highest genetic diversity indices for exons 5\u0026ndash;6 of the IRF3 gene which we believe must have contributed to its adaptability considering the transhumance activities it is exposed to. The study also revealed some of the polymorphisms identified were shared by the different breeds suggesting the possibility of common ancestors and similar functions. The polymorphic nature of the candidate gene in the three cattle breeds would also suggest a form of nature\u0026rsquo;s response to ability of pathogens to evolve to evade the immune system, considering the gene has been reported to perform various immune functions. The SNPs detected within exons 1\u0026ndash;2 and 5\u0026ndash;6 of the IRF3 gene can therefore be used as genetic markers for association studies in further researches.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eAppreciation goes to the Directors and staffers of\u0026nbsp;the Ipokia Local Government in Ogun State, the Federal Department of Livestock N\u0026rsquo;Dama Conservation Programme Ranch at Fashola in Oyo State,\u0026nbsp;The\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eInstitute of Agricultural Research and Training Moor Plantation, Ibadan, off-site ranch at Ilora in Oyo State Odeda Local Government in Ogun State, for their assistance in allowing us to sample their available Cattle. We also thank the directors and staffers of the Central Biotechnology laboratory for allowing us analyse the cattle blood samples, Cattle Production Venture(CPV) and Institute of food Security Environmental Resources and Agricultural Research (IFSERAR) all located at the \u0026nbsp;Federal University of Agriculture, Abeokuta, Ogun State, Nigeria for allowing us use their available cattle for this research work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement:\u0026nbsp;\u003c/strong\u003eThe authors declare that the research was not funded by any grants before, \u0026nbsp;during and after \u0026nbsp;the research and during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eData will be made readily available upon its request from the editorial board.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contribution: \u0026nbsp;\u003c/strong\u003eAll Authors (Abubakar A Mohammed, Michael O. Ozoje,, John S. De Campos, Christian O. Ikeobi, Talabi, A.O., Samuel O. Durosaro, Moses B. Ilori and Julius A. Aderoju,\u0026nbsp;contributed to the study conception and design. Material preparation, data collection and Analysis were performed by Abubakar A. Mohammed, John S. De Campos, Samuel O. Durosaro, Julius A. Aderoju and Moses B. Ilori. The first draft of the manuscript was written by Abubakar A. Mohammed and all authors commented on the manuscript. Michael O. Ozoje, Christian O. Ikeobi and Talabi, A.O. provided the scientific structured content and all authors (Abubakar A Mohammed, Michael O. Ozoje,, John S. De Campos, Christian O. Ikeobi, Talabi, A.O., Samuel O. Durosaro, Moses B. Ilori and Julius A. Aderoju)\u0026nbsp;read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards:\u0026nbsp;\u003c/strong\u003eThis study was approved by the University ethics Research committee with approval project number of FUNAAB/AEWC/2021/0019.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflict of Interest Statement:\u0026nbsp;\u003c/strong\u003eThe authors have no financial or non-financial interests to disclose. Therefore\u003cem\u003e,\u0026nbsp;\u003c/em\u003ethe authors declare that they have no conflict of interest and as such personal relationships that could have influenced the final report in this paper.\u003c/p\u003e\n\u003cp\u003eWe declare to the editor of this journal that this is our original work and has not been submitted for publication elsewhere. Animal welfare was not compromised anytime during this study and all our research protocols were cleared by the institution prior to embarking on the activities reported here.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdebambo, A. O. 2001. The Muturu: A rare sacred breed of cattle in Nigeria. Animal Genetic Resource Information 31: 27-36.\u003c/li\u003e\n\u003cli\u003eAkinwumi, J. A. and Ikpi, A. E. 1985. Trypano-tolerant cattle production in southern Nigeria. A report submitted to International Livestock Centre for Africa (ILCA) Adis Ababa \u0026ndash; Ethiopia, Pp 2-5.\u003c/li\u003e\n\u003cli\u003eAnderson, M.J. 2001. A new method for non-parametric multivariate analysis of variance. \u003cem\u003eAustralian Journal of Ecology\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e: 32\u0026ndash;46.\u003c/li\u003e\n\u003cli\u003eAu, W.C., Moore, P.A., Lowther, W., Juang, Y.T. and Pitha, P.M. 1995. Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. Proc. Natl. Acad. Sci. USA 92: 11657\u0026ndash;11661.\u003c/li\u003e\n\u003cli\u003eBehl, R., Behl, J. and Joshi, B\u003cem\u003e. \u003c/em\u003e2010\u003cem\u003e. \u003c/em\u003eHeat tolerance mechanisms in cattle\u0026mdash;status in zebu cattle: a review. \u003cem\u003eThe \u003c/em\u003e\u003cem\u003eIndian Journal of Animal Sciences\u003c/em\u003e 80(9): 1-12\u003c/li\u003e\n\u003cli\u003eBodmer, W. and Bonilla, C. 2008. Common and rare variants in multifactorial susceptibility to common diseases. \u003cem\u003eNature Genetics\u003c/em\u003e. \u003cem\u003e40(6): 695\u0026ndash;701. \u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eCarlson, B. 2008. \u0026quot;SNPs \u0026mdash; A Shortcut to Personalized Medicine\u0026quot;. Genetic Engineering and Biotechnology News (Mary Ann Liebert, Inc.) 28 (12). http://www.genengnews.com/gen-articles/snps-a-shortcut-to-personalized medicine/2507/. Retrieved 2017-07-06. \u003c/li\u003e\n\u003cli\u003eClaxton, J. and Leperre, P. 1991. Parasite burdens and host susceptibility of Zebu and N\u0026apos;Dama cattle in village herds in Gambia. \u003cem\u003eVeterinary Parasitology\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e:293-304.\u003c/li\u003e\n\u003cli\u003eCharles, G. H. 1991. \u003cem\u003eCattle Genetic Resources\u003c/em\u003e. Elsevier Health Sciences.p34\u003c/li\u003e\n\u003cli\u003eDesjaldins, P. and Conklin, D. 2010. Nanodrop microvolume quantitation of nucleic acid. \u003cem\u003eJournal of Visual Experiment\u003c/em\u003e \u003cem\u003e45: 2565-2570.\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eDobzhansky, T. 1970. \u003cem\u003eGenetics of the Evolutionary Process\u003c/em\u003e. New York: Columbia University Press. Pp505\u003c/li\u003e\n\u003cli\u003eEnsembl cow release 92.\u003cu\u003ehttp://m.ensembl.org\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eExcoffier, L., Smouse, P. E. and Quattro, J. M. 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data. \u003cem\u003eGenetics\u003c/em\u003e \u003cem\u003e131: 479-491.\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eFu, Y. X. 1997. Statistical test of neutrality and Mutation .\u003cem\u003eGenetics\u003c/em\u003e \u003cem\u003e147: 915\u0026ndash;925\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003e\u003ccite\u003eGoldstein, D.B., Allen, A., Keebler, J., Margulies, E.H., Petrou, S., Petrovski, S., Sunyaev, S. 2013. \u003c/cite\u003eSequencing studies in human genetics: design and interpretation \u003ccite\u003e. Nature Reviews Genetics 14(7): 460\u0026ndash;70\u003c/cite\u003e\u003c/li\u003e\n\u003cli\u003eHiscott, J., Pitha,P.; Genin, P., Nguyen, H., Heylbroeck, C., Mamane, Y., Algarte, M. and Lin,R.1999. Triggering the Interferon Response: The Role of IRF-3 Transcription Factor. \u003cem\u003eJournal of Interferon \u0026amp; Cytokine Research 19(1): 1-13.\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eJann ,O.C., King, A., Corrales, N.L., Anderson, S.I., Jensen, K., Ait-Ali, T., Tang, H., Wu, C., Cockett, N.E., Archibald, A.L. and Glass, E.J. 2009. Comparative genomics of toll-like receptor signaling in five species. \u003cem\u003eBMC Genomics \u003c/em\u003e10: 216.\u003c/li\u003e\n\u003cli\u003eMattioli, R.C., Pandey, V.S., Murray, M. and Fitzpatrick, J.L. 2000. Immunogenetic influences on tick resistance in African cattle with particular reference to trypanotolerant N\u0026rsquo;Dama (\u003cem\u003eBos taurus\u003c/em\u003e) and trypanosusceptible Gobra zebu (\u003cem\u003eBos indicus\u003c/em\u003e) cattle. \u003cem\u003eActa Tropica\u003c/em\u003e 75: 263\u0026ndash;277.\u003c/li\u003e\n\u003cli\u003eSaint Pierre, A. and G\u0026eacute;nin, E. 2014. How important are rare variants in common disease? \u003cem\u003eBrief Functional Genomics\u003c/em\u003e 13(5): 353-361.\u003c/li\u003e\n\u003cli\u003eStyslinger, M. 2011. N\u0026rsquo;Dama: Ancient West African cattle. \u003cu\u003eBlogs.worldwatch.org/nourishing the planet.\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eTajima, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. \u003cem\u003eGenetics \u003c/em\u003e123: 585\u0026ndash;595.\u003c/li\u003e\n\u003cli\u003eTaniguchi, T.; Ogasawara,K.; Takaoka,A. and Tanaka,N. 2001. IRF family of transcription factors as regulators of host defense. \u003cem\u003eAnnual Review\u003c/em\u003e \u003cem\u003eof Immunol\u003c/em\u003eogy 19: 623\u0026ndash;655.\u003c/li\u003e\n\u003cli\u003eTawah, C.L. and Rege, J.E.O. 1996. White Fulani cattle of west and central Africa.Anim. \u003cem\u003eGenet. Review. Info.Bull.17:137-158\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eThompson, J.D., Higgins, D.G. and Gibson, T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighing, position-specific gap penalties and weighting matrix choice. \u003cem\u003eNucleic Acid Research\u003c/em\u003e \u003cem\u003e22: 4673-1377\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eUdeh, I., Akporhuarho, P. O. and Onogbe, C. O. 2011. Phenotypic Correlations Among Body Measurements and Physiological Parameters In Muturu and Zebu cattle. \u003cem\u003eJournal of Agricultural \u0026amp; Biological Science 6(4):1\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eWilliam, J.; Ballard, O.and Kreitman,M. 1995. Is mitochondrial DNA a strictly neutral marker? \u003cem\u003eTrends in Ecology and Evolution\u003c/em\u003e \u003cem\u003e10: 485-488.\u003c/em\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Genetic diversity, Cattle, Interferon Regulatory Factor (IRF3), Polymorphisms","lastPublishedDoi":"10.21203/rs.3.rs-3917862/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3917862/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInterferon regulatory factor 3 (IRF3) is one of the strongest positional candidate genes implicated in a host of health-related phenotypes such as general disease resistance. The study was carried out to genetically characterize the IRF3 gene in the, N\u0026rsquo;Dama, Muturu and White Fulani cattle. DNA was extracted from the blood samples using the Zymo-spin extraction kit. ARLEQUIN 2.0001 software was used to estimate the basic population genetic statistics while DnaSP version 5.10.01 was used to estimate genetic diversity indices and test for deviation from neutrality. A total number of 18 and 29 Single nucleotide polymorphisms (SNPs) were observed after using the software called codon code aligner in exons 1\u0026ndash;2 and 5\u0026ndash;6 of the IRF3 gene respectively in the three cattle breeds after polymerase chain reaction and sequencing. In exons 1\u0026ndash;2, the Muturu (MU) possessed the highest value of SNPs (16) and genetic diversity indices, while the N\u0026rsquo;Dama (ND) possessed the least (1). In exons 5\u0026ndash;6, the highest value of SNPs (14) was observed in the White Fulani and the genetic diversity indices was also high while the Muturu had the least. Analysis of molecular variation (AMOVA) carried out for the loci under consideration revealed a higher level of variation among populations than within populations. It was therefore concluded that the IRF3 gene had many polymorphisms and was highly diversified in Nigerian cattle breeds.\u003c/p\u003e","manuscriptTitle":"Identification of Polymorphisms and Genetic Diversity Studies of Interferon Regulatory Factor 3(IRF3) Gene in Muturu,White Fulani and N’Dama Cattle","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-20 07:13:01","doi":"10.21203/rs.3.rs-3917862/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept with minor revision","date":"2024-04-02T04:22:28+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-02-21T08:12:31+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-16T01:13:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-05T04:15:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Animal Health and Production","date":"2024-02-04T03:06:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"55e45d3b-e00c-4907-acc2-1f2a9b9f9a9f","owner":[],"postedDate":"February 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-24T16:01:59+00:00","versionOfRecord":{"articleIdentity":"rs-3917862","link":"https://doi.org/10.1007/s11250-025-04356-0","journal":{"identity":"tropical-animal-health-and-production","isVorOnly":false,"title":"Tropical Animal Health and Production"},"publishedOn":"2025-03-19 15:57:45","publishedOnDateReadable":"March 19th, 2025"},"versionCreatedAt":"2024-02-20 07:13:01","video":"","vorDoi":"10.1007/s11250-025-04356-0","vorDoiUrl":"https://doi.org/10.1007/s11250-025-04356-0","workflowStages":[]},"version":"v1","identity":"rs-3917862","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3917862","identity":"rs-3917862","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}