Mutational and Evolutionary Analyses of MC1R Gene in Exotic and Nigerian Indigenous Turkeys with Different Colored Feathers
Received 25 Mar, 2024 |
Accepted 15 May, 2024 |
Published 31 Dec, 2024 |
Background and Objective: Understanding the genetic basis for breed variations is an important step toward achieving breed improvement. Investigations were conducted on the MC1R gene mutations and evolutionary assessments in Nigerian local and exotic turkeys with various colored feathers. Materials and Methods: The experiment, which lasted for 16 weeks, included 60 poults in total, 15 of which were each of three genotypes of indigenous Nigerian turkeys (white, black and lavender in color) and exotic turkeys. Quick-DNA Mini-prep Plus extraction kits were used to extract genomic DNA and product guidelines were followed to the letter. Using NCBI primer BLAST, a set of primers was developed to amplify exon 1 of the MC1R gene in Turkey. Results: In both Nigerian local and exotic turkeys, nine SNPs were found in exon 1 of the MC1R gene. The major allele frequency was in the range of 0.57 to 0.86. Seven haplotypes in all were found, with five belonging to the exotic, three to the Nigerian local white, one to the Nigerian local black and two to the lavender genotypes. In all genotypes, Tajima's D values were negative and non-significant (p>0.05), however, Fu's Fs values were negative in exotic turkey. According to the phylogenetic study, Nigerian local black and lavender turkeys have a closer relationship, whereas Nigerian local white turkeys are more closely related to exotics. Conclusion: Nigerian indigenous turkeys can be classified into distinct genotypes according to their plumage colours using the SNPs and haplotypes found in exon 1 of the MC1R gene.
Copyright © 2024 Oguntade et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
INTRODUCTION
Plumage colour plays an important role in the evolution of birds and therefore is a useful genetic marker in breed identification and population characterizations1-2. The plumage color of birds is mainly related to the pigment distribution or proportion of eumelanin and pheomelanin3. Since multiple genes might influence the same feature, the precise genetic mechanism underlying the formation of a certain type of color is still unclear4. While some genes have a direct impact on color, others function as regulators and modifiers to influence the pigment’s regional and zonal distribution as well as its distribution inside individual feathers such as banding, spotting, edging and other patterns5.
The pigments responsible for variations in feather colouration in birds include flavins, melanins, porphyrins, psittacofulvins, pterins, purines and turacin6 and they are majorly expressed in feathers and naked parts such as bills and legs. One of the key genes that regulate the biosynthesis of melanins is melanocortin 1 receptor (MC1R) gene7. This gene is responsible for the synthesis of eumelanin and pheomelanin pigments8, which are the two main types of melanins. Eumelanin gives rise to dark black, brown, or grey colorations while pheomelanin gives rise to lighter yellowish to reddish colorations9.
The melanocortin 1 receptor (MC1R) is encoded by the solid black color locus in birds; mutations in this locus alter receptor activation and result in distinct variants of the E-locus10.
A broad evolutionary distribution of the MC1R gene is demonstrated by the presence of banded hairs and/or pale ventral coloring in a variety of mammals, including several monotremes and marsupials11. Long noted by Jackson et al.12, loss-of-function of MC1R produces yellow pigment (pheomelanin), while gain-of-function of MC1R appears to produce black pigment (eumelanin). In certain research projects by Feelay and Munyard13 and Feeley et al.14, 2 linked missense mutations (positions 82 A/G and 901 C/T) were identified in the coding region of the MC1R gene in animals lacking black pigmentation.
Nigeria indigenous turkeys have been described as being hardy, tolerant to most infectious diseases in the tropics, can survive on low nutrient feed resources and are best adapted to prevailing tropical climatic conditions15. They are phenotypically classified along plumage colours into pure black; white and lavender15 with high genetic variability. The exotic turkey on the other hand has been selected and improved for relatively higher body weight and genetic characterizations. The MC1R gene has been proposed to play a vital role in coat colour genesis in mammals but their roles remain ambiguous in exotic and Nigerian indigenous turkeys.
MATERIALS AND METHODS
Description of experimental site: This project work was conducted at the Poultry Unit of the Teaching and Research Farm of the Faculty of Agriculture, Ambrose Alli University, Ekpoma Edo State. The laboratory analysis was carried out at African Biosciences Laboratory, Off Iwo road, Iyana agbala Ibadan Oyo State. The experiment lasted for 16 weeks between April and August, 2023.
Experimental birds: A total of 60 poults comprising 15 each of exotic and the three genotypes of Nigerian indigenous turkeys (white, black and lavender plumage colour) were used for this study. The poults were sourced from a reputable farm in Ibadan, Oyo State Nigeria. The poults were raised on a deep litter pen and brooded for four weeks. They were allowed access to feed and clean water ad libitum and subjected to the same management practices throughout the experimental period of 16 weeks.
Blood sample collection: At the end of the experiment (16th week), about 1mL of blood samples were collected with a needle and syringe through the brachial vein of each turkey and transferred into an anti-coagulant bottle (ethylene diamine tetra acetic acid-EDTA) for genomic DNA extraction.
DNA extraction: The genomic DNA was extracted using whole blood collected with Quick-DNA Miniprep Plus extraction kits. The manufacturer’s procedures for extraction were strictly followed during the extraction process.
The primer BLAST incorporated in NCBI was used to design a pair of primers that could amplify only one exon (945 bps) of MC1R gene in turkey (Ensembl release 109 Feb, 2023 © EMBL-EBI) for polymerase chain reaction.
Amplification and sequence of amplified DNA: Polymerase Chain Reaction (PCR) was carried out in a micro centrifuge tube using programmable thermocycler (Mastercycler pro by Eppendorf) to amplify the extracted DNA. The amplification reaction was performed in a volume of 20 μL mixture containing 12.8 μL of H2O MQ, 2.5 μL of 1XPCR reaction buffer, 1 μL dNTP’s, 1.5 μL MgCl2, pH 9, 1 μL each of forward and revers primers and 0.2 μL of Taq DNA polymerase. The PCR cycling protocol includes an initial denaturation at 96°C for 15 min followed by 40 cycles of denaturing at 95°C for 30 sec, annealing at 58°C for 30 sec, extension at 70°C for 1 min and 40 sec and final extension at 70°C for 5 min. The reliability of the PCR products was checked using electrophoresis. A volume of 20 μL was used for the sequencing. This was prepared using an ABI 3730 XL Capillary DNA Analyzer (Applied Biosystems, Foster City, USA) with approximately 20 ng of purified PCR product as template DNA, 3.2 Mol of primer, 8 μL of Big Dye Terminator Ready Reaction Mix (a combination of dNTPs, ddNTPs, buffer, enzyme and MgCl2), 8 μL of deionized water, 2 μL of primer and 2 μL of template DNA. The reaction was set up to occur 25 times, with intervals of 10 sec at 960C, 5 sec at 500C and 4 min at 600C. There was a quick thermal ramp to 400C following the last cycle, which was maintained until the sequencing product was purified.
Sequence editing and analysis: Bioedit® software was used to view, manually clean and align the sequences. The edited sequence was blasted (BLASTN)16 against other sequences in the GenBank to determine the similarity and identity with other turkey sequences of MC1R gene in the database. The edited sequences were further aligned using CLUSTALW for further analyses.
Identification and analyses of single nucleotide polymorphisms: The SNPs present in exon 1 of MC1R gene in Nigerian indigenous turkeys were identified by aligning with the reference exon downloaded from Ensembl database using Clustal W17. The SNPs were also confirmed using DnaSP18.
Evolutionary analysis: The frequency of nucleotides present in exon 1 of MC1R gene in Nigerian indigenous turkeys was determined using MEGA 6 software. Haplotype diversity was generated using HyPhy program incorporated in a program DNASP 4.1.0.818.
Heterozygosity of the SNPs was calculated using the formula19:
Polymorphism information content (PIC) of the SNPs was calculated using the formula20:
where, p is the major allele frequency, q is the minor allele frequency and He is the heterozygosity.
The evolutionary history was carried out using the UPGMA method21 to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method22 and are in the units of the number of base substitutions per site. The analysis involved 7 nucleotide sequences including exon 1 of MC1R gene in exotic (hybrid converter), Nigerian local black, Nigerian local white and lavender turkeys and other avian breeds (Red jungle fowl (Accession Number: AB201628), duck (Accession Number: 051783) and pheasant (Accession Number: EF360992)). Evolutionary analyses were conducted in MEGA523.
RESULTS
Single nucleotide polymorphisms identified, major allele frequency, heterozygosity and polymorphism information content of SNPs identified in exon 1 of MC1R gene in exotic and Nigerian indigenous turkeys: The major allele frequency, heterozygosity and polymorphism information content of SNPs identified in exon 1of MC1R gene in exotic and Nigerian indigenous turkeys are presented in Table 1. The major allele frequency of polymorphisms identified in exon 1 of MC1R gene in exotic turkey ranged from 0.57 to 0.86 with mutation 312A>G having the least major allele frequency. Also, the major allele frequency of SNPs identified in 1 of MC1R genes in Nigerian local white turkey ranged from 0.57 to 0.86 with the least major allele found in mutation 580G The highest heterozygosity value of 0.49 was obtained in SNPs 312A Haplotypes present in exon 1 of MC1R gene in exotic and Nigerian indigenous turkeys: Haplotypes present in exon 1 of MC1R gene in exotic and Nigerian indigenous turkeys are shown in Table 2. Seven haplotypes were identified in exon 1 of MC1R gene in exotic and Nigerian indigenous turkeys. Haplotype 3 was shared by exotic and Nigerian local white genotypes while haplotype 6 was shared by the three Nigerian indigenous turkeys (local white, local black and lavender turkeys). Haplotypes 1, 2, 4 and 5 were specific to exotic turkeys while haplotype 7 was specific to Nigerian local white turkeys. Haplotype diversity of exon 1 of MC1R gene in exotic and Nigerian indigenous turkeys: Table 3 shows the haplotype distribution in exon 1 of MC1R gene in exotic and the three genotypes of Nigerian indigenous turkeys. Seven haplotypes were found, comprising five exotic, three local white Nigerian, one local black Nigerian and two lavender genotypes. The highest haplotype diversity (0.952) was recorded in exotic turkeys. Among the Nigerian indigenous turkeys, the white turkeys had the highest haplotype diversity (0.524) while the black and lavender turkeys had a similar value (0.286) and the least. Also, nucleotide diversity was highest (0.00370) in exotic turkeys, followed by Nigerian local white (0.00317), Nigerian local black (0.00340) and lavender (0.00238) genotypes. The average number of nucleotide differences ranged from 1.714 in Nigerian local black and lavender turkeys to 2.667 in exotic turkeys while the sequence conservation ranged from 0.989 in white turkeys to 0.992 in Nigerian local black and lavender turkeys. Test of deviation of exon 1 of MC1R gene from neutrality: The test of deviation of exon 1 from neutrality is shown in Table 4. Non-significant (p>0.10) and negative values of Tajima’s D were estimated for exon 1 of MC1R gene in all the turkey genotypes. Negative Fu’s Fs value was observed in exon 1 of MC1R gene in exotic turkeys while positive values were observed in Nigerian local turkeys. The Fu and Li’s D and Fu and Li’s F were not significant (p<0.10) and the values were negative across the genotypes. The Achaz Y* value was negative and not significant (p>0.10) in exotic turkeys while no value was obtained in Nigerian indigenous turkeys. Phylogenetic relationship between exon 1 of MC1R gene in exotic and Nigerian indigenous turkey: Figure 1 shows the phylogenetic relationship between exon 1 of MC1R gene in exotic and Nigerian indigenous turkeys and other avian breeds. Two clades were formed with duck found on a single clade. The second clade gives rise to two sub-clades in which red jungle fowl and pheasant formed sister taxa on the first sub-clade while the second sub-clade split to form Nigerian indigenous white and exotic turkeys as sister taxa and Nigerian local black and lavender turkeys as sister taxa on the same sub-clade. Nine SNPs were identified in exon 1 of MC1R gene in exotic and Nigerian indigenous turkeys. The three of the SNPs identified are common to all the genotypes and are parsimonious while SNP 446G This study observed the highest heterozygosity value of 0.49 in SNPs 312A The polymorphism information content (PIC) ranged from 0.21 to 0.37 for the SNPs identified across the turkey genotypes. Chen et al.28 reported a maximum PIC of 0.38 for single nucleotide polymorphisms which are bi-allelic and 0.37 PIC was obtained for mutations 312A>G in exotic turkey and 580G Presence of four singleton variable sites out of 721 base pairs was an indication of pathogen/transcription signal pressure effect on the region. A singleton is a rare variant for which genetic variation is carried by a unique chromosome in a sample. Thus, singleton SNPs would enhance fitness in animals, although this was not covered in this study. Therefore, the singleton together with parsimonious SNPs identified in exon 1 of MC1R gene in exotic and Nigerian indigenous turkeys would provide useful information through phylogenic trees, on the origin of plumage colour variations among these turkey populations. Negative Tajima’s D values observed in exon 1 of MC1R gene across all the genotypes show an excess of rare variation which is characterised with population growth31. This was an indication of population size expansion which can result from purifying selection, bottleneck, or selective sweep32. Negative Tajima’s D values may be a result of presence of an excess of rare alleles. Four of the SNPs identified in exon 1 of MC1R gene in all the turkey genotypes were singletons which are rare alleles. The high haplotype diversity and low nucleotide diversity observed in the current study show that the genetic diversity of the exotic and Nigerian turkey populations is low. It then implies that both exotic and Nigerian indigenous turkey populations have remained stable with an old evolutionary history33. Therefore, this suggests rapid expansion after a period of a population bottleneck33. Thus, shows a mark of historical and rapid demographic expansion of improved turkey genotypes from a small effective population size34. More so, high genetic diversity and low nucleotide diversity of the regions of MC1R gene studied in this experiment might be the result of rich ancestral genetic variation35. Since the haplotype and nucleotide diversities were identified majorly in the coding regions of MC1R gene in the four turkey genotypes and the coding regions are subject to natural selection than coding region36, it, therefore, suggests that most of the variations in feather colour among the studied turkey populations especially the Nigerian indigenous turkeys might be due to natural selection which might have resulted from genetic drift or inbreeding36. Several authors also observed high haplotype and low nucleotide diversities in coat colour genes in some livestock species. For instance, in goats, Marín et al.37 observed high haplotype and low nucleotide diversities in coding regions of MC1R gene and coding/noncoding regions of ASIP gene in goats, while Shen et al.38 reported similar results in MC1R gene in Hs chicken. Phylogenetic analysis revealed that Nigerian local black and lavender turkeys were related based on exon 1 of MC1R gene while Nigerian local white is closely related to exotic turkeys. This close relationship between Nigerian local black and lavender turkeys and Nigerian local white and exotic turkeys imply high comparability and evolution from a most common ancestor. This can be harnessed for selection for genetic improvement of Nigerian indigenous turkeys. This study concluded that exon 1 of MC1R gene of exotic and Nigerian indigenous turkeys is polymorphic and the SNPs and haplotypes identified are informative and could explain the genetic basis for plumage colour variations among these turkey populations. The results obtained can also serve as basis for genetic characterizations of exotic and Nigerian indigenous turkeys into different genetic groups. The knowledge provided can further be used for marker-assisted selection for genetic improvement of Nigerian indigenous turkeys. A great deal of phenotypic variation exists between Nigerian indigenous turkeys and their exotic counterparts. These variations manifest in differences in productivities between these two turkey populations. This study looked at the evolution of Nigerian indigenous and exotic by examining the diversity of MC1R gene in the two populations in order to provide the basis for genetic characterization and improvement of Nigerian indigenous turkeys. Interestingly, the analysis of MC1R gene in both genotypes showed that Nigerian indigenous turkey with white plumage colour was more closely related to exotic turkey than the other two coloured variants (black and lavender). This result is therefore a high-throughput for genetic improvement of Nigerian indigenous white turkey.
Table 1:
Major allele frequency, heterozygosity and polymorphism information content of SNPs identified in exon 1 of MC1R gene in exotic and Nigerian indigenous turkeys
Table 2:
Haplotypes present in exon 1 of MC1R gene in exotic and Nigerian indigenous turkeys
Genotype
Haplotype
Haplotype sequence
Exotic
Local white
Local black
Lavender
Total
1
GGGCGCAGA
1 (0.17%)
0 (0.00%)
0 (0.00%)
0 (0.00%)
1
2
AGGCGCAAA
1 (0.17%)
0 (0.00%)
0 (0.00%)
0 (0.00%)
1
3
GAACGCAAA
2 (33.33%)
3 (50.00%)
0 (0.00%)
0 (0.00%)
5
4
AGGCGTAAA
1 (0.17%)
0 (00.00%)
0 (0.00%)
0 (00.00%)
1
5
GGGCGCAAA
1 (0.17%)
0 (00.00%)
0 (0.00%)
0 (00.00%)
1
6
GAACACAAA
0 (00.00%)
2 (33.33%)
6 (100.00%)
6 (100.00%)
14
7
GAACGCTGA
0 (00.00%)
1 (0.17%)
0 (00.00%)
1 (16.67%)
1
Table 3:
Haplotype diversity of exon 1 of MC1R gene in exotic and Nigerian indigenous turkeys
Indices
Exotic
White
Black
Lavender
Number of haplotypes
5
3
1
2
Haplotypes diversity (Hd)
0.952
0.524
0.286
0.286
Nucleotide diversity (Pi)
0.0037
0.00317
0.0034
0.00238
Average number of nucleotide difference (K)
2.667
2.286
1.714
1.714
Sequence conservation
0.99
0.989
0.992
0.992
Table 4:
Test of deviation of exon 1 of MC1R gene from neutrality
Test
Exotic
White
Black
Lavender
Tajima’s D
-0.345
-1.576
-0.1524
-1.524
Fu’s Fs
-2.522
1.598
2.92
2.92
Fu and Li’s D
-0.298
-1.666
-1.6088
-1.6088
Fu and Li’s F
-0.311
-1.66
-1.60315
-1.60315
Achaz Y*
-0.287
Na
Na
Na
Fig. 1:
Phylogenetic relationship between exon 1 of MC1R gene in
exotic and Nigerian indigenous turkeys and other avian
genotypesDISCUSSION
CONCLUSION
SIGNIFICANCE STATEMENT
REFERENCES
How to Cite this paper?
APA-7 Style
Oguntade,
D.O., Orose,
E., Noseregbe,
R., Ilori,
B.M., Durosaro,
S.O., Obasoyo,
D. (2024). Mutational and Evolutionary Analyses of MC1R Gene in Exotic and Nigerian Indigenous Turkeys with Different Colored Feathers. Asian Journal of Biological Sciences, 17(4), 505-513. https://doi.org/10.3923/ajbs.2024.505.513
ACS Style
Oguntade,
D.O.; Orose,
E.; Noseregbe,
R.; Ilori,
B.M.; Durosaro,
S.O.; Obasoyo,
D. Mutational and Evolutionary Analyses of MC1R Gene in Exotic and Nigerian Indigenous Turkeys with Different Colored Feathers. Asian J. Biol. Sci 2024, 17, 505-513. https://doi.org/10.3923/ajbs.2024.505.513
AMA Style
Oguntade
DO, Orose
E, Noseregbe
R, Ilori
BM, Durosaro
SO, Obasoyo
D. Mutational and Evolutionary Analyses of MC1R Gene in Exotic and Nigerian Indigenous Turkeys with Different Colored Feathers. Asian Journal of Biological Sciences. 2024; 17(4): 505-513. https://doi.org/10.3923/ajbs.2024.505.513
Chicago/Turabian Style
Oguntade, David, Oluwafemi, Ekinadose Orose, Ruth Noseregbe, Babatunde Moses Ilori, Samuel Olutunde Durosaro, and Dick Obasoyo.
2024. "Mutational and Evolutionary Analyses of MC1R Gene in Exotic and Nigerian Indigenous Turkeys with Different Colored Feathers" Asian Journal of Biological Sciences 17, no. 4: 505-513. https://doi.org/10.3923/ajbs.2024.505.513
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