GENETIC DIVERSITY AND EXPRESSION PROFILE OF TLR7 GENE IN THE NIGERIAN INDIGENOUS CHICKENS AND ISA BROWN COMMERCIAL LAYER CHICKEN

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ABSTRACT


This study was designed to assess genetic diversity of TLR7 gene and its expression profile in the Nigerian indigenous chickens and ISA Brown layer chicken. The study population comprises eight (8) groups (naked neck, normal and frizzled-feathered Nigerian indigenous chickens in rain forest and Guinea Savannah regions respectively, Fulani ecotype chicken and ISA Brown layer chicken). Five (5) blood samples were collected from each chicken group. Genomic DNA was isolated from each blood sample using the Zymo Quick-gDNATM Miniprep kit. The DNA sequencing of chTLR7 gene was done using the Sanger Sequencing Chemistry. Tissues from the thymus and the liver were aseptically collected from Two (2) clinically healthy chickens from each chicken group and were immediately transferred into separate 1.5 ml Eppendorf tubes containing 1 ml of RNALater solution. Total RNA was isolated using ISOLATE II RNA Mini kit. Complementary DNA (cDNA) was synthesized using SensiFASTTM cDNA synthesis kit. The expression of chTLR7 RNA was determined by qPCR assay; β-actin was used as the reference gene. 26 SNPs, two deletions and two insertions in the intronic region of TLR7 gene in the Nigerian indigenous chicken population and ISA Brown commercial layer chicken were found. Haplotype analysis revealed 13 haplotypes out of which nine (9) were unique to the Nigerian indigenous chickens; three (3) haplotypes were shared between ISA Brown layer chicken and the Nigerian indigenous chickens, while one (1) haplotype was unique to the Red jungle fowl. Nucleotide diversity estimates ranged from 0 to 0.019, which were close to zero and suggest that the chicken populations were not genetically differentiated at TLR7 locus. Estimates of gene flow ranged from -0.096 to 0.400 and were close to zero. Genetic distance estimates ranged from 0.007 to 0.054 and were close to zero, which suggests that the chickens have a close ancestor. The estimates of nearest-neighbour statistic ranged from 0.227 to 0.714, which showed that the chicken populations were part of the same panmictic population, hence were not genetically differentiated (P>0.05) at the TLR7 locus. Phylogenetic analysis of TLR7 gene sequences of the genetic groups and the Red jungle fowl revealed very close relationship at the TLR7 locus, which suggests that the TLR7 locus is highly conserved. TLR7 expression in the liver and thymus was significantly different (P<0.01) among the eight chicken groups; the Nigerian indigenous chickens expressed more TLR7 gene than ISA Brown layer chicken. Rain forest naked neck chicken had significantly (P<0.01) highest TLR7 expression of 2.07±0.07 fold. However, expression of TLR7 gene in the liver of rain forest frizzle-feathered and normal chicken, Guinea savannah naked neck, frizzle-feathered and normal chicken, and Fulani ecotype chicken were similar (P>0.05). Gene expression analysis of TLR7 RNA suggests that the Nigerian indigenous chickens could have comparatively more antiviral immune response than ISA Brown commercial layer chicken, hence could be used to develop chickens lines with good antiviral response. Polymorphisms observed at TLR7 gene in the Nigerian indigenous chickens could be used in marker-assisted selection to produce chicken lines with good antiviral response.








TABLE OF CONTENTS


Cover Page                                                                                                             i

Declaration                                                                                                 ii

Certification                                                                                              iii

Dedication                                                                                                 iv

Acknowledgement                                                                                      v

Table of Contents                                                                                                     vi

List of Tables                                                                                                         x

List of figures                                                                                                        xi

List of Plates                                                                                                        xii

Abstract                                                                                                   xiii


CHAPTER 1: INTRODUCTION                                                                                    1

1.1       Background Information on the Nigerian Indigenous Chickens                                                                                                     1

1.2       Brief Background on the ISA Brown Commercial Layer Chicken                                                                                                       4

1.3       Toll-like Receptor Seven (TLR7) Gene   5

1.4       Statement of Problem                                                                                                      7

1.5       Objectives of the Study   8

1.6             Justification                                                                                    8


CHAPTER 2: LITERATURE REVIEW                                                                                                  10

2.1       Origin of the Domestic Chicken                                                                                                     10

2.2       Genetic Diversity of the Domestic Chicken                                                                                                     10

2.3       Assessment of Genetic Diversity within and Between Chicken

            Populations                                                                                   11

2.4       Genetic Markers for Assessing Genetic Diversity                                                                                                   12

2.4.1    Random amplified polymorphic DNA (RAPD) markers                                                                                                     13

2.4.2    Restricted fragment-length polymorphism (RFLP) markers                                                                                                     14

2.4.3    Amplified fragment length polymorphism (AFLP)                                                                                                      15

2.4.4             Microsatellites                                                                              15

2.4.5    Mitochondrial DNA D-loop     16

2.4.6    Single nucleotide polymorphism (SNP)  16

2.4.6.1 Single nucleotide polymorphisms (SNPs) analyses in different chicken populations                                                                     18

2.5       Ecology and Genetic Profile of the Nigerian Indigenous Chicken

            Populations                                                                                   21

2.6       Phenotypic Characteristics of the Nigerian Indigenous Chickens                                                                                                   22

2.7       Genetic Analysis of the Nigerian Indigenous Chickens Using Physiological and Biochemical Markers                                                                                                         30

2.8       Molecular (DNA-based) Analyses of the Nigerian Indigenous Chickens                                                                                                   35

2.8.1    Molecular genetic analyses of the Nigerian indigenous chickens using microsatellite markers                                                                           36

2.8.2    Molecular genetic analyses of the Nigerian indigenous chickens at mitochondrial DNA D-loop region                                                                              37

2.8.3    Molecular genetics analyses of the Nigerian indigenous chickens using single nucleotide polymorphism (SNP) markers                                                                           38

2.9       Toll-like Receptors (TLRS)                                                                                                      39

2.9.1    The chicken toll-like receptor repertoire                                                                                                  40

2.9.2    Molecular structure of the chicken TLRs   41

2.9.3    The chicken toll-like receptor genes and their roles in pathogen

            recognition                                                                                    42

2.9.4    Molecular variants of TLR genes in avian species 44

2.9.5    The chicken toll-like receptor 7 (chTLR7) gene and its expression profile 47

2.9.6    Polymorphisms of chTLR7, and its association with viral diseases                                                                                                     50


CHAPTER 3: MATERIALS AND METHODS                                                                                              54

3.1       Experimental Populations                                                                                               54

3.2       Blood Sample Collection                                                                                                 55

3.3       Laboratory Analysis                                                                                                    55

3.4       Experiment 1: Analysis of Genetic Diversity at TLR7 Gene in the Nigerian Indigenous Chicken Populations and ISA Brown Commercial Layer Chicken Using DNA Sequencing                                                                       55

3.4.1    DNA extraction and protocol                                                                                                     55

3.4.2    DNA quantification and integrity                                                                                                    56

3.4.3    Polymerase chain reaction (PCR) and primers                                                                                                      56

3.4.4    Preparation of agarose gel       57

3.4.5    Electrophoresis of PCR products                                                                                                    57

3.4.6    Visualization of PCR products (amplicons)                                                                                               58

3.4.7    Cleaning of amplicons                                                                                                 58

3.4.8    Agarose gel electrophoresis of cleaned amplicons                                                                                                 59

3.4.9    Sequencing of PCR products                                                                                                    59

3.4.10 Alignment and editing of sequences                                                                                                  59

3.4.11 Single nucleotide polymorphism (SNP) identification and estimation of genetic diversity indices                                                                                59

3.4.12 Genetic distance estimation                                                                                                 60

3.4.13 Phylogenetic analysis                                                                                                     60

3.5       Experiment 2: Assessment of Evolutionary Relationship of TLR7 Gene in the Nigerian Indigenous Chickens, ISA Brown Layer Chicken and TLR7 Gene Sequences from Other Poultry Species in Genebank                                                                           60

3.5.1    Retrieval of DNA sequences from NCBI database                                                                                                    60

3.5.2    Multiple sequence alignment                                                                                                  61

3.5.3    Genetic distance estimation                                                                                                 61

3.5.4    Phylogenetic analysis                                                                                                     61

3.6       Experiment 3: Gene Expression Profile of TLR7 in Lymphoid Tissues of the Nigerian Indigenous Chickens and ISA Brown Commercial Layer Chicken                                                                              61

3.6.1    Experimental birds and management                                                                                             61

3.6.2    Tissue collection                                                                                                  62

3.6.3    RNA extraction                                                                                                  62

3.6.4    RNA quantification and integrity                                                                                                    63

3.6.5    Complementary DNA (cDNA) synthesis                                                                                                   64

3.6.6    Real-Time Polymerase Chain Reaction (qPCR)                                                                                                      64

3.6.7    Assembling of qPCR data and statistical analysis                                                                                                     65

 

CHAPTER 4: RESULTS AND DISCUSSION                                                                                          66

4.1       PCR Optimization of chTLR7 Gene   66

4.2       Genetic Diversity at TLR7 Gene of Nigerian Indigenous Chickens

            and ISA Brown Commercial Layer Chicken                                                                                                     66

4.2.1    Single nucleotide polymorphisms (SNPs) and INDELs of TLR7 gene in the Nigerian indigenous chickens and ISA brown commercial layer chickens                                                                             66

4.2.2    TLR7 haplotype variations, nucleotide diversity, genetic differentiation and nearest-neighbour statistic                                                                                                            75

4.2.3    Genetic distance and relationship of TLR7 gene sequences in Nigerian indigenous chickens and ISA brown layer chicken                                                                              83

4.2.4    Phylogenetic analysis of TLR7 gene in Nigerian indigenous chicken populations and ISA brown commercial layer chicken                                                                              88

4.3       Evolutionary Relationship of TLR7 Gene in Nigerian Indigenous Chickens, ISA Brown Commercial Layer Chicken and TLR7 Gene Sequence from Other Poultry Species in Genebank                                                                           90

4.4       Expression Profile of TLR7 RNA in Lymphoid Tissues of the Nigerian Indigenous Chickens and ISA Brown Commercial Layer Chicken                                                                              94

CHAPTER 5: CONCLUSION AND RECOMMENDATIONS                                                                       98

5.1             Conclusion                                                                                    98

5.2             Recommendations                                                                        99

References                                                                                             100

Appendices                                                                                                                117

 

 

 

 

 


LIST OF TABLES

                                                                                                                                    Page

2.1       Chicken TLR genes, their locations and length                                             42

2.2       Polymorphism statistics at ten TLR genes in Lesser Kestrels                     47

2.3       Polymorphism statistics at ten TLR genes in House Finches                         48

2.4       Expression pattern of chTLR7 mRNA in different tissues                            51

3.1       Chicken populations                                                                                       54

3.2       Primers and sequences for amplification of TLR7 gene                                57

3.3       Primers information for qPCR of TLR7 RNA                                               65

4.1       SNPs and INDELs of chTLR7 in the Nigerian indigenous chickens and

ISA Brown commercial layer chickens                                                          72

4.2       TLR7 gene haplotypes and their sequences                                                   78

4.3       TLR7 gene haplotype distribution among the eight chicken genetic groups

and the red jungle fowl                                                                                   79

4.4       Haplotype and nucleotide diversity of the chTLR7 gene in the Nigerian

indigenous chickens and ISA Brown commercial layer chicken                        80

4.5       Estimates of gene flow between the chicken populations                              81

4.6       Nearest-neighbour statistic, Snn between populations of Nigerian

indigenous chickens and ISA Brown commercial layer chicken                        82

4.7       Estimates of mean genetic distance within Nigerian indigenous chicken

Populations and ISA Brown commercial layer chicken                                85

4.8       Estimates of mean genetic distance between Nigerian indigenous chicken populations, ISA Brown commercial layer chicken                                     86

4.9       Estimates of mean genetic distance between the chicken genetic groups 87

4.10     Percent identity of chTLR7 gene sequence retrieved from NCBI database

and consensus sequence of Nigerian indigenous chickens and ISA Brown

layer                                                                                                                91

4.11     Genetic distance between the Nigerian indigenous chickens, ISA Brown

commercial layer chicken and the red jungle fowl (reference sequence)        92







 

LIST OF FIGURES

                                                                                                                                    Page

4.1       Phylogenetic (Neighbour-joining) tree constructed from TLR7 gene

sequences from the Nigerian indigenous chickens and ISA Brown

commercial layer chicken                                                                              89

4.2       Phylogenetic (Neighbour-joining) tree constructed from TLR7 gene

sequences of the Nigerian indigenous chickens, ISA Brown commercial

layer chickens, and TLR7 sequence belonging to the red jungle fowl     93

4.3       Expression profile of TLR7 RNA in liver tissues                                          96

4.4       Expression profile of TLR7 RNA in thymus tissues                                      97

 

 

 

 

 

 

LIST OF PLATES

                                                                                                                                    Page

1          PCR optimization of TLR7 gene in Nigerian indigenous chickens                        70

2          PCR optimization of TLR7 gene in ISA Brown commercial layer chicken           71

 

 

 

 


 

CHAPTER 1

INTRODUCTION

1.1 BACKGROUND INFORMATION ON THE NIGERIAN INDIGENOUS CHICKENS

Indigenous chickens are found in Nigeria and many parts of the world. They are kept and managed mostly by resource poor farmers in rural communities as excellent source of protein (egg and meat) and income. Their meat and eggs are preferred over that of exotic chickens (Dessie and Ogle, 2001). The Nigerian indigenous chickens constitute about 80 per cent of the 120 million poultry found in Nigeria (Fayeye et al., 2005). Almost every family in rural communities cheaply rears the Nigerian indigenous chickens under extensive system of management across the country. They are left to feed on household leftovers and often move about as scavenging birds. In some places, they are provided with little or no shelter at night. Despite their large number and economic importance as excellent source of protein and income to poor rural farmers, the Nigerian indigenous chickens have not been fully exploited for the purpose of genetic improvement.

Nigerian indigenous chickens are characterized by poor growth and small body size, which are not desirable in a competitive economic situation (Ibe, 1990; Ebang and Ibe, 1994). The indigenous chickens of Nigeria lay small sized eggs, and their egg production is dependent on the management system (Sonaiya and Olori, 1990; Ibe, 1990). Their exposure to extreme or harsh weather conditions, diseases and parasites partly account for their poor performance as evidenced by their small mature body weight and small egg size. However, the performance of Nigerian indigenous chickens can be enhanced by improving them genetically using various schemes of selection and improving their production environment. The Nigerian indigenous fowls possess great potentials for genetic improvement through breeding programmes (Ikeobi et al., 1996).

Nigerian indigenous chickens constitute an indispensable Animal Genetic Resource (AnGR) to the country. The uncontrolled distribution of exotic chickens in different parts of Nigeria, coupled with uncontrolled crossbreeding of exotic chickens with Nigerian indigenous chickens led to the dilution of the indigenous chicken genetic stock. If it is not controlled, the Nigerian indigenous chicken gene pool could be lost in the future before they are fully characterized genetically. This is supported with the FAO report (FAO, 1999) that animal genetic resources (AnGR) in developing countries are being eroded.

The Nigerian indigenous chickens are widely distributed in different geographical/ecological zones as classified by vegetation types such as rainforest zones of South-east, South-west and South-south; Savannah zones of North-central, North-east and North-western Nigeria. The Nigerian indigenous chickens found in each of the geographical zones are believed to constitute different genetic populations with limited inter-population gene flow, which could be attributed to long distances separating those (Ukwu et al., 2017b). These indigenous chickens may have evolved pronounced adaptabilities in response to combined influence of locally prevailing environmental conditions, uncontrolled breeding as well as forces of natural selection, mutation and random genetic drift. It is based on this background that some researchers have come up with phenotypic classification of Nigerian indigenous chickens ecologically into different ecotypes, for example Fulani ecotype, Yoruba ecotype, heavy and light ecotypes, Tiv ecotypes etc. Categorically, Sonaiya and Olori (1990) characterized Nigerian indigenous chickens based on ecotypes. Momoh et al. (2010) grouped Nigerian local chickens based on body size and body weight into heavy ecotype (HE) and light ecotype (LE). The heavy ecotype represents indigenous chickens from dry savannah (Guinea and Sahel savannah), Montane region and cattle kraals of the north with mature body weights range of 0.9 – 2.5kg (Momoh et al., 2010). The light ecotype represents indigenous chickens found in the swamp, rainforest and derived savannah zones with mature body weights of between 0.68 and 1.50kg.

However, characterization of Nigerian indigenous chickens based on their mean phenotypic values provides only a crude estimate of the average effects of the functional variants of genes possessed by the local chicken genetic resource. A better approach to characterization of Nigerian local chicken populations is the use of more informative methods of genetic analysis that employ DNA-based markers in high throughput genetic analysis of chicken populations. This is necessary in order to precisely delineate the Nigerian indigenous chicken genetic resource. Such techniques involving the use of microsatellites, single nucleotide polymorphisms (SNPs) and other DNA markers are more reliable and more informative than phenotypic measurements in genetic diversity studies. There is, therefore, need to perform further characterization of the Nigerian indigenous chicken genetic resource for information on genetic diversity, relatedness and phylogeny of the indigenous chicken populations as well as exotic broiler and layer chickens available in commercial farms in Nigeria using molecular genetics approach. Our central hypothesis is that genetic analysis of Nigerian indigenous chicken populations using molecular genetics techniques could uncover any DNA based polymorphism existing among the indigenous chicken populations across different regions in Nigeria. Polymorphisms at the level of the DNA can be markers of choice for molecular characterization of local chickens. If such polymorphisms at the level of the gene or DNA are uncovered, they can be exploited in marker-assisted selection (MAS) and gene-assisted selection (GAS) for the purpose of genetic improvement of Nigerian indigenous chickens. Genetic characterization is also useful to help conserve the valuable genetic variants inherent in the local chicken genetic resource.

Some researchers have attempted genetic diversity study of Nigerian indigenous chickens using molecular genetics approach. For example, Adeleke et al. (2011) carried out a preliminary screening of genetic lineage of Nigerian indigenous chickens based on blood protein polymorphisms. Ohwojakpor et al. (2012) have also carried out the use of microsatellite markers in genetic diversity study of Nigerian local chickens. Although randomly amplified polymorphic DNA (RAPD) markers are obsolete in genetic diversity studies, they had been used to investigate genetic lineage and relatedness of indigenous chickens in different countries (Rahimi et al., 2005; Olowofeso et al., 2006; Mollah et al., 2009; Choy and Kumaran, 2011).


1.2 BRIEF BACKGROUND ON THE ISA BROWN LAYER CHICKEN

The ISA Brown chicken is a hybrid type of sex-link chicken. The ISA Brown layer chicken Parent Stock (PS) is produced and globally distributed by HENDRIX Genetics, a multi-species Animal Breeding, Genetics and Technology Company.  The ISA Brown chicken is one among several commercial layer chickens distributed across Nigeria by Ajanla (CHI) Farms limited Ibadan, Oyo State, with exclusive franchise for HENDRIX Genetics (Europe).  This brown egg layer chicken has proven thirty-five (35) years of excellent performance as the best brown layer in the world and is capable of laying 420 high quality eggs for laying period between 18 to 90 weeks (ISA, 2019). This strain of layer chicken is a reliable and versatile layer with excellent feed conversion, which adapts to different climates, management systems and housing systems (ISA, 2019). The ISA Brown layer chicken is among different commercial chicken strains usually preferred by farmers due to their high egg production and their innate characteristics (Islam et al., 2015). However, the breeding programme from which ISA Brown layer chicken was developed is usually a guarded secret by the Breeding Company that developed it in order to avoid competition.


1.3 TOLL-LIKE RECEPTOR SEVEN (TLR7) GENE

One of the mechanisms by which the innate immune system of farm animals and humans sense the invasion of pathogenic micro-organisms is through the toll-like receptors (TLRs). Thus TLRs recognize specific molecular patterns known as pathogen associated molecular patterns (PAMPs) that are present in microbial components (Akira and Takeda, 2004). TLRs are membrane-bound pattern-recognizing receptors (PRRs) that play key roles in the phagocytosis and activation of pro-inflammatory signal transduction pathways (Underhill and Ozinsky, 2002) and are also essential for initiating adaptive immunity in vertebrates (Alcaide and Edwards, 2011). TLRs are proteins synthesized by toll-like receptor genes. Since these genes are implicated in the immune system, they could therefore be appealing candidates for examining the selection processes shaping genetic diversity (Downing et al., 2009). The identification of immunity related genes, which causes variations among individuals in a population, will provide a key element in characterization and further studies of disease resistance (Bulumulla et al., 2011).

Recent gene-targeting studies have revealed that TLRs are capable of sensing organisms such as bacteria, fungi, protozoa and viruses (Uematsu and Akira, 2008). In response to pathogen associated molecular patterns (PAMPs), which include various components of pathogens that are not expressed by hosts, TLRs induce the production of reactive oxygen and nitrogen intermediates (ROI and RNI), inflammatory cytokines and up regulates the expression of co-stimulatory molecules, subsequently initiating the adaptive immunity (Kannaki et al., 2010). The stimulation of different TLRs induces distinct patterns of gene expression, which not only leads to the activation of innate immunity but also instructs the development of antigen-specific acquired immunity (Akira and Takeda, 2004).

Toll-like receptor (TLR) genes are highly conserved group of DNA molecules (Boyd et al., 2007) present in a wide species of animals and plants (Temperley et al., 2008). The TLR multi-gene family comprises a large and variable number of genes (10 – 15 genes) with substantial differences within and between vertebrate groups (Roach et al., 2005; Werling et al., 2009). Since TLR genes are proven to be associated with resistance to infectious diseases, these genes can be exploited as molecular markers in genetic selection to develop disease resistant lines (Kannaki et al., 2010). TLR genes can also be candidate genes for genetic diversity studies of indigenous chickens to exploit SNPs that may be used in gene-assisted selection (GAS) and marker-assisted selection (MAS).

With recent advances in molecular biology and genomics, analyses of chicken expressed sequence tags (ESTs) and genomic sequences have revealed TLR genes in chicken (Leveque et al., 2003; Iqbal et al., 2005). TLRs and their corresponding genes in the domestic chicken (Gallus gallus domesticus) have been well characterized (Fukui et al., 2001; Lynn et al., 2003; Smith et al., 2004; Philbin et al., 2005; Iqbal et al., 2005; Yilmaz et al., 2005). Chicken toll-like receptor (chTLR) genes have been reported to be polymorphic among different avian breeds, suggesting a varied resistance across numerous chicken breeds (Ruan et al., 2012b).  Toll-like receptor TLR7 gene is a member of this multi-gene family, which is implicated in recognition of viral PAMPs. The chicken toll-like receptor 7 (chTLR7) gene encodes 1047 amino acid protein with 62 per cent identity to huTLR7 and a conserved pattern of predicted leucine-rich repeats (Philbin et al., 2005). Although TLR domains are highly conserved among species and among different members of the multi-gene family because of their crucial participation in signaling transduction (Barreiro et al., 2009), point mutation on their sequences can bring about single nucleotide polymorphisms (SNPs) hence creating genetic variants among populations of the same species. For example, four new single nucleotide polymorphisms (SNPs) of TLR7 gene were discovered in Chinese ducks (Zhu et al., 2011). In addition, three genotypes were all found in each mutation site on TLR7 gene in Chinese ducks (Zhu et al., 2011). Recent reports suggest that the ability of certain individuals to respond properly to TLR ligands may be impaired by single nucleotide polymorphisms (SNPs) within TLR genes, resulting in an altered susceptibility to, and course of infectious or inflammatory disease (Schroder and Schumann, 2005). However, TLR7 has not been investigated in the Nigerian local chicken populations.  An important goal in studies of DNA sequence variation is to identify loci that have been targets of natural selection and thus contribute to differences in fitness between individuals in a population (Akey et al., 2004).

Since TLR7 gene has been characterized and sequenced (Yilmaz et al., 2005; Bulumulla et al., 2011), it is an appealing candidate gene for genetic diversity study of Nigerian indigenous chicken populations as well as exotic broiler and commercial layer chickens in Nigeria. Thus, polymorphisms at TLR7 locus could be used in selection to develop chickens with excellent anti-viral response.


1.4 STATEMENT OF PROBLEM

The local chicken population in Nigeria constitutes a valuable genetic resource, which has not been fully harnessed. This valuable animal genetic resource (AnGR) is gradually being eroded due to extensive introduction and distribution of exotic chicken breeds into the local chicken gene pool. The use of molecular tools to assess the genetic characteristics of the local chicken population before they are completely eroded constitutes the problem to be addressed in this study.


1.5 OBJECTIVES OF STUDY

The objectives of this study are to:

·       Determine novel SNPs of TLR7 gene in Nigerian indigenous chicken populations and ISA Brown commercial layer chicken using DNA sequencing,

·       the haplotype variations that may exist among Nigerian indigenous chicken populations at TLR7 genes, and

·       the genetic distance and establish a phylogenetic relationship of TLR7 gene in Nigerian indigenous chicken populations and ISA Brown commercial layer chicken;

·       Compare the evolutionary relationship of TLR7 gene in Nigerian indigenous chickens ISA Brown commercial layer chicken and TLR7 gene sequences from other poultry species in Genebank, and

·       the expression profile of TLR7 RNA in the liver  and thymus tissues from Nigerian Indigenous chickens, and ISA Brown commercial layer chicken using real-time polymerase chain reaction (qPCR).


1.6 JUSTIFICATION

There is need to analyze the genetic potentials of the Nigerian local chicken using molecular genetics approach. Poultry, particularly local chickens are genetically diverse, so it is important to analyze their populations in order to identify functional variants, genotypes or individuals of particular merit. Report on phenotypic assessment of Nigerian indigenous chicken populations revealed a lot of phenotypic variations, which perhaps does not delimit the chickens into a breed. However, these reports need to be properly verified using DNA-based information in other to accurately delineate the genetic diversity status of the indigenous chicken populations in Nigeria, and probably classify them into a breed or different breeds. Chicken toll-like receptor (chTLR) genes have been found polymorphic among different avian breeds, which suggest a varied resistance across numerous breeds of chicken. Since toll-like receptors 7 (TLR7) gene is implicated in intracellular recognition of nucleic acids, especially viral ribonucleic acid (RNA), and has been sequenced, therefore, there is need to exploit this gene in the Nigerian indigenous chicken populations and ISA Brown commercial layer chicken. This is necessary in order to uncover hidden functional variants that may exist at TLR7 gene. Such functional variants at the level of the DNA, if uncovered, could be exploited in further studies of disease resistance and also used as molecular markers in genetic selection for the purpose of improvement of quantitative traits in the Nigerian local chickens.

 


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