Research Article Molecular Characterization of Sudanese ...downloads.hindawi.com/journals/gri/2014/928420.pdf · Research Article Molecular Characterization of Sudanese and Southern

Research ArticleMolecular Characterization of Sudanese and SouthernSudanese Chicken Breeds Using mtDNA D-Loop

Charles E Wani12 Ibrahim A Yousif1 Muntasir E Ibrahim3 and Hassan H Musa4

1 Department of Genetics and Animal Breeding Faculty of Animal Production University of Khartoum Sudan2 Faculty of Veterinary Sciences University of Bahr El Ghazal Sudan3 Institute of Endemic Diseases University of Khartoum Sudan4 Faculty of Medical Laboratory Sciences University of Khartoum Sudan

Correspondence should be addressed to Ibrahim A Yousief yousifi 2002yahoocom

Received 3 January 2014 Revised 25 February 2014 Accepted 18 March 2014 Published 24 April 2014

Academic Editor Arne Ludwig

Copyright copy 2014 Charles E Wani et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The objective of this study was to assess the genetic relationships and diversity and to estimate the amount of gene flow among thefive chicken populations from Sudan and South Sudan and commercial strain of egg line White Leghorn chickens The chickenpopulations were genotyped usingmtDNAD-loop as a molecular marker PCR product of the mtDNAD-loop segment was 600 bpand 14 haplotypes were identified The neighbor-joining phylogenetic tree indicated that the indigenous Sudanese chickens canbe grouped into two clades IV and IIIa only Median joining networks analysis showed that haplotype LBB49 has the highestfrequencyThe hierarchal analysis of molecular variance (AMOVA) showed that genetic variation within the population was 886and the differentiation among the population was 114 When the populations was redefined into two geographical zones richand poor Savanna the results were fractioned into three genetic variations between individuals within population 955 betweenpopulations within the group 075 and genetic variation between groups 375The pair wise 119865st showed high genetic differencebetween Betwil populations and the rest with 119865st ranging from 01492 to 02447 We found that there is large number of geneexchanges within the Sudanese indigenous chicken (Nm = 4622)

1 Introduction

Chicken genetic resources comprise a wide range of breedsand populations including red jungle fowl native and fancybreeds middle level food producers industrial stocks andspecialized lines Sudan possesses potential genetic resourcesof local poultry even though most of them have not beengenetically and phenotypically characterized Some effortshad been exerted by the Sudanese nationals [1 2] andinternational researchers and FAO investigated the geneticand phenotypic potentiality of the local chickens but stillfurther identification and characterization as an ultimateprerequisite for their conservation and utilization are neededThe assessment of genetic distance by means of molecularmarker techniques may provide useful information for initialevaluation of chicken genetic resources Microsatellites havebeen successfully used in chicken genetic diversity studies

Genetic diversity measures using the highly polymorphicvariable number of tandem repeat loci have yielded reliableand accurate information for the study of genetic relation-ships between chicken populations Sequencing a specificfragment of mtDNA (eg D-loop) gives more accurateinformation on evolution and genetic diversity [3] The D-loop region does not encode protein and evolves much fasterthan other region of the mtDNA genome For the past 20years mtDNA and particularly D-loop sequences have beenused in phylogenetic analysis [4] There is evidence frommtDNA D-loop variations in European African and Indiancattle breeds that indicate independent domestications ofBos taurus and Bos indicus cattle in two separate locations[5] Also D-loop sequences have been used in unravelingdomestication and diversity of dogs [6] horses [7] and goats[8] and in Australian red kangaroo and Macropus rufus [9]Study variation among 398 African indigenous chicken from

Hindawi Publishing CorporationGenetics Research InternationalVolume 2014 Article ID 928420 8 pageshttpdxdoiorg1011552014928420

2 Genetics Research International

12 countries was carried out using mtDNA D-loop regionand HVI domain and it was found that the polymorphicsites account for 1259 of the 397 sequenced base pairfragment [10] while variation rates among 25 individualsfrom six native Chinese chicken populations were recordedto be 705 and 554 respectively [11 12] Advantages ofthe mtDNA are that it plays a role in metabolism apoptosisdisease and aging and it is the site of oxidative phospho-rylation essential for the production of ATP and a varietyof other biochemical functions It is highly polymorphiccompared to the nuclear DNA evolutionary rate being 5ndash10times faster than nuclear genome [13] probably due to lackof replication repair mechanism [14] The understanding ofphylogeography will elucidate the demographic history ori-gin and expansion of livestock species Networks analysis hassupplemented phylogenetic trees to overcome the problem ofparallel mutations and lineage exchange between divergentpopulations [15] Therefore the aim of the present study is toevaluate the genetic variability within and between Sudanesenative chickens using mtDNA D-loop region

2 Materials and Methods

21 Experimental Animals The study was conducted in fourstates of Sudan chosen because they have only indigenouschicken and no exotic breeds have been introduced Atotal of 81 blood samples were collected from five chickenpopulations including Betwil (BTW 119899 = 21) from ElDilling Locality in South Kordofan State and Large Beladiof Bhari (LBB 119899 = 12) was collected from Khartoum NorthLocality Large Beladi of Abu-Neama (LB 119899 = 22) and BareNeck (BRN 119899 = 12) were both collected from Abu-NeamaLocality in Sinnar State The fifth population was (SUD 119899 =14) from Malakal Locality this sample was retrieved fromInternational livestock Research Institute (ILRI) data base Inaddition to a commercial strain of egg line White Leghorntype (COML 119899 = 9) was genotyped concurrently with theSudanese chickens

Genetic Analysis DNA was extracted using the ChloroformMethod [16] PCR was performed in 30 120583L reaction contain-ing 25mM of each dNTPs 14 pmol of each primer 15mMMgCl

2 1 times PCR buffer 125U Taq DNA polymerase (Roche

Applied Sciences Germany or Promega Madison USA)and 1 120583L Genomic DNA The mtDNA D-loop (HV1) regionwas amplified using specific primers based on the partialchicken mitochondrial genome GenBank accession number(AB098668) and complete chicken mitochondrial genomeGenBank accession number (NC 001323) (Table 1) PCRamplification was carried out on a Gene Amp PCR 9700(Applied Biosystems) thermo cycler PCR conditions wereas follows initial denaturation at 94∘C for 2min followedby 10 cycles at 94∘C for 15 s 58∘C for 30 s and 72∘C for40 s The amplified fragments were electrophoreses on 15agarose gel at 100 volts for one hour gel was stained with04 120583LmL ethidium bromide and detected under UV lightPCR products were purified using QIAquick PCR purifica-tion kit (QIAGEN GmbH Germany) Direct sequence ofHV1 segment of D-loop region was performed using two

Table 1 Primers used for the amplification and sequencing of HVIsegment of the D-loop

Primer type Primer name 51015840 to 31015840 Sequence

PCR primerL16750 (A)lowast AGGACTACGGCTTGAAAAGCH547 (D) ATGTGCCTGACCGAGGAACCAGCR1b (E) CCATACACGCAAACCGTCTC

Sequencingprimer

CR-For (B) TCTATATTCCACATTTCTCCR- Rev (C) GCGAGCATAACCAAATGG

A D E B and E indicate annealing point of the primer

Table 2 Reference chicken haplotypes

Haplotype name Code of haplotype Sampling siteClade I AF128344lowast ChinaClade II AB009436lowast Lombok Island IndonesiaClade IIIa FL17 ThailandClade IIIb DW07 ChinaClade IIIc DW02 ChinaClade IIId DC15 ChinaClade IV PKD15 Pakistan

internal primers CR-for and CR-rev as shown in Table 1 Thesequence was done using the BioDye Terminator version 31cycle sequencer kit (Applied Biosystems) with total volume of25 120583L comprising 20 ng of purified PCR product as templateDNA and 32 pmol of primer

22 The mtDNA Data Analysis The mtDNA sequence forthe first nucleotides (600 bp) of D-loop region [17] wasused for analysis after editing the sequences of amplified D-loop fragments Multiple alignments of the sequences wereperformed using ClustalX 183 [18] and Muscle 352 [19]programs

23 Phylogenetic and Molecular Evolution Analysis The evo-lutionary relationships of indigenous Sudanese chicken hap-lotypes were established using the molecular evolutionarygenetics analysis (MEGA) version 30 [20] Genetic distancesof the haplotypes were calculated using Kimurarsquos two param-etersmodel to construct a neighbor joining phylogenetic treeTwoD-loop sequences ofGalluswere included one fromwildjungle fowl Gallus gallus gallus and the other from Gallusgallus bankiva GenBank accession (number AB007720 andAB007718) respectively and seven Clade I II IIIa IIIb IIIcIIId and IV reference sequences that correspond to differentclades determined previously in Asian samples [10] (Table 2)

24 Haplotypes Diversity Haplotype diversity was illustratedusing network analysis implemented by NETWORK 4108[15] The DNA D-loop sequences diversity indices weredetermined to elucidate the sequence polymorphism andthe content of genetic variability in chicken populationThe populations indices include number of segregation sites(119878) number of haplotypes (119867) haplotype diversity (Hd)and nucleotide diversity (120587) as explained by Nei [21] The

Genetics Research International 3

analysis was conducted using DnaSP software version 40[22] Alignment gaps arising from a deletion event wereexcluded from the calculations The average number ofnucleotides differences per site between the two sequencesknown as nucleotide diversity (120587) is defined as 120587 = 119899(119899 minus1)Σ119909119894119909119895120587119894119895

or 120587 = Σ120587119894119895119899119888where 119899 is the number of

DNA sequences examined 119909119894and 119909

119895are the frequencies

of the 119894th and 119895th type of DNA sequences respectivelyin the sample 120587

119894119895is the proportion of nucleotides in the

respective types ofDNA sequences and 119899119888is the total number

of sequence comparisons [21] Average heterozygosity orhaplotype diversity ℎ is defined according to the formula ofNei [21] ℎ = 2119899(1 minus Σ119909

119894

2)(2119899 minus 1) where 119909

119894is the frequency

of haplotype and 119899 is the sample size The degree of geneticdifferentiation among the population was estimated usinggene or haplotype frequencies Population genetic structurewas investigated by119865st significance test [23] and119873st [24] usingArlequin software version 2000 [25]

25 Analysis of Molecular Variance (AMOVA) Maternalgenetic differentiationwas further quantified using hierarchi-cal analysis of molecular variance AMOVA [24] performedusingArlequin version 2000 software [25] Sudanese chickenpopulation was first considered as one single population andlater it was subdivided into two geographical areas rich andpoor Savanna

3 Results

31 Pattern of mtDNA D-Loop Variability The pattern of600 bp mtDNA D-loop variability revealed high variationsbetween nucleotide 212 and 397 Fourteen haplotypes wereidentified in the Sudanese and Southern Sudan indigenouschicken populations with variation at 19 sites and 235 ofthem are polymorphic (Figure 1)

Multiple sequence alignment was performed for thefourteen haplotypes identified in the Sudanese and SouthernSudan indigenous chicken Alignment of D-loop sequenceswas done to a reference sequence from Gene Bank acces-sion number (AB 098668) using Clustal-X 183 Two unitsof invariant tetradecamer 51015840-AACTATGAATGGTT-31015840 weredetected at positions 267 to 280 and 328 to 341 In the firstunit there were two transitions observed GA for SUD 71and TA for LB41 at position 268 and 272 respectively whilein the second unit of the tetradecamer TC transition wasobserved for SUD 40 and SUD 13 at position 330 In additionto that the following domains and motif were observed atthe 51015840 end of the D-loop An interrupted thymine string(AATTTTATTTTTT) was observed and found to be con-served in all the individuals studied There was also an inter-rupted poly-C sequence (51015840-CCCCCCCTTTCCCCCCC-31015840)which is widely conserved and downstream to this there isconserved sequence known as poly-G (51015840-AGGGGGGGT-31015840) Two conserved 51015840-TACAT-31015840 and 51015840-TATAT-31015840 were alsofound in all individuals There are six TATATmotifs and twoTACAT found within the 397 bases of the D-loop and werealso conserved The first 166 base pairs adjacent to tRNA Gluwere found to be highly conserved in all individuals except for

GTATCATACCTGTTTTCATTTCTACAC

GCCCCTCTCA

GCGCCCTCTC

CGCCCCTCTC

GGCCCCTCTC

GCCCCTAACTC

GCCCCTCTC

GCCCCTCTCT

TGCCCCTCTCT

GCACCCTCTC

GCACCCTCTCG

GCCCCCTCTC

CCCCTCTC

GCCCCTCC

GTCCCCCTGCTC

CTGTCCCCOML86

SUD71

BRN62

BRN48

LBB56

BTW4

BTW1

SUD40

SUD13

LBB49

LB41

LBB82

LB45

LB43

BTW14

Ref

011122222222222222222333333367911112333445566779111349377902785468364618266057021

Figure 1 Pattern of mtDNA D-loop variability Note nucleotidepolymorphisms observed in D-loop HV1 domain of 81 chickensequences Vertically oriented numbers indicate the site position andthe sequences shown are only the variable sites Dots () indicateidentity with the reference sequence and different base letters denotesubstitution

Table 3 Nucleotide substitutions in D-loop HVI region of indige-nous Sudanese and Southern Sudanese chicken

Substitution Variable sitesGT 33CA 317 and 391AG 177 212 218 238 268 and 342CT 167 199 210 217 234 236 254 310 315 and 330

one substitution a TG transversions in SUD 40 at position 33(data not shown)

The nucleotide substitutions found in the 14 variablehaplotypes comprised one GT and two CA transversionsand the rest were all transitions of which six were AG sub-stitutions and ten were CT substitutions This demonstratesa strong bias towards transition The CT substitutions aremore common than AG substitution (Table 3)

32 Phylogenetic Analysis of the Haplotypes A neighborjoining dendrogram showed the genetic relationships amongthe twelve haplotypes identified in Sudanese indigenouschicken from Sudan The egg line commercial strain chickenwas included and two haplotypes of genus Gallus (Gallusgallus gallus and Gallus gallus bankiva GenBank accessionnumber AB007720 and AB007718 resp) were retrieved fromGenBank and used as out groups and seven clade referencehaplotypes (Clade I II IIIa IIIb IIIc IIId and IV) werealso included The dendrogram revealed that 11 haplotypes


identified in the Sudanese indigenous chicken were placedinto two clusters with the domestic chicken Gallus gallusgallus This indicates a very close relationship between theSudanese indigenous chickens and Gallus gallus gallus whilethey are relatively genetically distanced from Gallus gallusbankiva Alignment with the reference lineage haplotypesfrom Asia showedthat all Sudanese indigenous chicken weregrouped into clade IV (Figure 2(a)) while the commercialegg line strainstudied concurrentlywith Sudanese indigenouschicken fell into clade IIIc When 3 haplotypes from UpperNile State of the Southern Sudan werecombined with 11haplotypes from the Sudan and aligned together again withGallus gallus gallus and Gallus gallus bankiva the dendro-gram constructed placed Sudanese chicken haplotypes intothree clusters with thedomestic haplotype from GenbankThis oncemore suggest that the Sudanese indigenous chickenis more closely related to Gallus gallus gallus while they arerelatively genetically distanced from Gallus gallus bankivaAlignment with reference haplotypes from Asia resultedin constructed neighbor-joining tree which grouped theSudanese indigenous chicken into two clade IV and cladeIIIa Figure 2(b)

33 Network Analysis Median-joining networks were drawnfor the 12 haplotypes identified from the Sudanese indige-nous chickens from Northern Sudan and one haplotypeof commercial layers based on the variable characters ofthe complete alignment using the computer program NET-WORK 4108 [15] The results showed that DNA sequenceof haplotype LBB49 has the highest frequencies and thishaplotype is connected to the frequencies of other haplotypesforming star-like connections with LBB49 in the centre Itwas also observed that there are mutational links to tenhaplotypes which include five singletons Therefore it can bereferred to as an interior or ancestry haplotype No medianvector (mv)lowast separating the clade was observed all the elevenhaplotypes identified in the Sudan region belong to clade IVmarked with yellow color as shown in Figure 3(a) while thecommercial egg line chicken haplotype belongs to differentclade which IIIc marked with green color and has sevenand eight mutation connection with BRN62 and LBB49respectively

Median-joining network analysis was carried out with thehaplotypes from the Southern States of the Sudan and theNorthern Sudan States The results illustrate that out of the14 identified Sudanese haplotypes only one haplotype (SUD71) from the South Sudan (Malakal) showed uniqueness Itfell into a different clade (IIIa) marked with red color whiletwo haplotypes (SUD13 and SUD40) are both sharing clade1V with other haplotypes from the Sudan regionmarked withyellow color Figure 3(b)

34 Population Diversity The diversity indices were calcu-lated for the five Sudanese indigenous chicken populationsfrom 81 D-loop sequences and 19 segregating sites The high-est number of haplotype (119867 = 6) was found in Large BeladiAbu-Neama populations from Sinnar State followed byBeladi Malakal chicken from Upper Nile State with (119867 = 5)

Betwil Beladi Bahri and Bare-Neck had equal number ofhaplotypes (119867 = 4) and are regarded as the lowest haplo-type number The gene haplotype diversity (Hd) was high inBetwil population (Hd = 0724) followed by Beladi Malakalwhile it was lower in Beladi Bahri and Bare Neck popu-lation (Hd = 0455) However the average overall hap-lotype diversity was approximately (0577) for the 81 chickenhaplotypes The average nucleotide diversity detected for81 D-loop sequences of the indigenous Sudanese chickenpopulation was estimated to be 000282 substitutions persite However the highest nucleotide diversity was found inMalakal population (000603 = 120587) followed by Betwil pop-ulation (000259 = 120587) and Beladi Neama (= 120587000179) whileBeladi Bahri and Bare recorded lowest nucleotide diversity(000126 = 120587) (Table 4)

35 Genetic Differentiation 119865st and 119873st were computedusing DnSP version 40 [22] the average 119865st and 119873st weresimilar (0098) while Nm was 4622 for both approachesThis indicates that 98 of maternal genetic differentiationestimated in Sudanese indigenous chicken resulted fromvariation among populations while 902 was due to con-tribution by genetic divergences among individuals withinpopulations The highest genetic differentiation betweenpopulations observedwas between Betwil population and therest of the populations with119873st value ranging from 01493 to02450 while the rest of populations showed a relatively littlematernal genetic subdivisions Table 5

36 Analysis of Molecular Variance (AMOVA) Maternalgenetic differentiation within population and among popula-tion within the Sudanese chicken was quantified using hier-archal analysis of molecular variance AMOVA on Kimura-2-parameter distance considering Sudanese populations asone single groupThe genetic variationwithin populationwas886 and the genetic differentiation among the populationswas 114 When Sudanese chicken population was oncemore subdivided into two geographical groups rich andpoor Savanna the resulting variation was partitioned intothree fractions The variation between individuals withinpopulations was 955 between populations within groupswas 075 and the genetic variation between groups was375 (Table 6)

4 Discussion

The complete alignment revealed a very high variabilityin the mtDNA D-loop region between 167ndash391 bases thisvariation constitutes 235 of the 81 sequences This rate isextremely high compared to the native chicken breeds 554ndash705 [11 12] Similarly the results were higher 1259 thanthose of 398African domestic chickens from 12 countries [10]This high rate mtDNA D-loop variation may be attributedto migration and exploratory movement of human intoSudan being as the largest country in Africa sharing borderswith nine countries The base composition of the Sudanesedomestic chicken D-loop HVI shows that A + T sequencecontent constitutes 5046 while G + C was 4954 similar


LB4179325016

50

52

66

39

22

9659

92 90

88

6271

LBB49LBB82LB43LB45BTW14

BRN62

LBB56BTW1

BTW4

BRN48

Clade IVClade I

Clade IIClade IIIa

Clade IIIbClade IIId

Clade IIIcCOML86

GallusgallusgallusAB007720BankivaAB007

0005

(a)

LB41

91

9663

8996

43

2663

6867

52

15506858

67

50

90

LBB82

LBB49

SUD13SUD40

SUD71

LB43

LB45BTW14

BRN62LBB56BTW1

BTW4BRN48

Clade IVClade I

Clade II

Clade IIIaClade IIIb

Clade IIIdClade IIIc

COML86GallusgallusgallusAB007720

BankivaAB

0005

(b)

Figure 2 (a) Neighbour-joining tree reconstructed usingMEGA 31 software from 11 haplotypes identified in Sudanese indigenous chickenstwo haplotypes of the genus Gallus retrieved from GenBank Gallus gallus gallus (GenBank accession number AB007720) and Gallus gallusbankiva (GenBank accession number AB007718) and seven clade reference haplotypes (Clade I II IIIa IIIb IIIc IIId and IV)The numbersat the nodes represent the percentage bootstrap values for interior branches after 1000 replications (b) Neighbour-joining tree reconstructedusing MEGA 31 software from 14 haplotypes identified in Sudanese indigenous chickens two haplotypes of the genus Gallus retrieved fromGenBank Gallus gallus gallus (GenBank accession number AB007720) and Gallus gallus bankiva (GenBank accession number AB007718)and seven clade reference haplotypes (Clade I II IIIa IIIb IIIc IIId and IV) The numbers at the nodes represent the percentage bootstrapvalues for interior branches after 1000 replications

LB41

BTW14

COML86167

210217

225243

256261

310

238 342

238

391272

276

212

218

199177

BRN62

LBB49

LBB82

LBB56

BTW1

BTW4

LB43

LB45

BRN48238

(a)

167

225243

256261

310

217210

234

217

236

254

268

317

315212218276

33

330

391199

238238

342

236

177

272

COML86LB43

LB41

LBB49

LB45

LBB56

LBB82BTW4

BTW1

SUD13

SUD40BRN48

BRN62

BTW14

SUD71

(b)

Figure 3 (a) Median-joining network (120576 = 0) for 11 haplotypes of the northern Sudan indigenous chickens and one haplotype of commerciallayers The yellow circles refer to clade IV while the green circle refers to clade IIIc and the green circle denotes clade IIIc The size of thecircles is proportional to the frequency of the respective haplotypes and the numbers between the haplotype nodes refer to the positions ofthe nucleotide mutations compared to reference sequence (GenBank accession AB098668) (b) The yellow circles refer to clade IV and thegreen circle refers to clade IIIc while the red circle denotes clade IIIa


Table 4mtDNAD-loop sequence diversity indices in Sudanese andSouthern Sudanese chicken populations based on 397 nucleotides

Number Population 119873 119878 119867 Hd 120587

1 LB 22 6 6 047619 0001792 LBB 12 3 4 045455 0001263 SUD 14 10 5 059341 0006034 BRN 12 3 4 045455 0001265 BTW 21 3 4 072381 000259

Total 81119873 number of sequences used 119878 number of segregation sites119867 number ofhypostyles Hd haplotypes diversity 120587 nucleotide diversity

Table 5 Pair wise119873st and 119865st values between 5 chicken populationsbased on D-loop sequences

LB LBB SUD BRN BTWLB mdash 00018 00514 minus00108 01493LBB 00018 mdash 00536 minus00285 02141SUD 00511 00536 mdash 00573 01356BRN minus00108 minus000286 00572 mdash 02450BTW 01492 02139 01351 02447 mdashPair wise 119873st values were above the diagonal and Pair wise 119865st values werebelow the diagonal LBN large Beladi Neama LBB Large Beladi Bahri BRNBare neck and BTW Betwil

results were noted by Ruokonen and Kvist [26]The two unitsof invariant tetradecamer 51015840-AACTATGAATGGTT-31015840 whichwas observed in this study at positions 267 to 280 and328 to 341 were found to be conserved in most of the 14haplotypes identified in the Sudanese indigenous chickenexcept for 4 haplotypes These four haplotypes were variedby one base substitution The substitutions were SUD71with GA LB41 with TC transition at positions 268 and272 respectively in the first tetradecamer unit and SUD13and SUD40 both with TC transition at position 330 inthe second tetradecamer unit This type of tetradecamerduplication was also observed by Fumihito et al [3] andfound to be a specific trait for genus Gallus gallus gallusHowever this result indicates the close genetic relationshipbetween Sudanese indigenous chicken and genus Gallus Onthe other hand at the 51015840 end of the D-loop HVI domainan interrupted thymine string (AATTTTATTTTTT) aninterrupted poly-C (51015840-CCCCCCCTTTCCCCCCC-31015840) andpoly-G (51015840-AGGGGGGGT-31015840) were widely conserved in allthe individualsThese conserved features have beendescribedacross many avian species other thanGalliformThey includeStruthioniformes Falconiformes and Sphenisciformes [27]However the presence of the cytosines and guanines strings inproximately to each other in D-loop segment sequence of theSudanese indigenous chickenmakes the formation of a stablehairpin structure possible [28] The conserved sequencemotifs of TACAT and TATA were found in all domesticchicken of SudanThese types ofmotifs are described asTASstermination-associated sequences elements involved in thetermination of mtDNA synthesis [29] The presence of theTASs in both Galliformesand mammals may suggest strong

structural function of D-loop region of the two genera whilethe lack of variation in TASs among the Galliformes maybe due to the selective functional constraints Phylogeneticanalysis of the 14 haplotypes identified from the Sudaneseindigenous chicken illustrated evolutionary relationships

All Sudanese chicken population from the Northernstates fell into two clusters However when a second phyloge-netic tree was reconstructed including populations from theUpper Nile State (Malakal) in South Sudan the dendrogramconstructed placed the Sudanese indigenous chicken intothree clusters and two different cladesmeaning that Sudanesechicken came from two different maternal lineages out ofthe seven clades Despite the fact that some populationshave more than one haplotype yet they fell in the samematernal lineage that is all Sudanese different haplotypesfall in Clade IV except haplotype SUD 71 which fell in CladeIIIa this result may suggest that these populations sharedthe same maternal ancestor and that their descendants haveaccumulated mutations to become distinct lineages

Total nucleotide diversity among the Sudanese chickenwas found to be (000282 = 120587) nucleotide substitutionsper site it was higher in Malakal and Betwil populationswhile it was lower in Beladi Bahri and Bare neck populationsThis low nucleotide diversity in Large Beladi of Bhari andBare neck populations may indicate loss of gene diversity forthese populations whichmay be attributed to relatively recentpopulation bottleneck On the other hand high nucleotidediversity in Malakal and Betwil populations may suggestthat the populations are more ancient [11] Network analysisshowed that DNA sequence of the haplotype of Large Beladi(LBB49) has the highest frequency and connected with thelargest number forming a star-like structure Such pattern ofstructure was found for different species of birds includingRed winged Blackbird Red poll finches and Greenfinch[30 31] The analysis also revealed convergent or reversemutation among haplotypes LBB40 LBB56 and BTW1 Thisconvergentmutation is commonwhere there is heterogeneitydue to unequal mutation rate for all nucleotide sites Undersuch circumstance accumulation of mutations at a smallnumber of fast sites leads to reverse mutation [32] Moreoverthe network analysis showed that there is probably morethan one maternal origin of Sudanese indigenous chickenpopulations as one haplotype from Malakal population fellinto a different (clade IIIa)

The hierarchical analysis of molecular variance AMOVAand 119865st or 119873st significance test indicate that 98 of mater-nal genetic differentiation in Sudanese indigenous chickenpopulations resulted fromvariation among populationswhile902 was due to contribution by genetic divergence amongindividual within population The highest observed geneticdifferentiation between populations was between Betwil pop-ulation and the rest of the populations followed by MalakalpopulationThe level of119865st value found in this study is close tothe value reported in African cattle breeds (119865st = 0060) [33]but smaller than that reported among 78 Chinese indigenouschicken breeds 119865st = 0106 [34] When using hierarchicalanalysis as a second tool to give more insight into geneticdifferentiation between individuals within the populationand to confirm119873st results the Sudanese chicken population


Table 6 Analysis of molecular variance (AMOVA) based on partial D-loop sequences of Sudanese and Southern Sudanese indigenouschicken populations

Samples Number of groups Number of populationsVariation ()

Withinpopulations

Amongpopulationswithin group

Among groups

All 81 chicken populations 2 5 955 075 375All 5 chicken populations as one group 1 5 886 1141 mdashRich Savanna 1 2 983 17 mdashPoor Savanna 1 3 9241 759 mdashAverage 119865st 009752 Nm 463

was first defined into two geographical groups or regions richand poor SavannaThe genetic variation between individualswithin populations was 955 and that occurring betweenpopulations within the groups was 075 while the geneticvariation between groups accounted for 375 The lowgenetic variation between the groups that were definedgeographically may suggest weak phylogeographic structureof Sudanese chicken and may be an indicator of commonmaternal origin When considering Sudanese chicken popu-lation as one single population group the genetic variationwithin the population accounted for 886 of the totalvariance while the proportion between populations was114 This indicates that the Sudanese chicken populationsare genetically differentiated along geographical localitiesFinally the study concludes that the region of mtDNA D-loop HVI which ranges from 167 to 397 has higher variationamong Sudanese domestic chicken population

Conflict of Interests

The authors declare that there is no conflict of interest in thisresearch

Acknowledgments

The authors acknowledge Dr Oliver Hanotte and Dr HanJianlin in the International Livestock Research Institute(ILRI) for their assistance to carry out the practical part ofthis work in their labs

References

[1] I A Yousif Phenotypic and genetic variation in body weight ofthe indigenous chicken [MS thesis] University of KhartoumKhartoum Sudan 1987

[2] M E F Suleiman Egg characteristics genetic and phenotypicrelationships of body weight at various ages in indigenous chicken[MS thesis] University of Khartoum Khartoum Sudan 1996

[3] A Fumihito T Miyake S Sumi M Takada S Ohno and NKondo ldquoOne subspecies of the red junglefowl (Gallus gallusgallus) suffices as the matriarchic ancestor of all domesticbreedsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 91 no 26 pp 12505ndash12509 1994

[4] W S Moore ldquoInferring phylogenies from mitochondrial DNAvariation mitochondrial-gene trees versus nuclear-gene treesrdquoEvolution vol 49 no 4 pp 718ndash726 1995

[5] R T Loftus D E MacHugh D G Bradley P M Sharp and PCunningham ldquoEvidence for two independent domesticationsof cattlerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 50 no 7 pp 672ndash679 1994

[6] P Savolainen Y P Zhang J Luo J Lundeberg and T LeitnerldquoGenetic evidence for an East Asian origin of domestic dogsrdquoScience vol 298 no 5598 pp 1610ndash1613 2002

[7] T Jansen P FosterMA Levine et al ldquoMitochondrialDNAandthe origins of the domestic horserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 11 pp424ndash429 2002

[8] G Luikart L Gielly L Excoffier J-D Vigne J Bouvet andP Taberlet ldquoMultiple maternal origins and weak phylogeo-graphic structure in domestic goatsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 98 no10 pp 5927ndash5932 2001

[9] S Clegg P Hale and CMoritz ldquoMolecular population geneticsof the red kangaroo (Macropus rufus) mtDNA variationrdquoMolecular Ecology vol 7 no 6 pp 679ndash686 1998

[10] A V Mobegi Genetic characterization of African chicken usingmitochondrial DNAD-loop sequences [MS thesis] University ofNairobi Nairobi Kenya 2006

[11] Z G Liu C Z Lei J Luo et al ldquoGenetic variability of mtDNAsequences in chinese native chicken breedsrdquoAsian-AustralasianJournal of Animal Sciences vol 17 no 7 pp 903ndash909 2004

[12] D Niu Y Fu J Luo et al ldquoThe origin and genetic diversity ofChinese native chicken breedsrdquo Biochemical Genetics vol 40no 5-6 pp 163ndash174 2002

[13] W M Brown E M Prager A Wang and A C WilsonldquoMitochondrial DNA sequences of primates tempo and modeof evolutionrdquo Journal of Molecular Evolution vol 18 no 4 pp225ndash239 1982

[14] D A Clayton ldquoReplication of animal mitochondrial DNArdquoCell vol 28 no 4 pp 693ndash705 1982

[15] H J Bandelt P Foster and A Rohl ldquoMedian-joining networksfor inferring intraspecific phylogeniesrdquo Molecular Biology andEvolution vol 16 no 1 pp 37ndash38 1999

[16] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratoryManual Cold SpringHarbor Laboratory Press ColdSpring Harbor NY USA 1989


[17] D M Buehler and A J Baker ldquoCharacterization of the red knot(Calidris canutus) mitochondrial control regionrdquo Genome vol46 no 4 pp 565ndash572 2003

[18] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[19] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[20] S Kumar K Tamura andMNei ldquoMEGA3 integrated softwarefor Molecular Evolutionary Genetics Analysis and sequencealignmentrdquo Briefings in Bioinformatics vol 5 no 2 pp 150ndash1632004

[21] M Nei Molecular Evolution Genetics Columbia UniversityPress New York NY USA 1987

[22] J Rozas J C Sanchez-DelBarrio X Messeguer and R RozasldquoDnaSP DNA polymorphism analyses by the coalescent andother methodsrdquo Bioinformatics vol 19 no 18 pp 2496ndash24972003

[23] S Wright ldquoThe genetical structure of populationsrdquo Annals ofEugenics vol 15 no 1 pp 323ndash354 1951

[24] L Excoffier P Smouse and J Quattro ldquoAnalysis of molecularvariance inferred from metric distances among DNA haplo-types application to human mitochondrial DNA restrictiondatardquo Genetics vol 131 no 2 pp 479ndash491 1992

[25] S SchneiderD Roessli and L ExcoffierArlequinVersion 2000Software for Population Genetics and Data Analysis Genet-ics and Biometry Laboratory University of Geneva GenevaSwitzerland 2000

[26] M Ruokonen and L Kvist ldquoStructure and evolution of theavian mitochondrial control regionrdquo Molecular Phylogeneticsand Evolution vol 23 no 3 pp 422ndash432 2002

[27] E Haring L Kruckenhauser A Gamauf J M Riesing and WPinsker ldquoThe complete sequence of the mitochondrial genomeof Buteo buteo (Aves Accipitridae) indicates an early split in thephylogeny of raptorsrdquoMolecular Biology and Evolution vol 18no 10 pp 1892ndash1904 2001

[28] T W Quinn and A C Wilson ldquoSequence evolution in andaround the mitochondrial control region in birdsrdquo Journal ofMolecular Evolution vol 37 no 4 pp 417ndash425 1993

[29] D R Foran J E Hixon and W M Brown ldquoComparisonsof ape and human sequences that regulate mitochondrialDNA transcription and D-loop DNA synthesisrdquo Nucleic AcidsResearch vol 16 no 13 pp 5841ndash5861 1988

[30] G Seutin L M Ratcliffe and P T Boag ldquoMitochondrialDNA homogeneity in the phenotypically diverse redpoll finchcomplex (Aves Carduelinae Carduelis flammea-hornemanni)rdquoEvolution vol 49 no 5 pp 962ndash973 1995

[31] JMerilaM Bjorklund andA J Baker ldquoHistorical demographyand present day population structure of the greenfinch Cardu-elis chlorismdashan analysis of mtDNA control-region sequencesrdquoEvolution vol 51 no 3 pp 946ndash956 1997

[32] J Wakeley ldquoSubstitution rate variation among sites in hyper-variable region 1 of human mitochondrial DNArdquo Journal ofMolecular Evolution vol 37 no 6 pp 613ndash623 1993

[33] E M Ibeagha-Awemu and G Erhardt ldquoGenetic structureand differentiation of 12 African Bos indicus and Bos tauruscattle breeds inferred from protein andmicrosatellite polymor-phismsrdquo Journal of Animal Breeding and Genetics vol 122 no1 pp 12ndash20 2005

[34] L J Qu X Y Li G E Xu et al ldquoEvaluation of genetic diversityin Chinese indigenous chicken breeds using microsatellitemarkersrdquo Science in China Series C Life Sciences vol 49 no 4pp 33ndash41 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


12 countries was carried out using mtDNA D-loop regionand HVI domain and it was found that the polymorphicsites account for 1259 of the 397 sequenced base pairfragment [10] while variation rates among 25 individualsfrom six native Chinese chicken populations were recordedto be 705 and 554 respectively [11 12] Advantages ofthe mtDNA are that it plays a role in metabolism apoptosisdisease and aging and it is the site of oxidative phospho-rylation essential for the production of ATP and a varietyof other biochemical functions It is highly polymorphiccompared to the nuclear DNA evolutionary rate being 5ndash10times faster than nuclear genome [13] probably due to lackof replication repair mechanism [14] The understanding ofphylogeography will elucidate the demographic history ori-gin and expansion of livestock species Networks analysis hassupplemented phylogenetic trees to overcome the problem ofparallel mutations and lineage exchange between divergentpopulations [15] Therefore the aim of the present study is toevaluate the genetic variability within and between Sudanesenative chickens using mtDNA D-loop region

2 Materials and Methods

21 Experimental Animals The study was conducted in fourstates of Sudan chosen because they have only indigenouschicken and no exotic breeds have been introduced Atotal of 81 blood samples were collected from five chickenpopulations including Betwil (BTW 119899 = 21) from ElDilling Locality in South Kordofan State and Large Beladiof Bhari (LBB 119899 = 12) was collected from Khartoum NorthLocality Large Beladi of Abu-Neama (LB 119899 = 22) and BareNeck (BRN 119899 = 12) were both collected from Abu-NeamaLocality in Sinnar State The fifth population was (SUD 119899 =14) from Malakal Locality this sample was retrieved fromInternational livestock Research Institute (ILRI) data base Inaddition to a commercial strain of egg line White Leghorntype (COML 119899 = 9) was genotyped concurrently with theSudanese chickens

Genetic Analysis DNA was extracted using the ChloroformMethod [16] PCR was performed in 30 120583L reaction contain-ing 25mM of each dNTPs 14 pmol of each primer 15mMMgCl

2 1 times PCR buffer 125U Taq DNA polymerase (Roche

Applied Sciences Germany or Promega Madison USA)and 1 120583L Genomic DNA The mtDNA D-loop (HV1) regionwas amplified using specific primers based on the partialchicken mitochondrial genome GenBank accession number(AB098668) and complete chicken mitochondrial genomeGenBank accession number (NC 001323) (Table 1) PCRamplification was carried out on a Gene Amp PCR 9700(Applied Biosystems) thermo cycler PCR conditions wereas follows initial denaturation at 94∘C for 2min followedby 10 cycles at 94∘C for 15 s 58∘C for 30 s and 72∘C for40 s The amplified fragments were electrophoreses on 15agarose gel at 100 volts for one hour gel was stained with04 120583LmL ethidium bromide and detected under UV lightPCR products were purified using QIAquick PCR purifica-tion kit (QIAGEN GmbH Germany) Direct sequence ofHV1 segment of D-loop region was performed using two

Table 1 Primers used for the amplification and sequencing of HVIsegment of the D-loop

Primer type Primer name 51015840 to 31015840 Sequence

PCR primerL16750 (A)lowast AGGACTACGGCTTGAAAAGCH547 (D) ATGTGCCTGACCGAGGAACCAGCR1b (E) CCATACACGCAAACCGTCTC

Sequencingprimer

CR-For (B) TCTATATTCCACATTTCTCCR- Rev (C) GCGAGCATAACCAAATGG

A D E B and E indicate annealing point of the primer

Table 2 Reference chicken haplotypes

Haplotype name Code of haplotype Sampling siteClade I AF128344lowast ChinaClade II AB009436lowast Lombok Island IndonesiaClade IIIa FL17 ThailandClade IIIb DW07 ChinaClade IIIc DW02 ChinaClade IIId DC15 ChinaClade IV PKD15 Pakistan

internal primers CR-for and CR-rev as shown in Table 1 Thesequence was done using the BioDye Terminator version 31cycle sequencer kit (Applied Biosystems) with total volume of25 120583L comprising 20 ng of purified PCR product as templateDNA and 32 pmol of primer

22 The mtDNA Data Analysis The mtDNA sequence forthe first nucleotides (600 bp) of D-loop region [17] wasused for analysis after editing the sequences of amplified D-loop fragments Multiple alignments of the sequences wereperformed using ClustalX 183 [18] and Muscle 352 [19]programs

23 Phylogenetic and Molecular Evolution Analysis The evo-lutionary relationships of indigenous Sudanese chicken hap-lotypes were established using the molecular evolutionarygenetics analysis (MEGA) version 30 [20] Genetic distancesof the haplotypes were calculated using Kimurarsquos two param-etersmodel to construct a neighbor joining phylogenetic treeTwoD-loop sequences ofGalluswere included one fromwildjungle fowl Gallus gallus gallus and the other from Gallusgallus bankiva GenBank accession (number AB007720 andAB007718) respectively and seven Clade I II IIIa IIIb IIIcIIId and IV reference sequences that correspond to differentclades determined previously in Asian samples [10] (Table 2)

24 Haplotypes Diversity Haplotype diversity was illustratedusing network analysis implemented by NETWORK 4108[15] The DNA D-loop sequences diversity indices weredetermined to elucidate the sequence polymorphism andthe content of genetic variability in chicken populationThe populations indices include number of segregation sites(119878) number of haplotypes (119867) haplotype diversity (Hd)and nucleotide diversity (120587) as explained by Nei [21] The










119894

2)(2119899 minus 1) where 119909




3 Results




GCCCCTCTCA

GCGCCCTCTC

CGCCCCTCTC

GGCCCCTCTC

GCCCCTAACTC

GCCCCTCTC

GCCCCTCTCT

TGCCCCTCTCT

GCACCCTCTC

GCACCCTCTCG

GCCCCCTCTC

CCCCTCTC

GCCCCTCC

GTCCCCCTGCTC

CTGTCCCCOML86

SUD71

BRN62

BRN48

LBB56

BTW4

BTW1

SUD40

SUD13

LBB49

LB41

LBB82

LB45

LB43

BTW14

Ref

011122222222222222222333333367911112333445566779111349377902785468364618266057021















4 Discussion



LB4179325016

50

52

66

39

22

9659

92 90

88

6271


BRN62

LBB56BTW1

BTW4

BRN48

Clade IVClade I

Clade IIClade IIIa


Clade IIIcCOML86


0005

(a)

LB41

91

9663

8996

43

2663

6867

52

15506858

67

50

90

LBB82

LBB49

SUD13SUD40

SUD71

LB43

LB45BTW14

BRN62LBB56BTW1

BTW4BRN48

Clade IVClade I

Clade II




BankivaAB

0005

(b)


LB41

BTW14

COML86167

210217

225243

256261

310

238 342

238

391272

276

212

218

199177

BRN62

LBB49

LBB82

LBB56

BTW1

BTW4

LB43

LB45

BRN48238

(a)

167

225243

256261

310

217210

234

217

236

254

268

317

315212218276

33

330

391199

238238

342

236

177

272

COML86LB43

LB41

LBB49

LB45

LBB56

LBB82BTW4

BTW1

SUD13

SUD40BRN48

BRN62

BTW14

SUD71

(b)





1 LB 22 6 6 047619 0001792 LBB 12 3 4 045455 0001263 SUD 14 10 5 059341 0006034 BRN 12 3 4 045455 0001265 BTW 21 3 4 072381 000259












Withinpopulations


Among groups





Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










119894

2)(2119899 minus 1) where 119909




3 Results




GCCCCTCTCA

GCGCCCTCTC

CGCCCCTCTC

GGCCCCTCTC

GCCCCTAACTC

GCCCCTCTC

GCCCCTCTCT

TGCCCCTCTCT

GCACCCTCTC

GCACCCTCTCG

GCCCCCTCTC

CCCCTCTC

GCCCCTCC

GTCCCCCTGCTC

CTGTCCCCOML86

SUD71

BRN62

BRN48

LBB56

BTW4

BTW1

SUD40

SUD13

LBB49

LB41

LBB82

LB45

LB43

BTW14

Ref

011122222222222222222333333367911112333445566779111349377902785468364618266057021















4 Discussion



LB4179325016

50

52

66

39

22

9659

92 90

88

6271


BRN62

LBB56BTW1

BTW4

BRN48

Clade IVClade I

Clade IIClade IIIa


Clade IIIcCOML86


0005

(a)

LB41

91

9663

8996

43

2663

6867

52

15506858

67

50

90

LBB82

LBB49

SUD13SUD40

SUD71

LB43

LB45BTW14

BRN62LBB56BTW1

BTW4BRN48

Clade IVClade I

Clade II




BankivaAB

0005

(b)


LB41

BTW14

COML86167

210217

225243

256261

310

238 342

238

391272

276

212

218

199177

BRN62

LBB49

LBB82

LBB56

BTW1

BTW4

LB43

LB45

BRN48238

(a)

167

225243

256261

310

217210

234

217

236

254

268

317

315212218276

33

330

391199

238238

342

236

177

272

COML86LB43

LB41

LBB49

LB45

LBB56

LBB82BTW4

BTW1

SUD13

SUD40BRN48

BRN62

BTW14

SUD71

(b)





1 LB 22 6 6 047619 0001792 LBB 12 3 4 045455 0001263 SUD 14 10 5 059341 0006034 BRN 12 3 4 045455 0001265 BTW 21 3 4 072381 000259












Withinpopulations


Among groups





Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









4 Discussion



LB4179325016

50

52

66

39

22

9659

92 90

88

6271


BRN62

LBB56BTW1

BTW4

BRN48

Clade IVClade I

Clade IIClade IIIa


Clade IIIcCOML86


0005

(a)

LB41

91

9663

8996

43

2663

6867

52

15506858

67

50

90

LBB82

LBB49

SUD13SUD40

SUD71

LB43

LB45BTW14

BRN62LBB56BTW1

BTW4BRN48

Clade IVClade I

Clade II




BankivaAB

0005

(b)


LB41

BTW14

COML86167

210217

225243

256261

310

238 342

238

391272

276

212

218

199177

BRN62

LBB49

LBB82

LBB56

BTW1

BTW4

LB43

LB45

BRN48238

(a)

167

225243

256261

310

217210

234

217

236

254

268

317

315212218276

33

330

391199

238238

342

236

177

272

COML86LB43

LB41

LBB49

LB45

LBB56

LBB82BTW4

BTW1

SUD13

SUD40BRN48

BRN62

BTW14

SUD71

(b)





1 LB 22 6 6 047619 0001792 LBB 12 3 4 045455 0001263 SUD 14 10 5 059341 0006034 BRN 12 3 4 045455 0001265 BTW 21 3 4 072381 000259












Withinpopulations


Among groups





Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


LB4179325016

50

52

66

39

22

9659

92 90

88

6271


BRN62

LBB56BTW1

BTW4

BRN48

Clade IVClade I

Clade IIClade IIIa


Clade IIIcCOML86


0005

(a)

LB41

91

9663

8996

43

2663

6867

52

15506858

67

50

90

LBB82

LBB49

SUD13SUD40

SUD71

LB43

LB45BTW14

BRN62LBB56BTW1

BTW4BRN48

Clade IVClade I

Clade II




BankivaAB

0005

(b)


LB41

BTW14

COML86167

210217

225243

256261

310

238 342

238

391272

276

212

218

199177

BRN62

LBB49

LBB82

LBB56

BTW1

BTW4

LB43

LB45

BRN48238

(a)

167

225243

256261

310

217210

234

217

236

254

268

317

315212218276

33

330

391199

238238

342

236

177

272

COML86LB43

LB41

LBB49

LB45

LBB56

LBB82BTW4

BTW1

SUD13

SUD40BRN48

BRN62

BTW14

SUD71

(b)





1 LB 22 6 6 047619 0001792 LBB 12 3 4 045455 0001263 SUD 14 10 5 059341 0006034 BRN 12 3 4 045455 0001265 BTW 21 3 4 072381 000259












Withinpopulations


Among groups





Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




1 LB 22 6 6 047619 0001792 LBB 12 3 4 045455 0001263 SUD 14 10 5 059341 0006034 BRN 12 3 4 045455 0001265 BTW 21 3 4 072381 000259












Withinpopulations


Among groups





Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Withinpopulations


Among groups





Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Molecular Characterization of Sudanese ...downloads.hindawi.com/journals/gri/2014/928420.pdf · Research Article Molecular Characterization of Sudanese and Southern