Molecular Characterization of Balochi Sheep by using Microsatellite Markers in Pakistan

Mudassar Jehan1, Masroor Ahmed Bajwa2, Mohammad Masood Tariq2, Asim Faraz3*, Abdul Samaad2, Jameel Ahmad1 and Yousaf Hassan Barozai2 1Livestock and Dairy Development Department Balochistan, Quetta, Pakistan 2Center for Advanced Studies in Vaccinology and Biotechnology, University of Balochistan, Quetta, Pakistan 3Department of Livestock and Poultry Production, Bahauddin Zakariya University, Multan, Pakistan


INTRODUCTION
G enetic diversity studies are based on DNA molecular markers which are commonly employed to understand polymorphism. Among the first developments, PCR based markers were Random Amplified Polymorphic DNA (RAPD) in which sequences of DNA were amplified using short oligonucleotide, from 10 to 12 base pair (bp) long, for the amplification of random DNA sections (Williams et al., 1990).Overall, the reliability of RAPD markers is low due to the success of amplification of any DNA fragment which could be due to many factors such as DNA template quality, PCR conditions, reagents and equipment (Edwards and Mc-Couch, 2007). Restriction Fragment Length Polymorphisms (RFLP's) have been the first genomic DNA-based molecular markers produced from a specific application of Southern blot analysis.
Amplified fragment length polymorphisms (AFLPs) are more sophisticated than RAPD markers, which provide the amplification of restriction fragments ligated to specific adaptors. Only a portion of restriction fragments within the range of 50 and 350 bp in size can be amplified by PCR and visualized on polyacrylamide gels. Large genomes usually require additional selective bases to the primers to reduce the number of co-amplified bands (Zhang and Hewitt, 2003). In animal cells, besides the nuclear DNA, Mitochondrial DNA (mtDNA) is also found. The study of this kind of DNA in population genetic is famous because it can only be transferred from mother to off springs in most species. The second feature of this DNA is that it grows faster than nuclear DNA, however, having small size about 15 to 20 kilobase (kb) in length, containing around 37 genes which are found in duplicates in the cells of the eukaryotic organisms (Othman et al., 2015). Microsatellite markers contain DNA section, which is only a few nucleotides long (2 to 6 bp), it occurs frequently many times in tandem (CACACACACACACACA) (Teneva, 2009). Microsatellites loci are also recognized

O n l i n e F i r s t A r t i c l e
as simple sequence repeats (SSR's) and sequence tagged microsatellites (STMS) (Mburu and Hanotte, 2005). The recurrent fragment can be a mono, di, tri or tetra nucleotide; however, the most common is di-repeats. Molecular markers are suggested in order to overcome the limitations related with RFLP and RAPD (Garcia et al., 2004). These markers serve as a useful tool for animal classification and genetic distance estimation (Baker and Bradley, 2006). This is first time designed to study Balochi sheep breed at molecular level, using a set of 15 ovine specific SSR markers, in Balochistan. The findings of the current study may help in understanding the scope of genetic variability and would be helpful in making better approaches for conservation and future development in sheep breeding programmed.

MATERIALS AND METHODS
Balochi sheep (n=30) of both sexes (from different pedigrees) present on different sites were sampled for their whole blood to extract DNA and it was kept in view that they should not be genetically related to each other in order to have diversity of samples. The animals under study were present at Sheep Research Center Yetabad at Loralai, Balochistan Pakistan and they were randomly selected for sampling. The whole blood samples were obtained in specific tubes (vacutainers) having anti-coagulant and their volumes measured to be nearly 7 millilitters. The anticoagulant contained in vacutaniers was ethylene diamine tetra acetic acid (EDTA). After collection the samples were carried to the processing labs in cold containers avoiding them from extreme heat conditions which could have otherwise destroyed the samples. The blood samples were kept cold and caution was taken to prevent exposing them to extreme temperatures keeping in view standard sampling carrying protocols. The collected samples then were stored at -20 o C until they were processed for DNA extraction and further analysis at the Hi-Tech Laboratory in the Centre for Advanced Studies in Vaccinology and Biotechnology (CASVAB), University of Balochistan, Quetta.

DNA extraction
For DNA extraction from the blood, 300µl of blood and 1 ml of lysis buffer were taken. After that samples were vertexed for five minutes. After vertex mixing samples were centrifuged at 13000 rpm for fifteen minutes. The process was repeated 2 to 3 times. White colorless pellet formed at the bottom of the Eppendrof tube, discarded the remaining solution save the pellet. Then we added 20µl protienase kinase (PK), 80µl Sodium Dodecyl Sulfate (SDS) and 250µl buffer solution and vertex mixed for five minutes. Now the samples were left overnight at 58ºC for digestion in water bath, afterwards added 300µl PCI and vertexed for five minutes. After vertexing, the sample was centrifuged at 13000 rpm for fifteen minutes at 4 o C. Then top aqueous layer was separated in a new tube and small amount of isopropanol was added with a ratio of 1:1 and incubated at room temperature for 5 min. Samples were centrifuged at 13000rpm for 10 min at 4ºC. For washing, 200µl ethanol was added in samples and centrifuged at 13000 rpm for 10 min at 4 o C, ethanol was discarded and the pellet was air dried. After adding 150-200 µl water, the DNA was stored at -20ºC for further analysis.

Polymerase chain reaction (PCR)
All PCR reactions (DNA sample of 30 Balochi sheep) were carried out in 25 pl reaction vessels containing 100 ng total genomic DNA, 0.5 pM of each primer, 200 pM of dNTPs, 50mMKCl, 10 mM Tris, 2.0 mM MgCl 2 and 1.0 unit of Taq DNA polymerase. Different conditions for amplification used were denaturation, annealing and extension respectively at 95 o C for 7 min, followed by 30 cycles, for 30 sec. at 94 o C for 30 sec (annealing) and an extension step of 1 min at 72 o C. The final cycle was followed by l0 min extension at 72 o C. All amplification reactions were performed using Palm Cycler PCR system (Corbett Research) Programmable Thermo cycler. For the amplification of each individual DNA sample all the 15 Ovine specific SSR markers (OarFCB128, OarCP34, OarJMP58, OarAE129, BM1329, BM8125, HUJ616, DYMS1, SRCRSP9, OarFCB226, SRCRSP5, ILSTS11, and ILSTS28) were employed ( Table I). The annealing temperature for each primer was fixed accordingly. A total of 2240 reactions were done on PCR and the products of PCR were separated on 5% agarose gel using Thermo Scientific 0' Range Ruler 5bp DNA ladder.

Statistical analysis
The data obtained were fed into POPGENE software in binary format as ASCII files and analysis were carried out that generated several parameters including observed number of alleles (Na), effective number of alleles (Ne), Shannon's information index, observed homozygosity (obs. Hom.), observed heterozygosity (obs. Het.), expected homozygosity (Exp. Hom.), expected heterozygosity (Exp. Het.) and average Heterozygosity (Ave. Het.) (Kimura and Crow, 1964;Nei, 1973). Other genetic diversity related estimates and fixation indices, FIS (within population inbreeding estimates), FIT (total inbreeding estimates) and FST (measurement of population differentiation) proposed by Wright (1978) were also computed.
As shown in Figure 2, minimum and maximum polymorphism was observed in case of primer BM1329 and primer BM8125, respectively.
Various characteristics of genic polymorphism (Table  II), homozygosity and heterozygosity (Table III) along with F-statistics (Table IV) have shown phenomenal attributes, genetic diversity and multiple allelomorphism in Balochi sheep characterizing particular breed markers that might be helpful in identification and breed improvement programs.     During the present study the mean number of observed alleles (Na) was 3.7333 ±0.869 (range 1.0-4.0), while the average number of effective alleles (Ne) was 1.8237±0.064 (range 1.0-3.509) which is significantly less than reported by Musavi et al. (2011) 6.296 and 4.394 in Hazaragie sheep, Ahmed et al. (2014) 5.2727 in Indian sheep breed Changthangi and Munjal. The Shannon lndex (I) found in the present study was 0.7413 ± 0.353 which is markedly less than those reported by other researchers such as 0.87 (Musavi et al., 2011);1.445 (Ahmed et al., 2014) and 1.419 in Harnai sheep.

O n l i n e F i r s t A r t i c l e
The average observed heterozygosity (Ho) in the population under study was 0.372+0.219 which can be compared with the findings of various workers viz; 0.825 by Musavi et al. (2011), 1.445 by Ahmed et al. (2014, 0.712 by Yama et al. (2011). Mean expected heterozygosity (He) was 0.405±0.219 during the study which was higher than Ho at most of the loci; however, the primers ILSTS28, OarCP38, OarFCB34, HUJ616, DYMS1, SRCRSP9, OARFCB128 showed higher He then Ho. The finding 0.405 and 0.372 of the present study were less than those reported by Musavi et al. (2011) 0.778, Ahmed et al. (2014 0.7185, Wajid et al. (2014) 6.23 and 6.255 in Balochi and Rakhshani breeds, respectively.
The average heterozygosity and gene diversity were found to be 0.4056±0.209 and 0.4067±0.209, respectively during the study. These values were quite smaller than those reported by Musavi et al. (2011) 0.757 and 0.772, whereas, higher values for gene diversity were reported by Dalvit et al. (2008) 0.806, 0.801, 0.796 and 0.801 in four Italian sheep breeds Alpagota, Brogna, Foza and Lamon, respectively value of F-statistic found during the study ranging from 0.2851 to 0.9132 for SSR markers, with an average value of 0.0856±0.021, showed that majority of the marker yielded higher than average expected reduction in heterozygosity. The standard errors found during the study were usually low indicating that homozygosity prevails in the population under study. This might be due to intense inbreeding in this flock of Balochi sheep.
Al- Barzinji et al. (2011) reported 0.469 inbreeding value in Hamdani sheep, whereas in contrast Musavi et al. (2011) reported negative (-0.069) value for inbreeding estimates. However positive, but less than ours (0.0525) and (0.253) inbreeding estimates were reported by Ahmed et al. (2014) in the Kail sheep of Azad Jammu and Kashmir and Indian Bellary sheep populations, respectively. The findings of Wajid et al. (2014) about the inbreeding estimates of Balochi and Rakhshani sheep breeds of Balochistan were 0.0292 and 0.0084, respectively.

CONCLUSIONS
The Balochi is a unique sheep that due its local adaption have evolved some peculiar characteristics and needs more attention. The presence and absence of horns and the fleece color were not the effect of adaptations to the environment but have a communal' importance and therefore, could not be revealing of the genetic relationships. The latest use of microsatellite markers in various sheep breeds showed to be very beneficial for the determination of genetic relationships among populations. It is concluded from the study that a set of 15 FAO recommended microsatellite markers, most of them have shown their effectiveness for the significant genetic diversity in the population of Balochi sheep breed in Balochistan. The results of the current study may be helpful contribution in the understanding of genetic structure of the indigenous breeds of the province. Furthermore, the results may be helpful for the comparison and conservation of local genetic resource. Balcohi breed improvement program needs to be launched to improve the breed in the native place.