DISCUSSION

Signatures of selection detection in both wild and domesticated species is considered a critical step toward understanding the molecular basis of adaptive evolution. This information can be used to identify genomic regions and underlying biological mechanisms affecting their essential traits. There are different methods of detecting selection signatures. However, two complementary approaches (composite likelihood ratio test (CLR) and integrated haplotype score (iHS)) that were found to have power > 70% to detect selection signatures were applied in this study. Unfortunately, there is currently no information on the effects of selection on the wild African harlequin quail, despite the incessant harvesting by rural smallholder farmers, effects of habitat destruction, and climate change, among other challenges. Moreover, there is limited information on selection signatures detected in the domestic Japanese quail, the most common domesticated quail species reared in Kenya. It has experienced constant evolutionary and genetic changes in its phenotype and genome architecture due to modern breeding methods. Such information could have been a foundation for understanding biological pathways governing important traits in wild quails and how modern breeding methods will likely affect them if breeding initiatives were to be made.
The application of CLR and iHS in detecting selective sweeps in wild African harlequin and domestic Japanese quail genomes was successful. Several complementary methods for signatures of selection detection allow for improved testing as each statistical test captures patterns in data differently (Fariello et al., 2013). The linkage disequilibrium (LD)-based iHS approach, which requires haplotypes per individual of one population to detect selection, was anticipated to be more reliable for this study due to the single populations that were sampled and the lack of a good reference population about quail (Voight et al., 2006). In addition, the CLR method was also adopted for this study as it uses allele frequency data to compare a neutral and a selective sweep model, and it is not highly sensitive to assumptions about the underlying recombination rate or recombination hotspots (Williamson et al., 2007). Furthermore, the CLR method can detect a beneficial mutation that spread in the entire population. In contrast, methods based on extended haplotype length and high linkage disequilibrium only detect the beneficial mutation that has yet to spread throughout the entire population (Voight et al., 2006; Wang et al., 2006; Williamson et al., 2007).
Both signatures of selection detection methods identified candidate genes associated with crucial biological, molecular, and cellular processes such as immune response, growth, reproduction, and morphological and behavioral traits. Some essential candidate genes involved in various immune response processes and pathways include MAPK1, MAPK13, MAPK14, CREB1, DYNLL2, RAC1, and ITGB3. The MAPK signaling pathway genes were enriched in the wild African harlequin and domestic Japanese quail. Mitogen-activated protein kinases (MAPKs) are a group of serine/threonine protein kinases that are highly conserved and have essential roles in cellular processes, such as proliferation, stress responses, apoptosis, and immune response regulation (Liu et al., 2007). The MAPK13 and MAPK14 (p38 MAPK pathway) genes, which play an important role in inflammatory responses, were positively selected in the wild African harlequin quail. The p38 pathway participates in the innate and adaptive immune response process by controlling the production of inflammatory cytokines (TNFα, interleukin (IL)-1, IL-10, and IL-12) by specialized dendritic and macrophage cells, CD40-induced gene expression, and proliferation of B cells, antigen processing in CD8+ conventional dendritic cells, and T cell homeostasis and function (Arthur & Ley, 2013; Soares-Silva et al., 2016; Han et al., 2020).
In addition to the p38 pathway, the extracellular signal-regulated kinase (ERK1/2) is also a MAPK pathway involved in positive selection and the differentiation of DP thymocytes to either CD4 or CD8 T cells (Fischer et al., 2005). The MAPK1, a key component of the ERK1/2 pathway, was positively selected in domestic Japanese quails. It was shown to participate in adaptive immune responses during bacterial infection in Nile tilapia (Wei et al., 2020). MAPK1, through the MAPK/ERK pathway, facilitates the activation and proliferation of immune cells, including T cells, B cells, and macrophages (Sun et al., 2015). Bacterial pathogens such as Salmonella , Shigella ,Yersinia and Escherichia species are known to manipulate pathways like the MAPK in their hosts to propagate infection (Krachler et al., 2011). Another positively selected candidate gene in domestic Japanese quails associated with Salmonella infection was the DYNLL2 gene, which is predicted to be involved in cytoskeletal motor activity and cytoskeletal protein binding. An observed increase in DYNLL2 protein in gamma delta T-lymphocytes (γδ T-lymphocytes) of chickens infected with Salmonella Enteritidis showed its role in regulating immune response (Sekelova et al., 2017). Like commercial chicken, domestic Japanese quails are reared on farms under different housing systems such as battery cages, free-range and floor-raised systems, among others. Choice of the housing system in connection to other production factors such as infrastructure, stocking density (farm and flock size), manure collection, disease status of the flock, and rodent and insect load does determine the risk of Salmonellainfection (Van Hoorebeke et al., 2011).
The ITGB3 gene, which was positively selected in the wild African harlequin quail, is involved in the blood coagulation pathway through adrenergic signaling in platelet activation. ITGB3 encodes for glycoprotein IIIa (GPIIIa) and, along with the alpha IIb chain, forms the platelet adhesive protein receptor complex glycoprotein IIb/IIIa (GP IIb/IIIa), which mediates platelet aggregation by acting as a receptor for fibrinogen (Cerhan et al., 2007). It plays a critical role in many cellular processes, such as cytoskeletal organization, cell adhesion, migration, proliferation, and survival (Ridley et al., 1992; Arthur et al., 2002; Guo et al., 2008). Its immune roles in the T cell development (Luo et al., 2013), B cell development and signaling (Walmsley et al., 2003), epidermal homeostasis resulting in wound healing (Winge & Marinkovich, 2019), and phagocytosis ( Lee et al., 2000; Han et al., 2019) are continuously being studied. Phagocytosis is a necessary component of the innate immune response. It plays an essential role in host-defense mechanisms by enabling the uptake and destruction of infectious pathogens through specialized cell types like macrophages, neutrophils, and monocytes (Lee et al., 2020). Similar to the ITGB3 gene, the RAC1 candidate gene that was detected in domestic Japanese quail is also associated with wound healing by regulating the innate immune response in keratinocytes (Bustelo et al., 2007; Pedersen et al., 2012; Winge & Marinkovich, 2019).
PPP1CA, WNT5A, GRIA1, CREB1 and ADCY8 candidate genes, associated with adaptation and behavior, were detected in the wild African harlequin quail. Melanogenesis and ear morphogenesis biological processes are crucial for wild African harlequin quail breeding behavior, social interactions, and survival. Melanogenesis involves melanin production, which plays a significant role in structural plumage in birds (Jeon et al., 2021). Plumage color and patterns are helpful in camouflage, mating, and differentiating between male and female wild African harlequin quails (Mason & Bowie, 2020). Melanin-based patterns are influenced by melanocyte migration, differentiation, cell death, and/or interaction with neighboring skin cells at the cellular level (Inaba & Chuong, 2020). The wild African harlequin quail songs and calls are also essential to their communication and behavior; hence, ear morphogenesis is a vital biological process for their survival and reproduction. The WNT5A gene, among other genes, was implicated in melanogenesis, ear morphogenesis, sex differentiation, and muscle development. Through activating multiple intracellular signaling cascades, the WNT gene family controls cell proliferation, differentiation, apoptosis, survival, migration, and polarity (Kikuchi et al., 2012). According to Kikuchi et al. (2012), regulating cellular functions, including migration and differentiation, makes the WNT5A gene a target for selection.
The GluA1 subunit has been implicated in the regulation of circadian rhythms and behavior (Ang et al., 2021). The Glutamate receptor 1 (GluA1), encoded by the GRIA1 gene, forms part of the AMPA receptor that mediates fast glutamate signaling in the central nervous system (synaptic plasticity). Positive selection of genes associated with behavioral response to stress and circadian entrainment (GRIA1, CREB1, ADCY8) could contribute to the observed timely seasonal migration and behavioral patterns of wild African harlequin quails (Bossu et al., 2022). The PPP1CA gene encodes for a protein that is part of the three catalytic subunits of protein phosphatase 1 (PP1). The PP1 protein was found to be a key regulator of period and light-induced resetting of the circadian clock in the common fruit fly (Fang et al., 2007) and different mammals (Eide et al., 2005; Gallego et al., 2006; Schmutz et al., 2011). It regulates the circadian period length, in counterbalance with casein kinase 1δ and ε (CK1δ/ε), through the regulation of the speed and rhythmicity of period circadian regulator 1 and 2 (PER1 and 2) phosphorylation (Lee et al., 2011; Schmutz et al., 2011).
Candidate genes associated with growth and reproduction were detected in the wild African harlequin and domestic Japanese quail. The Wnt–Hippo signaling pathway-related genes identified in wild African harlequin quails include SOX2, FZD7, WNT3, NKD1, and WNT4. The Hippo signaling pathway plays a crucial role in cellular differentiation, tissue, and organ development by controlling organ size through the regulation of cell proliferation and apoptosis (Justice et al., 1995; Xu et al., 1995; Heallen et al., 2011; Wu & Guan, 2021). It has also been implicated in other diverse roles, such as tissue homeostasis, wound healing and regeneration, immunity, tumorigenesis, and embryogenesis (Wu & Guan, 2021). The transcription factor SOX2 is involved in osteoblast differentiation (Seo et al., 2013), inner ear development (Kiernan et al., 2005), neurogenesis, and the proliferation and/or maintenance of stem cells (Oesterle et al., 2008), among others. The positive selection of genes associated with tissue and organ size, among other factors, could contribute to how the wild African harlequin quails have managed to maintain their small body size, supporting their growth and survival in the wild. The wild African harlequin quail is known to fly longer distances during migration and at faster speeds (Wamuyu et al., 2017).
CREB1 gene is involved in growth hormone synthesis, secretion, and action. CREB1 encodes a phosphorylation-dependent transcription factor that mediates the response to various cellular processes, including regulation of transcription, signal transduction, glucose homeostasis, and growth-factor-dependent cell survival, proliferation, and memory (Kinjo et al., 2005). CREB1 stimulates transcription upon binding to the DNA cAMP response element (CRE) located in the promoter region of target genes, leading to the recruitment of transcriptional coactivators and the initiation of gene transcription (Shankar et al., 2005). In humans, the CREB/CREB1 gene is involved in immune function (Cerhan et al., 2007) as it promotes proliferation and survival and differentially regulates Th1, Th2, and Th17 responses (Wen et al., 2010). It has also been shown to be a critical driver of vaccine efficacy in non-human primates (Tomalka et al., 2021).
Several candidate genes associated with lipid and cholesterol transport and metabolism in domestic Japanese quails, such as the APOA (APOA1 and APOA4) and ABCA (ABCA2, ABCA5, ABCA7) gene families, were identified (Albrecht & Viturro, 2007; Dominiczak & Caslake, 2011). Additionally, muscle cell differentiation, tissue, and structure development genes (COL6A3, SLC9A1, SMARCD3, MSX2, and PRF1) were identified. The positively selected DYNLL2 gene, apart from its role in immune response, was also found to be a regulator of chicken myogenesis, providing insights into breast muscle development in chickens and other birds (Li et al., 2022). VIPR2, one of the positively selected cAMP signaling pathway genes identified in domestic Japanese quails, has been implicated in the regulation of egg production as it has been associated with brooding behavior in chicken and geese (Luan et al., 2014; Huang et al., 2022).
In this study, only two genes (CBFB and RET) overlapped between the CLR and iHS tests in the domestic Japanese quail, in contrast to the wild African harlequin quail, where no overlapped genes were observed. CBFB protein is mainly associated with definitive hemopoiesis (Speck et al., 1999). In contrast, RET is known for its protein kinase activity, leading to the activation of signaling pathways involved in cell growth, differentiation, and survival, such as MAPK and AKT (Schuchardt et al., 1994; Taraviras et al., 1999).