research article genetic dissection of new genotypes of...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 604598 6 pageshttpdxdoiorg1011552013604598

Research ArticleGenetic Dissection of New Genotypes of DrumstickTree (Moringa oleifera Lam) Using Random AmplifiedPolymorphic DNA Marker

Shamsuddeen Rufai1 M M Hanafi1 M Y Rafii1 S Ahmad2

I W Arolu1 and Jannatul Ferdous1

1 Food Crops Laboratory Institute of Tropical Agriculture Universiti Putra Malaysia43400 Serdang Selangor Malaysia

2 Department of Crop Science Faculty of Agriculture Universiti Putra Malaysia43400 Serdang Selangor Malaysia

Correspondence should be addressed to M M Hanafi mmhanafiagriupmedumy

Received 18 January 2013 Revised 12 March 2013 Accepted 27 March 2013

Academic Editor Kok Tat Tan

Copyright copy 2013 Shamsuddeen Rufai et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The knowledge of genetic diversity of tree crop is very important for breeding and improvement program for the purpose ofimproving the yield and quality of its produce Genetic diversity study and analysis of genetic relationship among 20 Moringaoleifera were carried out with the aid of twelve primers from random amplified polymorphic DNA marker The seeds of twentyM oleifera genotypes from various origins were collected and germinated and raised in nursery before transplanting to the field atUniversity Agricultural Park (TPU) Genetic diversity parameter such as Shannonrsquos information index and expected heterozygosityrevealed the presence of high genetic divergence with value of 180 and 013 for Malaysian population and 030 and 019 for theinternational population respectively Mean of Neirsquos gene diversity index for the two populations was estimated to be 020 Inaddition a dendrogram constructed using UPGMA cluster analysis based onNeirsquos genetic distance grouped the twentyM oleiferainto five distinct clusters The study revealed a great extent of variation which is essential for successful breeding and improvementprogram From this studyM oleifera genotypes of wide genetic origin such as T-01 T-06 M-01 and M-02 are recommended tobe used as parent in future breeding program

1 Introduction

Drumstick tree (Moringa oleifera Lam) a short to mediumheight tree with luxurious evergreen leaves was said to haveoriginated from Himalayan tract in northwestern part ofIndia [1ndash4] The tree has a true diploid chromosome 2n =28 with a distinguished tripinnate leaves having yellow orwhite petiole streaks [5 6] Moringa is potentially one of theplanetrsquos most valuable plants at least in humanitarian terms[7] and has been regarded as a wonder tree due to its greateconomic importance and uses [3 7] Its pods were reportedto have a protein content ranging from 20 to 30 with a highvitamin C content The moringa seeds were found to exhibitthe property of natural coagulantsflocculants which allowsfor growing of the tree for the purpose of usage by water and

sewage treatment plant to clear turbidity in drinking waterand sludge in sewage [8] Similarly the nutritive value of thisplant for animals has been documented by Mendieta-Araicaet al [1] who reported that moringa contains large amount ofcrude protein iron zinc and high concentration of vitaminsA B and C in its foliage sample which makes it a very goodfeed and fodder for animals to browse and graze upon [9]

With respect to oil quality M oleifera seed concentratecontains about 35ndash45 seed oil having odourless and colour-less physical properties [10]The edible oil is highly nutritiousand is extracted by boiling the seeds with water and collectingthe oil from the surface of the water [9 11] The seed oil hashigh concentration of oleic acid (gt73) coupled with lowpolyunsaturated fatty acid which gives the oil an outstandingand remarkable oxidative stability properties The suitability

2 BioMed Research International

ofM oleifera seed oil as biodiesel feed source has been testedand recommended by Da Silva et al [12] who reported thatthe oil could be used as pure biodiesel or petrodiesel mixtureon engine after converting it to fatty acid methyl esters(FAME) through the process of transesterification in thepresence of sodium hydroxide (NaOH) as catalyst

Moreover despite the great economic importance ofthis plant in terms of nutritional social and environmentalbenefits the genetic diversity pattern genetic makeup andagronomical requirement needed for successful breeding andimprovement domestication and large scale cultivation areyet to be established This obstacle is an impediment to asuccessful production and commercialization ofmoringa andits related products [6] Also the knowledge of genetic diver-sity of tree crop is very important for rational planning ofconventional modern breeding and improvement programfor the purpose of improving the yield and quality of itsproduce [9 13]

In other words the use of molecular markers such asinter-simple sequence repeat (ISSR) random amplified pol-ymorphic DNA (RAPD) and simple sequence repeat (SSR)has gained popularity as a genetic diversity assessment meth-ods of tree and oil seed crops [14ndash16] Molecular methods ofgenetic diversity study are a fast efficient reliable and simplemeans of establishing genetic diversity pattern in plant [17]The RAPD as one of the numerous molecular markers hasbeen reported to be a reliable reproducible cost effectivefast and less tedious marker which is widely used in thefield of plant breeding and molecular genetics due to itsoutstanding quality [18]

Therefore this research work will study the genetic diver-sity of twenty new genotypes of Moringa oleifera from twopopulations with the aim of studying genetic diversity patternin relation to their geographical origin and dissection ofgermplasms as a means of initiating the breeding programmein the nearest future

2 Materials and Methods

21 Plant Materials These were made up of seeds of twentynew genotypes of M oleifera collected from six differentcountries (Table 1)Themoringa genotypes prior to their col-lection were found in the wild growing in their natural formThe countries of origin are Virgin Island USA ThailandIndia Tanzania Taiwan and Malaysia The collection wasprincipally made by the Asia Vegetable Research and Devel-opment Center (AVRDC) orWorldVegetable Centre Taiwan(15 accessions classified as international population) and theInstitute of Tropical Agriculture universiti Putra Malaysia (5accessions classified as Malaysian population) The collectedseeds were germinated and raised in nursery UniversitiPutra Malaysia Agricultural Park (TPU) for two monthsexposed to the hardening process in the last ten days ofnursery then transplanted out to the Universityrsquos agriculturalexperimental farm in Puchong (02∘N590351015840 101∘E389131015840)Selangor Malaysia Young and disease-free leaves of Moleifera were collected for each of the genotypes during theearly hours of the day the leaves sample were wrapped inaluminum foil and labeled and kept in the freezer at minus10∘C

Table 1 List of moringa genotypes and their countries of origin

Serialnumber

GenotypesID Pedigreecultivar name Origin

country1 T01 Virgin Islands Drum Stick USA2 T02 Ma Rum01 Thailand3 T03 Ma Rum02 Thailand4 T04 Ma Rum03 Thailand5 T05 Ma Rum04 Thailand6 T06 Ma Rum05 Thailand7 T07 Ma Rum Khaw Nheaw Thailand8 T08 Ma Rum06 Thailand9 T09 Ma Rum07 Thailand10 T10 Ma Rum K Thailand11 T11 Tnau-1 India12 T12 Rca Moringa Tanzania13 T13 Ma Rum C Thailand14 T14 Drumstick Tree Pkm-1 India15 T15 La-MuW Taiwan16 M01 ITA-UPM01 Malaysia17 M02 ITA-UPM02 Malaysia18 M03 ITA-UPM03 Malaysia19 M04 ITA-UPM04 Malaysia20 M05 ITA-UPM05 Malaysia

22 DNA Extraction Protocol Six hundred 120583L of extractionbuffer (100mM of Tris-HCl 20mM of EDTA 14M NaCland 5 SDS) was added to 10mg leaf sample and groundwith mortar and pestle without liquid nitrogen accordingto Ferdous et al [19] The finely ground leaf tissue wastransferred into 2mL centrifuge tube Four hundred 120583L of2X CTAB solution 100mM of Tris-HCl 20mM of ethylene-diaminetetraacetic acid di-sodium salt (EDTA) 14M ofsodium chloride (NaCl) 2 (wv) CTAB and 1 (wv) ofpolyvinyl pyrrolidone (PVP) and 400 120583L chloroform isoamylalcohol phenol (24 1 5) mixture were then added to theleaf tissue containing the extraction buffer After mixingthrough vortex and centrifuge the supernatant was trans-ferred to another tube Two-third of volume of isopropanolwas added and incubated at room temperature for 10 to15min Centrifuged supernatant was then removed and theDNA pellet was washed using 70 ethanol afterwards theDNA pellet was air-dried and dissolved into 50 120583L TE buffer

23 DNA Quantification and Dilution The quantification ofDNAwas carried out using NanoDrop ND-1000 spectropho-tometer (NanoDrop Technologies Wilmington USA) TheDNAwas again requantified by running it through 1agarosegel electrophoresis with 1timesTAE buffer for 30min and viewedunder UV light after staining it withMidori green DNA stain(Nippon Genetics Inc Germany) The dilution was donewith sterile distilled water to ensure that all of the DNAsamples have equal concentration of 100 ng120583L

BioMed Research International 3

24 RAPD Polymerase Chain Reaction Procedure Accordingto the company instruction (Promega) 5120583L of 5X GreenGoTaq Flexi Buffer 3 120583L MgCl

2solution (25mM) 05 120583L

PCRnucleotidemix (10mMeach) 02120583Lprimers (04120583mol)and 10U ofTaqDNApolymerase were used for 25 120583L of PCRreaction including 1120583L DNA template directly used afterextraction [19] In RAPD analysis the following conditionwas used initial denaturation at 94∘C for 1min followed by45 cycles of denaturation done at 94∘C for 1min annealingwas done at 34∘C for 15min and extension was done at 72∘Cfor 2min and a final extension at 72∘C for 5min [20] Theamplified PCR products were subjected to electrophoresis on3 (wv) MetaPhor agarose gel at 75 volt for 70 minutes Thegel was stained with ethidium bromide and visualized underultraviolet (UV) light

25 Band Scoring The image of the gel acquired in JPEGformatwas imported intoUVIdoc 9902 for band scoringTheband sizes were estimated based on DNA ladder (PromegaInc) The absence and presence of band were scored in abinary model of 0 and 1 respectively Band scoring was car-ried out only on those bands that are clear and reproducibleand then those that are gt50 bp The data obtained at the endof the scoring was transferred and saved in Microsoft excelsheet

26 Data Analysis Data of the twelve primers were analyzedto obtain the information on genetic diversity of the 20moringa accessions (Table 2) Genetic similarity among thegenotypes and principal component analysis (PCA) werecalculated using NTSYS-pc 21 Cluster analysis was alsocarried out using the unweighted pair-group method witharithmetic average (UPGMA) based on the Neirsquos geneticdistancematrix and dendrogramwas drawn to show the clus-tering pattern of the different genotypes usingNTSYS-pcThepercentage polymorphismof the bands (PPB) effective alleles(ne) genetic diversity index (h) Shannonrsquos information index(I) and Neirsquos gene diversity were calculated using POPGEN131 software Analysis of molecular variance was conductedusing GeneAIEx 65 to partition the variation present in thegermplasm and at the same time test the variance componentfor RAPD phenotype

3 Results

31 Screening of Primers A total of 24 RAPD primers wereused to study the genetic diversity of twenty genotypes ofM oleifera (Figure 1) Out of these primers only 12 showedas distinct reproducible polymorphic bands A total of 108polymorphic fragments were generated by these 12 primerswith an average of 90

Genetic Diversity within the Two Populations The meanpercentage polymorphic loci in the two populations (inter-national and Malaysian) were calculated to be 7573 and3270 respectively (Table 3) The observed number of allelesin the two populations from Taiwan and Malaysia is 150 and071 respectively with 126 as the mean value for effective

Table 2 RAPD polymorphic primers and their sequence ID

No Synthesis ID Sequence 120583g nmol1 OPA 17 51015840-GAC CGC TTG T-31015840 101 3352 OPA 19 51015840-CAA ACG TCG G-31015840 93 3453 OPB 17 51015840-AGG GAA CGA G-31015840 85 2714 OPBC 10 51015840-AAC GTC GAG G-31015840 160 525 OPBD 18 51015840-ACG CAC ACT C-31015840 179 6066 OPBD 19 51015840-GGT TCC TCT C-31015840 181 6117 OPF 20 51015840-GAG GAT CCC T-31015840 95 3138 OPH 19 51015840-CTG ACC AGC C-31015840 100 3379 OPO 3 51015840-CTG TTG CTA C-31015840 103 34610 OPM 6 51015840-CTG GGC AAC T-31015840 100 32911 OPM 8 51015840-TCT GTT CCC C-31015840 110 37512 OPQ 2 51015840-TCT GTC GGT C-31015840 104 345

Table 3 Genetic diversity in M oleifera germplasm as revealed byRAPD

Population 119873 Na Ne 119868 He 119875()International 15000 1500 1308 0295 0188 7273Malaysia 5000 0709 1220 0184 0125 3273Grand total Mean 10000 1105 1264 0239 0156 5273

SE 0338 0065 0024 0018 0013 2000119873 number of genotypes per each populationsNa observed number of allelesNe effective number of alleles Kimura and Crow [21]119868 Shannonrsquos information index Lewontin [22]He expected heterozygosity119875() Percentage of polymorphism

alleles for the two countries With the Hardy Weinberg equi-librium assumption in place Shannonrsquos information indexand expected heterozygosity for Taiwan population were 03and 019 and those of Malaysian population were 018 and013 respectively while mean Neirsquos gene diversity for the twopopulations were estimated to be 020

Furthermore in order know the source of genetic varia-tion for theseMoringa genotypes RAPDprofile was analyzedusing AMOVAThis was aimed to partition all the sources ofvariation existing in the germplasm into two major groupsThe result revealed that 95 of the total genetic variationoccurred as a result of variation within the population whilevariation among the populations accounted for the remaining5 of the total genetic variance (Table 4) Also the geneticvariance among the population as indicated by the result(119865st = 016) was significant at 5 probability level whenpermutation test was conducted

32 Cluster Analysis Cluster analysis based on Jaccardrsquosgenetic similarity coefficient showed high level of geneticvariation among the genotypes from the two countries Thesimilarity coefficient ranged from 038 to 089 with T-11and T-15 genotypes found to have highest genetic similarity(089) while T-06 together with T-07 possessed least similar-ity coefficient (Figure 2)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20M

Figure 1 Gel picture of twenty genotypes ofMoringa oleifera RAPD profile

Table 4 Genetic divergence differentiation with analysis of molecular variance

Source Degree offreedom Sum square Mean square Estimated

variance variation () 119865st 119875 value

Among population 1 15733 15733 0558 5 0160 lt0001Within population 18 207867 11548 11548 95 lt0001Total 19 20 233600 12106 100

In addition a dendrogram was constructed usingUPGMA cluster analysis to show the genetic relationshipamong the twenty genotypes from different geographicalbackgrounds From this dendrogram the twenty genotypeswere grouped into five major clusters at a coefficient of063 Cluster III with highest number of members had 14genotypes followed by cluster I (T-01 and T-03) and clusterIV (M-01 andM-04) with two genotypes each Clusters II (T-07) and V (T-06) have one genotype each and were rankedthe least populated clusters (Table 5)

33 Principal Component Analysis Theprincipal componentanalysis (PCA) carried outwithRAPDM oleiferaprofile clas-sified the twenty genotypes ofMoringa into fivemajor groupswith two genotypes fromMalaysia occupying one groupThisgrouping pattern corresponds with that of clustering analysisas shown by the dendrogram Dimension one of the PCAranges from 049 to 092 while dimension two ranged fromminus065 to 026 and dimension three ranged from minus065 to 068(Figure 3)

4 Discussion

Effective and efficient genotyping of any plant speciesthrough RAPD requires a careful selection of suitable primercombination in order to get detail and informative resultHigh level of genetic polymorphism detected by these mark-ers is in agreementwith the assumption that outcrossing plantspecies from natural population will have higher level ofgenetic diversity when compared to in-breeding plantspecies This finding agrees with earlier report on similarout-crossing plant species such as Jatropha [23] and otheroil plant species High value of Shannonrsquos information

Table 5 Name of clusters and their corresponding genotypes

Cluster I T-01 T-03Cluster II T-07

Cluster III T-02 T-04 T-05 T-08 T-09 T-10 T-11 T-12 T-13T-14 T-15 M-2 M-03 M-04

Cluster IV M-01 M-04Cluster V T-06

index (0295) for international population as compared toMalaysian population (0184) suggests that members of thispopulation are more diverse This is also obvious from theway the accessions cluster together

Additionally high level of genetic differentiation in thesetwo populations as reflected by the genetic diversity parame-ters such as Shannonrsquos information index expected heterozy-gosity percentage polymorphism and others are pointing tothe fact that there is wild variability in these populations ofMoringa and this is very important for successful crossing andimprovement programs in future This observation follows asimilar trend with the result of genetic diversity study on 75accessions from Sudan and Guinea savanna zones of Nigeriawhere six polymorphic primers of RAPD origin gave a totalof 42 polymorphic bands [9]

Furthermore interaction between various ecological andbiological factors such as genetic drift gene flow selectionand mating system affects the genetic structure of any plantpopulations [24] The overall genetic variability and differen-tiation pattern observed in these M oleifera populations arein agreement with those of other outcrossing plant species[14 25 26] As shown by these results the two populations ofM oleifera exhibit moderate119865st value in order to demonstrate


038 050 063 076 089

T-01

T-01 T-03 T-07 T-02 T-09 T-04 T-05 T-08 T-10 M-02 T-11 T-15 T-14 T-12 T-13 M-03 M-05 M-01 M-04 T-06

Coefficient

I

II

III

IV

V

Figure 2 A dendrogram display of twenty accessions ofMoringa oleifera

M-05

M-04

M-03

M-02

M-01

T-15

T-14 T-13T-12

T-11T-10

T-09

T-08

T-07

T-06

T-05

T-04

T-03

T-02

T-01

026003

Dim-2049

060

Dim-3

022

Dim-1 070

045

068

081092

minus065

minus042

minus020

000

Figure 3 Three-dimensional principal components analysis of 20accessions ofMoringa oleifera

low significant genetic differentiation among the populationsHowever higher genetic differentiation and diversity wereobserved within the populations of M oleifera and thisindicates a relatively restricted variability as expected Thispattern of population structure has been previously reportedin other out crossing plant species [27 28]

Moreover clustering analysis showing wide range ofsimilarity coefficient showed that there is high level of geneticvariation within the two populations The M oleifera geno-types from the two populations were seen clustering togetherin the same group This shows that there is no any distinctrelationship between the geographical origin and the geneticdistance as shown in the dendrogram This finding implies

that the genetic divergence within and between these twopopulations could not be explained by their geographicaldistance This finding also means that isolation as a resultof distance cannot be said to have been responsible for thedivergence observed in these population [24]

In conclusion these findings have proven that geneticdivergence is very high in these populations and it can there-fore be inferred from the data that the Moringa populationswill be a very good germplasm material for the future breed-ing and improvement of this economically important treecrop Genotypes that are far apart based on their genetic simi-larity coefficient (like T-01 T-06 M-01 and M-02) should beselected for future breeding

Acknowledgments

The authors are grateful to Professor M C Palada and DrAndreas Ebert from the World Vegetable Centre (Asia Veg-etable Research and Development Center) Taiwan for theprovision of germplasm The authors also wish to expresstheir sincere gratitude to the Research Management Unit(RMC) of Universiti Putra Malaysia (UPM) for the fundingof this research and to the management of Bayero UniversityKano Nigeria for granting study fellowship to the SR

References

[1] B Mendieta-Araica E Sporndly N Reyes-Sanchez F Salm-eron-Miranda and M Halling ldquoBiomass production andchemical composition ofMoringa oleifera under different plant-ing densities and levels of nitrogen fertilizationrdquo AgroforestrySystems vol 87 no 1 pp 81ndash92 2012

[2] A Pandey K Pradheep R Gupta E Roshini Nayar andD C Bhandari ldquolsquoDrumstick treersquo (Moringa oleifera Lam) amultipurpose potential species in Indiardquo Genetic Resources andCrop Evolution vol 58 no 3 pp 453ndash460 2011


[3] H J von Maydell Trees and Shrubs of the Sahel Their Charac-teristics and Uses Verlag Josef Margraf 1990

[4] M R Berger M Habs S A A Jahn and D SchmahlldquoToxicological assessment of seeds from Moringa oleifera andMoringa stenopetala two highly efficient primary coagulants fordomestic water treatment of tropical raw watersrdquo East AfricanMedical Journal vol 61 no 9 pp 712ndash716 1984

[5] C Ramachandran K V Peter and P Gopalakrishnan ldquo(Mor-inga oleifera) economicrdquo Botany vol 34 pp 276ndash283 1980

[6] M G Mgendi M K Manoko and A M Nyomora ldquoGeneticdiversity between cultivated and non-cultivated Moringa oleif-era Lam provenances assessed by RAPD markersrdquo Journal ofCell and Molecular Biology vol 8 no 2 pp 95ndash102 2010

[7] National Research Council Lost Crops of Africa vol 1 NationalAcademy Press Washington DC USA 2006

[8] G S Madrona G B Serpelloni A M S Vieira L Nishi KC Cardoso and R Bergamasco ldquoStudy of the effect of salinesolution on the extraction of the Moringa oleifera seedrsquos activecomponent for water treatmentrdquoWater Air and Soil Pollutionvol 211 no 1ndash4 pp 409ndash415 2010

[9] B Y Abubakar R Wusirika S MuArsquozu A U Khan and A KAdamu ldquoDetection of genetic variability using random ampli-fied polymorphic DNA marker in some accession of Moringaoleifera Lam from Northern Nigeriardquo International Journal ofBotany vol 7 pp 237ndash242 2011

[10] R Ayerza (h) ldquoSeed and oil yields of Moringa oleifera varietyPeriyakalum-1 introduced for oil production in four ecosystemsof South Americardquo Industrial Crops and Products vol 36 no 1pp 70ndash73 2012

[11] M A Somali M A Bajneid and S S Al-Fhaimani ldquoChemicalcomposition and characteristics ofMoringa peregrina seeds andseeds oilrdquo Journal of the American Oil Chemistsrsquo Society vol 61no 1 pp 85ndash86 1984

[12] J P V Da Silva T M Serra M Gossmann C R Wolf MR Meneghetti and S M P Meneghetti ldquoMoringa oleifera oilstudies of characterization and biodiesel productionrdquo Biomassand Bioenergy vol 34 no 10 pp 1527ndash1530 2010

[13] MY Rafii IWAroluMHAOmar andMA Latif ldquoGeneticvariation and heritability estimation in Jatropha curcas L pop-ulation for seed yield and vegetative traitsrdquo Journal of MedicinalPlants Research vol 6 pp 2178ndash2183 2012

[14] M Y Rafii M Shabanimofrad M W Puteri Edaroyati and MA Latif ldquoAnalysis of the genetic diversity of physic nut Jatrophacurcas L accessions using RAPD markersrdquo Molecular BiologyReports vol 36 no 6 pp 6505ndash6511 2012

[15] Y Wang Y Qin and Z Du G Yan ldquoGenetic diversity anddifferentiation of the endangered tree Elaeagnus mollis Diels(Elaeagnus L) as revealed by simple sequence repeat (SSR)markersrdquo Biochemical Systematics and Ecology vol 40 pp 25ndash33 2012

[16] P Tanya P Taeprayoon Y Hadkam and P Srinives ldquoGeneticdiversity among Jatropha and Jatropha-related species based onISSR markersrdquo Plant Molecular Biology Reporter vol 29 no 1pp 252ndash264 2011

[17] H M Abdelmigid ldquoEfficiency of random amplified polymor-phic DNA (RAPD) and inter-simple sequence repeats (ISSR)markers for genotype fingerprinting and genetic diversity stud-ies in canola (Brassica napus)rdquoAfrican Journal of Biotechnologyvol 11 pp 6409ndash6419 2012

[18] F Bardakci ldquoRandom amplified polymorphic DNA (RAPD)markersrdquo Turkish Journal of Biology vol 25 pp 185ndash196 2001

[19] J Ferdous M M Hanafi M Y Rafii and K MuhammadldquoA quick DNA extraction protocol without liquid nitrogen inambient temperaturerdquo African Journal of Biotechnology vol 11no 27 pp 6956ndash6964 2012

[20] D S Resmi V A Celine L Rajamony and K B Sony ldquoDetec-tion of genetic variability in Dramstick (Moringa oleifera Lam)usingRAPDmarkersrdquo inRecent Trends inHorticultural Biotech-nology R Keshvachandran and PA Nazeem Eds pp 587ndash592New India Publishing Agency New Delhi India 2007

[21] M Kimura and J F Crow ldquoThe number of alleles that can bemaintained in a finite populationrdquo Genetics vol 49 pp 725ndash738 1964

[22] R C Lewontine ldquoTesting the theory of natural selectionrdquoNature vol 236 pp 181ndash182 1972

[23] I W Arolu M Y Rafii M M Hanafi T M M Mahmudand M A Latif ldquoMolecular characterization of Jatropha curcasgermplasm using inter simple sequence repeat (ISSR) markersin Peninsular Malaysiardquo Australian Journal of Crop Science vol6 no 12 pp 1666ndash1673 2012

[24] S Verma and T S Rana ldquoGenetic diversity within and amongthe wild populations of Murraya koenigii (L) Spreng asrevealed by ISSR analysisrdquo Biochemical Systematics and Ecologyvol 39 no 2 pp 139ndash144 2011

[25] I B El Hadj Ali A Guetat andM Boussaid ldquoGenetic diversitypopulation structure and relationships of Tunisian Thymusalgeriensis Boiss et Reut and Thymus capitatus Hoffm et linkassessed by isozymesrdquo Industrial Crops and Products vol 36 no1 pp 149ndash163 2012

[26] F Nejatzadeh-Barandozi M R Naghavi S T Enferadi AMousavi Y Mostofi and M E Hassani ldquoGenetic diversity ofaccessions of IranianAloe vera based on horticultural traits andRAPDmarkersrdquo Industrial Crops and Products vol 37 no 1 pp347ndash351 2012

[27] S Kumar S Kumaria S K Sharma S R Rao and P TandonldquoGenetic diversity assessment of Jatropha curcas L germplasmfromNortheast Indiardquo Biomass and Bioenergy vol 35 no 7 pp3063ndash3070 2011

[28] M ParvareshM Talebi and B E Sayed-Tabatabaei ldquoMoleculardiversity and genetic relationship of pomegranate (Punicagranatum L) genotypes using microsatellite markersrdquo ScientiaHorticulturae vol 138 pp 244ndash252 2012

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ofM oleifera seed oil as biodiesel feed source has been testedand recommended by Da Silva et al [12] who reported thatthe oil could be used as pure biodiesel or petrodiesel mixtureon engine after converting it to fatty acid methyl esters(FAME) through the process of transesterification in thepresence of sodium hydroxide (NaOH) as catalyst

Moreover despite the great economic importance ofthis plant in terms of nutritional social and environmentalbenefits the genetic diversity pattern genetic makeup andagronomical requirement needed for successful breeding andimprovement domestication and large scale cultivation areyet to be established This obstacle is an impediment to asuccessful production and commercialization ofmoringa andits related products [6] Also the knowledge of genetic diver-sity of tree crop is very important for rational planning ofconventional modern breeding and improvement programfor the purpose of improving the yield and quality of itsproduce [9 13]

In other words the use of molecular markers such asinter-simple sequence repeat (ISSR) random amplified pol-ymorphic DNA (RAPD) and simple sequence repeat (SSR)has gained popularity as a genetic diversity assessment meth-ods of tree and oil seed crops [14ndash16] Molecular methods ofgenetic diversity study are a fast efficient reliable and simplemeans of establishing genetic diversity pattern in plant [17]The RAPD as one of the numerous molecular markers hasbeen reported to be a reliable reproducible cost effectivefast and less tedious marker which is widely used in thefield of plant breeding and molecular genetics due to itsoutstanding quality [18]

Therefore this research work will study the genetic diver-sity of twenty new genotypes of Moringa oleifera from twopopulations with the aim of studying genetic diversity patternin relation to their geographical origin and dissection ofgermplasms as a means of initiating the breeding programmein the nearest future

2 Materials and Methods

21 Plant Materials These were made up of seeds of twentynew genotypes of M oleifera collected from six differentcountries (Table 1)Themoringa genotypes prior to their col-lection were found in the wild growing in their natural formThe countries of origin are Virgin Island USA ThailandIndia Tanzania Taiwan and Malaysia The collection wasprincipally made by the Asia Vegetable Research and Devel-opment Center (AVRDC) orWorldVegetable Centre Taiwan(15 accessions classified as international population) and theInstitute of Tropical Agriculture universiti Putra Malaysia (5accessions classified as Malaysian population) The collectedseeds were germinated and raised in nursery UniversitiPutra Malaysia Agricultural Park (TPU) for two monthsexposed to the hardening process in the last ten days ofnursery then transplanted out to the Universityrsquos agriculturalexperimental farm in Puchong (02∘N590351015840 101∘E389131015840)Selangor Malaysia Young and disease-free leaves of Moleifera were collected for each of the genotypes during theearly hours of the day the leaves sample were wrapped inaluminum foil and labeled and kept in the freezer at minus10∘C

Table 1 List of moringa genotypes and their countries of origin

Serialnumber

GenotypesID Pedigreecultivar name Origin

country1 T01 Virgin Islands Drum Stick USA2 T02 Ma Rum01 Thailand3 T03 Ma Rum02 Thailand4 T04 Ma Rum03 Thailand5 T05 Ma Rum04 Thailand6 T06 Ma Rum05 Thailand7 T07 Ma Rum Khaw Nheaw Thailand8 T08 Ma Rum06 Thailand9 T09 Ma Rum07 Thailand10 T10 Ma Rum K Thailand11 T11 Tnau-1 India12 T12 Rca Moringa Tanzania13 T13 Ma Rum C Thailand14 T14 Drumstick Tree Pkm-1 India15 T15 La-MuW Taiwan16 M01 ITA-UPM01 Malaysia17 M02 ITA-UPM02 Malaysia18 M03 ITA-UPM03 Malaysia19 M04 ITA-UPM04 Malaysia20 M05 ITA-UPM05 Malaysia

22 DNA Extraction Protocol Six hundred 120583L of extractionbuffer (100mM of Tris-HCl 20mM of EDTA 14M NaCland 5 SDS) was added to 10mg leaf sample and groundwith mortar and pestle without liquid nitrogen accordingto Ferdous et al [19] The finely ground leaf tissue wastransferred into 2mL centrifuge tube Four hundred 120583L of2X CTAB solution 100mM of Tris-HCl 20mM of ethylene-diaminetetraacetic acid di-sodium salt (EDTA) 14M ofsodium chloride (NaCl) 2 (wv) CTAB and 1 (wv) ofpolyvinyl pyrrolidone (PVP) and 400 120583L chloroform isoamylalcohol phenol (24 1 5) mixture were then added to theleaf tissue containing the extraction buffer After mixingthrough vortex and centrifuge the supernatant was trans-ferred to another tube Two-third of volume of isopropanolwas added and incubated at room temperature for 10 to15min Centrifuged supernatant was then removed and theDNA pellet was washed using 70 ethanol afterwards theDNA pellet was air-dried and dissolved into 50 120583L TE buffer

23 DNA Quantification and Dilution The quantification ofDNAwas carried out using NanoDrop ND-1000 spectropho-tometer (NanoDrop Technologies Wilmington USA) TheDNAwas again requantified by running it through 1agarosegel electrophoresis with 1timesTAE buffer for 30min and viewedunder UV light after staining it withMidori green DNA stain(Nippon Genetics Inc Germany) The dilution was donewith sterile distilled water to ensure that all of the DNAsamples have equal concentration of 100 ng120583L







3 Results












1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20M








4 Discussion










038 050 063 076 089

T-01


Coefficient

I

II

III

IV

V


M-05

M-04

M-03

M-02

M-01

T-15

T-14 T-13T-12

T-11T-10

T-09

T-08

T-07

T-06

T-05

T-04

T-03

T-02

T-01

026003

Dim-2049

060

Dim-3

022

Dim-1 070

045

068

081092

minus065

minus042

minus020

000






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







3 Results












1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20M








4 Discussion










038 050 063 076 089

T-01


Coefficient

I

II

III

IV

V


M-05

M-04

M-03

M-02

M-01

T-15

T-14 T-13T-12

T-11T-10

T-09

T-08

T-07

T-06

T-05

T-04

T-03

T-02

T-01

026003

Dim-2049

060

Dim-3

022

Dim-1 070

045

068

081092

minus065

minus042

minus020

000






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20M








4 Discussion










038 050 063 076 089

T-01


Coefficient

I

II

III

IV

V


M-05

M-04

M-03

M-02

M-01

T-15

T-14 T-13T-12

T-11T-10

T-09

T-08

T-07

T-06

T-05

T-04

T-03

T-02

T-01

026003

Dim-2049

060

Dim-3

022

Dim-1 070

045

068

081092

minus065

minus042

minus020

000






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


038 050 063 076 089

T-01


Coefficient

I

II

III

IV

V


M-05

M-04

M-03

M-02

M-01

T-15

T-14 T-13T-12

T-11T-10

T-09

T-08

T-07

T-06

T-05

T-04

T-03

T-02

T-01

026003

Dim-2049

060

Dim-3

022

Dim-1 070

045

068

081092

minus065

minus042

minus020

000






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

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