research article genetic diversity of parkia biglobosa from...

Research ArticleGenetic Diversity of Parkia biglobosa from DifferentAgroecological Zones of Nigeria Using RAPD Markers

Oluwafemi Amusa12 Adenubi Adesoye2 Adebayo Ogunkanmi1 Ojobo Omoche2

Olumayowa Olowe2 Solomon Akinyosoye3 and Taiwo Omodele3

1 Department of Cell Biology and Genetics University of Lagos Akoka Nigeria2 Department of Botany University of Ibadan Ibadan Nigeria3 Institute of Agricultural Research and Training Moor Plantation Ibadan Nigeria

Correspondence should be addressed to Oluwafemi Amusa femisworldyahoocom and Adenubi Adesoye aadesoyeyahoocom

Received 13 August 2014 Accepted 16 October 2014 Published 9 November 2014

Academic Editor Alexandre Sebbenn

Copyright copy 2014 Oluwafemi Amusa 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

Parkia biglobosa (Jacq) is an important leguminous tree crop in the African Savannahs useful to the natives where it is found fordomestic use Previous diversity studies on this tree crop had been majorly on morphological and biochemical analysis In order tocapture the maximum diversity not obtained by previous research the study aimed at evaluating the genetic diversity of accessionsof this crop in the different agroecological zones in Nigeria using RAPD markers A total of 81 scorable bands with an averageof 81 bands per primer were amplified among the accessions studied Intrazonal genetic diversity analysis showed a percentagepolymorphism with a range of 1111 to 6543 among the agroecological zones studied Although gene diversity was highestwithin Humid forest agroecological zone a low genetic distance and high genetic similarity between the agroecological zones wereobserved Cluster analysis indicated six main groups of which four groups had single accessions while the two groups clustered theremaining accessions indicating a narrowed genetic base from the 23 accessions studied

1 Introduction

TheAfrican locust bean plant (Parkia biglobosa) is a perennialtree legume belonging to the subfamily Mimosoideae of thefamily Fabaceae It is one of the thirty-four known species ofthe genus Parkia whose centre of origin is South AmericaDistributed in a belt between latitudes 5∘N and 15∘N andlongitudes 16∘Wand 32∘W from the Atlantic coast in Senegalto Southern Sudan and Northern Uganda in the Africancontinent it has its greatest belt (about 800Km) in WestAfrica and narrows to the east [1 2]

Aside from its ecological role in cycling of nutrients [3 4]it is a valuable source of food especially the seedswhich servesas a source of useful spices for cooking [1 5 6] Kwon-Ndunget al [7] reported that it is a very important tree crop in theAfrica Savannas where the natives use it as medicine glazefor ceramic pots fodder firewood and charcoal productionParkia tree is also used as timber for making pestles mortars

bows hoe handles and seats [8 9] while the husks and podsare good food for livestock [10]

Previously studies on diversity within this speciesfocused on accessions from West African countries likeBurkina Faso Benin and Uganda with only three accessionscollected from the Southern Guinea Savanna in Nigeria[11ndash13] These studies dealt majorly with phenotypic andanatomical diversity within species of the plant P biglobosais however known to occur in diverse agroecological zonesin Nigeria ranging from tropical rain forests to arid zonesfrom the lower Sudan Savanna to Derived Savanna and theLowland forest Adesoye et al [14] reported variations inaccessions of P biglobosa collected across different agroeco-logical zones (AEZ) of Nigeria based on seedling morpho-logical characteristics evaluated

Beyond the phenotypic and anatomical estimations ofdiversity the heritable variations observed in plants (termedplant genetic diversity) can also be evaluated using agro-morphological biochemical and molecular data Molecular

Hindawi Publishing CorporationInternational Journal of BiodiversityVolume 2014 Article ID 457309 6 pageshttpdxdoiorg1011552014457309

2 International Journal of Biodiversity

diversity however outweighs others since it is less affected byenvironmental factors Maintaining genetic diversity withinspecies is important for effective conservation of geneticresources ensuring variation for crop improvement andadaptability to the ever-changing environmental challenges

Owing to the limited information on the species diver-sity in Nigeria this study sought to elucidate the geneticdiversity among the Nigerian accessions of P biglobosa fromseven AEZ using rapid amplified polymorphic DNA (RAPD)markers Although RAPD markers are criticized for theirlack of reproducibility yet they are thought to be goodmarkers for elucidating overall genetic diversity especiallywhere the diversity of such species has not been previouslyknown It has been used for genetic diversity assessmentand for identifying germplasm in a number of plant species[15 16] Subramanyamet al [17] reported that RAPDprovidesinformation that can help define the distinctiveness of speciesand phylogenetic relationships at molecular level

2 Materials and Methods

21 Plant Material To carry out our genetic study open-pollinated seeds were collected from twenty-three (23) acces-sions of P biglobosa collected from different states andlocations within the seven (7) agroecological zones in Nigeria(Table 1 Figure 1)

Dry seeds of each accession per zone were tested forviability using the simple floating test The seeds consideredviable after the test were collected for planting To breakthe seed dormancy the selected seeds were immersed inconcentrated hydrogen tetraoxosulphate (VI) acid for 15minutesThe seeds were rinsed trice in distilled water and airdried at room temperature before planting in plastic bags

22 DNA Extraction and RAPD Analysis Sample DNA wasextracted from 03 g young leaves using modified Dellaportaet al [18] protocol The extracted DNA was dissolved inlow Tris EDTA buffer and stored at minus20∘C until use Tendecamer primers were used to evaluate genetic diversityamong the accessions collected PCR reaction was carried outin a 25 120583L volume reaction (25 120583L 10x buffer 12 120583L MgCl

2

20 120583L dNTPs 10 120583L DMSO 10 120583L primer 02 120583L Taq 10 120583LDNA template and 161 120583L dH

2O) The mixture was overlaid

with 20120583L of sterile mineral oilDNA amplification reaction was carried out in a Perkin

Elmer Centus Thermocycler For each amplification pro-cess a preheating denaturation of DNA at 94∘C for 3minwas done followed by 44 amplification cycles (each 94∘Cfor 20 secs 35∘C for 40 secs and 72∘C for 1min) A finalextension of 7min at 72∘C was performed The amplifiedfragments were then run on 15 agarose in 05M TBEbuffer at 100V for 3 hours Gels were stained with 05 120583gmLethidium bromide and visualized under a high-performanceUV light The observed bands were scored 1 where presentand 0 where absent The data generated were analyzed usingNumerical Taxonomic System of Statistics (NTSYS) andPopgene 131 software Nei and Li [19] formula was used toestimate the genetic similarities distances and percentage

Table 1 P biglobosa collections from different agroecological zonesof Nigeria

State Location Accession numberAbuja Municipal P3Bauchi Katagum P22Benue Vandiekya P5Benue Akpagher Gboko P11Delta Kaboro P9Gombe Kaltungo P14Kaduna Chikun P23Kaduna Zaira P24Kano Nasarawa P15Katsina Mani P13Niger Doko Village P6Ogun Abeokuta Abeokuta North P4Ogun Ago Sasa Ipokia P36Ondo Oba Akoko P2Ondo Akungba Akoko P7Ondo Enpare Akoko P8Oyo NIHORT Ibadan P1Oyo Saki Saki East P12Plateau Isaya Bassa P10Plateau Guanawuri Riyom P31Plateau Soukwa P32Yobe Bade Gashua P16Yobe Ubu P28

Table 2 List of RAPD primers used

Primers Sequence 51015840 rarr 31015840 Polymorphic bandsOPD-17 -TTTCCCACGG- 12OPAD-05 -ACCGCATGGG- 10OPAE-11 -AAGACCGGGA- 6OPAE-14 -GAGAGGCTCC- 6OPAC-11 -CCTGGGTCAG- 6OPAE-09 -TGCCACGAGG- 4OPB-01 -GTTTCGCTCC- 11OPB-02 -TGATCCCTGG- 7OPF-10 -GGAAGCTTGG- 10OPG-03 -GAGCCCTCCA- 9

polymorphisms within and between the accessions The totalnumber of alleles observed and alleles effective in detectingpolymorphismwere also recorded Dendrogram showing therelationship between the accessions was constructed fromJaccardrsquos similarity coefficients using unweighted pair groupmethod of arithmetic means (UPGMA)

3 ResultsThe bands generated ranged from 700 to 6000 base pairs (bp)with majority falling between 1000 and 4000 bp (Figure 2)

The polymorphic bands observed ranged from 6 to 12(Table 2) with an average of 81 bands per primer A total of81 scorable bands were amplified among the 23 accessions ofP biglobosa studied

International Journal of Biodiversity 3

P13 P16

P28P22P15

P24

P23

P3

P6

P14P10P32P31

P12

P1

P4

P36

P8 P2P7

P5P11

Sokoto

Kebbi ZamfaraKatsina

Kano

Jigawa

Yobe

Borno

GombeAdamawa

TarabaBenue

EnuguAnambra

AbiaImd

AkwaIbgmRiversBayelsa

EbonyiCrossRiver

Delta

EdoOndoOgun

Lagos

OsunEkiti Kogi

Kwara

Nassarawa

Niger

Oyo

Kaduna

Plateau

Bauchi

Lake

Chad

N

Accession locationState boundaryLake

Agroecological zone

Sahel savannaSudan savannaNorthren Guinea savannaMidaltitudeSouthern Guinea savannaDerived savannaHumid forest

150 75 0 150 300(km)

3∘09984000998400998400E 6

∘09984000998400998400E 9

∘09984000998400998400E 12

∘09984000998400998400E

3∘09984000998400998400E 6

∘09984000998400998400E 9

∘09984000998400998400E 12

∘09984000998400998400E

6∘09984000998400998400N

9∘09984000998400998400N

12∘09984000998400998400N

6∘09984000998400998400N

9∘09984000998400998400N

12∘09984000998400998400N

Figure 1 Geographical locations of Parkia accessions used for study analysis

The percentage polymorphism ranged from 1111 to6543 (Table 3) with the two agroecological zones (DerivedSavanna and Humid forest) showing the highest number(53) of polymorphic loci and percentage polymorphism(6543) while North Guinea Savanna had the least num-ber of (9) polymorphic loci and percentage polymorphism(1111)

Within the agroecological zones two agroecologicalzones showed the highest number of observable alleles of 165plusmn 048 with 121 plusmn 024 and 125 plusmn 024 effective number ofalleles for derived Savanna and for Humid forest respectively(Table 3) However North Guinea Savanna showed the leastobserved number of alleles (111 plusmn 032) with 109 plusmn 026effective number of alleles Analysis showed that the greatestgene diversity was found in Humid forest (018 plusmn 014) andleast diversity in Sudan Savanna (005 plusmn 014) and NorthGuinea Savanna (005 plusmn 013)

Interzonal genetic diversity between the various AEZsindicated a high genetic similarity and consequently a lowgenetic distance between the various AEZs consideredAccessions from Sudan Savanna showed the highest percent-age genetic similaritywith those collected fromNorthGuineaSavanna (9951) and Southern Guinea Savanna Neirsquos coeffi-cient showed the highest genetic distance accessions of SudanSavanna andDerived Savannawith genetic distance of 1454(Table 4)

Cluster analysis of the dendrogram (Figure 3) con-structed from Jaccardrsquos similarity coefficient showed sixgroups four contained one accession each one group con-tained two accessions (P3 and P4) and the last groupcontained the remaining seventeen (17) accessions at Jaccardrsquoscoefficient of 070 The accessions P31 and P32 showed thehighest similarity followed by P5 and P13 with Jaccardrsquossimilarity coefficient of 099 and 095 respectively


Table 3 Genetic diversity analysis within each agroecological zone

AEZ ONA ENA GD NPL PP ()1 132 plusmn 047 117 plusmn 028 011 plusmn 016 26 32102 114 plusmn 034 110 plusmn 024 006 plusmn 014 11 13583 111 plusmn 032 109 plusmn 026 005 plusmn 014 9 11114 114 plusmn 034 108 plusmn 023 005 plusmn 013 11 13585 130 plusmn 046 121 plusmn 032 012 plusmn 019 24 29636 165 plusmn 048 121 plusmn 024 015 plusmn 014 53 65437 165 plusmn 048 125 plusmn 024 018 plusmn 014 53 6543Values are represented inmean plusmn SD AEZ Agroecological zones ONA observed number of alleles ENA effective number of alleles GD gene diversity NPLnumber of polymorphic loci PP percentage polymorphism 1 Sahel Savanna 2 Sudan Savanna 3 North Guinea Savanna 4 Midaltitude 5 South GuineaSavanna 6 Derived Savanna 7 Humid forest

Table 4 Genetic correlation between agroecological zones

AEZ 1 2 3 4 5 6 71 099 099 098 089 097 0962 001 099 099 086 098 0993 002 001 098 088 098 0984 002 001 020 087 097 0975 012 014 012 014 091 0886 004 002 002 002 009 0987 004 001 003 003 012 002Neirsquos genetic similarity index (above diagonal) and genetic distance index(below diagonal) AEZ agroecological zones 1 Sahel Savanna 2 SudanSavanna 3 North Guinea Savanna 4 Midaltitude 5 South Guinea Savanna6 Derived Savanna 7 Humid forest

4 Discussion

The results obtained from RAPD analysis of P biglobosaindicate intra- and interpopulation variations from the acces-sions evaluations were low Similar results were reported inthe inter- and intrapopulation variation study of P biglobosagenetic diversity using isoenzymatic electrophoresis by Sina[13] The ten RAPD primers used generated 81 bands anda polymorphic range of 1111ndash6543 similar to that ofThangjam et al [20] which reported 95 scorable bands fromtwelve primers and a polymorphic range of 0ndash5714 in astudy of genetic diversity of a close relative P timoriana Theresult shows that high polymorphism exists in the accessionsof P biglobosa studied Monteleone et al [21] also reported64 polymorphic bands from ten RAPD markers in theirstudy and were able to detect genetic diversity differences infifteen populations of subspecies of Pinus mugo from theirevaluation

Ouedraogo [11] reported a large variation in P biglobosabetween the West African provenances in terms of adapt-ability and morphology attributed to several factors amongwhich are genetics and environment Adesoye et al [14] alsoreported a wide genetic variance among the accessions ofP biglobosa evaluated using seedling morphological traitswhich may be attributed to the different environmentalinfluence from the various AEZs Neirsquos genetic diversityanalysis from this present study indicated an overall lowgenetic diversitywithin theAEZs with theHumid forest zone

having the highest gene diversity of 018 plusmn 014 This resultcorroborates Hamrick and Godt [22] that species with smallgeographic ranges tend tomaintain less genetic diversity thangeographically widespread species The highest percentagepolymorphism observed in both the derived Savanna andHumid forestmay be attributed to the fact that they constitutethe largest AEZs in Nigeria as well as the zone with thehighest number of accessions studied Likewise among theAEZs studied the genetic diversity was generally low thusindicating a narrow genetic base among the accessions ofthe different zones This has implications in considerationsfor the conservation In this regard Schaal et al [23] statedthat genetic diversity is a prerequisite for future adaptivechange and evolution and has a profound effect for speciesconservation The genetic distance was least between theSudan Savanna and Northern Guinea Savanna These zonesare close in terms of location and P biglobosa being an openpollinating plant allows for gene flow between populationscorroborating the work of Sayed et al [24] that there ispossibly free or random pollen flow and fertilization in closenatural populations hence narrowing the genetic distancebetween them

The present study shows a low genetic distance (001 to020) from the RAPD marker analysis indicating a commonancestry Sina [13] reported a similar result (genetic distancerange 0 to 024) by means of isoenzymatic analysis andindicated that the presence of a low genetic distance showsthat the populations were similar enough to belong tothe same genetic group The genetic diversity and geneticstructure of P biglobosa result from its evolutionary historywithout bottleneck sequence the reproductive biology withhigh flowering synchronism and outcrossing rates and theP biglobosa parkland systems that human developed throughyears and as such have facilitated gene flow between popula-tions [13]

The distribution of genotypes among clusters did notcorrespond accurately with their geographical pattern Clus-ter analysis shows that the subclusters were more or lessheterogeneous and contained accessions from different AEZswith a relatively high Jaccardrsquos similarity coefficient of 070Similar result was reported by Adesoye et al [14] fromtheir evaluation of morphological differences in seedlingtraits of Parkia accessions collected from different NigerianAEZsThey suggested that the situation was possible because


10000

8000

6000

5000

4000

3000

2500

2000

1500

1000

700

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23(bp)

Figure 2 Amplified RAPD polymorphic bands of OPF-10 across the 23 accessions of P biglobosa

P1P5P13P14P36P16P9P10P31P32P22P23P24P15P11P8P6P12P7P3P4P28P2

057 068 078 088 099

Coefficient

Dendrogram of 23 cultivars of parkia using UPGMA option in NTSAYSpc

Figure 3 Dendrogram results from RAPD analysis showing the relationship between the 23 accessions of P biglobosa P1-Oyo (Derived) P2-Ondo (Humid) P3-Abuja (Derived) P4-Ogun (Derived) P5-Benue (Derived) P6-Niger (S Guinea) P7-Ondo (Humid) P8-Ondo (Humid)P9-Delta (Derived) P10-Plateau (Midaltitude) P11-Benue (Derived) P12-Oyo (Derived) P13-Katsina (Sudan) P14-Gombe (N Guinea) P15-Kano (Sudan) P16-Yobe (Sahel) P22-Bauchi (N Guinea) P23-Kaduna (N Guinea) P24-Kaduna (N Guinea) P28-Yobe (N Guinea) P31-Plateau (Midaltitude) P32-Plateau (Midaltitude) and P36-Ogun (Humid)


of different genetic makeup of genotypes and subsequentnatural selection during their development The crossingbetween genotypes belonging to same clusters is not likely togive heterotic desirable segregants as suggested by Adesoyeet al [14] Additionally the frequency of seed exchangeover wide geographic-ethnic regions is thought to also beresponsible for this outcome as Ntudu [25] from survey ofseed markets assessment reported that seeds are generallyexchanged between small farmers and marketers in Africa

In conclusion while the occurrence of a narrow geneticbase observed between the accessions of P biglobosa maymean that they resulted from a common gene pool it isalso possible that this high genetic similarity may also resultfrom its outcrossing nature over a long time resulting in highhomozygosity This makes it important for further researchefforts using more specific molecular markers to ensure abetter understanding of the diversity in P biglobosa so asto enhance the selection of germplasm in conservation ofthe species as well as fostering a better understanding of thediversity within the species

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] G Campbell-Platt ldquoAfrican locust bean and itrsquos West Africanfermented products-Dadawardquo Ecology of Food and Nutritionvol 9 pp 123ndash132 1980

[2] B Hopkins ldquoThe taxonomy reproductive biology and eco-nomic potentials of Parkia in Africa and Madagascarrdquo BotanyJournal of Linnean Society vol 87 pp 135ndash167 1983

[3] A E Gbadamosi ldquoGenetic variation in Enantia chlorantha(oliv) A medicinal plantrdquo Journal of Food Agriculture andEnvironment vol 3 no 1 pp 153ndash156 2005

[4] C E Udobi J A Onaolapo and A Agunu ldquoAntibacterialpotentials of the methanolic extract and aqueous fraction ofthe stem bark of the African locust bean (Parkia biglobosa)rdquoEuropean Journal of Scientific Research vol 43 no 4 pp 590ndash602 2010

[5] D A Alabi O R Akinsulire and M A Sanyaolu ldquoNutritiveand industrial utility values of African locust bean seeds Parkiabiglobosa (Jacq) Benthrdquo Proceedings of Science Association ofNigeria vol 25 pp 105ndash110 2004

[6] C Orwa A Mutua R Kindt R Jamnadass and A SimonsldquoAgroforestree Database a tree reference and selection guideversion 40rdquo httpwwwworldagroforestryorgsitestreedbstreedatabasesasp

[7] E H C Kwon-Ndung C D Chup N A Binbol and AIsmaila ldquoEcological implications of climate change on thegenetic diversity and distribution of African locust bean parkiabiglobossa in Central Nigeriardquo IOPConference Series Earth andEnvironmental Science vol 6 no 37 Article ID 372026 2009

[8] F R Irvine Woody Plants of Ghana with Special References toTheir Trees Oxford University Press London UK 1961

[9] T H Hagos ldquoA revision of genius Parkia (Mim) in Africardquo ActaBotanica Nearthandica vol 11 pp 231ndash265 1962

[10] I C Obizobia Fermentation of African Locust Bean Text onNutritional Quality of Plant Fruits Edited by A U Osagieand O U Eka Post-Harvest Research Unit Department ofBiochemistry University of Benin Benin City Nigeria 2000

[11] A S Ouedraogo Parkia biglobosa (Leguminosae) en Afrique delrsquoOuest biosystematique et amelioration [PhD thesis] Instituteof Forestry and Natural Resources Wageningen The Nether-lands 1995

[12] Z Teklehaimanot J Lanek and H F Tomlinson ldquoProvenancevariation inmorphology and leaflet anatomy ofParkia biglobosaand its relation to drought tolerancerdquoTrees vol 13 no 2 pp 96ndash102 1998

[13] S Sina Reproduction et diversite genetique chez Parkia biglobosa(Jacq) [G Don doctoral thesis] Wageningen University andResearch Centre Wageningen The Neitherlands 2006

[14] A I Adesoye C O Ogunremi and O O Aina ldquoGenetic varia-tion and heritability of seedling traits in African locust BeanmdashParkia biglobosa (Jacq) RBr Ex GDonrdquo Legume Research vol36 no 2 pp 89ndash97 2013

[15] J Welsh and M McClelland ldquoFingerprinting genomes usingPCR with arbitrary primersrdquoNucleic Acids Research vol 18 no24 pp 7213ndash7218 1990

[16] J Kapteyn and J E Simon ldquoThe use of RAPDs for assessmentof identity diversity and quality of Echinaceardquo inTrends in NewCrops and New Uses A Janick and A Whipkey Eds pp 509ndash513 ASHS Press Alexandria Va USA 2002

[17] K Subramanyam D Muralidhararao and N DevannaldquoGenetic diversity assessment of wild and cultivated varieties ofJatropha curcas (L) in India by RAPD analysisrdquo African Journalof Biotechnology vol 8 no 9 pp 1900ndash1910 2009

[18] S L Dellaporta J Wood and J B Hicks ldquoA plant DNAminipreparation version IIrdquo Plant Molecular Biology Reportervol 1 no 4 pp 19ndash21 1983

[19] M Nei andW H Li ldquoMathematical model for studying geneticvariation in terms of restriction endonucleasesrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 76 no 10 pp 5269ndash5273 1979

[20] R Thangjam D Maibam and J G Sharma ldquoDetection ofgenetic diversity in Parkia timoriana (DC) Merr using ran-domly amplified polymorphic DNA analysisrdquo Journal of FoodAgricultural Environment vol 1 no 3-4 pp 46ndash49 2003

[21] I Monteleone D Ferrazzini and P Belletti ldquoEffectiveness ofneutral RAPD markers to detect genetic divergence betweenthe subspecies uncinata and mugo of Pinus mugo Turrardquo SilvaFennica vol 40 no 3 pp 391ndash406 2006

[22] J L Hamrick and M J W Godt ldquoAllozyme diversity in plantsrdquoin Plant Population Genetics Breeding and Genetic Resources DA H Brown M T Clegg A L Kahler and B S Weir Eds pp43ndash63 Sinauer Sunderland Mass USA 1989

[23] B A Schaal D A Hayworth K M Olsen J T Rauscher andW A Smith ldquoPhylogeographic studies in plants problems andprospectsrdquoMolecular Ecology vol 7 no 4 pp 465ndash474 1998

[24] M Z H Sayed S B S SMohammed and M S RamisahldquoAnalysis of random amplified polymorphic DNA (RAPD) ofArtemisia capillaries (Worm wood capillary) in east coast ofPenisular Malaysiardquo World Applied Science Journal vol 6 no7 pp 976ndash986 2009

[25] W H Ntudu Genetic diversity of Bambara groundnut (Vignasubterraneea (L) Verdc) in Tanzania [PhD thesis] The RoyalVeteriary and Agricultural University Copenhagen Denmark2002

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


diversity however outweighs others since it is less affected byenvironmental factors Maintaining genetic diversity withinspecies is important for effective conservation of geneticresources ensuring variation for crop improvement andadaptability to the ever-changing environmental challenges

Owing to the limited information on the species diver-sity in Nigeria this study sought to elucidate the geneticdiversity among the Nigerian accessions of P biglobosa fromseven AEZ using rapid amplified polymorphic DNA (RAPD)markers Although RAPD markers are criticized for theirlack of reproducibility yet they are thought to be goodmarkers for elucidating overall genetic diversity especiallywhere the diversity of such species has not been previouslyknown It has been used for genetic diversity assessmentand for identifying germplasm in a number of plant species[15 16] Subramanyamet al [17] reported that RAPDprovidesinformation that can help define the distinctiveness of speciesand phylogenetic relationships at molecular level

2 Materials and Methods

21 Plant Material To carry out our genetic study open-pollinated seeds were collected from twenty-three (23) acces-sions of P biglobosa collected from different states andlocations within the seven (7) agroecological zones in Nigeria(Table 1 Figure 1)

Dry seeds of each accession per zone were tested forviability using the simple floating test The seeds consideredviable after the test were collected for planting To breakthe seed dormancy the selected seeds were immersed inconcentrated hydrogen tetraoxosulphate (VI) acid for 15minutesThe seeds were rinsed trice in distilled water and airdried at room temperature before planting in plastic bags

22 DNA Extraction and RAPD Analysis Sample DNA wasextracted from 03 g young leaves using modified Dellaportaet al [18] protocol The extracted DNA was dissolved inlow Tris EDTA buffer and stored at minus20∘C until use Tendecamer primers were used to evaluate genetic diversityamong the accessions collected PCR reaction was carried outin a 25 120583L volume reaction (25 120583L 10x buffer 12 120583L MgCl

2

20 120583L dNTPs 10 120583L DMSO 10 120583L primer 02 120583L Taq 10 120583LDNA template and 161 120583L dH

2O) The mixture was overlaid

with 20120583L of sterile mineral oilDNA amplification reaction was carried out in a Perkin

Elmer Centus Thermocycler For each amplification pro-cess a preheating denaturation of DNA at 94∘C for 3minwas done followed by 44 amplification cycles (each 94∘Cfor 20 secs 35∘C for 40 secs and 72∘C for 1min) A finalextension of 7min at 72∘C was performed The amplifiedfragments were then run on 15 agarose in 05M TBEbuffer at 100V for 3 hours Gels were stained with 05 120583gmLethidium bromide and visualized under a high-performanceUV light The observed bands were scored 1 where presentand 0 where absent The data generated were analyzed usingNumerical Taxonomic System of Statistics (NTSYS) andPopgene 131 software Nei and Li [19] formula was used toestimate the genetic similarities distances and percentage

Table 1 P biglobosa collections from different agroecological zonesof Nigeria

State Location Accession numberAbuja Municipal P3Bauchi Katagum P22Benue Vandiekya P5Benue Akpagher Gboko P11Delta Kaboro P9Gombe Kaltungo P14Kaduna Chikun P23Kaduna Zaira P24Kano Nasarawa P15Katsina Mani P13Niger Doko Village P6Ogun Abeokuta Abeokuta North P4Ogun Ago Sasa Ipokia P36Ondo Oba Akoko P2Ondo Akungba Akoko P7Ondo Enpare Akoko P8Oyo NIHORT Ibadan P1Oyo Saki Saki East P12Plateau Isaya Bassa P10Plateau Guanawuri Riyom P31Plateau Soukwa P32Yobe Bade Gashua P16Yobe Ubu P28

Table 2 List of RAPD primers used

Primers Sequence 51015840 rarr 31015840 Polymorphic bandsOPD-17 -TTTCCCACGG- 12OPAD-05 -ACCGCATGGG- 10OPAE-11 -AAGACCGGGA- 6OPAE-14 -GAGAGGCTCC- 6OPAC-11 -CCTGGGTCAG- 6OPAE-09 -TGCCACGAGG- 4OPB-01 -GTTTCGCTCC- 11OPB-02 -TGATCCCTGG- 7OPF-10 -GGAAGCTTGG- 10OPG-03 -GAGCCCTCCA- 9

polymorphisms within and between the accessions The totalnumber of alleles observed and alleles effective in detectingpolymorphismwere also recorded Dendrogram showing therelationship between the accessions was constructed fromJaccardrsquos similarity coefficients using unweighted pair groupmethod of arithmetic means (UPGMA)

3 ResultsThe bands generated ranged from 700 to 6000 base pairs (bp)with majority falling between 1000 and 4000 bp (Figure 2)

The polymorphic bands observed ranged from 6 to 12(Table 2) with an average of 81 bands per primer A total of81 scorable bands were amplified among the 23 accessions ofP biglobosa studied


P13 P16

P28P22P15

P24

P23

P3

P6

P14P10P32P31

P12

P1

P4

P36

P8 P2P7

P5P11

Sokoto


Kano

Jigawa

Yobe

Borno

GombeAdamawa

TarabaBenue

EnuguAnambra

AbiaImd


EbonyiCrossRiver

Delta

EdoOndoOgun

Lagos

OsunEkiti Kogi

Kwara

Nassarawa

Niger

Oyo

Kaduna

Plateau

Bauchi

Lake

Chad

N


Agroecological zone


150 75 0 150 300(km)

3∘09984000998400998400E 6

∘09984000998400998400E 9

∘09984000998400998400E 12

∘09984000998400998400E

3∘09984000998400998400E 6

∘09984000998400998400E 9

∘09984000998400998400E 12

∘09984000998400998400E

6∘09984000998400998400N

9∘09984000998400998400N

12∘09984000998400998400N

6∘09984000998400998400N

9∘09984000998400998400N

12∘09984000998400998400N











4 Discussion







10000

8000

6000

5000

4000

3000

2500

2000

1500

1000

700

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23(bp)



057 068 078 088 099

Coefficient








References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


P13 P16

P28P22P15

P24

P23

P3

P6

P14P10P32P31

P12

P1

P4

P36

P8 P2P7

P5P11

Sokoto


Kano

Jigawa

Yobe

Borno

GombeAdamawa

TarabaBenue

EnuguAnambra

AbiaImd


EbonyiCrossRiver

Delta

EdoOndoOgun

Lagos

OsunEkiti Kogi

Kwara

Nassarawa

Niger

Oyo

Kaduna

Plateau

Bauchi

Lake

Chad

N


Agroecological zone


150 75 0 150 300(km)

3∘09984000998400998400E 6

∘09984000998400998400E 9

∘09984000998400998400E 12

∘09984000998400998400E

3∘09984000998400998400E 6

∘09984000998400998400E 9

∘09984000998400998400E 12

∘09984000998400998400E

6∘09984000998400998400N

9∘09984000998400998400N

12∘09984000998400998400N

6∘09984000998400998400N

9∘09984000998400998400N

12∘09984000998400998400N











4 Discussion







10000

8000

6000

5000

4000

3000

2500

2000

1500

1000

700

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23(bp)



057 068 078 088 099

Coefficient








References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






4 Discussion







10000

8000

6000

5000

4000

3000

2500

2000

1500

1000

700

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23(bp)



057 068 078 088 099

Coefficient








References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


10000

8000

6000

5000

4000

3000

2500

2000

1500

1000

700

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23(bp)



057 068 078 088 099

Coefficient








References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






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

research article genetic diversity of parkia biglobosa from...

Documents