GENETIC DIVERSITY OF FIVE POPULATIONS OF THE NIGERIAN LOCAL BREEDS OF GOAT USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKERS

ABSTRACTS

Genetic diversity and genetic distance of five populations of the Nigerian breeds of goat were investigated using random amplified polymorphic DNA markers. Five breeds of goat were used  for the study  and each breed  constituted  a population,  hence,  the  five populations considered in the study. The populations were: Sokoto Red (SR), Sahel (SH), Kano Brown (KB),  Bornu  White  (BW)  and West  African  Dwarf  (WAD)  goats.  The  experiment  was conducted within four geographical zones of Nigeria: South east, North west, North east and North central. One hundred and twenty (120) blood samples were randomly collected from various locations across the four geographical zones in Nigeria. The samples were collected from the stocks at the central markets, which served as collection point from all the localities and from household goat keepers. Varying numbers of sample sizes (Sokoto Red = 23, Kano Brown = 21, Bornu White = 23, WAD = 26 and Sahel = 27) were collected from each breed. The blood samples were collected from the jugular vein of the animals through a process known as venipuncture. Approximately, 5 ml of blood was collected aseptically from each animal into an EDTA container, using 23 gauge sterile needle and syringe, and was stored at

-20oC using (ethylene-di-amine-tetra-acetic  acid, EDTA) as anticoagulant  containers.  The

DNA samples were isolated and purified from the 120 blood samples following protocol as recommended         by         (ZYMO         RESEARCH         CORPORATION,         e-mail: ).  The RAPD-PCR reaction followed the procedure described by El Hentati et al., (2012). Seven random primers were used for this study  but only three (3) primers  produced clearly polymorphic  and reproducible  bands  whereas four (4) failed to produce any band. Allele frequency per population per loci under consideration exceeded the minimum allele frequency (MAF) limit 10%. Level of polymorphism per primer varied from

43 – 80%. Total of 20 scoreable bands were obtained and thirteen (13) out of which were

polymorphic  to arrive  at a total  of 65% polymorphism.  Genetic  diversity  of the  studied populations   was   measured   with   three   indices:   (Nei’s   genetic   diversity,   Shannon’s information index, Observed and Effective number of alleles). Observed  number of allele (Na) was 2.0000 across the populations while Effective number of alleles (Ne) varied from

1.9003 in WAD to 1.9900 in Sahel population. However, SR, KB and BW had Ne values of

1.9897, 1.9287 and 1.9836 respectively. Nei’s heterozygosity varied across the populations with highest  values  0.4975  and 0.4974  obtained  in Sahel  and Kano  Brown  respectively whereas lowest value 0.4736 was obtained in WAD. Shannon’s Information index followed similar trend across with average of 0.6822. The mean of coefficient of gene differentiation Gst was (0.0139) and mean of gene flow Nm across populations was (35.3710). The highest genetic  similarity  (0.9995)  and  lowest  genetic  distance  (0.0005)  was  recorded  between Sokoto Red and Sahel, while the lowest  similarity  (0.9505)  and highest  genetic  distance (0.0507) was recorded between Bornu White and WAD. Populations with higher similarity indicated that they are of closer descent and closer geographical locations. The unweighted pair  group  method  of  arithmetic  means  (UPGMA)  dendrogram  based  on  Nei’s  genetic distance clearly separated the five populations into two clusters. The closest relationship was observed  between  Sokoto  Red,  Sahel  and Bornu  White  goat  populations/breeds  and the farthest  relationship  was  observed  between  WAD  and  Sokoto  Red  populations.  It  was concluded  that  the  high  similarity  obtained  in  this  study  was  as  a  result  of  loss  of heterozygosis  in  the  populatons  of  Nigerian  breeds  of  goat  which  may  have  originated among mates in each population/breed.  However, the results of this  experiment can offer some crucial scientific data useful for breeding programme of Nigerian breeds of goats.

CHAPTER ONE

1.0. INTRODUCTION

Nigeria is a country with heavy human population  of about 168.8 million  (U.S.C.B.,

2012), and this population is continuously on the increase. The increase has led to the high demand for the available animal and animal products in all parts of the country, to meet up with the minimum animal protein requirement per individual per day. The protein intake of an  average  Nigeria  is  45.5g  per  head  per  day  as  against  the  Food  and  Agricultural Organization’s recommended minimum intake of 70g per head per day, of which 35g should be of animal source (FAOSTAT, 2010). The inadequate supply of animal protein in Nigeria can be attributed to inadequate production potentials of the most common sources of meat which include poultry, goat, cattle, pig, sheep, rabbit etc. Gambo et al. (2004) reported that among the cheapest and mostly affordable protein source for this ever increasing population is mainly the poultry products and chevrons (goat meat).

Goat  (Capra  hircus)  is  one  of  the  smallest  domesticated  ruminants  which  are managed for the production of milk, meat, wool and leather particularly in arid, semi-tropical or mountainous countries (Morand-Fehr, 2004). Goat is the most prolific ruminant among all domesticated  ruminant  under  tropical  and  subtropical  conditions.  It  is a resourceful  and efficient ruminant producing meat, milk, skin and hair (Morand-Fehr, 2004).

Goats constitute  the largest group of small ruminant livestock in Nigeria  totalling about  53.8  million  and  also  constituting  6.2  percent  of  the  World’s  goat  population (FAOSTAT,  2011). Surveys have shown that up to 85 percent of rural  households,  poor farmers and small-time business people of all age groups and sexes  keep goat (FDLPCS,

2007). The ability of goats to tolerate harsh climates, the presence of trypanotolerance  in some breeds (Salako, 2004), suitability to traditional systems on account of small size, short generation interval (Abdul-Aziz, 2010) and ability to thrive on poor quality diets provided by scarce grazing on marginal lands (Adedeji et al., 2011) all combine to make small ruminants strategic to increasing livestock  productivity  in rural agricultural  systems (Adedeji et al.,

2011). Despite these advantages, little attention has been paid to the genetic characterization and possible improvement of small ruminants in Nigeria.

Detailed knowledge of population structures among and within breeds of livestock is essential for establishing conservation priorities and strategies (Caballero and Toro, 2002). Livestock  production  is  vital  to  subsistence  and  economic  development  (Yakubu  and Ibrahim,  2011).  The  ever  increasing  demand  for  livestock  production  to  cater  for  the

nutritional   needs   of   rapidly   growing   human   population   has   led   to   indiscriminate crossbreeding in an effort to improve productivity. It has been estimated that up to 1.6 billion people rely on livestock to supply part of their entire daily needs. Consequently, there is the need for conservation and sustainable use of the indigenous livestock genetic resources that are found in the Africa continent (FAO, 2000), precisely Nigeria.

Molecular  markers  are  important  tools  in  tagging  desirable  loci  underlying  the expression  of  traits  which  have  breeding  importance.  Estimations  of  genetic  variation increasingly  are  being  based  upon  information  at  the  DNA  level  by  various  molecular markers  such  as,  Randomly  amplified  Polymorphic  DNA  (RAPD),  Amplified  Fragment Length Polymorphism (AFLP), Restriction Fragment Length Polymorphism (RFLP) (Rincon et al., 2000),  Simple  Sequence  Repeat  (SSR)  or  Microsatellite  (Dalvit et al., 2008) etc. Among them, RAPD markers, generated by the polymerase chain reaction (PCR) is widely used since the 1990’s to assess infra-specific genetic variation at nuclear level (Welsh and McClelland, 1990; Williams et al., 1990).

In recent years, a range of innovations in molecular genetics have been developed for the study of genetic variation and evolution  of populations  using DNA marker  genotype information. The study of genetic variation plays an important role in  developing rational breeding strategies for economical animal species (Maudet et al., 2002). Genetic analysis of livestock  species  have been performed  using of  polymorphic markers  such as restriction fragment length polymorphisms (RFLPs) and microsatellites (Rincon et al., 2000; Dalvit et al.,  2008).  However,  their  use  is  limited  since  designation  of  these  genetic  markers  is expensive, technically demanding and  is time consuming (Beuzen et al., 2000). However, Random  amplified  polymorphic  DNA  (RAPD)  assay  which  uses  short  oligonucleotide primers  of  arbitrary  sequence  to amplify  genomic  DNA  by Polymerase  Chain  Reaction (PCR) enables an approach for identifying polymorphic and genetic markers faster (Cushwa and Medrano, 1996). A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze the process, DNA polymerase, can only add new nucleotides to an existing strand of DNA. These markers have been used for genotype identification (Tinker et al., 1993), construction of genetic maps (Maddox and Cockett, 2007), etc. The RAPD technique has also been used in analysis of genetic variations between different breeds of animals such as fish (Ambak et al., 2006), chicken (Okomus and Kaya, 2005), cattle (Hassen et al., 2007), buffaloes (Abdel- Rahman and Elsayed, 2007), goat (Yadav and Yadav, 2007) and sheep (Kunene et al., 2009).

Random amplified polymorphic DNA (RAPD) molecular markers, has proved to be an efficient  tool in the quantification  of genetic diversity  and genetic  characterization  of various  species  and  populations  (Rahman  et  al.,  2006;  Barker  et  al.,  2001).  Random amplified  polymorphic  DNA (RAPD)  was developed  by  Williams  et al. (1990) and was proved  to be a powerful  tool in different  genetic  analyses.  This approach  detects  DNA polymorphisms based on amplification using single DNA fragments. They are specific and quick and do not require previous DNA sequence information (Williams et al., 1990; Ragot and Hoisington, 1993).

Genetic diversity may be measured through genetic markers. These have been used to determine evolutionary relationship within and between species, genera or higher taxonomic categories  (Paterson  et  al.,  1991).  However,  breeders  tend  to  concentrate  on  specific genotypes for determination of genetic diversity which combine traits of interest and may be used as progenitors in several breeding programmes in order to introduce important genetic traits. In an attempt to solve the problem of  maintaining  pure breeds using the observed morphological  characteristics  that  require  a  lot of time  and effort,  the use of molecular markers in maintaining goat breeds is more suitable and less time consuming.

Gene diversity is an appropriate measure of genetic variability within a population. Genetic  variation  between  and  within  breeds  is  described  as  diversity.   Low  genetic variability may be caused by selection practices in farms. Populations showing higher intra- breed  similarity   and  lower  proportion   of  polymorphic   loci   are  likely  to  have  less heterozygosity, i.e. possess lower level of genetic variation compared to those showing less intra-breed similarity and high proportion of  polymorphic  loci. In other words, genotypes having higher similarity have more homozygous groups (Yasmin et al., 2006).

Genetic diversity is shaped by past populations and the future. The maintenance of genetic diversity is a key to the long-term survival of most species (Hall and Bradley, 1995). Farm  animal  genetic  diversity  is  required  to  meet  current  production  needs  in  various environments, to allow sustained genetic improvement and to facilitate rapid adaptation to changing breeding objective (Kumar et al., 2006). It is essential to characterize a breed for its conservation. If genetic diversity is very low, none of the individuals in a population may have the characteristics needed to cope with the new environmental conditions or challenges. Such a population could be suddenly wiped out. Low amount of genetic diversity increases the vulnerability of populations to catastrophic events such as disease outbreaks. Also, low genetic  diversity  may indicate  high levels  of inbreeding  with its associated  problems  of expression of deleterious alleles or loss of over-dominance. Change in the distribution of the

pattern  of  genetic  diversity  can  destroy  local  adaptation  and  break  up co-adapted  gene complexes. These problems combined, lead to an increasing poorer ‘match’ of the population to its habitat,  and eventually  lead to the  probability  of population  or  species  extinction (Bizhan et al., 2010).

The genetic diversity of goat population in Nigeria needs a thorough investigation so as to identify the undisclosed genetic potentials associated with these animals, conserve the genetic resource and develop better breeding strategies. Molecular markers (RAPD markers) are the useful tools for the divulgence of these genetic attributes and can also be employed in formulating long term inference or plans for genetic improvement programmes.

1.1. Statement of Problem

Production performances of Nigerian breeds are limited compared to those of their counterparts  reared  in  temperate  countries.  The  limited  performances  may  not  only  be explained by the breeds’ genetic potentials but also traditional management practices. On the other hand, local breeds are well adapted to the rather difficult  conditions.  These unique characteristics are results of the evolutionary forces and their interactions over long periods of  time.  However,  resistance  and  adaptation  capabilities  might  have  declined  due  to indiscriminate  mating, crossbreeding, sub-structuring and consequent genetic drift in these goat populations over time.

For more than four decades now, the efforts of breeders in Nigeria to improve  the performance  of our native  breeds of goats have virtually  proven abortive  due to  limited knowledge  of the genetic  diversity  of these animals,  hence, the inevitability  role of this experiment.  Furthermore,  as the demand for certified  products  such as  meat and milk is increasing,   the  focus  is  now  on  local  indigenous   breeds.   Consequently,   indigenous populations   will  be  at  a  higher  risk  of  genetic   erosion   due  to  selection   pressure, indiscriminate  mating  and  lack  of  characterization,  unless  adequate  steps  are  taken  to characterize  and  conserve  their  genetic  diversity.  A  detail  genetic  study  of  local  goat populations  is therefore  imperative  in  quantifying their structure  of genetic  diversity and optimizing conservation and utilization strategies.

Majority of countries in the developed world have the genetic information/status of their local breeds stored in the genebank to be accessed by everybody throughout the world, reason  being that  there  have  been  substantial  research  works  carried  out on  the subject matter, but such information is lacking with regard to Nigerian local breeds of goats due the fact that there are limited research work. Unfortunately, this is due to non-involvement  in

biotechnologically  oriented researches as a result of their high cost, lack of  governmental incentives on research and lack of interest.

Establishment of genebank in Nigeria is of paramount importance as this could serve as benchmark or reference point for accessibility of phenotypic and genotypic information on Nigerian native breed of goats. However, to feed the rapidly increasing population in Nigeria as well as to counter the challenges imposed by global climate  change and fragile natural resource  base, animal  production  need to be doubled  in  the  next three decades,  through exploration of their genetic potentials and marker assisted selection (MAS).

Therefore, the investigation  of genetic diversity and similarity between and  within breeds is necessary to provide useful genetic information essential for developing effective management   plans  for  the  conservation   and  improvement   of   the  genetic  resources. Furthermore, there is a worldwide recognition of the need for the conservation of livestock diversity  (FAO,  1995)  and  for  the  genetic  characterization  of  breeds  and  populations including their genetic differentiation and relationships.

1.2. Objective of the study

General Objective of the study

The  general  objective  of this study was to evaluate  the genetic  diversity  of  five populations  of Nigerian  breeds  of goat  (Sahel,  West  African  Dwarf,  Red  Sokoto, Kano Brown and Bornu white), using random amplified polymorphic DNA markers.

Specific Objectives of the study

i.         To evaluate the allele frequencies of the populations and primer loci used

ii.        To  evaluate  the  genetic  diversity  of  Nigerian  local  breeds  of  goat  across populations and marker loci.

iii.       To determine the gene differentiation and gene flow in the five goat populations.

iv.       To  determine  the  between  and  within  breed  genetic  similarity  and  genetic distance among goat populations, and classify them using dendrogram into their most likely populations of origin.

1.3. Justifications of the study

Goats,  particularly  Nigerian  breeds,  are  genetically  diverse,  so it is important  to analyze their genetic diversity in order to identify populations and individuals of particular merit.  Genetic  diversity  of  indigenous  goat  breeds  in  Nigeria  has  not  been  sufficiently studied or understood clearly. Considering the fact that much research works have not been

carried out on the genetic diversity of the Nigerian breeds of goat, coupled with the fact that this nature of research is a novel and still at its infancy stage in the country, it  becomes imperative that this study be carried out to create an insight on the genetic integrity of the Nigerian breeds of goat, which can also serve as a benchmark to conservation, selection and improvement  strategies.  For instance,  low  genetic  distance  among  the populations  is an indication of incessant inbreeding which on the contrary  reduces heterosis, whereas, high genetic distance is as a result of long term divergence and lack of genetic mixing and inter- migration   between   the   breeds,   groups   and   populations.   Uncontrolled   breeding   and indiscriminate mating of these animals to other breeds (exotic or local) of goat has led to the dilution  of  these  enormous  genetic  potentials.  Therefore,  the  genetic  study  of  the  goat populations in Nigeria is crucial, so as to integrate the result into the livestock sector data base and as well conserve the  superior genotypes, and also, to provide information at the molecular level about genetic structure and diversity of Nigerian goat breeds reared across the six geographical zones in Nigeria, employing RAPD-PCR molecular markers.

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