Genetic Variation and Phylogenetic relationships of Commercial Pigs and Indigenous Pigs in Southwestern Nigeria

Abstract

This study examined genetic variations among indigenous and commercial pigs in Southwestern Nigeria. Sixty (60) individuals including Nigerian indigenous pigs (NIP) (n = 25), exotic (n = 25), and crosses between the exotic pigs (n = 10) were genotyped based on eight microsatellite markers. The mean number of alleles (Na) observed in the overall population of the three populations was 3.63 ± 0.36. Allele frequency ranged from 0.00 to 1.00 from the 39 alleles obtained within the population. Other allele frequency results obtained from the interpretation for the microsatellite loci across the three populations were polymorphic except for S0101 that was monomorphic. The mean number of effective allele (Ne) was 2.22 ± 0.17. The means obtained in this study for heterozygosities (Ho and He) were 0.529 and 0.501 respectively and the F-statistics showed the reduction in heterozygosity at various loci across the population studied i.e F_IS and F_IT. The mean values for F_IS and F_IT were − 0.06 ± 0.08 and − 0.02 ± 0.08, respectively. The mean level of gene flow (Nm) among the population was estimated to be 9.18 ± 3.59. The pairwise F_ST values among the three populations ranged from 0.02 to 0.04. The very low genetic differentiation between NIP and other populations reflects high gene flow. Molecular variance analysis revealed that there were 2% variance among the populations, 48% among the individual and 51% within the populations, with estimated variance of 0.04, 1.24 and 1.31 respectively. The phylogenetic tree separated the NIP from the remaining two populations while the exotic and the crosses clustered closely. This study revealed that genomes of pure indigenous pigs in Southwestern Nigeria are threatened by genetic erosion and recommend quick actions for sustainable conservation strategies.

Introduction

There are three common populations of pigs available in Southwestern Nigeria; these are the Nigerian indigenous pigs (NIP), exotic pigs and crossbred. The NIP is a small size animal that adapted well to the tropical condition of the region. It is a good genetic resource with good trait of disease resistance, heat stress, high parasitic load and good mothering ability. It has small body size and low liter size. The exotic pig was first imported into the region in the 1970s and have thrived thereafter. There are more than 90 breeds in the world but the most common ones in Southwest Nigeria are large white, Landrace. and Duroc. The microsatellite loci have been widely used to study genetic diversity and population structure of livestock among other types of molecular markers (Jordana et al., 2003; Yang et al., 2003; Parker et al., 2004; Martínez et al., 2006; Peter et al., 2007; Li et al., 2004; Kim et al., 2005; Behl et al., 2006; Solero et al., 2008). Chaiwatanasin et al. (2002) defined Microsatellites (MS) as simple tandem repeats that have been widely used for genetic relationship and variation in animals. It is very polymorphic and provides extremely useful markers for comparative studies of genetic variation, parentage assessment, traceability and studies of gene flow and hybridization.

This study aims to evaluate the genetic diversity and relationships of NIP in southwestern Nigeria and their genetic differentiation with commercial pigs using microsatellite markers". These results would serve a baseline information useful for the genetic improvement and conservation of the NIP.

Materials And Methods

Genotyping Of Microsatellite Markers

Table 1 showed the eight microsatellite markers used and their chromosome number and size ranges. Polymerase chain reaction (PCR) of 15µl volume each containing of 2 µl of genomic DNA, 8.25 µl of primermix, 1.5 µl of 10XPCR buffer (10mM Tris-HCl, 50 mM KCl, 1.5mM MgCl₂, pH 8.3), 1.2µl dNTPs (2.5 mM), 0.4µl Taq polymerase (10 unit/µl) was used. Multiplex PCR was done with initial denaturation at 95°C for 15 min; 5 cycles of 94°C for 1min; 55°C for 1min; 72°C for 1min, 10 cycles at 94°C for 1min; 54°C for 75min; 72°C for 1min and 24 cycles at 94°C for 1min; 53°C for 75min; 72°C for 1min and a final elongation at 65°C for 30min. The amplified products were electrophoresed in 1% low-melting point agarose gels (Promega) in 1X TAE and the resulting bands were viewed under transilluminator and genotyped using GeneMapper® Software Version 4.0 Microsatellite Analysis.

Microsatellite data analysis. Genotypic, allelic frequencies, and Hard-Weinberg equilibriums as well as Analysis of Molecular variance, were estimated using GenAlEX 6.41 software.

The Phylogenetic Tree Construction

The phylogenetic tree was constructed by downloading of Poptree2® software (http://www.med.kagawau.ac.jp/~genomelb/takezaki/poptree2/download.html#download) and the analysis was done by using the Poptree2 user guide (Poptree2, 2015).

Results And Discussions

The microsatelite markers (MSM), the chromosome position of each marker is shown in Table 1. Thirty nine alleles in total were obtained from the eight microsatellite markers used with a size range of 99 to 260 bp detected across the three breeds analyzed. All the eight MSM used in this study belong to the list of microsatellite markers recommended by FAO/ISAG (2011).

The genetic variation among population and individual of the total genetic variance were 2% and 48% respectively, while 51% of the genetic variation was attributed to within population genetic diversity as shown in Table 2.

The observed allele sizes and their frequencies for the microsatellite markers in each breed’s population in Fig. 1 shows the allele frequency ranged from 0.00 to 1.00 from the 39 alleles obtained within the population. Other allele frequency results obtained from the interpretation of Fig. 1 for the microsatellite loci across the three breeds were polymorphic with the exception of S0101 that was monomorphic.

The mean number of alleles (Na) observed in the overall population of the three populations is 3.63 ± 0.36 (Table 3). The mean of Na obtained in this study was higher than black Slovan pigs(ref) but lower than those obtained for Benin pig (8.94 ± 2.64) (Djimènou et al., 2021), Ghanaian Pigs (7.65) (Ayizanga et al., 2016), 4.20 for Philipine pigs (Jay Don Oh et al., 2014) and 14.59 for Thai pigs (Rangusun et al., 2019). In the subpopulation as shown in Table 4, P1 and P2 (Exotic and NP) have the same highest Na of 8, while P3 (Hybrid) had the lowest Na of 6. In Ghanian pig it was 5.48 for Berkshire and 10.6 for Papu indigenous ghanian pig (Ayizanga et al., 2016); 8.23 for Jeju black pig in Korea and 5.03 for Berkshire (Jay Don Oh et al., 2014). High Na has been reported to have high allelic diversity caused by crossbreeding or admixture among the population. The low total means of Na obtained in this study may be as a result of low allelic diversity of the markers used because some can be more polymorphic than others.

The mean number of effective allele (Ne) was 2.22 ± 0.17. SW122 marker had the highest Ne 3.43 ± 0.39, Sharon index (I) 1.44 ± 0.16, Observed heterozygocity (Ho) 0.85 ± 0.09, expected heterozygosity (He) 0.70 ± 0.03 and uHe 0.73 ± 0.02 as shown in Table 3, while the lowest was S01001 because it was monomorphic. The Ne ranged (1.00 to 3.43) obtained in this study was lower than the one obtained for Brazilian pig that ranged from 1.17 to 8.84 as reported by Sollero et al. (2008), Ghanian pigs ranged from 5.23 to 5.71 (Ayizanga et al., 2016); Thai Pig ranged from 2.62 to 7.15 (Rangusun et al., 2019).

The means obtained in this study for Ho and He were 0.529 and 0.501 respectively. These results were closer to values (0.51 and 0.53) obtained for Berkshire (Jay Don Ho et al., 2014a) and 0.54 and 0.54 (Jay Don Ho et al., 2014b). IThe values are higher than the Philippine pig values of 0.30 and 0.40 (Jay Don Ho et al., 2014b). Higher values were reported for Ghanian pig 0.467 and 0.711 (Ayizanga et al., 2016); 0.68 and 0.67 for Jeju pigs (Jay Don Ho et al., 2014); Thai pigs were 0.679 and 0.710 (Rangsun Charoensook et al., 2019); Portuguese pigs were 0.667 and 0.621 (Vincente et al., 2007); 0.576 and 0.697 for Iberian pigs (Fabuele et al., 2004); 0.534 and 0.696 for Mexican pigs (Chaiwatanasin et al., 2001).

The P1 had the highest Ne, I and He within the three populations, P3 had the highest Ho and uHe. The P2 had the least figures in all with the exception of F as shown in Table 5. The Ne is an important genetic variation parameter that determines alleic diversity among the breeds. It also determines the diversity of the MSM polymorphism. The Ho values were higher than He in 4 loci (SW026, S009, SW122, S0226) while He was higher than Ho in 3 (SW24, S0227 and SW632). It has been reported that He higher than Ho revealed the existence of population structure (Jyoshi et al., 2012). It has also been proved that He had been widely used as most parameters to measure the genetic diversity across and within the populations (Adeoye et al., 2021). Ho and He are one of the parameters used for selection of MSM for pig breed identification (Oh et al., 2014). Takezaki et al. (1996) and Oh et al. (2014) reported that markers can only be useful for measuring genetic variation, when they have an average heterozygosity between 0.3 and 0.8 in the population. Therefore, from this study Ho and He ranged from 0.30 to 0.85 and 0.32 to 0.70 respectively that made the markers good enough for diversity study. The Shannon information index (I) determines the genetic diversity in the populations. The low value range of I show low genetic diversity between the 3 populations.

Departure from Hardy-Weinberg equilibrium (HWE) was tested across the 3 populations within the loci studied. There was significant deviations from HWE observed in Locus S0226, S0227 (P < 0.05) and S0632 (P < 0.001) for P1; SW122, S0632 (P < 0.001) and S0126 (P < 0.05) locus for P2; while others were non-significant as shown in Table 6.

The F-statistics showed the reduction in heterozygosity at various loci across the population studied i.e Fis, Fit and Fst as shown in Table 7. The mean values for Fis and Fit were − 0.06 ± 0.08 and − 0.02 ± 0.08, respectively. The Fis values ranged from − 0.017 (S0026) to 0.119 (SW24) while the Fit ranged from − 0.034 (SW122) to 0.174 (SW24). The Fst value ranged from 0.01 at locus S0027 and 0.07 at locus SW227 as shown in Table 7. The mean Fst (0.04) can be translated to 4% and 96% for among/inter-population and within/intra-population variation respectively. The FST values up to 0.05 indicate negligible genetic variations, while values greater than 0.25 indicate large genetic differentiation among populations (Weir and Cockerham, 2014; Awobajo et al., 2022). This also show the low genetic variations among the populations studied. The mean level of gene flow (Nm) among the population was estimated to be 9.18 ± 3.59 (Table 7). The pairwise Fst values among the three populations were also ranged from 0.02 to 0.04 (Table 8).

Table 8 shows the pairwise F_st values among the 3 populations ranged from 0.02 to 0.04. These values almost corroborated with mean value of F_st (0.04) result obtained (Table 7).

Table 9 show the Pairwise Population Matrix of Nei Genetic Distance (below diag-onal) and Pairwise Population Matrix of Nei Genetic Identity (above diagonal) among the 3 pig populations. The genetic similar coefficients varied from 0.98 to 1.01 with an average of 0.99. The Genetic distances between the three pigs were 0.02 (Exotic and NIP), 0.01 (NIP and Crosses) and 0.00 (Exotic and crosses) respectively. This shows that the crosses were hybrid of the exotic pigs and not of the NIP.

A phylogenetic tree was estimated based on the equation of Nei et al. (1983), by the distribution of allele sharing by genetic distance (D) using POPTREE2 software. Figure 2 shows that the three pig populations originated from the same ancestor and branched into two where Exotic and crosses clustered together while the NIP clustered alone.

Figure 3 shows the outcome of the PCA conducted among the three pig populations in this study. The principal coordinate plot show clear clustering of Commercial pigs consisting of exotic, hybrids and Southwestern Nigerian indigenous pig. Percentage of variation explained by the first 3 axes were in 33.45, 47.04 and 59.23 percentages respectively. All the breeds dispersed well in all the coordinates as seen in Fig. 3.

Conclusion

This study is the first genetic characterization of indigenous pigs in Southwestern using Microsatellite markers. The study revealed enough genetic variation among the three populations. There was enough polymorphism among the eight microsatellites except for S01001 that was monomorphic. The low genetic differentiation between populations of NIP and crosses reflects the extent of genetic erosion of NIP genomes. The phylogenetic tree clustered the NIP from exotic and crosses. It is recommended to further investigate the diversity within the pure NIP itself and test the markers for traceability of coat color, pig products such as meat, milk etc to identify the individual breed. Limitation due to the sample size does not permit us to draw any further conclusions. Future studies require extensive samplings of indigenous from other regions of Nigeria to discern their distribution areas for conservation of pure NIP.

Declarations

Acknowledgement

“The laboratory aspects of this project was partly/fully supported by the BecA-ILRI Hub through the Africa Biosciences Challenge Fund (ABCF) program and Africa women in Agriculture and development (AWARD). The ABCF Program was funded by the Australian Department for Foreign Affairs and Trade (DFAT) through the BecA-CSIRO partnership; the Syngenta Foundation for Sustainable Agriculture (SFSA); the Bill & Melinda Gates Foundation (BMGF); the UK Department for International Development (DFID) and; the Swedish International Development Cooperation Agency (Sida).”

Authors Contributions

Oluwole Olufunke Oluwakemi conceived the project, performed the analysis, analysed the data and wrote the paper . Okoth Edward gave conceptual support during the period of experimentation. Ogugo Moses partook in laboratory analysis and technical support. Adeola Charles A. reviewed and appraised the manuscript. 

Funding

“The authors declare that no funds, grants or other support were received during the preparation of the manuscript”.

Competing Interest

The authors declare no competing interests.

Data Availability

“The data can be provided upon request.

Ethics Approval

The study did not require ethics approval

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