MICROSATELLITE MARKERS POLYMORPHISM BETWEEN TWO EGYPTIAN GOAT POPULATIONS (Capra hircus)

oats are considered one of the important economic sources of meat in Egypt. The majority of Egyptian goats belong to the Baladi breed, which is distributed all over the Nile valley and Delta region, while Barki goats are found throughout the coastal zone of the Western Desert. Zaraibi goats are a prolific breed originated in Nubia with high dairy potential and they have shown encouraging response to selection for dairy production while maintaining their high prolificacy. Other breeds, Saidi and Wahati goats are concentrated in the Southern region, while, Black Sinai goats can be found only in the Negev and Sinai deserts (Elbeltagy, 2012). While, Galal et al. (2005) had reported that goats are distributed across the country, especially dense in the Nile valley and Delta and with lower concentration in the North-Western coastal region and at the Oases. They further pointed out that in the Nile valley goats are usually found in small holdings as mixed flocks with sheep and other farm animals like cattle and buffaloes, while in the North-Western Mediterranean coast they are in large herds mixed with sheep. Egypt is predominantly desert and arid and semi-arid rangelands and can be divided into four major physical regions; the Nile Valley and Delta, Western Desert, Eastern Desert and Sinai Peninsula (Fig. 1). The New Valley Oases (El-Kharga, ElDakhla, El-Farafra and Siwa) in the western desert are geographically isolated regions. The climate is hot and the temperature is cold at night in certain seasons. Accordingly, these oases have farm animal genetic resources (cattle, sheep, goat and camels) that are able to survive and are adapted to the prevailing conditions in these areas. Unique individuals of these animal species need to be screened, evaluated, collected and maintained as nucleus herds to benefit from them in sire centers.


M. A. EL-SAYED 1 , A. AL-SOUDY 1 AND A. A. EL BADAWY 2
oats are considered one of the important economic sources of meat in Egypt. The majority of Egyptian goats belong to the Baladi breed, which is distributed all over the Nile valley and Delta region, while Barki goats are found throughout the coastal zone of the Western Desert. Zaraibi goats are a prolific breed originated in Nubia with high dairy potential and they have shown encouraging response to selection for dairy production while maintaining their high prolificacy. Other breeds, Saidi and Wahati goats are concentrated in the Southern region, while, Black Sinai goats can be found only in the Negev and Sinai deserts (Elbeltagy, 2012). While, Galal et al. (2005) had reported that goats are distributed across the country, especially dense in the Nile valley and Delta and with lower concentration in the North-Western coastal region and at the Oases. They further pointed out that in the Nile valley goats are usually found in small holdings as mixed flocks with sheep and other farm animals like cattle and buffaloes, while in the North-Western Mediterranean coast they are in large herds mixed with sheep. Egypt is predominantly desert and arid and semi-arid rangelands and can be divided into four major physical regions; the Nile Valley and Delta, Western Desert, Eastern Desert and Sinai Peninsula (Fig.  1). The New Valley Oases (El-Kharga, El-Dakhla, El-Farafra and Siwa) in the western desert are geographically isolated regions. The climate is hot and the temperature is cold at night in certain seasons. Accordingly, these oases have farm animal genetic resources (cattle, sheep, goat and camels) that are able to survive and are adapted to the prevailing conditions in these areas. Unique individuals of these animal species need to be screened, evaluated, collected and maintained as nucleus herds to benefit from them in sire centers.
Genetic diversity studies based on molecular genetic markers would help in understanding the genetic relationship and diversity within and among local Egyptian breeds and populations. Many researchers employed the random amplified polymorphic DNA markers technique (RAPD) to characterize and estimate genetic distances among different genotypes (Williams et al., 1990;Welsh and McClelland, 1990;Nyamsamba et al., 2002;Ouafi et al., 2002). With the further development and application of various DNA marker techniques, microsatellites and/or simple sequence markers were found to be useful in the study of genetic diversity within and among goat populations and breeds (Li et G al., 1999;Meng-Hua et al., 2002;El-Seoudy et al., 2005;Agha et al., 2008;Takahashi et al., 2008;Mahmoudi et al., 2010;Siwek and Knol, 2010;Visser et al., 2011;Marini et al., 2014;Thiruvenkadan et al., 2014).
The determination of genetic variation based on DNA markers provides accurate information for genetic distance analysis that allows a ranking of populations according to the level of phylogenetic distinction (May et al., 1995). Liu et al. (2008) mentioned that the inbreeding coefficient (FIS) measures the relative heterozygote deficiencies and non-random mating in populations and breeds. Its value ranges between -1 (all individuals heterozygote), 0 (random association of alleles) and 1 (all individuals homozygote). If inbreeding is avoided, F=0; negative F indices result from selection in favor of the heterozygotes, while positive values indicate that the considered population has an inbreeding system of mating. Therefore, the aim of this study is to determine the degree of polymorphism and genetic diversity between two Egyptian goat populations found in the western desert oases (El-Farafra and Siwa) using microsatellite markers, to assess their inbreeding coefficients and to identify SSR markers that could be useful in goat breeding programs.

MATERIALS AND METHODS
This work was implemented as part of a project focused on sustainable utilization of agriculture biodiversity, develop-ment of agricultural products and improvement of rural livelihood in the western desert of Egypt namely, in the oases where farm animal genetic resources (AnGR), are adapted to harsh environments.

Animal sampling and DNA extraction
A total of 34 blood samples were randomly collected from two goat populations located in El-Farafra and Siwa Oases (14 and 20 samples, respectively). The volume of blood sample was 15 ml from each individual. DNA isolation was carried out according to the salting out method by Sambrook et al. (1989). Ten  microsatellite  markers  (ILSTS28,  ILSTS029,  SRCRSP5,  MAF70, SRCRSP23, OarFCB48, SRCRSP9, OarAE54, SRCRSP8 and SPS113) are shown in Table ( 1). The microsatellite primers were selected based on the degree of polymorphism and genome coverage that have been recommended by the Measurement of Domestic Animals Diversity (MoDAD) (FAO, 2004 and and published on the FAO website (www.dad.fao.org/en/refer/library/guideli n/marker.pdf). The PCR reaction included: 50 ng template DNA, 10 pmol from each primer, 10 µl Master Mix (Emerald AMP GT PCR Master Mix, Takara Bio.Inc. composed of DNA polymerase, optimized reaction buffer, dNTPs, and a density reagent. The premix also contains a vivid green dye that separates into blue and yellow dye fronts. The PCR reactions was carried out under the following conditions: an initial denaturation step for 2 minute at 95C, followed by 35 cycles of denaturation for 30 seconds at 95C; annealing temperature (53-65C) for 60 seconds at optimized primer annealing temperature as shown in Table ( 1). Extension for 60 seconds at 72C and final extension step at 72C for 60 seconds. Amplified fragments were analyzed on a 10% polyacrylamide gel and stained with Ethidium bromide. The gel was photographed and the image analyzed using Gel Documentation System (Alphaimager TM 2200, Cell Biosciences).

Statistical analyses
Gels were visualized and scored with Alphaimager 2200 software Version 4.0.1. All scored microsatellite data was first adjusted using a Tandem Repeat Analyzer software package to estimate each allele size according to its number of repeats for each marker. Then, a spread sheet program (Microsoft Excel) was used to arrange the data for each population regarding each locus. Data was analyzed employing the Arlequin 3.11 software package after data conversion using CONVERT program. POPGENE software package (Yeh et al., 1999) was used to calculate allele frequencies; observed expected number of alleles and effective number of alleles (Kimura and Crow, 1964).
The number of alleles per population ranged from 2 (loci MAF70, SRCRSP8 and SPS113) to 4 (loci ILSTS028, OarFCB48 and SRCRSP9) in Farafra goats, and 2 (loci MAF70 and SRCRSP23) to 7 (locus SRCRSP8) in Siwa goat population ( Table 2). The numbers of alleles of the two goat populations were lower than those reported for the Mongolian Goat Populations (Takahashi et al., 2008), Girgentana goat breed (Siwek and Knol, 2010), South African Angora goats (Visser et al., 2011), Malaysian Goat Breeds (Marini et al., 2013) and Salem Black goat breed (Thiruvenkadan et al., 2014). The mean number of observed alleles per locus in Farafra (3.00) and Siwa (3.4) goats were also low compared to the mean number of alleles per locus reported for the Katjang, Jamnapari, Boer and Savanna goat breeds (5.43, 5.70, 5.90 and 5.70, respectively) by Marini et al. (2014). Barker (1994) suggested that loci with at least four alleles should be used in diversity studies to reduce the standard error of the estimated distance. In the present study only four loci, ILSTS029, SRCRSP5, MAF70 and SPS113 were found to have less than four alleles. The low number of alleles observed could be due to the small sample size, low number of polymorphic loci or the effect of inbreeding (Pandey et al., 2006;Maletsanake et al., 2013).  Table   (2). While the Barki and Zaraibi breeds had smaller numbers of alleles (Agha et al., 2008). The mean number of alleles shared between Barki and Ardi was 3.  Table ( 3). Similar to the present study, the observed heterozygosities of Malaysian goat breeds were lower than the expected heterozygosities (Marini et al., 2014). High heterozygosity values indicate high genetic diversity as well as a high degree of genetic variation. Araújo (2006)  The effective number of alleles (Table 3) varied from 1.65 (SRCRSP8) to 3.24 (SRCRSP9) with a mean value of 2.19 in Farafra goats and varied from 1.60 (SRCRSP23) to 3.85 (OarFCB48) with a mean value of 2.60 in Siwa goats which was lower than the Kannaiadu (4.22), Osmanabadi (3.25) and Sangamneri (3.30) breeds studied by Dixit et al. (2010) and from Sangamneri (4.04) by Verma et al. (2011) and from Katjang (3.56), Jamnapari (3.81), Boer (3.96) and Savanna (3.92) by Marini et al. (2013).
Analysis of molecular variance AMOVA (Table 4) of Farafra and Siwa goat populations based on microsatellite DNA variation showed that the majority of the genetic diversity obtained in the current study is presented by among individuals within populations (46.93%) and within individuals (29.37%). Population fixation indices give an idea about the population structure in terms of inbreeding coefficient and population differentiation. Population Fixation indices revealed a 0.7063 of variation referring to differences among individuals versus total variance (Fit). While, among populations differences versus total variance was the lowest fixation index (Fst = 0.2371) indicating low level of population differentiation. A pair wise difference among Frafra and Siwa goat populations was 0.6151 based on among breeds F index (Fis) as shown in Table ( Allele size in base pair, their frequencies for each locus, average of allele frequencies and polymorphism information content (PIC) values for each locus per population as observed in the present study are given in Table (5). The highest allele frequency overall loci was 0.750 for allele 87 at locus SRCRSP23 in the case of Siwa goat population. While, the lowest one was 0.053 associated with Siwa goat population at two loci ILSTS029 (for allele 135) and SRCRSP8 (for alleles, 170 and 266), respectively. The highest average of allele frequency estimated was (0.500) for Farafra population at loci MAF70, SRCRSP8 and SPS113 and for Siwa goat population (0.500) at loci MAF70 and SRCRSP23. On the other hand, the lowest one was 0.143 in the case of locus SRCRSP8 for Siwa goat population. Visser et al. (2011) reported that allele frequency ranged from -0.017 (BM 1818) to 0.150 (SRCRSP 8) with an average of 0.043 in South African Angora goats. The highest frequency of a specific allele was 0.178 for ILSTS087 (155 bp) in Maltese goats (Agha et al., 2008). However, Thiruvenkadan et al. (2014) found that the alleles with the lowest frequency (0.001-0.1) were found to be abundant in Salem Black goat breed.
The polymorphic information content (PIC) was an ideal indicator for the measurement of allele polymorphism. Loci were considered highly polymorphic when PIC>0.5; moderately polymorphic when PIC ranged from 0.25 to <0.5 and low polymorphic when PIC<0.25 (Botstein et al., 1980). Table (5) shows the highest PIC was (0.791) for SRCRSP8 and the lowest PIC was (0.375) for SRCRSP23. All markers exceeded 0.5 except MAF70 and SPS113 in both Siwa and Farafra goat populations. Meanwhile, PIC value was lower than 0.5 in Farafra goats for loci OarFCB48 and SRCRSP8 and also for Siwa goats for locus SRCRSP23. The overall average PIC per population of the ten markers were 0.530 and 0.570 for Farafra and Siwa goat populations, respectively, which indicated that the ten microsatellite markers contained high polymorphic loci in both Egyptian goat populations. In the genetic diversity analysis, microsatellite markers with PIC>0.7 were taken as the most ideal selected markers. From the selected mi-crosatellite markers in the present study, the PIC of SRCRSP8 and OarFCB48 for Siwa goats exceeded 0.7 which indicated that these loci could be used as genetic markers for genetic diversity analysis of Siwa goat population. Hassanane et al. (2010) reported that the PIC of (ILSTS087) exceeded 0.7 in Egyptian Zaraibi breed. The markers SRCRSP8 and OarFCB48 could be used in marker assisted selection (MAS) to improve the performance of Egyptian goat populations.
A further conclusion is that the microsatellites analyzed in this study were informative and should be used in future genetic diversity studies of goats in Egypt. Also, the importance of the conservation of genetic diversity should be considered by goat breeders in the interest of the long-term improvement of Egyptian breeds.
In conclusion, the present investigation proved the usefulness of the ten microsatellite markers used herein in discriminating between the two Farafra and Siwa goat populations. Among 42 alleles across the two populations a total of 20 unique alleles were obtained (8 alleles for Farafra and 12 alleles for Siwa goat populations). In this regard, SRCRSP8 produced the highest number (5) of unique alleles compared to the other markers. This result could be utilized as a molecular tool in fingerprint analysis of Siwa goat population. The present work suggests using wide genome scan analysis based on more recommended microsatellites covering goat genome which could be further employed in MAS (marker assisted selection) and QTL (Quantitative Trait Loci) programs. A further studies with increasing the population sample size is recommended to characterize and measure genetic diversity in the New Valley oases.

SUMMARY
Two native goat populations present in El-Farafra and Siwa oases, located in the Western Desert of Egypt were genotyped using ten microsatellite molecular markers (SSR). Blood samples taken from a total of 34 individual goats, 14 from Farafra and 20 from Siwa, were subjected to DNA extraction and subsequently to SSR-PCR amplification. The number of alleles ranged from two for MAF70 marker to seven for OarFCB48 and SRCRSP8 loci, the average number per population for Farafra goats was 3.0 and 3.4 for Siwa goats with a total number of a 42 alleles for both populations. The mean observed heterozygosity (Ho) and expected heterozygosity (HE) for both populations varied from 0.21 to 0.24 and 0.56 to 0.59, respectively. Fixation indices revealed a 0.7063 variation referring to differences among individuals versus total variance (Fit). While, among populations differences versus total variance had a lower fixation index (Fst = 0.2371) indicating low level of genetic differentiation between Farafra and Siwa populations. A pair wise difference between Farafra and Siwa goat populations was (0.6151) based on among breeds F index (Fis).The highest PIC was observed for SRCRSP8 mi-crosatellite marker (0.791) and the lowest PIC was 0.375 for SRCRSP23. The average PIC of the ten markers was 0.530 and 0.570 for Farafra and Siwa goat populations, respectively, which indicated that the ten microsatellite markers contained highly polymorphic loci in both Egyptian goat populations. In the genetic diversity analysis, microsatellite markers with PIC>0.7 were taken as the most ideal selected markers. From the selected microsatellite markers in the present study, the PIC of OarFCB48 and SRCRSP8 (Siwa) exceeded 0.7 which indicated that these loci could be used as genetic markers for genetic diversity analysis of Siwa goat population. The markers generated by OarFCB48 and SRCRSP8 loci could be utilized in marker assisted selection (MAS) to improve the performance of Egyptian goat populations. FAO, (2004