Genetic Diversity and Population Structure of Three Strains of Indigenous Tswana Chickens and Commercial Broiler Using Single Nucleotide Polymormophic (SNP) Markers

The Tswana chicken is native to Botswana and comprises strains such as the naked neck, normal, dwarf, frizzled, and rumples. The origins of the different strains of Tswana chicken remain unknown and it is not yet clear if the different strains represent distinct breeds within the large Tswana chicken population. Genetic characterization of different strains of Tswana chickens using SNP arrays can elucidate their genetic relationships and ascertain if the strains represent distinct breeds of Tswana chicken population. The aim of this study was therefore to investigate population structure and diversity and to estimate genetic distances/identity between the naked neck, normal and dwarf strains of Tswana chickens. A total of 96 chickens (normal strain (n = 39), naked neck strain (n = 32), dwarf strain (n = 13) and commercial broiler (n = 12)) were used in the study. SNP genotyping was carried out using the Illumina chicken iSelect SNP 60 Bead chip using the Infinium assay compatible with the Illumina HiScan SQ genotyping platform. The observed heterozygosity (H o ) values were 0.610 ± 0.012, 0.611 ± 0.014, 0.613 ± 0.0006 for normal, naked neck and dwarf strains of Tswana chickens respectively structure of indigenous Tswana chickens. The first two principal components revealed a set of three clusters. The normal strain of Tswana chicken and commercial broiler clustered together in one group. The dwarf strain clustered separately in one group and the naked neck and normal strains clustered together in the last group. The separate clustering of the dwarf strain from the rest of Tswana chicken strains suggests significant genetic uniqueness of the dwarf strain and very close genetic similarities between the normal and naked neck strains. The clustering pattern was confirmed by less genetic differentiation and less genetic distances between the naked neck and normal strains of Tswana chicken than between the two strains and the dwarf strain of Tswana chicken.


Introduction
Chickens have more distinct use and benefits to the household in different developing countries [1]. Indigenous Tswana chickens are one of the most important livestock species which provide most of the protein in the form of eggs and meat and improve the rural economy of subsistence farmers through sales of eggs as well as live birds. The chicken products (meat and eggs) are preferred by many people in rural areas due to their taste, leanness, palatability, and appropriateness for exceptional dishes [2] [3] [4]. Indigenous Tswana chickens contribute to food security in the rural areas and also generate emergency cash income for women since indigenous Tswana chickens are mostly owned by women. The Tswana chickens play a significant role in the sociocultural life of the rural population. Indigenous chickens also have roles in traditional ceremonies and other customs as gift payments [5]. Nonetheless, the growth rate of indigenous Tswana chickens is relatively low as compared to the commercial broiler due to poor nutritional support, poor housing, poor health care, and lack of selection for growth potential under the scavenging management system [6].
Generally, indigenous chickens are kept in small flocks (2 to 20 chickens) of varied ages under traditional scavenging management system with basic supplementary feeding, housing, and healthcare [6]. They possess important positive characteristics such as hardiness, the ability to tolerate the harsh environmental condition, and poor husbandry practices (climate, handling, watering, and feeding) without much loss in production [7]. Indigenous chickens grow slowly and normally require up to 12 months to reach slaughter age [8] and age at first lay is approximately 7 months [9]). [10] Desta reported a mating ratio of 1 cock to 2 hens for indigenous chickens' population in Ethiopia; but the recommended mating ratio is 1 cock to 5 -10 hens [9].
The dwarf, frizzled and rumpless strains are found at a relatively low frequency within the indigenous Tswana chicken population and the normal strain is by far the most common strain [13]. [

Collection of Blood Samples
Blood samples were collected from the medial metatarsal vein located on the leg of a chicken better suited for puncture using a 23-gauge, 1-in needle. The alternative site for blood collection was the brachial vein on the wings and for puncture, feathers in this area were plucked for smooth insertion of needle on the

DNA Extraction
24 µl of NucleoMag® B-Beads and 360 µl MB2 Buffer were then added to the square-well Block and mixed by pipetting up and down, shaking for 5 minutes at room temperature. Magnetic beads were then separated against the wells by placing the square-well block on the NucleoMag SEP magnetic separator for at least 2 minutes. The supernatant was then removed from the wells and discarded by pipetting. The square-well block was then removed from the NucleoMag SEP magnetic separator and 600 µl of MB3 buffer was added to each of the wells, accompanied by shaking to completely resuspend the beads. Magnetic beads were again separated against the wells by placing the square-well block on the Nuc-leoMag SEP magnetic separator for at least 2 minutes. The supernatant was again removed and discarded by pipetting. The square-well block was removed again from NucleoMag SEP magnetic separator. 600 µl of MB4 buffer was then added to each of the wells and the beads were resuspended by shaking for 5 minutes. Magnetic beads were again separated by placing the square-well block on the NucleoMag SEP magnetic separator for at least 2 minutes and supernatant was removed and discarded by pipetting. 900 µl of MB5 buffer was then added to each of the wells while the beads were still attracted to magnets. After an incubation period of 50 seconds, the supernatant was aspirated and discarded. The square-well block was then removed from the NucleoMag SEP magnetic separator. 50 µl of DNA elution buffer was then added to each of the wells and shaking for 10 minutes at 56˚C to resuspend the beads. Magnetic beads were again separated by placing the square-well block on the NucleoMag SEP magnetic separator for at least 2 minutes. The supernatant containing purified genomic DNA was then transferred to the elution plate for SNP genotyping.

SNP Genotyping and Data Preparation
SNP genotyping was carried out at Agricultural Research Council-Biotechnology Platform in Pretoria according to the protocols described by [

Population Structure
A complete SNP data set with all four populations was filtered to remove SNPs that were on sex chromosomes or had their positions unmapped. Markers with missing data > 5%; that had a MAF ≤ 2% or were monomorphic were removed from the complete data set. SNPs that were in high linkage disequilibrium at a threshold of LD ≥ 0.2 were also filtered out of the complete data set. Individuals with missing genotypes of more than 5% and those that were closely related, as inferred by a kinship estimator ≥ 0.45 were also excluded from the analysis.
A principal component analysis (PCA) was then performed to establish relationships among different strains of Tswana chickens and the commercial broiler line using the Golden helix SNP variation suit (SVS) version 8.1 [19]. Fur-Open Journal of Animal Sciences thermore, the Admixture 1.23 software [20] was used to estimate the most probable number of ancestral populations based on the SNP genotype data as described by [16] Khanyile et al. Admixture was run from K = 2 to K = 4 and the optimal number of clusters (K-value) was determined as that which had the lowest cross-validation error (CV error).

Population Differentiation and Genetic Distances
Pairwise identity by state (IBS) distances between all four chicken populations (naked neck, normal and dwarf strains of Tswana chicken and the commercial broiler) were calculated using PLINK v1.9. Genetic distances between the four populations were evaluated based on Nie's (1987) unbiased. Genetic distance uses the R-package [21]. To evaluate pair-wise genetic differentiation, the fixation index Fst [22] 4 was calculated for all pairs of chicken populations.

Linkage Disequilibrium
Complete SNP data for the individual populations were filtered to remove SNPs on sex chromosomes or those were not mapped, those with MAF ≤ 5%, those that deviated from Hardy-Weinberg equilibrium (HWE) (P ≤ 0.001) and individual chickens with missing genotypes (>5%) and those with very close kinship (IBD ≥ 0.45) using PLINK (v1.07) [18]. Pairwise r 2 estimation was used to measure LD between pairs of SNPs within a chromosome and population using PLINK (v1.07) program [18] 2007 for SNPs on chromosomes 1 -28 that had passed quality control tests detailed above. According to [23] Lu et al., the r 2 measure, defined as the squared correlation coefficient of alleles at two loci was chosen because it is independent of allele frequency. Briefly, its calculation, considers two loci, A and B, each locus having two alleles (denoted A1, A2; B1, B2, respectively) [24]. The frequencies of the haplotypes will then be denoted as F11, F12, F21, and F22 for haplotypes A1B1, A1B2 and A2B2, respectively and as FA1, FA2, FB1 and FB2 for A1, A2, B1 and B2 alleles, respectively. From this, r 2 according to [16] PLINK by default only reports r 2 -values above 0.2 and to allow reporting of all r 2 -values observed in the populations, the −r 2 -window-ld0 option was used. An additional option, −r 2 -window-snp 5000 kb 10,000 described by [16] Khanyile et al., allowed for estimation of r 2 for SNP marker pairs separated by at most 5000 Open Journal of Animal Sciences SNPs and within a 10 MB SNP interval.

Effective Population Size
The effective population size trends were estimated using the procedure described by [16] Khanyile et al. Briefly, the relationship between N e , recombination frequency, and expected LD (r 2 ) was determined using the equation from [25] Corbin et al. shown in Formula (2): where α = 1 when assuming no mutations and 2 if mutation was considered, 2 2 1 2 adj r r n = − , c was the recombination rate, and n was the chromosomal sample size. The effective population size N e , as 1/2c generations, was estimated from the adjusted 2 adj r values related to a given genetic distance d in Morgans, assuming c = d [24]. For each pair of SNPs on each chromosome, recombination rate was estimated by converting physical marker interval length x i (MB) to the corresponding genetic length c i using the formula: c i = ṓ i x i , where ṓ i is the average ratio of Morgans per kilo base pair on chromosome I, which was taken from physical lengths of the chicken genome v74 [26]. The genetic length of chromosomes was adopted from [27]. The r 2 -values range from 0 and 1, whereby a zero value indicates uncorrelated SNPs while a value of one reflects SNPs that are perfectly correlated [24]. The trends in effective population sizes for each of the defined subpopulations were then estimated by setting bins at 10, 20, 40, 60, 100, 200, 500, 1000, 2000 and 5000 kb. The bins were designed to cover the genome in tens, hundreds, thousands and hundred thousand base pairs.  [28] Al-Atiyat and Abudabos, who reported higher gene diversity in indigenous chickens of Jordan than in Ross broiler chickens (H e of 0.54 vs 0.09). Higher genetic diversity in indigenous Tswana chickens than commercial broiler chickens might be due to inherent traditional breeding practices of natural and random mating of indigenous chickens. Indigenous Tswana chickens are also not subjected to intensive selection in various traits of economic importance which tends to promote diversity than uniformity. Lower genetic diversity in commercial broiler compared to indigenous Tswana chickens might be due to artificial selection for traits of economic importance such as meat production [28].

Basic Population Genetic Parameters
In Intensive selection during development of commercial broiler chickens reduced diversity and increased uniformity partially as result of inbreeding.
The minor allele frequency (MAF) was also presented in Table 2

Population Structure
Principal component analysis (PCA) was used to get an insight into the population structure of indigenous Tswana chickens. The first two principal components

Admixture Analysis
The graphic results of the clustering analysis for K = 2 to 4 are illustrated in    The lowest cross-validation error was observed at K = 3, which represented the number of ancestors in indigenous Tswana chicken strains and the commercial broiler strain (Figure 3).

Population Differentiation (FST)
Pairwise population (F ST ) was calculated from filtered SNPs to investigate population differentiation among different strains of Tswana chickens. F ST values are shown in Table 3  Generally higher genetic differentiation and genetic distance between strains of

Linkage Disequilibrium (LD) Estimates and the Effect of Strain
A summary of r 2 values for the 28 chicken autosomal chromosomes in the three strains of Tswana chickens and commercial broiler chicken are shown in Table  4 Consistent with [16] Khanyile et al., the current study also indicates that evolutionary forces affecting LD act differently on different chromosomes and different strains. Commercial broiler chicken had higher LD compared to the three strains of indigenous Tswana chickens probably because of the effects of artificial selection for higher meat yield. On the other hand, natural selection could be a major evolutionary force in the three strains of Tswana raised under free-running management systems with minimal artificial selection [16]. There was no significant difference in LD between the normal and naked neck strains of Tswana chickens. However, the two strains of Tswana chickens had significantly lower LD than dwarf strain of Tswana chicken. Of the four chicken strains, the commercial broiler chicken had significantly higher LD compared to the three strains of Tswana chickens. Higher LD in commercial broiler compared to the three strains of Tswana chickens is consistent with [16] Khanyile et al. who found significantly higher LD in conservation flocks compared village chicken populations kept by small holder farmers. Differences in LD between commercial broiler and the three strains of Tswana chickens could be due to their different evolutionary histories under the influence of random genetic drift, selection, and mutations [16]. The dwarf strain of Tswana had higher LD across the 28 autosomal chromosomes compared to normal and naked neck strains of Tswana chickens. Higher LD in the dwarf strain compared to the naked neck and normal strains of Tswana

Trends in Effective Population Size (Ne)
Plots In comparison with the three strains of indigenous Tswana chickens, the commercial broiler chicken had higher N e values at all generations than the dwarf strain. The LD patterns are consistent with effective population size and diversity patterns in the commercial broiler and the three strains of Tswana chickens. Generally, higher LD patterns are associated with low effective population sizes and lower diversity in the populations.

Conclusion
The naked neck, normal and dwarf strains of Tswana chicken had similar, mod-Open Journal of Animal Sciences erate genetic diversity measures (observed and expected heterozygosity which was significantly higher than those of the modern commercial broiler chicken).