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Molecular Characterization of Balochi Sheep by using Microsatellite Markers in Pakistan

PJZ_54_5_2295-2300

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

ABSTRACT

Balochi sheep is one among the established sheep breeds of Balochistan province in Pakistan. As far as its size is concerned, it is medium sized, having fat tail and inhabits in the Northern regions in Balochistan. Molecular studies are the base of breed characterization. Hence, nearly 25 unrelated (including both male and female) animals of Balochi breed were sampled for DNA extraction. Some specific markers (15 out of available 30 SSR markers) were employed in the present study to highlight genetic polymorphism. The PCR was utilized for amplification of individual DNA samples. All of the 15 SSR markers were amplified. After gel documentation, a total number of 97 alleles were recognized having 1 to 5 polymorphic forms (OARFCB193, OARJMP29, MAF33) to 4 (OARHH47, DYMS1, SRCRSP5). For total loci, number of alleles averaged 2.1162±0.3769. Shannon’s Information index (I) 0.6184±0.2447 and the effective number of alleles (Ne) averaged 1.6251±0.4604. Average observed, expected and average heterozygosities were found to be 0.5815±0.1059, 0.4330±0.5811 and 0.4331±0.5811, respectively. It was noted that the F-statistic was ranging from 0.2166 to 0.95182 for the microsatellite markers employed in the study. Expected reduction in heterozygosity was higher than mean value in case of most of the markers with lower standard errors showing the prevalence of homozugous Balochi sheep population. The main cause reason behind prevalence of homozygous population might be inbreeding as only few rams had been used for breeding the flocks. This study would provide basis for breed characterization and lead to breed improvement program.


Article Information

Received 10 March 2021

Revised 12 May 2021

Accepted 05 June 2021

Available online 07 December 2021

(early access)

Published 13 June 2022

Authors’ Contribution

MJ conducted research. MAB and MMT supervised the work. AS and JA helped in write up. AF and YHB helped in write up and reviewed the article.

Key words

Balochi sheep, Genetic characterization, Molecular markers, Genetic diversity

DOI: https://dx.doi.org/10.17582/journal.pjz/20210310070338

* Corresponding author: drasimfaraz@bzu.edu.pk

0030-9923/2022/0005-2295 $ 9.00/0

Copyright 2022 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).



INTRODUCTION

Genetic 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 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 -20oC 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 4oC. 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 oC, 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 MgCl2 and 1.0 unit of Taq DNA polymerase. Different conditions for amplification used were denaturation, annealing and extension respectively at 95oC for 7 min, followed by 30 cycles, for 30 sec. at 94oC for 30 sec (annealing) and an extension step of 1 min at 72oC. The final cycle was followed by l0 min extension at 72oC. 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.

 

Table I. List of ovine markers employed in current research work.

Primer

Primer Sequence (5 → 3)

OarFCB128

 

F: ATAAAGCATCTTCTCTTTATTTCCTCGC

R: CAGCTGAGCAACTAAGACATACATGCG

OarCP34

 

F: GCTGAACAATGTGATATGTTCAGG

R: GGGACAATACTGTCTTAGATGCTGC

OarCP38

 

F: CAATTTGGTGCATATTCAAGGTTGC

R: GCAGTCGCAGCAGGCTGAAGAGG

OarJMP58

 

F: GAAGTCATTGAGGGGTCGCTAACC

R: CTTCATGTTCACAGGGTCAGGG

OarFCB304

 

F: CCCTAGGAGCTTTCAATAAAGAATCGG

R: CGCTGCTGTCAACTGGGTCAGGG

OarAE129

 

F: AATCCAGTGTGTGAAAGACTAATCCAG

R: GTAGATCAAGATATAGAATATTTTTCA ACACC

BM1329

 

F: TTGTTTAGGCAAGTCCAAAGTC

R: AACACCGCAGCTTCATCC

BM8125

 

F: CTCTATCTGTGGAAAAGGTGGG

R: GGGGGTTAGACTTCAACATACG

HUJ616

 

F: TTCAAATACACATTGACAGGG

R: GGACCTTTGGCAATGGAAGG

DYMS1

 

F: AACAACATCAAACAGTAAGAG

R: CATAGTAACAGATCTTCCTACA

SRCRSP9

 

F: AGAGGATTGGAAATGGAATC

R: GCACTCTTTTCAGCCCTAATG

OarFCB226

 

F: CTATATGTTGCCTTTCCCTTCCTGC

R: GTGAGTCCCATAGAGCATAAGCTC

SRCRSP5

 

F: GGACTCTACCAACTGAGCTACAAG

R: GTTTCTTTGAAATGAAGCTAAAGCAATG C

ILSTS11

 

F: GCTTGCTACATGGAAAGTGC

R: CTAAAAGTCAGAGCCCTACC

ILSTS28

 

F: TCCAGATTTTGTACCAGACC

 

R: GTCATGTCATACCTTTGAGC

 

RESULTS AND DISCUSSION

Banding pattern of 15 ovine SSR markers against DNA sample of Balochi sheep breed in Balochistan, using 50bp DNA ladder (50-1350bp) on 5% agarose gel are shown in Figure 1.

 

The figure illustrates that Primer 1 (OarFCB128) amplified two bands (50,45 bp) suggesting two alleles, Primer 2 (OarCP34) amplified two bands (45,43 bp) suggesting two alleles, Primer 3 (OarCP38) amplified two bands (50, 55bp) suggesting two alleles, primer 4 (OarJMP58) amplified one band (60 bp) suggesting only one allele, Primers 5 (OarFCB304), primer 6 (OarAE129) and primer 7 (BM1329) amplified no bands, suggesting no alleles and primer 8 (BM8125) amplified four bands (45, 50, 60 and 100 bp) suggesting four alleles.

Primer 9 (HUJ616) amplified one band (50 bp) suggesting one allele, Primer 10 (DYMS1) amplified two bands (60 and 65bp) suggesting two alleles, Primer 11 (SRCRSP9) amplified only one band (45 bp) suggesting one allele, Primers 12 (OarFCB226) and 13(SRCRSP5) showed no amplification of bands, signifying no alleles, Primer 14 (ILSTS11) amplified two bands (90, 100 bp) suggesting two alleles, and Primer 15 (ILSTS28) amplified one band (above 100 bp) suggesting only one allele.

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.

 

Table II. Genic variation statistics for all locii.

Locus

Sample size

na

ne

I

OARFCB128

124

3

1.1027

0.2245

OarCP34

124

4

1.239

0.4227

OarCP38

124

4

1.4293

0.6398

OarJMP58

124

5

1.1592

0.333

OarFCB304

124

3

2.1541

0.891

OarAE129

124

4

1.3554

0.5737

BM1329

124

4

1.7096

0.74

BM8125

124

4

2.3234

0.9616

HUJ616

124

4

1.6786

0.8085

DYMS1

124

2

1.7009

0.6024

SRCRSP9

124

4

2.8182

1.164

OarFCB226

124

4

2.0773

0.9326

SRCRSP5

124

4

2.1699

0.9117

ILSTS11

124

3

1.8638

0.7823

ILSTS28

124

4

2.5738

1.132

Mean

124

3.7333

1.8237

0.7413

St. Dev

 

0.7037

0.5226

0.2761

 

na, Observed number of alleles; * ne, Effective number of alleles [Kimura and Crow (1964)]; * I, Shannon’s Information index [Lewontin (1972)]

 

Table III. Heterozygosity statistics for all loci.

Locus

n

Obs. Hom

Obs_Het

Exp_Hom*

Exp_Het*

Nei**

Ave_Het

OARFCB128

124

0.9355

0.0645

0.9061

0.0939

0.0931

0.0936

OarCP34

124

0.7903

0.2097

0.8055

0.1945

0.1929

0.193

OarCP38

124

0.7742

0.2258

0.6972

0.3028

0.3003

0.298

OarJMP58

124

0.8548

0.1452

0.8615

0.1385

0.1374

0.1381

OarFCB304

124

0.5968

0.4032

0.4599

0.5401

0.5358

0.535

OarAE129

124

0.7097

0.2903

0.7356

0.2644

0.2622

0.2609

BM1329

124

0.4677

0.5323

0.5816

0.4184

0.4151

0.4151

BM8125

124

0.1935

0.8065

0.4258

0.5742

0.5696

0.5652

HUJ616

124

0.7097

0.2903

0.5924

0.4076

0.4043

0.4048

DYMS1

124

1

0

0.5846

0.4154

0.4121

0.4122

SRCRSP9

124

0.5806

0.4194

0.3496

0.6504

0.6452

0.6449

OarFCB226

124

0.4032

0.5968

0.4772

0.5228

0.5186

0.518

SRCRSP5

124

0.3065

0.6935

0.4565

0.5435

0.5392

0.5366

ILSTS11

124

0.5

0.5

0.5328

0.4672

0.4634

0.4616

ILSTS28

124

0.5968

0.4032

0.3836

0.6164

0.6115

0.6072

Mean

124

0.628

0.372

0.590

0.410

0.407

0.406

St. Dev

 

0.229

0.229

0.1755

0.1755

0.174

0.173

 

*Expected homozygosity and heterozygosity were computed using Levene (1949), ** Nei’s (1973) expected heterozygosity.

 

Table IV. F-statistics and gene flow for all loci.

Locus

Sample size

Fis

Fit

Fst

Nm*

OARFCB128

124

0.3036

0.312

0.012

20.5411

OarCP34

124

-0.0988

-0.0881

0.0097

25.4356

OarCP38

124

0.2423

0.2553

0.0171

14.3387

OarJMP58

124

-0.0643

-0.0573

0.0066

37.5956

OarFCB304

124

0.2439

0.2446

0.001

247.9324

OarAE129

124

-0.109

-0.1068

0.002

123.0179

BM1329

124

-0.2885

-0.284

0.0035

70.6853

BM8125

124

-0.4316

-0.415

0.0116

21.2962

HUJ616

124

0.2813

0.2816

0.0004

648.2206

DYMS1

124

1

1

0.0008

311.75

SRCRSP9

124

0.3493

0.3495

0.0004

616.0954

OarFCB226

124

-0.1525

-0.1497

0.0024

104.4611

SRCRSP5

124

-0.2903

-0.2866

0.0029

86.8323

ILSTS11

124

-0.0798

-0.0753

0.0042

59.6357

ILSTS28

124

0.3363

0.3403

0.0061

40.8444

Mean

124

0.0815

0.0856

0.0045

55.4463

 

FIS (within population inbreeding estimates), FIT (total inbreeding estimates) and FST (measurement of population differentiation).

 

 

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 and 3.9471 in Kail sheep, 63 and 3.658 in Indian Bellary sheep, 8.76 and 4.54 and 8.64 and 4.57 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.

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.

Acknowledgement

The help extended by Sheep Research Center Yetabad at Loralai, Balochistan Pakistan is gratefully acknowledged.

Statement of conflict of interest

The authors have declared no conflict of interest.

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