Genetic characterization of three ovine breeds in Tunisia using randomly amplified polymorphic DNA markers

Genetic structure and diversity were investigated by random amplified polymorphic DNA (RAPD) markers in three Tunisian sheep breeds. These breeds were: Barbarine, Queue Fine de l’Ouest and D’man. The DNA samples were isolated from 160 animals from the three breeds. Twenty random primers were used for this study.

Only 9 primers produced clearly polymorphic and reproducible bands. Level of polymorphism varied from 71.42 to 88.88 per primer and from 75.43 to 83.02 per breed. Genetic variation in studied breeds was measured with three indices (Nei’s gene diversity, Shannon’s information and level of polymorphic loci) and showed that the highest diversity was found in the exotic D’man breed while the lowest diversity was obtained for both the Barbarine and Queue Fine de l’Ouest native breeds. The inter breed similarity indices and the UPGMA dendrogram, based on genetic distance clearly separated the three breeds. The closest relationship was observed between Tunisian Barbarine and Queue Fine de l’Ouest breeds. The AMOVA analysis indicated that the largest part of the genetic variability (97.86%) originated from differences among individuals within breeds.

Introduction

Sheep populations represent the most important domestic livestock species in Tunisia and constitute the principal source of meat. There are several breeds reared under different ecological zones and production systems. The first most important (2555600 heads) (Ministry of Agriculture and hydraulic resources 2006) is the Barbarine (BR), locally known as “Nejdi” or “fat tailed sheep”. The BR is a meat breed widespread in the country (mostly in the middle) and is traditionally managed under extensive production systems. It is a sprig of the population with big tail originating from the Asian steppes that has been implanted in the country since the Punic time. The second most important (1264500 heads) breed (Ministry of Agriculture and hydraulic resources 2006) is the Queue Fine de l’Ouest (QF) known as “Bergui” or “Western Fine Tail”, which is a dual purpose (for milk and meat production) breed. The latter originates from Algeria, where is known as “Ouled Djellal”, and is essentially found in the western region of Tunisia. The black of Thibar “noire de Tiber” is the third breed. The latter is specialized in meat production and is solely found in the Béja region, in the north of the country, and is managed in a relatively more intensive production system. The Black of Thibar breed was the result from crossing of the western Fine Tail and the Merino of Arles at the beginning of the XX^th century (Khaldi 1984). Finally, a new exotic meat breed was introduced from Morocco the last decade into the Tunisian oasis, named D’man (DM), and is known by its high prolificacy (Rekik et al 2002).

Production performances of Tunisian breeds are limited compared to those of their counterparts reared in temperate countries. These limited performances may not only be explained by breeds’ genetic potentials but also traditional management practices. On the other hand, local breeds are well adapted to the rather difficult conditions (harsh climatic conditions, poor-quality food, etc.). These unique characteristics are results of the evolutionary forces and their interactions over long periods of time. However, resistance and adaptation capabilities might have been reduced because of intermixing, sub-structuring and/or consequent genetic drift in these sheep population over time. 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 these 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.

Genetic analysis of livestock species can be performed by the use 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 (Williams et al 1990, Appa Rao et al 1996, Beuzen et al 2000). Random amplified polymorphic DNA (RAPD) assay which uses short oligonucleotide primers of arbitrary sequence to amplify genomic DNA by PCR enables an approach for identifying polymorphic and genetic markers (Williams et al 1990, Cushwa and Medrano 1996). These markers have been used for genotype identification (Welsh et al 1991, Tinker et al 1993), construction of genetic maps (Tingey and Del Tufo 1993, Maddox and Cockett 2007), analysis of the genetic relationships within and between plant species (Salhi-Hanachi et al 2006), and measurement of genetic diversity of many crop plants (Hussein et al 2005, Saker 2005). The RAPD technique has also been used in analysis of genetic variations between different breeds of fish (Ambak et al 2006), chicken (Okomus and Kaya 2005), ducks (El Gendy and Helal 2005, Gholizadeh et al 2007), cattle (Lee et al 2000, Parejo et al 2002, Devrim and Kaya 2006, Hassen et al 2007), buffaloes (Saifi et al 2004, Abdel-Rahman and Elsayed 2007), goat (Rahman et al 2006, Blundell and Felice 2006, Yadav and Yadav 2007) and sheep (Paiva et al 2005, Devrim et al 2007, Okumus and Mercan 2007, Kumar et al 2008 , Mahfouz and Othman 2008, Kunene et al 2009).

Genetic diversity of indigenous sheep breeds in Tunisia has not been sufficiently studied. The objective of this study was to provide information at the molecular level about genetic structure and diversity of some Tunisian breeds reared in the south-west of Tunisia, employing RAPD-PCR molecular markers.

Materials and methods

Blood sampling and DNA extraction

Sheep breeds (BR, QF and DM) from the Gafsa and Tozeur regions in the south west of Tunisia were used in this study (Figure 1). They included BR and QFG (the letter G refers to Gafsa) populations in Gafsa and DM and QFT (the letter T refers to Tozeur) populations in Tozeur.

Blood samples were collected from adult and presumably unrelated animals of both sexes. Before collecting blood samples from animals, owners were asked if there were apparent relatedness among sampled animals to avoid sampling rams and their progenies. Only one sample from small flocks (flock size < 10) and a maximum of three samples from large flocks (flock size >= 10) were taken. A total of 160 individual samples were collected on all populations (40 for BR,40 for DM,40 for QFG and 40 for QFT).

Whole blood was collected from the jugular vein into 8 ml Vacutainer tubes containing the K3EDTA anticoagulant. Blood samples were kept cold at 4°C. DNA extraction was carried out using two DNA purification Kit, “Wizard® Genomic DNA Purification Kit” (Promega Corporation, Madison, USA) and “blood DNA Preparation Kit” (Jena Bioscience, Loebstedter, Jena, Germany) on 3 ml of total blood. DNA concentration and purity was determined using a UV spectrophotometer at 260 and 280 nm optical densities.

Primer selection

A panel of 20 decamer arbitrary primers synthesized from Operon Technologies (Operon Technologies Inc., Alameda, Calif., kits OPE) was screened using DNA samples of each breed. Only 9 primers were selected based on their distinct polymorphism and revealing a pattern with identifiable amplified band. The sequence guanine and cytosine (GC) contents of selected oligonucleotide primers are given in Table 1.

Table 1. Characteristics of selected primers
Primer	Primer sequence	GC content, %
OPE1	CCCAAGGTCC	70
OPE2	GGTGCGGGAA	70
OPE4	GTGACATGCC	60
OPE5	TCAGGGAGGT	60
OPE7	AGATGCAGCC	60
OPE8	TCACCACGGT	60
OPE9	CTTCACCCGA	60
OPE10	CACCAGGTGA	60
OPE13	CCCGATTCGG	70

PCR amplification

Polymerase chain reaction (PCR) was carried out in 25 µl reaction mixtures containing 2µl of template DNA (~50 ng), 200 µM each of dNTPs, 0.5µM of each primer, 1.5mM MgCl2, 1 unit of Taq DNA polymerase (Promega, Madison, USA), 2.5 μL of 10 X Taq DNA Polymerase buffer. MilliQ sterilized water was added to complete the total (25 µl) volume.

Amplification of DNA was carried out using T personal thermocycler (Biometra). Polymerase chain reaction cycling conditions were: initial denaturation for 5 min at 94°C, followed by 40 consecutive cycles. Each cycle included denaturation at 94°C for 25 seconds, annealing at 35°C for 35 seconds and extension at 72°C for 1 min. Theses cycles were followed by a final extension of 5 min at 72°C.

The amplification products were separated by electrophoresis on 2% agarose gels in 0.5x TBE buffer in the presence of ethidium bromide (0.5µg/µl) for 2.5 hours at 100 V. A 1 Kb DNA (Promega® USA) was used as molecular size marker. The RAPD patterns were visualized by UV illumination and the images of each gel were photographed for documentation and further analysis.

Data analysis

All amplified and reproducible bands were scored for their presence (1) or absence (0) in the RAPD profile of individuals from all studied breeds. The scores were then pooled for constructing a single data matrix. The statistical analysis of RAPD data was performed using the computer program POPGENE (version 1.31) (Yeh et al 1999). Genetic diversity was estimated by number and percentage of polymorphic loci, genetic diversity of Nei (1973) and Shanon’s information index. Genetic similarity and genetic distance were calculated on the basis of Nei’s (1972) method of genetic identity and genetic distance, respectively. Relationships among the studied breeds were analyzed by generating dendrogram using Nei genetic distance matrices (1972) with UPGMA (Unweighted Pair Group Method of Arithmetic Means) technique. Finally, analysis of molecular variance (AMOVA) was used to split the variance between and within analyzed breeds using WINAMOVA (version 1.55) software (Excoffier 1993). To ensure that the amplified DNA bands originated from genomic DNA and not primer artifacts, negative control was carried out for each primer/breed combination. No amplified was detected in control reaction. All amplification product were found to be reproducible when reaction were repeated using the same reaction conditions.

Results

Nine of twenty primers (45%) were successfully amplified polymorphic bands among the studied breeds. Figure 2 and Table 2 summarize amplification results with polymorphic primers.

Table 2. Amplification results for used primers in the studied Barbarine, Queue fine de l’Ouest and D’man sheep breeds from Tunisia
	Total of amplified bands	Polymorphic bands across all sampled animals	% Polymorphism
OPE1	7	5	71.4
OPE2	8	6	75
OPE4	5	4	80
OPE5	6	5	83.3
OPE7	7	6	85.7
OPE8	8	6	75
OPE9	5	4	80
OPE10	9	8	88.9
OPE13	4	3	75
Total	59	47	79.6

The number of bands amplified with these primers ranged from 4 to 9 and had sizes ranging from 200 to 2050 bp. A total of 59 loci were amplified, out of which 47 (79.66%) were polymorphic with an average of 5.44 bands per primer. The maximum number of fragment bands were produced by the primer OPE10 (9) with 88.9% polymorphism while the minimum number of amplified bands was recorded for the OPE13 primer with 75 % polymorphism. Gene diversity index, Shanon’s index and the level of polymorphism per breed and for all the sampled animals are given in Table 3.

Table 3. Estimates of genetic variability by breed and for all sampled animals
Breeds	Nei’s gene diversity	Shanon’s index	Proportion of polymorphic loci
BR	33.7	49.0	81.1
QFG	33.1	47.9	77.4
QFT	33.1	47.5	75.5
DM	36.1	51.9	83.0
All sampled animals	42.8	61.7	83.1
BR: Barbarine, QFG: Queue Fine de l’Ouest in Gafsa, QFT: Queue Fine de l’Ouest in Tozeur; and DM: D’man

Nei’s gene diversity in BR, QFG, QFT and DM populations were 33.7, 33.1, 33.1 and 36.1, respectively. The average gene diversity was 42.8 across all sampled populations, whereas Shanon’s index values were 48.9, 47.8, 47.5 and 51.9 for BR, QFG, QFT and DM, respectively; with an overall populations mean value of 61.6. Genetic distance and genetic identity ranged from 0.05 to 0.29 and from 0.74 to 0.95, respectively (Table 4).

Table 4. Nei’s genetic distance (below diagonal) and genetic identity (above diagonal) among sheep breeds based on RAPD analysis
Population	BR	QFG	QFT	DM
BR	0.000	0.849	0.866	0.757
QFG	0.163	0.000	0.952	0.761
QFT	0.144	0.049	0.000	0.743
DM	0.248	0.273	0.297	0.000
BR: Barbarine, QFG: Queue Fine de l’Ouest in Gafsa, QFT: Queue Fine de l’Ouest in Tozeur; and DM: D’man

The highest genetic distance was found between QFT and DM breeds, whereas the lowest distance was observed between the two sub- populations of the QF breed (QFT and QFG). Genetic distance between native breeds was lower than that observed between Tunisian and exotic breeds. On the other hand, genetic identity was the highest between the QFT and QFG sub- populations (I = 0.95) and the lowest between QFT and DM breeds (I = 0.743).

The UPGMA dendrogram, based on Nei’s genetic distance, depicts the relationship among the investigated sheep breeds (Figure. 3).

The dendrogram revealed the existence of three branches indicating the separation of the populations studied into three breeds while the QFG and QFT were clustering together as a unique breed. The BR and QF breeds had a close genetic relationship whereas DM breed was distinctly different from other breeds.

The ANOVA analysis (Table 5) showed that approximately 2.1 % of the genetic variability in the analyzed animals is due to differences among breeds and that the largest part of that variability (97.9%) is due to differences within breeds.

Table 5. Analysis of molecular variance for the studied Barbarine, Que fine de l’Ouest and D’man sheep breeds
Source of variation	df	Sum of squares	Variance components	% of total variance	Fixation indice ΦST
Among breeds	3	23,7	0,3	2,1	0,021***
Within breeds	156	12,6	12,6	97,9
***: P<0.001

A higher and significant differentiation characterize individuals between population (Φ-statistic = 0.021; P<0.001 after 1000 permutations).

Discussion

Random amplified polymorphic DNA (RAPD) developed by Williams et al (1990) 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). In this study, only 9 primers out of 20 primers, screened using DNA samples from BR, QF and DM sheep breeds, generated reproducible and distinct RAPD profiles and were used to evaluate genomic variability due to their clear RAPD bands for each of the investigated breeds. Similar results were found by others investigating sheep diversity by RAPD technique, According to Ali (2003) , 5 from 19 primers used to investigate genetic similarity among four Egyptian breeds generated polymorphic and reproducible bands. Likewise, Yadav and Yadav (2007) used a total of 40 primers to study genetic diversity in six breeds of Indian goats, but they found only 10 primers that generated polymorphic bands. Primers screened varied in the extent of information they generated with some producing highly polymorphic patterns whereas others produced less polymorphic products. The polymorphism found within breeds varied between primers and breeds. In this study, a relatively highly proportion of the total bands amplified from the 9 selected primers were polymorphic. Level of polymorphism varied from 71.4 to 88.9 per primer and from 75.4 to 83.0 per breed. These observations revealed comparatively high level of genetic variation between sheep breeds. Similar levels of polymorphism were reported by earlier on sheep populations (Paiva et al 2005, Devrim et al 2007, Mahfouz and Othman 2008) and also in goat populations (Blundell and Felice 2006).

Gene diversity is an appropriate measure of genetic variability with in a population. The genetic variation in the studied breeds is illustrated by Nei’s gene diversity, Shannon’s information and level of polymorphic loci (Table 3). The highest means of heterozygosity, Shanon’s index and level of polymorphism were found in the DM breed while the lowest values were found in the QF breed (QFG and QFT sub- populations). These results indicate that the greatest diversity was found in the exotic DM breed indicating a high level of heterozygosity. Inversely, the two native breeds (BR and QF) showed the least genetic diversity or variation (higher similarity) among its individuals indicating low heterozygosity levels. Low genetic variability may be caused by selection practices in farms from where samples were collected. 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). Similar results were reported by Mahfouz and Othman (2008) in five Egyptian sheep breeds and by Paiva et al (2005) in Brazilian hair sheep breeds.

The inter breed similarity indices and the UPGMA dendrogram, based on genetic distance, depict the relationship among the investigated sheep breeds, separate clearly the three breeds. The closest relationship was observed between native Tunisian breeds (the BR and QF breed) while this relationship was loose between Tunisian and exotic breeds. The exotic breed (DM) has a different geographic origin, different reproductive performance and different morphologic appearance. On the other hand, the close relationship observed between native breeds may be explained by a possible cross migration in the past between these two breeds. This cross migration may have occurred because of short geographic distances between areas where BR and QF breeds are distributed. Migration has a great effect on the reduction of genetic differentiation between populations (Laval et al 2000). There is a significant genetic structure within breeds (Table 5) with an average of 97.9 % suggesting a low migration rate or exchange of animals between studied breeds. These results corroborate the clusters observed in the dendrogram of the three sheep breeds and suggest that there is a population substructure defined by breeds and subgroups of the breeds.

Conclusions

Acknowledgment

We are grateful to the farmers who permitted us to obtain blood samples from their sheep. We also wish to express our sincere gratitude to Miss Ben Dhifi Monia and Miss Smaoui Faten for their kind helpe. This work was funded by the collaboration of Research Center in Oases Agriculture and Faculty of Sciences of Gafsa.

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Genetic characterization of three ovine breeds in Tunisia using randomly amplified polymorphic DNA markers

Z Khaldi, B Rekik, B Haddad, L Zourgui and S Souid***

Abstract

Introduction

Materials and methods

Blood sampling and DNA extraction

Primer selection

PCR amplification

Data analysis

Results

Discussion

Conclusions

Acknowledgment

References

Genetic characterization of three ovine breeds in Tunisia using randomly amplified polymorphic DNA markers

Z Khaldi, B Rekik*, B Haddad**, L Zourgui*** and S Souid***

Abstract

Introduction

Materials and methods

Blood sampling and DNA extraction

Primer selection

PCR amplification

Data analysis

Results

Discussion

Conclusions

Acknowledgment

References

Z Khaldi, B Rekik, B Haddad, L Zourgui and S Souid***