Single Nucleotide Polymorphisms in Insulin-like Growth Factor (Igf) Genes and Their Associations With Growth in Local Guinea Fowls (Numida Meleagris) of Ghana



Due to the pivotal role of Insulin-like Growth Factor 1 (IGF1) and Insulin-like Growth Factor 2 (IGF2) in growth regulation of poultry, the IGF1 gene (gIGF1) and IGF2 gene (gIGF2) in guinea fowl were examined as candidate genes for early growth in helmeted guinea fowls (Numida meleagris) from three populations of Northern Ghana (TPNG). Keets hatched from eggs collected from 32 sample locations comprising 11 subpopulations across three main populations located in Upper East Region, Upper West Region, Northern Region, North East Region, Savannah Region and an experimental flock maintained at Animal Research Institute (ARI) were raised and appraised for body weight and growth rate traits up to 11 weeks. Protein coding exons of gIGF1 and selected targets of gIGF2 were sequenced, aligned for discovery of novel Single Nucleotide Polymorphisms (SNPs) and genotyping. Effect of the genotypes at each SNP and gIGF1 haplogroups were estimated using linear models. Birds from the TPNG did not vary in body weights and weekly growth rates among the populations and with that of ARI flock from the fourth week (p > 0.05). However, birds from subpopulations within the three main populations varied significantly (p < 0.05) in weekly body weights and growth rates from the second week, with between subpopulation variations becoming pronounced after the sixth week. Although ARI flock did not vary with other three populations in terms of body weight and growth rate, they demonstrated remarkably high survivability. In total six novel SNPs were identified within gIGF1 including two SNPs within 5’ Untranslated Region (UTR), one SNP in the second protein coding exon and three SNPs within 3’UTR. These SNPs were distributed among seven haplotypes and eight haplogroups among local guinea fowls from Northern Ghana. SNPs within the 5’UTR and 3’UTR had significant effects on body


weights and weekly growth rates from the second and fourth week, respectively, indicating possible roles for these polymorphisms influencing IGF1 synthesis at the translation level, and need to be further investigated to decipher the underlying molecular mechanisms. The only synonymous SNP located at the second protein coding region in gIGF1 and both SNPs identified in gIGF2 did not influence early growth in local guinea fowls from TPNG. The study provides baseline information on novel SNPs in gIGF1 and their associations with body weight traits and early growth rates in local guinea fowls from Northern Ghana. It is recommended that the effect of the SNPs residing within 5’UTR and 3’UTR in gIGF1 should be further investigated up to slaughter stage in structured breeding programmes aimed at developing fast growing guinea fowl breeds with pedigree data to facilitate their use in Marker Assisted Selection.




1.1.   Background


Proteins are important biopolymers made up of amino acid monomers in all living organisms. By far, proteins are the most versatile biomolecules with diverse functions from cellular organization to modulating most complex biological functions such as human intelligence and immunity. At cellular level, they are important structural constituents of membranes and organelles. They also mediate intermediary metabolism by acting as receptors, enzymes and transporters. Highly specialized tissues in humans such as muscles, brain and skeleton are largely made up of proteins. At systemic level they play important roles as hormones and immunoglobulins, with no biological function possible without proteins (Buxbaum, 2015).

All proteins of the human body are produced from endogenous or dietary amino acids. A group of amino acids referred to as essential amino acids cannot be synthesized by the body and can only be supplied by the diet. Therefore, inadequate dietary protein, rich in essential amino acids, has negative effects on tissue development, cellular and physiological functions from conception to adulthood. Adequate but moderate level of proteins is also important in management of emerging non communicable conditions like diabetes and obesity. Therefore, a healthy human needs an optimum age dependent level of quality proteins for growth, maintenance, immunity and for optimal mental and physical functions (Nelson and Cox, 2005).

Food from animal sources are rich sources of essential amino acids and micronutrients with a higher bioavailability compared to plant based protein sources (Gibson, 1994). A diet deficient in animal source proteins, due to low income or due to spiritual believes in


affluent countries results in stunted growth, decreased cognitive development and decreased immunity during childhood (Dagnelie et al., 1991; Neumann et al., 2002). Although the overall protein requirement reduces from childhood to adulthood, a healthy adult needs an adequate quantity of quality proteins from diverse sources. In the recent past, association of high intake of red meat from beef, chevron, mutton or pork with greater risk of cardiovascular heart disease and colon cancer has been suggested and debated (McAfee et al., 2010). Epidemiological studies conducted across the globe suggest lean poultry meat as the healthiest meat options available (Marangoni et al., 2015).

According to dietary and physical activity guidelines prescribed by the Ministry of Health of Ghana, both men and women need an average of 0.8 gram of protein/kilogram of body weight while children, pregnant and lactating mothers have greater protein requirements. Therefore, the ranges for Recommended Dietary Allowances (RDAs) for protein are 16- 28, 45-63, and 46-50 g for children, adult males and adult females, respectively, while for pregnant and lactating mothers the RDAs are 60 g and 62 g respectively (MOH, 2010). Over the past decades, the country has gained significant improvements, reaching an average per capita protein consumption of 59g /person/day at national level (FAO-STAT, 2010).

Although the average national protein intake has demonstrated modest gains over the years, reaching the recommended levels, an extensive review of trends of per capita protein intake by regions, age groups and the quality of proteins, points out several disparities in quantities and quality of protein consumed by the Ghanaian populace. Aside an encouraging per capita protein intake on a quantitative scale, the quality of protein consumed by vast majority of Ghanaians deserves greater attention. About 75% of protein


in Ghanaian diet comes from starchy foods such as cereals, roots and plantains while intake of animal proteins which are rich sources of essential amino acids and micronutrients still remain deficient in the diet of vast majority of the populace (FAO-STAT, 2010). Therefore, at the national level more effort is needed to increase the proportion of animal protein by increasing accessibility and affordability to animal protein. Although the national daily average protein intake seems adequate for the whole population, nearly a quarter of school children are reported to be stunted (FAO, 2009). The prevalence rate of stunting was greater in rural areas (28%) against a prevalence rate of 13% in urban centers (GSS, 2007). Low levels of quality proteins in the diets in these children results in reduced cognitive development and increased susceptibility to diseases. Carbohydrate-rich diet with low quality protein increases mortalities and reduces growth during childhood. It also increases morbidity in adults by increasing susceptibility to infections, obesity and other non- communicable diseases (FAO, 2009). Therefore, improving quality protein intake seems still relevant in all age groups.

There are disparities in protein intake and distribution of malnutrition across the different regions of the country. According to FAO (2009), malnutrition characterized by stunting and wasting largely due to low level dietary protein intake is rated as “medium” at the national level, but higher in the former Northern Region (now North East, Savannah and Northern Regions) and Upper East Regions. Therefore strategic interventions are needed to improve protein intake particularly in Northern Ghana (FAO, 2009).

Interventions to address these disparities should focus on increasing accessibility and affordability of healthy animal protein to the larger population with particular attention to Northern Ghana (NG) and children.


1.2.   Justification


Ghanaians have derived dietary animal proteins from diverse sources over centuries. Presently fish ranks the most consumed animal protein, while poultry ranks the most preferred meat (FAO, 2009). However both of these products largely come from imported frozen fish and meat which are listed among the top ten imported commodities to Ghana (UN , 2017). In the year 2013 Ghana imported 170,600 metric tons of frozen poultry products (UN Comtrade, 2016) worth 200.4 Million US Dollars (UN, 2017). The prices of these are subject to frequent increases due to higher rate of inflation (Al-Hassan et al., 2014) making animal protein less affordable to populations particularly in the rural areas where poverty is more prevalent.

In this regard, the most appropriate intervention remains increasing and diversifying of domestic livestock and poultry production which currently account for only 8.9 % of Agricultural Gross Domestic Product (AGDP) and 1.1 % of total Gross Domestic Product (GSS, 2018). Greater preference of Ghanaian consumers for poultry informs all stakeholders to prioritize policy and technological interventions to revolutionize the poultry sub-sector in Ghana. Poultry include chicken, guinea fowl, duck and turkey with their significance varying based on the region.

The helmeted guinea fowl (Numida meleagris) is considered the most common poultry species in Northern Ghana (Agbolosu et al., 2012a), which is home to over 80% of domestic guinea fowl population (FAO, 2014). Helmeted guinea fowl is the most common type of fowl, within a group of Galliforms collectively known as “guinea fowls” (Frank


and Wright, 2006). Therefore the description “guinea fowls” refers to “helmeted guinea fowls (HLGF)” (Numida meleagris) throughout this thesis.

Guinea fowl production plays vital roles in the lives of guinea fowl farmers particularly in NG where protein related malnourishment is most prevalent. Almost every house hold in Northern Ghana keeps some guinea fowls (Dei and Karbo, 2004; Naazie et al., 2007) while some raise them in larger numbers as profitable agribusinesses. The meat and eggs from guinea fowls are important sources of animal protein and other essential nutrients in rural households in NG (Agbolosu et al., 2012b). However, in most cases, the primary purpose of rearing guinea fowls remains cash returns (Issaka and Yeboah, 2016). Rural farmers in the North recorded greater profits from guinea fowl rearing as compared to chicken (Avornyo et al., 2016). Issaka and Yeboah (2016) indicated that guinea fowl farmers in Tolon and Busila North districts in Upper East recorded average net returns from 532 to 1750 USD per annum with a cost:benefit ratio of 8.2. Revenue generated from sale of meat and eggs is used to meet important financial needs such as paying school fees, medical treatment or as direct investment for crop farming (Avornyo et al., 2016). Therefore, HLGF is not only an important protein source but plays a central role in the livelihood, wellbeing and socioeconomic growth of guinea fowl farmers and the general population in Northern Ghana.

Due to increasing preference for leaner meat, the demand is growing in the urban centers creating greater potential for commercialization than before (Issaka and Yeboah, 2016) with demand already exceeding supply (Karbo and Avornyo, 2006). Guinea fowl also has important socio-cultural importance as they are used in welcoming guests and payment of dowries (Teye and Gyawu, 2001; Issaka and Yeboah, 2016). Due to the aforementioned


nutritional, socioeconomic and cultural roles of local guinea fowls, the local guinea fowl varieties represent an important Animal Genetic Resource (AnGR) for the people of Northern Ghana.

The significance of local guinea fowl as an important AnGR goes beyond Northern Ghana for several emerging global trends. Due to the origin of HLGF in the Guinea Coast of Africa (Crowe, 1978), the guinea fowl populations found in NG are expected to represent most of their ancestral gene pool making them an important AnGR to the global guinea fowl industry which is growing (Nahashon et al., 2006). Having survived extreme weather conditions prevalent in the Guinea Savannah zone, their genomes are also expected to be rich in adaptive genes that will make them an important genetic resource in the face of emerging threats of climate change that threatens other livestock species. Guinea fowls also exhibit greater resistance to common poultry diseases beyond the keet stage (Sayila, 2009) and can be reared with minimal farm inputs (Avornyo et al., 2016). This also is becoming an advantage amidst growing demand for organic poultry meat produced with minimal chemical inputs.

Amidst the socioeconomic significance, emerging advantages as an AnGR and increased demand due to recent preference for leaner meat, there are several challenges for commercialization of guinea fowl production in Africa (Moreki and Radikara, 2013). Slow growth rate and limited baseline data to facilitate genetic improvement programmes have been identified as two of the key challenges for the guinea fowl industry in tropical Africa (Agbolosu et al., 2012b) although poultry farmers in African countries identified body size and faster growth among the most preferred traits to be improved in their breeds (Okeno et al., 2011).


The majority of guinea fowl farmers in NG (98%) depend on local varieties of guinea fowls (Avornyo et al., 2016) that have neither been characterized nor maintained as separate breeds. There is no evidence of dedicated breeding programmes to improve local guinea fowl breeds.

Selecting for faster growth has by far been the focus in majority of the poultry breeding programmes of the last century (ALBC, 2007). Poultry farmers desire breeds that grow faster to maximize their production capacity within a given period of time. Most long term breeding programmes that aimed at developing divergent lines for growth have used body weight at the eighth week as the selection criterion (Flisar et al., 2014). Like many other quantitative traits, growth in poultry is influenced by genetic factors, environmental factors and their interactions (Lawrence and Fowler, 2012). It is a complex biological function tightly regulated by multiple neuro-endocrine factors. At the heart of this regulation and the coordination of environmental and genetic factors is the somatotropic axis (Kim, 2010).

Somatotropic axis modulates its physiological functions by two key proteins that share varying levels of similarities with insulin in structure and functions namely, the Insulin- like Growth Factor 1 (IGF1) and Insulin-like Growth Factor 2 (IGF2) (McMurtry et al., 1998). IGF1 and IGF2 bind to cell surface receptors on target cells and initiate signal transduction mechanisms that ultimately result in cellular reactions that culminate in growth at cellular, tissue and organ levels. Due to direct involvement of IGF1 and IGF2 in signal transduction of growth regulation, the genes that code for them are considered as important candidate genes for growth and body weight traits in poultry (Amills et al., 2003; Nie et al., 2005; Xu et al., 2013). The polymorphic genotypes arising from these gene sequences have been linked to phenotypic variations of growth (Amills et al., 2003; Wang


et al., 2005) and body weight (Bian et al., 2008; Bhattacharya et al., 2015) making them ideal molecular markers for marker assisted selection schemes desiring genetic progress in growth.

Breed improvement can be achieved by breed replacement, cross-breeding with improved exotic strains or by improvement of local breeds through long term selection. Due to the relatively shorter time taken to achieve genetic progress and other factors, most African countries have largely opted for cross-breeding in past poultry breeding programmes (FAO, 2007a). Unfortunately, most of these programmes have failed due to low level of maintenance of genetic gain beyond a few generations, inbreeding and inability of offspring to adapt to conditions of tropical Africa and prevalent production systems (FAO, 2015). Therefore, the most sustainable option for breed improvement in African countries remain breed improvement through selection from indigenous breeds. Although African countries are endowed with rich AnGR, improving farm animals by selection programmes using indigenous AnGR is least considered largely due to the long time taken to achieve desired genetic progress.

Recent advances in the application of molecular genetic markers to complement phenotypic selection using Marker Assisted Selection (MAS) have the potential to significantly reduce the time taken and to increase selection intensity to achieve desired genetic progress (Eggen, 2012). However, application of MAS using markers such as Restriction Fragment length Polymorphisms (RFLPs) and Microsatellites remained low until the wide utilization of Single Nucleotide Polymorphisms (SNPs) (Stock and Reents, 2013). SNPs are polymorphisms arising from changes in DNA at single nucleotide positions (Vignal et al., 2002). Abundant genome-wide distribution, low cost and speed in


genotyping with high density genotyping platforms make SNPs ideal markers for Genomic Selection (GS) (Dekkers, 2012). Particularly with the reducing cost of genotyping, SNPs have the potential to be utilized in MAS in the developing world (FAO, 2007b). Although SNPs within functional regions such as exons, 5’ and 3’ untranslated regions (UTRs) are relatively rare compared to introns, their utility in MAS is high due to greater probability of association with phenotypes (Schmid et al., 2005).

Several workers have reported SNPs in IGF1 gene (Amills et al., 2003; Wang et al., 2004; Nie et al., 2005; Gouda and Essawy, 2010; Pandey et al., 2013; Bhattacharya et al., 2015; Ilori et al., 2016) and IGF2 (Amills et al., 2003; Nie et al., 2005; Wang et al., 2005; Tang et al., 2010; Yan et al., 2017) in chicken, the most studied avian model. Associations between these SNPs and growth rate have also been reported (Amills et al., 2003; Zhou et al., 2005; Pandey et al., 2013; Bhattacharya et al., 2015). However, there have been no previous reports on SNPs within IGF1 gene and IGF2 gene in guinea fowls. Recent release of Whole Genome Sequence of guinea fowl by Vignal et al. (2017) is a major milestone in realizing the potential of genomic selection in this species. Therefore, identification of SNPs associated with growth in local guinea fowls will make significant contributions to facilitate MAS. Identification of these SNPs will also facilitate representation of genotypes present in local birds in high density SNP panels in the future to make genomic selection useful for breed improvement programmes in Africa.

Therefore, this study seeks to identify SNPs within exonic regions of IGF1 and selected regions in IGF2 genes in guinea fowls and to determine associations between them and early growth and body weight traits in three indigenous guinea fowl populations originating from Northern Ghana (NG).


1.3.   Objectives


1.3.1.    Main Objective


The main objective of the study was to identify Single Nucleotide Polymorphic DNA markers within exonic regions of Insulin-like growth factor 1 gene (IGF1) and selected regions of Insulin-like growth factor 2 gene (IGF2) in local guinea fowls (Numida meleagris) from the three regions of Northern Ghana (i.e Upper-East, Upper-West and former Northern Region) and to determine their associations with early growth and body weight traits during the keet and grower stages.

1.3.2.    Specific objectives



The study was conducted with the following specific objectives


  1. To compare three main guinea fowl populations from Northern Ghana (Upper East Region, former Northern Region and Upper West Region) for early growth and body weight traits during the keet and grower
  2. To identify SNPs within exonic regions of guinea fowl IGF1 gene (gIGF1) among the three populations of local guinea fowls of Northern Ghana and to determine their distribution among the three
  3. To identify SNPs within guinea fowl IGF2 gene (gIGF2) among three local guinea fowl populations from Northern Ghana and to determine their distribution among the three
  4. To establish associations between the body weight and early growth traits with the identified SNPs within gIGF1 and gIGF2 in local guinea fowls from the three populations of Norther


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